Meat Science and Applications

Meat Science and

Applications edited by

Y H. Hu. Science Technology System West Sacramento, California

Wai-Kit Nip University of Hawaii at Manoa Honolulu, Hawaii

Robert W. Rogers Mississippi State University Mississippi State, Mississippi

Owen A. Young MIRINZ Centre AgResearch Hamilton, New Zealand

MARCEL DEKKER, INC. D E K K E R

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ISBN: 0-8247-0548-3 This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc. 270 Madison Avenue, New York, NY 10016 tel: 212-696-9000; fax: 212-685-4540 Eastern Hemisphere Distribution Marcel Dekker AG Hutgasse 4, Postfach 812, CH-4001 Basel, Switzerland tel: 41-61-261-8482; fax: 41-61-261-8896 World Wide Web http://www.dekker.com The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the headquarters address above.

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In Honor and Memory of Mary Demas Rogers February 3, 1941 to October 24, 1999 Great appreciation is expressed to the authors and editors for allowing me the privilege of dedicating this book in honor and memory of my late wife, Mary Demas Rogers, who became ill and died during the book’s preparation. She was a dear and true friend in addition to being an extraordinary Christian wife, mother, grandmother, and nurse. She was a wonderful, caring person, befriended by many, old and young alike. Well known for her compassion, Mary carried herself in a way that exuded all the above attributes, and never faltered in her quest to provide for the needs of others. A steadfast person, always a lady; she is sorely missed and remembered by so many with true love and respect. Robert W. Rogers

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Preface

Consumption of red meat and meat products has a long history in most cultures. Meat is a source of nutrients, as well as a sign of wealth in some countries. Various techniques have been developed in different parts of the world over the centuries to preserve meat for extended shelf life and enjoyment. Even nonedible parts of animals are used for various reasons. Thus, meat, meat products, and by-products are important to our daily life. In the past two decades, many books on the science and processing of meats and meat products have been published. Many of these were useful reference and classroom texts. However, most of these books are limited in their intended focus. Meat Science and Applications is a professional reference book organized similarly to a classroom text. The volume covers the following major areas: science, safety, slaughtering, carcass evaluation, meat processing, workers’ safety, and waste management. However, this book differs from others in the market in several aspects. It offers comprehensive coverage in depth and breadth; separate yet integrated approaches; and discussion of the most recent science, technology, and applications. This reference book will be useful to research professionals in government, industry, and academia. Worldwide, many scientists and technologists join the meat-packing industry with degrees in basic or applied sciences, such as chemistry, food science, and engineering, and with only rudimentary understanding of meat properties and processing. These scientists are steeped in scientific principles but lack industrial experience. This book bridges this gap and links the science of meat and meat processing to today’s technology. The fundamentals of slaughter and processing have changed little over the centuries, except for the introduction and use of refrigeration. A key difference between meat processing and many other industrial practices is the inherent variability of animals and their meat. Application of science and technology to the meat industry has been slow. To help put it in perspective, consider that new technologies to measure parameters associated with meat processing mean that the application of control at critical points is feasible, and modern computing helps make the statistical approach to control easier.

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With the participation of over 45 contributors, we took on the challenge of assembling in one volume up-to-date information on major topics related to meat processing. In 27 chapters, this work provides the readers with a convenient reference book. We have drawn international expertise from professionals in five countries to realize this goal. Y. H. Hui Wai-Kit Nip Robert W. Rogers Owen A. Young

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Acknowledgments

The production of a volume of this size could not have been accomplished without the excellent cooperation of the production team at Marcel Dekker, Inc. Our editorial team appreciates their assistance, especially that of Ms. Theresa Dominick in her coordinating effort during the production phase of this handbook. Y. H. Hui I’d to take this opportunity to thank some of my former students and colleagues for agreeing to contribute chapters to this book, as well as my family, who have been very understanding during this time. The assistance of the Hamilton Library of the University of Hawaii at Manoa on the literature search for “Intermediate-Moisture Meat and Dehydrated Meat” (Chapter 17) is gratefully acknowledged. Wai-Kit Nip I wish to personally thank the authors for all the hard work they did in preparing the manuscripts for this book. Also, I thank Mr. J. Byron Williams, Mr. Keith Remy, Mr. Joshua Herring, Dr. T. G. Althen, Ms. Kay Talbot, Ms. Lou Adams, Ms. Sandy Babb, and Ms. Sara Liddell for their assistance in proofreading, typing, indexing, and reviewing the manuscripts. Robert W. Rogers Much of the information presented in certain chapters of this book, particularly those relating to carcass processing, arise directly from research funds supplied by Meat New Zealand and New Zealand’s Foundation for Research, Science and Technology, or their

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antecedents. This funding is gratefully acknowledged. For the past 50 years much of this funding was directed to MIRINZ Incorporated, whose research activity is currently continued by AgResearch, a government-owned research and development company. However, meat-related work of AgResearch is still associated with the name MIRINZ. Most activity stems from the MIRINZ Centre, located in Hamilton, New Zealand. Owen A. Young

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Contents

Preface Acknowledgments Contributors I. MEAT SCIENCE: CHEMISTRY, BIOCHEMISTRY, AND BIOTECHNOLOGY 1.

Meat Composition Robert G. Kauffman

2.

Postmortem Muscle Chemistry Marion L. Greaser

3.

Meat Color Owen A. Young and John West

4.

Flavors of Meat Products Tzou-Chi Huang and Chi-Tang Ho

5.

Analytical Methods Owen A. Young, Deborah A. Frost, John West, and Terry J. Braggins

6.

Meat Biotechnology M. B. Solomon

II.

MEAT SAFETY

7.

Microbiology of Meats Douglas L. Marshall and M. Farid A. Bal’a

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8.

Meat Safety Daniel Y. C. Fung, Maha N. Hajmeer, Curtis L. Kastner, Justin J. Kastner, James L. Marsden, Karen P. Penner, Randall K. Phebus, J. Scott Smith, and Martha A. Vanier

9.

Drug Residues in Meat: Emerging Issues Sherri B. Turnipseed

III.

SLAUGHTERING AND CARCASS PROCESSING

10.

Antemortem Handling and Welfare Temple Grandin

11.

Slaughtering and Processing Equipment María de Lourdes Pérez-Chabela and Isabel Guerrero Legarreta

12.

Carcass Processing: Factors Affecting Quality Owen A. Young and Neville G. Gregory

13.

Carcass Processing: Quality Controls Owen A. Young, Simon J. Lovatt, Nicola J. Simmons, and Carrick E. Devine

14.

Electrical Inputs and Meat Processing Philip E. Petch

IV.

PROCESSING MEATS

15.

Meat and Meat Products Youling L. Xiong and William Benjy Mikel

16.

Spices and Flavorings for Meat and Meat Products Patti C. Coggins

17.

Intermediate-Moisture Meat and Dehydrated Meat Tzou-Chi Huang and Wai-Kit Nip

18.

Manufacturing of Reduced-Fat, Low-Fat, and Fat-Free Emulsion Sausage Robert W. Rogers

19.

Meat Packaging: Protection, Preservation, and Presentation R. Graham Bell

20.

Meat Curing Technology Mike Martin

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21.

Meat Smoking Technology Douglas F. Ellis

22.

Meat Canning Technology Isabel Guerrero Legarreta

23.

Meat Fermentation Technology Fidel Toldrá, Yolanda Sanz, and Mónica Flores

V. MEAT PRODUCTION BY-PRODUCTS, WORKERS’ SAFETY, AND WASTE MANAGENENT 24.

Meat Production Yong-Soo Kim

25.

Meat Co-Products Deng-Cheng Liu and Herbert W. Ockerman

26.

Occupational Safety Tin Shing Chao and Ahmad C. K. Yu

27.

Waste Management Albert J. van Oostrom

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Contributors

M. Farid A. Bal’a Department of Food Science and Technology, Mississippi State University, Mississippi State, Mississippi R. Graham Bell Food Safety, MIRINZ Centre AgResearch, Hamilton, New Zealand Terry J. Braggins Food Systems and Technology, MIRINZ Centre AgResearch, Hamilton, New Zealand Tin Shing Chao Occupational Health Branch, Occupational Safety and Health Division, Department of Labor and Industrial Relations, State of Hawaii, Honolulu, Hawaii Patti C. Coggins Department of Food Science and Technology, Mississippi State University, Mississippi State, Mississippi Carrick E. Devine New Zealand

Technology Development Group, HortResearch, Hamilton,

Douglas F. Ellis Research and Development, Bryan Foods, Inc., West Point, Mississippi Mónica Flores Department of Food Science, Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Burjassot (Valencia), Spain Deborah A. Frost Nutrition and Behavior, MIRINZ Centre AgResearch, Hamilton, New Zealand Daniel Y. C. Fung Department of Animal Sciences and Industry, Kansas State University, Manhattan, Kansas Temple Grandin Collins, Colorado

Department of Animal Sciences, Colorado State University, Fort

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Marion L. Greaser Department of Animal Sciences, University of Wisconsin–Madison, Madison, Wisconsin Neville G. Gregory Flaxley Agricultural Centre, South Australian Research and Development Institute (SARDI), Flaxley, South Australia, Australia Maha N. Hajmeer Department of Animal Sciences and Industry, Kansas State University, Manhattan, Kansas Chi-Tang Ho New Jersey

Department of Food Science, Rutgers University, New Brunswick,

Tzou-Chi Huang Department of Food Science, National Pingtung University of Science and Technology, Pingtung, Taiwan Curtis L. Kastner Department of Animal Sciences and Industry, Kansas State University, Manhattan, Kansas Justin J. Kastner Kansas State University, Manhattan, Kansas Robert G. Kauffman Department of Animal Sciences, University of Wisconsin– Madison, Madison, Wisconsin Yong-Soo Kim Department of Human Nutrition, Food and Animal Sciences, University of Hawaii at Manoa, Honolulu, Hawaii Isabel Guerrero Legarreta Departamento de Biotecnología, Universidad Autónoma Metropolitana–Iztapalapa, Mexico City, Mexico Deng-Cheng Liu Taichung, Taiwan

Department of Animal Science, National Chung-Hsing University,

Simon J. Lovatt Processing and Preservation Technology, Food Systems and Technology, MIRINZ Centre AgResearch, Hamilton, New Zealand James L. Marsden Kansas State University, Manhattan, Kansas Douglas L. Marshall Department of Food Science and Technology, Mississippi State University, Mississippi State, Mississippi Mike Martin Research and Development, Bryan Foods, Inc., West Point, Mississippi William Benjy Mikel Lexington, Kentucky

Department of Animal Sciences, University of Kentucky,

Wai-Kit Nip Department of Molecular Biosciences and Biosystems Engineering, University of Hawaii at Manoa, Honolulu, Hawaii Herbert W. Ockerman Department of Animal Sciences, The Ohio State University, Columbus, Ohio

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Karen P. Penner Department of Animal Sciences and Industry, Kansas State University, Manhattan, Kansas María de Lourdes Pérez-Chabela Departamento de Biotecnología, Universidad Autónoma Metropolitana–Iztapalapa, Mexico City, Mexico Philip E. Petch Measurement and Electronic Technology, Food Systems and Technology, MIRINZ Centre AgResearch, Hamilton, New Zealand Randall K. Phebus Kansas State University, Manhattan, Kansas Robert W. Rogers Animal and Dairy Sciences Department, and Food Science and Technology Department, College of Agriculture and Life Sciences, Mississippi State University, Mississippi State, Mississippi Yolanda Sanz Department of Food Science, Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Burjassot (Valencia), Spain Nicola J. Simmons Meat Science, Food Systems and Technology, MIRINZ Centre AgResearch, Hamilton, New Zealand J. Scott Smith Department of Animal Sciences and Industry, Kansas State University, Manhattan, Kansas M. B. Solomon Meat Science Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland Fidel Toldrá Department of Food Science, Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Burjassot (Valencia), Spain Sherri B. Turnipseed Animal Drug Research Center, U.S. Food and Drug Administration, Denver, Colorado Albert J. van Oostrom Albert van Oostrom and Associates, Hamilton, New Zealand Martha A. Vanier Department of Animal Sciences and Industry, Kansas State University, Manhattan, Kansas John West Zealand

Nutrition and Behavior, MIRINZ Centre AgResearch, Hamilton, New

Youling L. Xiong Department of Animal Sciences, University of Kentucky, Lexington, Kentucky Owen A. Young Food Systems and Technology, MIRINZ Centre AgResearch, Hamilton, New Zealand Ahmad C. K. Yu Food and Cosmetic Group, Aloha Hawaii Enterprises, LLC, Keaau, Hawaii

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1 Meat Composition ROBERT G. KAUFFMAN University of Wisconsin–Madison, Madison, Wisconsin

I. II.

PREFACE INTRODUCTION A. Definitions and Measurements

III. DESCRIPTION AND COMPOSITION OF MUSCLE AND ITS MODIFIERS A. Description B. Gross Composition C. Molecular Composition D. Modifiers of Muscle Composition IV. DESCRIPTION AND COMPOSITION OF FAT AND ITS MODIFIERS A. Description B. Gross and Molecular Composition C. Modifiers of Fat Composition V.

DESCRIPTION AND COMPOSITION OF BONE AND ITS MODIFIERS A. Description B. Gross and Molecular Composition of Bone and Its Modifiers

VI. THE COMPOSITION–QUALITY PARADOX OF MEAT ACKNOWLEDGMENTS REFERENCES

I. PREFACE Anyone who has an interest in meat should know something about what it consists of . . . what the pieces are, how much of the whole each piece represents, and how to measure each piece. Furthermore, the interested and educated person really should know from where meat originates and how to measure its composition and what causes it to vary. This is what this chapter is about. When you have finished reading it, I hope you will have a clear picture of where meat comes from, what it consists of, why its consistency varies, and

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how to assess it. This information should help provide a better understanding of how to use meat as a food, how to make it taste better and be more safe for consumption, and how its properties can be best utilized for further processing, storage, and distribution. Specific references, primarily by the author, are given for further details on specific topics (1–6). II. INTRODUCTION A. Definitions and Measurements In the broadest sense, meat is the edible postmortem component originating from live animals. For the purposes of this text, these animals include domesticated cattle, hogs, sheep, goats, and poultry, as well as wildlife such as deer, rabbit, and fish. It is reasonable for the definition of meat to include such organs as heart and liver (often defined as variety meats), but the focus of this chapter is on meat defined as those tissues exclusively originating from an animal’s carcass—a proportion amounting to about one-half to three-fourths of the animal’s live weight. This carcass proportion of the live animal weight is classically calculated as dressing percentage and can vary considerably. Some species, such as the turkey, can yield a carcass weighing about 80% of the live weight, whereas a market lamb’s yield is closer to 50%. Animals with small and empty gastrointestinal tracts (such as hogs or poultry rather than ruminants) that are not pregnant, that are more heavily muscled and fatter, that do not have long fleeces or dirty hides, and that have been slaughtered in a manner that leaves the skin and feet intact with the carcasses (hogs), will have higher dressing percentages. Excluding the skin, the carcass component of live animals basically consists of three parts: muscle, fat, and bone. Of these, muscle is the most important, constitutes the majority of the weight, and often is considered unequivocally synonymous with “meat.” This can be a reasonable assumption, but fat deposits and some bones are often processed, merchandized, and used along with muscle and must be included in the broader definition of meat. Figure 1 is included as an example of the relative composition (in specific detail) of market animals and is representative of a live mature beef steer. From this information, one can calculate the proportions of any one part to the various larger component parts. For instance, the longissimus muscle represents (approximately) 51% of the back muscles, 12% of all carcass muscles, 7% of the carcass, and 4% of the live animal. These values can vary depending on species, degree of fatness, and other similar factors affecting dressing percentage. However, it provides a relative guide that reflects the composition of live animals and how it is related to the meat component. Furthermore, this figure indicates that “meat” has its origin in many muscles of the carcass. In closer observation, one can deduct that some muscles contribute considerably more to meat than others, and that is because they vary in size and shape, dependent directly on biological functionality. Composition is defined as the aggregate of ingredients, their arrangement, and the integrated interrelationship that forms a unified, harmonious whole. Figure 1 is an example of this. For market animals raised to produce meat for humans, the greatest emphasis is on the musculature and its relationship to everything else. The proportion of the animal’s musculature is related to several criteria, but the three most important are dressing yield, fatness, and muscling (expressed in terms of ratio of muscle to bone). Realistic averages of composition for most meat animals are included in Table 1. Muscle varies from 25% (lamb) to 50% (turkey) of the live weight and muscle to bone ratio varies from 1.8 (chicken) to 5.0 (venison).

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Figure 1 Live animal composition. Items within dotted lines are components found in both carcass and non-carcass parts of animals.

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Table 1 Gross Compositional Variations Among Animal Species Live weight, kg Average proportions of live weight Non-carcass, % Carcass skin, % Carcass fat, % Carcass bone, % Carcass muscle, % TOTAL Dressing yield, % Carcass muscle/bone ratio a

Tom turkey 15

Broiler chicken 2

Farm-raised catfish 0.7

23 9 7 22 39

37

17 10 25

18 9 6 17 50

12 51

100

100

100

100

Beef 550

Veal 160

Pork 110

Venison 70

Lamb 50

38

46

48

a

a

a

17 10 35

7 15 32

27 5 23 9 36

42

a

10 8 40

100

100

100

100

62 3.5

54 2.1

73 4.0

58 5.0

52 2.5

82 2.9

77 1.8

a a

63 4.3

Included with non-carcass component.

There are several arithmatic approaches to expressing quantitative composition, but the one most commonly used is the weight of a part expressed as a percentage of a larger part, such as % muscle of a retail cut of meat, or % protein of a muscle. Another lessused technique is to express the part and the whole as logarithmic functions of each other. Measuring composition can vary from subjective techniques to ones precisely objective. Even when a technique is considered objective, at least some subjectivity inadvertently prevails. Here are some of the more commonly used approaches for determining gross composition (% lean) of meat cuts. 1. Visual Appraisal Every time consumers purchase cuts of meat, they often select them on the basis of their lean/fat/bone ratios as estimated by visual inspection. This is simply accomplished through visual comparisons. Quantitatively the method lacks accuracy, but for practical purposes in meat selection, it is effective, especially when compositional variations are large. On a more detailed basis, visual scores can be established with photographs and then the meat cuts can be scored on proportions of lean. However, scoring is too subjective to reflect quantitative differences and is too difficult to standardize to be consistently applied over time. Perhaps the one greatest value of visual inspection is in the estimation of proportions of intramuscular fat (marbling) when determining gross composition by dissection (in which dissecting marbling is impossible). 2. Linear Measurements A simple, inexpensive ruler can be used to measure subcutaneous fat thickness, muscle depth and width, and bone length and thickness. From these measurements, areas of each component can be estimated and then expressed as a proportion of the whole. Unfortunately, the areas are not exactly accurate, nor are unexposed bones and seam fat of different dimensions (as well as marbling) included in the estimate. 3. Area Measurements By tracing the areas of the exposed muscles, bones, and fat on acetate paper and then measuring the exact areas of each component with a compensating polar planimeter, composiCopyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

tion can be estimated. Also, such areas can be assessed using photometric, electronic, and computerized imaging techniques. Even though this is more accurate in determining the exposed areas, it has the same limitations as visual appraisal or linear measures because unexposed bone and seam fat as well as marbling cannot be accounted for. 4. Density The Archimedean principle suggests that cuts of meat displace a volume equal to their own. Because the density of fat is less than that of muscle, such a technique, even though destructive if water displacement is used and expensive if gas displacement is used, would provide an accurate estimate of lean. However, for meat cuts containing bones, the technique would not be satisfactory. The density of bone is nearly twice that of muscle and would bias the estimate of muscle. 5. Anyl-ray This is based on x-ray attenuation as an index of tissue fatness. It generates electromagnetic waves of a character sensitive to absorption and reflection or back scatter by the elements in ground meat. The radiation is directed through a sample where size, shape, compaction, and weight must be constant while sample composition varies. When calibrated intensity of radiation is directed through a sample, this energy intensity is directly proportional to sample composition. A carefully mixed and selected ground meat sample is subjected to a minute amount of carefully controlled x-rays. Because lean absorbs more x-rays than fat, there is a difference in energy transmitted. The penetrating rays are collected by a radiation-measuring device, which in turn energizes a calibrated digital percentage fat meter. It is used regularly to determine fat content in ground meat used for processing. The method is fast, requires a sample that can be reutilized for processing after measurement, and has a high degree of accuracy. The instrument correctly evaluates meat at any temperature, provided it can be properly compacted in the container. Further grinding or the addition of warm water may be necessary to achieve proper compaction for frozen samples. However, it is a relatively expensive method for determining composition, and it cannot be used for small meat cuts or ones containing bone, and it estimates only fat and no other specific chemical components. 6. Dissection This is the one most effective method of determining the gross composition of whole carcasses or individual wholesale or retail cuts. The method can be standardized and is highly repeatable in application. The method requires knowledge of anatomy and the patience and care to separate each component, preventing weight loss through evaporation and drip, and weighing and recording accurately. However, it does not account for variations of marbling, which would have to be assessed visually or subjected to chemical evaluation. 7. Proximate Analysis For animal tissues, the primary chemical components used as a follow-up to or an alternative for physical dissection, are moisture, protein, lipid, and ash. The procedures for chemically analyzing each of these are described in the AOAC (7). A major concern in using this method is adequate mixing and sampling of the tissues to be analyzed. Another limitation is in chemically analyzing bone because of the difficulty in grinding and sampling (this does not apply for the ash determined in muscle). Also, mixing ground components of soft tissues creates problems of fat collecting on the sides of the mixer. Finally, when moisture Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

is being assessed, large errors will occur when muscles are soft and watery or when excessive evaporation of moisture is lost from the surfaces of unprotected samples. To determine the detailed chemical composition of muscle, fat, or bone, such as specific minerals, myofibrilar proteins, fatty acids, individual vitamins, and bound vs. free water, numerous detailed and often extremely difficult, expensive, and sensitive chemical and spectrophotometric procedures are required. These procedures are not identified and described here because of their complexities and the need to maintain brevity. III. DESCRIPTION AND COMPOSITION OF MUSCLE AND ITS MODIFIERS A. Description Meat animals contain, as a majority of their carcass weight, many muscles distributed in an unusually designed pattern to move the skeleton, for posture control, and for more specialized functions such as respiration, swallowing, and peristalsis. This musculature is categorized into two major types: striated and nonstriated. The less voluminous non-striated or smooth muscles have some similar functions as striated muscles but possess different histological structures. Smooth muscles are primarily found in the linings of the gastrointestinal tract and the circulatory system as well as in specialized organs such as the gizzard of birds. Striated muscles are categorized as either cardiac or skeletal. Cardiac muscles are confined to the heart and have the continuous responsibility of distributing and collecting blood throughout the body. Structurally, they are similar to skeletal muscles, except that they are more highly aerobic in their metabolic properties and therefore require higher concentrations of oxygen for their rhythmic contractions. Skeletal muscles are, as the name implies, associated with the skeleton; they either lie next to a bone or are attached to various bones, either closely or indirectly through their connective tissue fascia that may attach directly or indirectly to distant bones. Depending on function and needs, skeletal muscles contract and relax and have very exacting cross-banding patterns. Skeletal muscles play the major role in locomotion and posture control as well as in protecting vital organs. On average, the meat animal carcass contains about 100 bilaterally symetrical pairs of individually structured muscles. There are large ones and small ones, depending on function and location. They have different shapes, colors, and concentrations of tendons. Many have a fusiform, multipennate shape, having a large middle potion that tapers at the ends. The attachments contain large quantities of tendinous connective tissue that attaches to bone. The long head of the triceps brachii would be an example of a fusiform-shaped muscle. Other shapes include flat or sheet-like muscles such as the cutaneus trunci, round-shaped muscles such as the quadriceps femoris, and irregular shapes such as the tensor fasciae latae, which has more than two attachments and is somewhat triangular shaped with thick and thin portions. In the more distal portions of the limbs, small muscles are uniquely attached to tendons for the specific purpose of either flexing or extending the feet and legs. In the more proximal locations, the muscles are larger and primarily serve as major sources of power. This is particularly true of the pelvic limb musculature. There are less than 10 major pelvic muscles, whereas there are twice as many of smaller size in the thoracic limb. The longissimus thoracis et lumborum is the longest and largest muscle in many species and is located in the back to support the axial skeleton and to extend and erect the vertebral column. The flat muscles, generally located in the ab-

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dominal wall, support the abdominal cavity and its contents. Commercially, the flat muscles have less economic importance but they are the only ones found in bacon. Skeletal muscles have a complex composition because they contain, in addition to muscle fibers, large quantities of supportive connective tissue, a complete vascular supply, and a nerve supply controlling each of the billions of muscle fibers. Also, skeletal muscles serve as storage depots for lipids and contain considerable quantities of extracellular fluids, primarily consisting of water. Postmortem muscles vary in color, ranging from a dark purplish-red to a pale, light gray. This variation is primarily the result of myoglobin concentration as well as other biological factors such as pH. Myoglobin is a protein physiologically important in the transfer of oxygen and carbon dioxide to and from muscles during their normal metabolic activities. Breast muscles of poultry (pectorales superficiales) are very pale or white in color and contain low quantities of myoglobin, whereas leg muscles of venison are extremely dark purple and contain more than twice as much myoglobin. Striated muscles are multinucleated, distinguishing them from smooth muscles, which are mononucleated. These nuclei are near the sarcolemma; in smooth muscles, the nuclei are more centrally positioned. Skeletal muscles contain mitochondria, but not as many as are found in cardiac muscle. Other organelles such as ribosomes and the Golgi apparatus are also found in muscle fibers. Each fiber is surrounded by an intricate membrane, the sarcolemma, which surrounds the sarcoplasm that bathes the myofibrils, which are the contractile units of the fiber. Lipid particles in the form of neutral droplets and free fatty acids as well as glycogen granules are distributed throughout the sarcoplasm (in postmortem muscles, glycogen is metabolized to lactic acid). Enzymes are located in mitochondria and in other portions of the sarcoplasm. The sarcoplasmic reticulum and transverse tubules are responsible for the storage and transportation of calcium for contraction. To permit muscles to function properly as moving forces, they are harnessed to the skeleton through a unique set of connective tissue structures. This connective tissue “harness” circumvents the entire muscle and is called the epimysium; it winds its way through each muscle, dividing fibers into groups called fascicular bundles. The connective tissue at this level is perimysium. The perimysium subdivides further into endomysium, which lines each fiber. The vascular system, which winds its way through muscles to supply the nutrients and remove toxic wastes, is closely related to individual fibers. In both the extracellular spaces and within fibers there are fluids high in water content. In addition to the water, there are minerals, some water-soluble proteins, non-protein nitrogenous materials, and other organic entities. Lipid in the form of neutral triglycerides is stored in the adipose tissue cells, which accumulate around venules and arterioles in the interfascicular spaces. This fat, when visible, is called marbling. Excluding water, the major components in muscle are the contractile proteins, which make up the myofibrils. B. Gross Composition A simpler approach to assessing the composition of muscles is to use proximate analyses to quantitate moisture, protein, lipid, ash, and carbohydrate. Muscles vary considerably in these components, and the accumulation of lipid is the most influential on this variation. On average, most muscles should contain about 1% ash (primarily represented by the elements potassium, phosphorus, sodium, chloride, magnesium, calcium, and iron), 1% carbohydrate (primarily glycogen antemortem, and lactic acid postmortem), 5% lipid, 21% nitrogenous compounds (predominantly proteins), and the rest (72%) as moisture. These

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values are compared to the composition of fat and bone as shown in Table 2. Some muscles may contain as much as 15% lipid (fresh weight basis), whereas others may contain less than 2%. Regardless of the lipid content, the protein/moisture ratio of about 0.3 remains quite constant for mature muscles. If time and expenses are limited, one may quickly, easily, and somewhat accurately assess proximate composition of muscles by making a few assumptions, using moisture analysis for the only determination. If it is assumed that ash and carbohydrate will not vary greatly and that their sum contribution is estimated at 2%, and if it is assumed that the protein/water relationship is 0.3, then if water is determined by homogenizing the sample and drying it, the only unknown left to be estimated is lipid content. This is calculated by difference. For example, if a sample (analyzed for moisture content) contained 70% moisture (M), then protein (P) content would be equal to P/M
0.3, or P/70
0.3. Therefore, P
21 or 21% protein. By subtracting the sum [2% (ash & carbohydrate)  70% (M)  21% (P)] from 100%, then lipid would be 7% or [100  (2  70  21)]. C. Molecular Composition There are a host of chemical compounds in muscles. They include free fatty acids, glycerol, triglycerides, phospholipids, non-protein nitrogenous components such as DNA, RNA, ammonia, amine groups, and vitamins. There are glycogen granules and ATP. Myoglobin is present. Several minerals are present in minute quantities. Most important from a quantitative perspective, there are the various proteins of each fiber. These proteins are classified into four groups, the largest of which is myofibrillar. Myofibrillar proteins represent about 60% of the total proteins, whereas sarcoplasmic proteins represent 29%, stroma proteins 6%, and granular proteins 5%. Figure 2 is included to provide a detailed overview of the complexity of muscle composition. It is not intended to be precisely accurate nor to be memorized, but to serve as a guide to identify the various components of muscle and their quantitative contributions to its mass. It is assumed that these values represent mature, postrigor muscles of various species. Of all information presented, this figure should receive the highest priority for your attention because it is a detailed summary of the most important features of meat composition. (It required more time and effort to construct than everything else combined in this chapter!) It should be understood that the methods of analTable 2 A Comparative and Approximate Gross Composition of Muscle, Fat, and Bonea Muscle Moisture, % Nitrogenous compounds, % (primarily protein) Lipid, % Ash, % Carbohydrate, %

Fat

Bone

72 21

9 1

25 10

5 1 1

90  

20 45 

100

100

100

a

Proximate analysis expressed on a fresh basis for mature, postmortem tissues representing various anatomical locations.  Less than 0.5%.

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Figure 2 Fresh muscle composition. ysis used to determine most of the components of this figure greatly affect the quantities reported. The myofibrillar proteins are responsible for the contractile mechanisms and thus shorten or lengthen the muscle for movement and support functions. Sarcoplasmic proteins are primarily represented by enzymes and myoglobin. Stroma proteins originate from the connective tissue structure found as a part of muscle, the most important quantitatively being collagen. Collagen is resistant to most enzymatic reactions except collagenase. When heated in water, collagen is converted to gelatin, which is readily hydrolyzed by several en-

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Kauffman

zymes. About one-third of collagen’s amino acid residues consist of glycine, whereas another one-fifth is proline and hydroxyproline. It is the only protein known which contains hydroxyproline, with the possible exception of reticulin. Hydroxyproline analysis is often used as a measure for determining total connective tissue in muscles. Another stroma protein of less concentration is elastin. It is even more resistant to degradation: to degrade, it must be subjected to high temperatures in the presence of strong bases or acids. Elastin contains about one-third of its amino acid residues as glycine and over one-tenth as proline. Reticulin is the other major stroma protein. Its amino acid composition is similar to that of collagen, and it is often considered a form of collagen that contains lipids and carbohydrates. There are nine known major myofibrillar proteins, as illustrated in Figure 2. Quantitatively, the one most important protein is myosin. In referring to Figure 2, myosin represents 43% of the myofibrillar proteins, 26% of all muscle proteins, 23% of all nitrogenous compounds, and 5% of the fresh muscle mass. Myosin is the thick strand of protein that appears in the sarcomere structure. Actin represents about 22% of myofibrillar proteins and is the thin filament within this same contractile formation. The other seven proteins represent much smaller compositional fractions, but play equally important roles in contraction. Titin represents 8% and has by far the largest molecular weight and is considered more structural than metabolic in function. Tropomyosin and troponin each contribute about 5% and can be found attached to the actin molecule and are primarily responsible for initiating contraction after calcium has been released by the sarcoplasmic reticulum. All the other proteins combined represent less than 20% of the weight. All the above mentioned proteins are composed of the 22 amino acids shown in Figure 2. Each amino acid is different according to the molecular characteristics of its side chain. The 10 essential and 12 nonessential amino acids and their mole contributions to muscle mass are included in Figure 2. In addition to the proteins, there are other important nitrogenous constituents in muscle. First are the vitamins, which are divided into two classes based on their solubility in either aqueous or non-aqueous solutions. The lipid-soluble vitamins are minimal because of the small quantities of fat normally deposited in most muscles. However, water-soluble vitamins, primarily the B vitamins, are present in substantive enough quantities to serve as appropriate sources to meet daily dietary requirements for humans. They include thiamin, riboflavin, niacin, pyridoxine, pantothenic acid, biotin, folic acid, and B12. Ascorbic acid [vitamin C] (as well as calcium) is essentially absent in muscles, and because of this, muscles are not considered a perfect food from a nutritional perspective. The nitrogenous, nonprotein extractives include creatine, nucleotides, ammonia, methylamines, free amino acids, and other derivatives of proteins. Two of the components in highest concentrations are carnosine and anserine. Other extractives include volatile organic carbonyls, such as acetyl aldehyde, acetone, carbon dioxide, and formaldehyde, all of which have been found in muscles. Various sulfur compounds include hydrogen sulfide, methylmercaptans, and methyl sulfides. The elemental components include carbon, hydrogen, and oxygen in great abundance either because of their molecular weight or number of molecules and are listed in Figure 2. In addition, nitrogen is abundant because it is a component of all proteins. Some minute quantities of sulfur are present in the form of the amino acids cystine, cysteine, and methionine. Inorganic ions include calcium, magnesium, sodium, potassium, chlorine, phosphorus, and iron, but their contributions to mass are minimal. In assessing the various elements in the various components of muscles, in most instances—whether proteins, lipids,

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Meat Composition

11

carbohydrates, vitamins, or nucleic acids—the elements carbon, hydrogen and oxygen are always present. The unique compositional difference among proteins, nucleic acids, and vitamins is that in proteins, nitrogen molecules are in the side chains; in the other two groups, nitrogen molecules are incorporated into the ring structures. The protein myoglobin is somewhat of an exception in structure in that it contains a heme group as well as a globular protein fraction and contains iron as its central ion in the heme ring. The iron element in myoglobin is paralleled by the cobalt element in vitamin B12. D. Modifiers of Muscle Composition General fatness of the animal influences the composition of muscles. Individual muscle fibers remain constant in their composition, but fresh muscle may vary from 1% to 15% in lipid content. This variation is due to such factors as genetics, stage of growth, sex of animal, and amount of physical exercise. As animals mature and muscles stop growing, intramuscular fat may accumulate around the vascular system, thus decreasing the relative mass of other components. The nature of the connective tissue matrix also affects the accumulation of fat. Loosely arranged muscles such as the latissimus dorsi, having parallel connective tissue strands, contain more fat than tightly compacted muscles such as the peroneus longus. The latter’s connective tissue strands are thicker and more tightly structured, thus physically preventing excess fat accumulation. Nutrition affects muscle composition simply by controlling the total lipid accumulation, depending on the total caloric intake and expenditures. In submaintenance diets, fat is mobilized (rather than deposited) from muscles. Quality of nutrition can also affect the mineral and vitamin content of muscles, but not to the extent that fat deposition is affected. Stage of growth affects the protein/moisture relationship of muscles. In very young animals, this ratio is low (~0.1), whereas at maturity, the relationship is about 0.3. As already indicated, this remains reasonably constant throughout the animal’s lifetime and serves as a reliable guide in estimating composition. In addition to the structural differences in connective tissue, anatomical location of muscles affects composition because some muscles contain higher concentrations of tendon and epimysial sheaths of connective tissue. Because of this, there is a difference in quantity of stroma proteins as compared to myofibrillar, sarcoplasmic, and granular proteins. For example, lower limb muscles have higher concentrations of connective tissue proteins than do supportive back muscles. Even though the molecular nature of stroma proteins changes during growth, the absolute quantities do not change. Some muscles such as the gluteus medius and longissimus have proportionately more white fibers requiring less oxygen. Therefore, their energy needs for muscle contraction are more anaerobic than that of muscles containing more red fibers. Consequently myoglobin concentration is lower and this may be true for fat content as well. An exception to this is the trapezius. It contains over 60% red fibers but also contains high amounts of lipid. The semitendinosus contains two clearly defined portions, one having predominantly red fibers and the other predominantly white fibers. As a result, molecular composition within this muscle varies considerably. However, in this example, the white fiber portion contains considerably more lipid than the red fiber portion, suggesting that muscle location and function affect composition more than fiber type per se. Perhaps fiber type affects composition primarily by its effect on postmortem tissue characteristics. The postmortem musculature originating from short-term stressed animals (especially those genetically susceptible to stress) become soft and watery and are much more susceptible to exudation during processing. Therefore, composition is

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Kauffman

readily affected if processing is considered. Dark, firm, and dry (DFD) muscles that contain high concentrations of red fibers (often the result of long-term antemortem stress) are less susceptible to such abnormal postmortem shrinkage. Disease influences composition. Portions of muscles may be eroded away by muscular dystrophy and replaced with fat. Certain inorganic elements are lost from the tissues during stressful conditions related to disease. Certain central nervous system diseases also affect the general composition of muscle, primarily affecting the fat component. Injury to muscles affects composition. When major nerves are severed (accidentally or experimentally) the muscle atrophies and fat accumulates in the vacated spaces. Exercise stimulates fiber hypertrophy and mobilization of lipid within muscles. However, there is little evidence suggesting changes in other chemical components. Genetics affects fatty accumulation in muscles because of its relation to rate of maturity. Certain species of animals, such as the domestic duck, deposit very little fat in muscles. The rabbit has similar tendencies, whereas certain breeds of pigs, cattle, and sheep deposit large quantities of intramuscular fat. Within species, some breeds have greater tendencies to deposit intramuscular fat. Duroc swine appear to contain more intramuscular fat for a given degree of body fatness and age. Hereford and Charolais cattle do not deposit as much intramuscular fat at a given physiological state of maturity as do Angus cattle. Fat in muscles is related to the total fatness of the body. When carcasses from obese animals are examined, there is generally more intramuscular fat than from those possessing leaner carcasses. However, some animals have a very high potential for developing intramuscular fat as compared to total body fat per se (for example, Japanese Wygue cattle producing Kobe beef), whereas other animals deposit large quantities of subcutaneous fat but deposit very little intramuscular fat. Muscles grow at different rates and mature at different physiological times. This in itself affects composition. These differences are small, but if a muscle matures earlier and also has the structural potential for accumulating fat, then it will have a higher fat content at a given age than another muscle that matures at a later stage. This variation is also responsible for differences in protein/moisture ratios among muscles. Control of various body processes by the endocrine system affects fat deposition in muscles. Thus by the presence or absence of testosterone, fat deposition is regulated in muscle. Bulls, rams, and boars possess muscles containing less intramuscular fat than steers, wethers, and barrows. When cattle are fed diethylstilbestrol (a synthetically produced hormone that is currently banned from use), it changes the composition of muscles simply by slowing down the animal’s physiological time clock. The supplementation of certain hormones will increase fat mobilization in muscles; however, most of these changes are small. IV. DESCRIPTION AND COMPOSITION OF FAT AND ITS MODIFIERS A. Description Fat is often associated with such words as obese, plump, oily, wasty, greasy, big, and thick. Also it has been defined as the excess “white stuff” that is trimmed from a piece of meat, or the grease that lubricates a skillet, or some of the juices that flow out of hamburgers during cooking, or the foodstuff that is responsible for making foods “rich” because of its high caloric content. Fat has probably received more attention from consumers, and has been referred to more often, than any other single biological substance.

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Meat Composition

13

Epithelial, nervous, muscular, and connective are the four basic tissues involved in the processes of postnatal growth. Connective tissue’s primary function is structural support: it is responsible for the physical shape of such biological substances as bone, cartilage, muscle, and fat. Adipose tissue is a type of connective tissue that surrounds synthesized lipids, which serve as heat-cold insulators and as reserve supplies of body energy. Therefore, fat is defined as a collection of adipose cells suspended in a matrix of connective tissue distended with cytoplasmic lipids, water, and other constituents. Often fat and lipids are used interchangeably. Generally this is appropriate, but specifically it is an incorrect concept. Adipose or fatty tissue contains lipids, but lipids per se do not contain connective tissue, water, enzymes, and other constituents present in fat. However, because lipids are the major components of fat, it is important to describe these lipids in greater detail. Lipids include that group of nonpolar compounds soluble in organic solvents but insoluble in water. Pure lipids are colorless, odorless, and flavorless and can be classified as follows: 1. Simple Lipids Simple lipids are esters of fatty acids with certain alcohols such as glycerol. If lipids are solid at room temperature, they are called fats; if liquid, oils. Waxes are simple lipids that are esters of fatty acids with long-chain aliphatic alcohols or with cyclic alcohols. Examples of waxes include esters of cholesterol and the vitamins A and D. 2. Compound or Conjugate Lipids Compound or conjugate lipids are esters of fatty acids that, on hydrolysis, yield such substances as phosphoric acid, amino acids, choline, carbohydrates, and sulfuric acid, in addition to fatty acids and an alcohol. Examples include phospholipids, glycolipids, sulfolipids, and lipoproteins. 3. Derived Lipids Derived lipids are formed in the hydrolysis of simple or compound lipids. Examples include saturated and unsaturated fatty acids, aliphatic alcohols, sterols, alcohols containing the Beta-ionone ring, aliphatic hydrocarbons, carotenoids, squalene, and the vitamins D, E, and K. Fat is found in nearly every anatomical location imaginable, but the great majority of it occurs subcutaneously, inter- and intramuscularly, in the mesentery, on the walls of the thoracic, abdominal and pelvic cavities, and in the bone marrow (intraskeletal). Fat is deposited in the udders of females and in the scrotal sacs of male castrates. Fat is deposited in brain, liver, and kidney, and the quantity may be excessive under abnormal conditions. Lipids are found in some form in all body cells because phospholipids contribute to the structure of every cell wall. Blood and lymph contain lipids, the quantity varying greatly with time after an animal consumes a fatty meal. All dietary fats are transported to body tissues via one of these routes. Although adipose tissue is ubiquitous, it is not evenly and universally distributed in obesity, but is deposited in certain preferential sites while others are spared. For example, feet, eyelids, nose, ears, and genitalia seldom accumulate excess fat. B. Gross and Molecular Composition Quantitatively, Table 3 represents an example of variations in proportionality of fat in different anatomical locations and it is expressed on both a dissectable and an extractible ba-

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Kauffman

Table 3 Distribution of Pig Body Fat or Lipid During Growth Stage of growth (days of age) 21–43

64–106

Dissectable fat basis Total fat as % of empty body

15

20

25

Distribution by location Cavity wall, % Mesentary, % Intermuscular, % Subcutaneous, %

5 8 29 58

5 13 25 57

7 8 28 57

100

100

100

7

10

16

2 2 18 51 20 7

3 4 16 54 14 9

6 4 18 52 11 9

100

100

100

Sum Extractable lipid basis Total lipid as % of empty body Distribution by location Cavity wall, % Mesentary, % Intermuscular, % Subcutaneous, % Intramuscular, % Intraskeletal, % Sum

149–213

sis. For this example, as the animal matures, the total fat increases when compared to other body tissues, but for the most part the proportionate distribution remains reasonably constant. On an extractible basis, the internal cavity fats (cavity wall and mesentary) represent about one-tenth, intermuscular about one-fifth, intramuscular and intraskeletal about onefourth and subcutaneous over one-half of the total lipids deposited, regardless of stage of growth. Even though intramuscular and intraskeletal lipids are not included in the dissectable allocation, the total representation of extractible lipids (as a percentage of empty body weight) are lower than for the dissectable fat values because water is not included when extractible portions are expressed. One must remember that fat contains significant quantities of water. Table 2 includes the gross composition of fat and how it compares to that of muscle and bone. Adipose cells vary in size depending on such factors as age, species, and state of nutrition. The increase in numbers of adipose cells does not necessarily dictate the quantitative amount of fat deposited. For mature pigs, about 45% of the total adipose cells are in intramuscular fat but this fat component represents less than 15% of the volume of extractable lipid. This is verification that cell hypertrophy contributes more to volume than does cell hyperplasia. Lipids dominate in their contribution to adipose volume and weight. However, other constituents are present, too. In immature tissues, there is a significant quantity of water. Also, because adipose tissue is structurally supported by a connective tissue matrix, the extracellular protein collagen is present. Other substances include enzymes responsible for li-

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Meat Composition

15

pogenesis and lipolysis, traces of certain minerals and minute quantities of glycerol, glucose and glycogen, and nerves. Adipose tissue lipids are primarily present as mono-, di-, or triglycerides. Each is composed of a molecule of glycerol bonded to one, two, or three fatty acids. These acids are either synthesized in the adipose cell or are synthesized in the liver and subsequently transported to the adipose cell via the circulatory system. Fatty acids in adipose tissue usually contain 16 or more carbon atoms, but there are a few that are shorter. The carbon chain may be completely saturated with hydrogen atoms, or there may be some double bonds, and these are called unsaturated fatty acids. The dietary origin of the fat dictates the variation expected and the iodine and saponification values reflect such variations. Oleic, palmitic, and stearic acids represent over 80% of the composition of meat animal lipids. However, the primary difference in physical properties of lard as compared to beef tallow is the higher quantity of linoleic acid that is more unsaturated and occurring in higher proportions in lard, giving it a softer structure at room temperature. Also, this higher degree of unsaturation results in a fat more susceptible to oxidative rancidity. C. Modifiers of Fat Composition The discussions that follow will parallel some of those already covered in the muscle section and thus will be limited to unique differences pertaining exclusively to fat. When compared with mature animals, young ones contain adipose tissue having considerably more water. Also, the phospholipid component is proportionally higher in young animals as compared with their triglyceride content. For ruminants, most dietary fats are digested in the rumen where ingested unsaturated fatty acids are hydrogenated and then absorbed into the circulatory system. However, for monogastric animals, ingested unsaturated fatty acids are not hydrogenated and are absorbed and deposited in adipose tissue in their original structures. Therefore this explains why pork fat is softer than beef tallow. The release of some hormones will stimulate mobilization of fatty acids. If this persists, the triglyceride fraction will be significantly reduced. Anatomical location of fat is important in determining its composition. The number of intramuscular adipose cells represents nearly half the total adipose cell population of the body, yet the amount of extractable lipid is less than 10%. These cells are smaller and contain more water than do cells located subcutaneously. Also, within subcutaneous tissue, that portion located at the base of the pelvic limb contains less extractable lipid than the cells located over the back. Furthermore, the three distinct layers of subcutaneous fat over the back of pigs vary in fatty acid composition. The outer layer contains greater proportions of unsaturated fatty acids as compared with the other layers. Mesentary adipose tissue contains more saturated fatty acids than subcutaneous tissue; udder fatty tissues contain more fluid, nonlipid material and less extractable lipids than other adipose tissues. Finally, certain organs such as the bovine kidney contains 30% oleic acid and 33% stearic acid as compared with bovine subcutaneous fat, which contains 40% oleic acid and less than 20% stearic acid. Genetic variables influence the quantity of fat and its composition. Some examples include (a) more hydrogenated fatty acids in ruminants than monogastric species, (b) double-muscled cattle do not deposit fat as quickly as normal cattle, (c) some breeds of sheep accumulate greater quantities of fat over the rump, and (d) pigs contain more subcutaneous and less intermuscular fat than sheep or cattle. Females have the capacity to lactate, which

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Kauffman

is a unique process of fat accumulation in milk. Intact males of most species contain less fat than castrate males or females of similar chronological ages. Heifers contain more fat than steers at a given age whereas gilts contain less fat than barrows at a similar age. This observation may simply reflect variations in stages of compositional, physiological, and sexual maturity. Atypical conditions such as obesity and steatosis (excessive fat deposition in muscles) increases the quantity of lipids deposited. Conversely, exercise and various environmental stresses reduce lipid deposition. V. DESCRIPTION AND COMPOSITION OF BONE AND ITS MODIFIERS A. Description Bone is a complex tissue and subject to continual metabolic activity. A most obvious difference when compared to muscle and fat is its dense, hard, mineralized, cellular type tissue. The three cellular components of bone are of one cell type and may change in morphological characteristics directly according to specific functional needs of the tissue. The cells involved include osteocytes which are responsible for maintenance; osteoplasts, which are involved in formation of new bone; and osteoclasts, which are responsible for mobilization and reabsorption of bone material. Histologically bone is characterized by its branching lacunae, which are cavity-like membranous materials, and by canaliculi, which are fine-structured canals. Bone contains a dense matrix of collagenous fibrous bundles in a ground substance encased with calcium and phosphorus. Bone is capable of structural alterations to accommodate stresses due to mechanical changes and biological demands incurred by pressure and by vascular, nerve, endocrine, and nutritional influences. Most of the rigid material in the skeletons of meat animals is either compact or cancellous bone. This indicates that there are different degrees of mineral density in the bone including available porous spaces that provide for the accumulation and maintenance of the marrow. Another method of classifying bone is on the basis of bone formation. Some bones develop within mesenchymal tissue such as the skull, which is known as membrane bone. Other bones depend on prior cartilaginous scaffolding, such as the vertebrae, and this is called cartilage bone. This cartilage-type bone contains collagen and polysacchrides. There are more than 200 individual bones in meat animals, and they are either on the axial skeleton or the appendicular skeletons (limbs). Figure 1 includes the major bones and their proportionate masses in the live animal. They all include bone marrow, which produces the majority of the red blood cells. They store minerals and mobilize them as needed for other body tissues. They repair themselves after an injury. They are designed to provide the greatest support with a minimum amount of weight; this is why most bones have hollow structures. B. Gross and Molecular Composition of Bone and Its Modifiers Bone basically contains mineral deposited in an organic matrix. The matrix includes not only calcium, phosphorus, and carbonate, but also citrate, water, and small amounts of sodium, magnesium, potassium, fluorine, and chlorine. The crystals of bone mineral have a chemical composition similar to that of fluoral apatite. Fibers of collagen run throughout the matrix. The remaining space in the bone “mortar” is filled with a semiliquid substance that exchanges materials to and from the bone mineral via the circulating blood.

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Meat Composition

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More than 99% of the body calcium is in bones. Collagen is about 93% of the total organic portion of bone. There are small amounts of insoluble sclera proteins and ground substance that are composed of mucopolysaccharides and mucoproteins. In fat-free analyzed bone, the mineral content accounts for about two-thirds of the mass, whereas in fresh bone it is about two-fifths. As included in Table 2, water represents about one-fourth the mass, protein about one-tenth, and the remaining one-fifth portion (which is the most variable) is lipid. As indicated, type of bone, age of animal, and species are three factors that most affect bone composition. Bone ash is composed mostly of calcium and phosphorus and much lesser quantities of magnesium, sodium, potassium, chlorine, and fluorine. When comparing bone to fat and muscle as shown in Table 2, the average composition is considerably different. If bone is compared to the Achilles tendon (almost entirely connective tissue), the tendon consists of two-thirds water and one-third organic solids with very little inorganic material. This compositional profile is quite similar to muscle. For the ligamentum nuchae, which is slightly more similar to bone, water content is about 57% and organic solids make up most of the remainder, but the elastin content is considerably higher than that for tendon. The factors modifying the composition of bone are quite similar to that of muscle and fat and will not be repeated here. As illustrated in Table 4, age, species, and type of bone are three major modifiers of bone composition. Other unique factors modifying bone composition are (a) absence of Vitamins D and A in the diet, (b) abnormalities in endocrine secretions (both low and high quantities), (c) lack of mineral supplementation in the diet (especially calcium and phosphorus), and (d) wasting type diseases that mobilize mineral content from the bone, creating brittle and friable structures that have been significantly altered in composition. Table 4 Variation of Bone Composition when Comparing Species, Age and Bone Type Moisture, %

Lipid, %

Ash, %

Protein, %

Sum, %

Cattle

2–3 months 48–96 months Costae Femur

55 27 37 34

7 17 9 21

19 47 37 39

19 9 17 6

100 100 100 100

Pigs

6–8 months 24–48 months Costae Femur

37 25 33 26

18 23 18 28

33 43 37 43

12 9 12 3

100 100 100 100

Sheep

5–6 months 48–96 months Costae Femur

39 23 32 24

11 16 9 23

33 49 39 47

17 12 20 6

100 100 100 100

Chickens

2–3 months 12–13 months Costae Femur

50 39 42 44

11 9 10 12

24 34 29 30

15 18 19 14

100 100 100 100

Source: Data extrapolated from Field et al. (8).

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Kauffman

VI. THE COMPOSITION–QUALITY PARADOX OF MEAT When evaluating meat, both composition and quality are important. Leanness (as contrasted to fatness) is virtuous, but by itself, it fails to meet ultimate expectations of consumers. The nutrient density of muscle is higher in lean meat and nutritive value is a part of quality. Therefore, from this perspective, composition affects quality. However, quality is more than just nutrient density. Wholesomeness, appearance, water-holding capacity and palatability are quality virtues too! Marbling contributes to juiciness and flavor, however, more marbling reduces nutrient density. Furthermore, the exterior fat covering of fresh meat cuts is related to marbling. The association is not strong, but fatter cuts usually exhibit muscles containing more marbling. The paradox is that meat animals are fed to heavier weights, for longer times, and to ultimately less favorable “feed-to-meat” ratios so that muscles will contain more marbling to ultimately satisfy consumer demands. This negative relationship between marbling and leanness is difficult to compromise and is one of the reasons why beef and lamb cuts may be too fat. In pork and turkey, trim and heavily muscled carcasses appear to be more susceptible to the pale, soft, and exudative (PSE) condition. Meat cuts from such carcasses possess lean, heavily muscled cuts containing minimum quantities of fat. Nevertheless, the muscles often shrink excessively during processing. Fresh cuts of pork (loin and ham) and turkey (breast) that are exceptionally lean may be pale in color, soft in texture, and watery, all of which detract from appearance and ultimately their acceptance by consumers. Therefore, quality of all meat products must be considered along with composition when assessing overall value. The conflict between composition and quality continues to challenge scientists to discover new genetic combinations, different feeding and management programs, and more satisfactory postmortem processing technologies to ensure an ideal meat product that meets consumer demands. ACKNOWLEDGMENTS The author appreciated being invited to prepare this chapter. He thanks the dedicated scientists and teachers that contributed resources and advice and is especially indebted to the Department of Animal Sciences and the College of Agricultural and Life Sciences, University of Wisconsin–Madison, for providing financial assistance, support, and encouragement. Finally, you are encouraged to take one last look at Figures 1 and 2. They summarize this chapter and serve as foundations for those that follow! REFERENCES 1.

2. 3. 4.

5.

CE Allen, DC Beitz, DA Cramer, RG Kauffman. Biology of Fat in Meat Animals. North Central Regional Research Publication # 234, College of Agricultural and Life Sciences, University of Wisconsin, Madison, WI, 1976, pp. 1–74. HB Hedrick, ED Aberle, JC Forrest, MD Judge, RA Merkel. Principles of Meat Science. 3rd ed. Dubuque, IA: Kendall/Hunt, 1993, pp. 11–78. RG Kauffman. Variation in gross composition of meat animals. Proc Recip Meat Conf 24:292–303, 1971. RG Kauffman, TD Crenshaw, JJ Rutledge, DH Hull, BS Grisdale, J Penalba. Porcine Growth: Postnatal Development of Major Body Components in the Boar. University of Wisconsin, College of Agricultural and Life Sciences Research Report R3355, 1986, pp. 1–25. RG Kauffman, LE St. Clair. Porcine Myology. University of Illinois College of Agriculture Experiment Station Bulletin 715, Urbana, IL, 1978, pp. 1–64.

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Meat Composition 6.

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DM Kinsman, AW Kotula, BC Breidenstein. Muscle Foods: Meat, Poultry and Seafood Technology. New York: Chapman & Hall, 1994, pp. 224–247. 7. AOAC. Official Methods of Analysis, 16th ed. 6th rev. Vol. II. Gaithersburg, MD: AOAC International, 1999, pp. 39.1–39.23. 8. RA Field, ML Riley, FC Mello, MH Corbridge, AW Kotula. Bone composition in cattle, pigs, sheep and poultry. J Anim Sci 39:493–499, 1974.

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Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

2 Postmortem Muscle Chemistry MARION L. GREASER University of Wisconsin–Madison, Madison, Wisconsin

I. INTRODUCTION II.

III.

STRUCTURE AND FUNCTION OF LIVING MUSCLE A. Microstructure B. Contraction Mechanism C. Muscle Metabolism POSTMORTEM CHANGES IN MUSCLE A. Biochemical Alterations B. Physical Alterations C. Unusual Patterns of Postmortem Metabolism

IV. SUMMARY REFERENCES

I. INTRODUCTION The conversion of muscle to meat is a complex process involving many biochemical and physical changes. Muscle tissue is converted from an extensible, metabolically active system to one that is inextensible and quiescent in regard to its biochemical reactions. The speed and extent of postmortem metabolism has a profound effect on the properties of the muscle and its subsequent use for food. Also the extent of shortening during the postmortem period affects meat’s textural properties. This chapter summarizes our understanding of muscle’s structural components, its metabolic pathways, and its behavior postmortem. Several books (1,18,37,41,46) and reviews (5,10,13,21,22,23,29) may be consulted for more extensive coverage of these topics.

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II. STRUCTURE AND FUNCTION OF LIVING MUSCLE A. Microstructure 1. Light Microscope Level Skeletal muscle is the largest tissue component of most meat animal species. A diagram showing the organization of muscle at various levels is shown in Fig. 1. Whole muscle is usually attached to bone by a tough, nearly inextensible connective tissue layer called the epimysium. This layer is composed primarily of the protein collagen. The muscle is divided into bundles by thinner layers called the perimysium. Finally, each muscle cell or fiber is encased in a thin layer referred to as the endomysium. Muscle cells, when viewed longitudinally in the microscope, have a striped or striated appearance. They are formed during embryonic development by the fusion of many precursor cells. The resulting muscle cells are typically long and cylindrical and contain nu-

Figure 1 Diagram showing the levels of organization of muscle. (From Ref. 22, used by permission.)

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Postmortem Muscle Chemistry

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Figure 2 Light micrograph of a longitudinal view of several muscle fibers. This micrograph was prepared from muscle tissue that had been fixed, paraffin embedded, sectioned, and stained with hematoxylin and eosin. The double-headed arrow demarcates a single muscle fiber. The typical striped or striated appearance is visible. Each muscle fiber has multiple nuclei; one is shown with an N. Each muscle cell is adjacent to one or more capillaries (arrowhead) that supply oxygen and substrates for metabolism. Scale bar
100 micrometers.

merous nuclei. The number of muscle cells remains relatively constant after birth. The large increase in muscle mass during growth is due to large increases in cell length and diameter. Some of these precursor-type cells persist in the adult and are referred to as satellite cells. The satellite cells lie on the surface of the true muscle cells. A longitudinal view of a single muscle cell (double-headed arrow) is shown in Fig. 2; this photograph is from muscle that has been fixed, paraffin embedded, sectioned, and stained. Note the nuclei (N) and the stripes that run perpendicular to the fiber long axis. This figure also shows an adjacent capillary and blood cells (arrowhead). The alternating dark and light stripes are the result of the presence of myofibrils inside the fiber. The individual myofibrils also have alternating stripes, and the striations in the fiber occur because the adjacent myofibrils have their respective light and dark bands aligned. The dark bands are called the A-bands and the light bands the I-bands (Figure 1). A thin perpendicular line referred to as the Z line bisects the I-bands. The banding structure is somewhat obscure in whole fibers and sections because the cell is too thick for everything in the picture to be in focus. However, the myofibrils can be separately observed after disrupting muscle by homogenization and their band patterns are much clearer (Fig. 3). The region between successive Z lines is called a sarcomere and it is the smallest functional

Figure 3 Light micrograph of a bovine psoas myofibril. The banding patterns visible in the intact cells are also seen in the myofibril. Alternating dark A bands and light I bands can be observed. The I band is bisected by a thin line called the Z line. The section of a myofibril between a pair of Z lines is called a sarcomere. Scale bar
1 micrometer. Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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unit of the myofibril. The length of sarcomeres varies in suspensions of myofibrils because they are derived from muscle cells that have varying degrees of shortening. The A-bands, if visible, always have the same length, but the I-bands decrease in length in myofibrils with shorter sarcomeres. Myofibrils with long sarcomeres have a zone in their middle that has somewhat lower intensity; this region is called the H zone. 2. Electron Microscope Level An understanding of the filament arrangement that is responsible for these patterns became apparent with the advent of electron microscopy. The elegant work of Hugh Huxley showed that the sarcomere is composed of two major types of filaments (24). The thick filaments (about 1.6 micron in length) are found in the A-band and they interdigitate with thinner filaments (about 1 micron in length) that attach to the Z lines (Figs. 1 and 4). Muscle contracts by a relative sliding of these two filaments over one another. The filaments are in turn composed of proteins; myosin is the major constituent of the thick filaments and actin, troponin, and tropomyosin make up the bulk of the thin filaments. Two other narrow, filamentous proteins are present in the sarcomere but are not typically visible by electron microscopy of intact muscle. Titin, a giant protein that extends from the middle of the sarcomere to the Z line (Fig. 1), is elastic in nature and believed to be important for myofibril assembly and for protecting the muscle from overstretch. Nebulin is attached to the Z lines and is postulated to regulate the length of the thin filaments. These latter two proteins are sometime referred to as cytoskeletal proteins. The sarcomere and its filaments are highly ordered in cross-section as well as longitudinally. The thick filaments are arranged in a hexagonal pattern. Six thin filaments surround each thick filament, and each thin filament is centered between three thick filaments. 3. Muscle Cell Heterogeneity Despite the high degree of filament order in muscle, there is considerable heterogeneity in the properties of the individual muscle cells. The striking color difference between the breast muscle of a chicken or turkey compared to the muscle of the thigh or leg is readily apparent. Early classification work grouped muscle cells into two groups, red and white (15). The red muscle cells have more myoglobin (responsible for the color), have slower contraction speed, and typically rely on oxidative metabolism for ATP generation. The

Figure 4 Electron micrograph of a longitudinal view of muscle. The banding pattern can be explained by the positions of the thick and thin filaments. The thin filament length is approximately 1 micrometer.

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Figure 5 Light micrograph of pig muscle in cross-section. A. Succinic dehydrogenase stained. B. Myosin ATPase after pre-incubation at pH 4.7. In pig muscle the dark type I fibers tend to be more clustered together than with muscles from other species. Scale bar
100 micrometers.

white muscles have more glycolytic enzyme content, have a faster contraction speed, and have an energy metabolism that depends on glycolysis. However, it soon became apparent that this simple two-type system for muscle fiber classification was inadequate. The best current fiber type classification system is based on myosin isoforms (using either type-specific antibodies or histochemistry after various acid or alkaline pretreatment of muscle cross-sections). Type I fibers belong to the red group; type IIA, type IIB, and type IIX (or IID) constitute the white group. However, even this nomenclature is muddied by the fact that many fibers contain more than one myosin type. In most mammalian muscles the fiber types are mixed even down to the fiber bundle level. An example of fiber-type heterogeneity is shown with cross-sections from pig muscle (Fig. 5). In A, a frozen muscle section has been stained for the mitochondrial enzyme succinic dehydrogenase. The red, oxidative type I fibers have a darker appearance whereas the type II fibers are more lightly stained. In B, the muscle section has been incubated at pH 4.7 prior to exposure to ATP and a phosphate-precipitating agent. The lighter fibers (in this case also the type II fibers) have had their myosin inactivated by the low pH treatment, while the darker staining fibers (type I) have stronger ATPase activity remaining. The proportions of the different fiber types are different between muscles, between different regions of the same muscle, and different between species. These divergent properties need to be kept in mind in the ingredient mixtures used in meat processing. B. Contraction Mechanism The primary function of muscle is locomotion. The muscle cell is packed full of contractile myofibrils. Contraction is activated by a nerve impulse that passes from the spinal cord to

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a specialized region on the muscle cell termed the motor end plate. A single axon connects with several hundred muscle fibers in a group called a motor unit. At the end plate a small amount of acetyl choline is release from the end of the axon, and this acetyl choline diffuses to the muscle cell surface and binds to acetyl choline receptors embedded in the muscle cell membrane. The receptors cause a local depolarization of the cell membrane, and this depolarization wave spreads over the surface of the muscle cell. In addition the depolarization wave passes down special perpendicular invaginations called T tubules that penetrate to the center of the muscle cell. The T tubules are attached to a specialized intracellular membrane system termed the sarcoplasmic reticulum. The attachment involves a protein called the ryanodine receptor (this protein is so named because of its affinity for the plant alkaloid ryanodine). When the depolarization reaches the T tubule–sarcoplasmic reticulum junction, the ryanodine receptor opens, and calcium is released into the cell cytosol. The calcium diffuses to the myofibrils and binds to troponin on the thin filaments. Calcium causes a shape change in the troponin; this in turn causes tropomyosin to move deeper into the groove of the actin. With the tropomyosin movement, a binding site for the heads of the myosin on the surface of the actin is exposed, and the myosin binds and pulls or pushes the actin a small distance (about 10 nm). The myosin head then releases and it can re-attach to another actin. This sliding of the filaments requires energy that is provided by ATP. The terminal phosphate bond can store this energy and release it to do the mechanical work. The myosin head is believed to bend in its middle region, with the portion near the thick filament shaft acting as a lever arm. The calcium required for activation is pumped back inside the sarcoplasmic reticulum by an ATP powered process. The cell membrane polarity is reestablished by the sodium—potassium pump found in the outer cell membrane. It too requires ATP to move the sodium and potassium against their concentration gradients. A single muscle contraction is called a twitch, and it only requires about 200 milliseconds to complete. C. Muscle Metabolism An overview of the major metabolic pathways involved in muscle energy conversions is shown in Fig. 6. The pathways illustrated all revolve around the production and utilization of ATP. The major fuels include glycogen, glucose, and fatty acids. In addition, small

Figure 6 Overview of metabolism of muscle.

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amounts of amino acids can be metabolized under certain conditions. Glucose and fatty acids enter muscle cells by escape from the capillaries, diffusion through the extracellular space, and active transport across the muscle cell membrane. Glycogen is a polymer of glucose that is stored in muscle in preparation for ATP generation. Resting muscle or muscle undergoing mild activity relies primarily on fatty acid metabolism for ATP synthesis. However, intense work may require more ATP-generating capacity than can be provided by lipolyis and fatty acid transport from the bloodstream (13). There is a small supply of triglycerides in the muscle cell that could liberate fatty acids, but this pathway does not appear to be significant for producing ATP. Both the glycolysis from glycogen and glucose as well as degradation of fatty acids result in the generation of pyruvate. In living muscle, pyruvate is usually transported to the mitochondria for further oxidation to carbon dioxide and water. The mitochondrial pathway yields a much larger amount of ATP than the glycolysis steps (indicated by the dense arrow in the figure). The greatest rate of ATP utilization in the muscle cell occurs during contraction. However, there is a continual need for ATP to power the calcium and sodiumpotassium pumps in the cell. An additional backup source of high-energy phosphate bonds can be provided by creatine phosphate (CP) (Fig. 6). The enzyme creatine phosphokinase catalyzes the reaction of ADP plus CP to generate ATP plus creatine (C). When the ATP and C levels are high, the enzyme operates in the reverse direction to generate new CP. ATP levels are typically about 5 mMolar in the muscle cell, but CP levels may reach 20 to 30 mMolar in quiescent muscle. The transfer of the phosphate from CP to ATP is very rapid. Historically there was much controversy about whether ATP really was required for muscle contraction because no decline in ATP levels could be detected during a twitch—the creatine phosphokinase reaction restores ATP to resting levels so quickly that the ATP level stays essentially constant. However, the limited quantity of CP (probably no more than 5-fold greater than the ATP levels) means that it rapidly becomes depleted during short periods of intense work. The steps in the glycolysis pathway are illustrated in Fig. 7. The enzymes catalyzing the various steps are shown in italics. This is a remarkable pathway because the flux of metabolites can be varied nearly 100-fold with little or no change in the concentrations of the various intermediates (23). Intense effort for many years has been exerted to determine what controls the rate of glycolysis. It is now clear that stress can activate the glycolytic rate by a cascade of steps involving formation of cyclic AMP and ultimately the conversion of the relatively inactive phosphorylase a to the much more active phosphorylase b (Fig. 8). The enzyme phosphofructokinase is also activated and inhibited by various metabolites that change in concentration between active and inactive cells. However, the key control of the glycolytic pathway is the supply of ADP and phosphate (5). Thus, glycolysis will proceed rapidly when ATP breakdown rates are high and virtually stop when the ATP requirements are minimal. III. POSTMORTEM CHANGES IN MUSCLE A. Biochemical Alterations 1. Small Molecule Changes Muscle does not cease to function at the time an animal dies. However, the metabolic functions are markedly altered. The dashed arrows in Fig. 6 indicate the reaction pathways that

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Figure 7 Glycolysis pathway. become blocked within a few minutes postmortem. First, the cessation of the blood supply means that there is no longer a renewable supply of glucose or fatty acids from the bloodstream. Second, the supply of oxygen is cut off. Thus the major ATP generating source for muscle is abolished after death. The pyruvate that is generated as an end product of glycolysis is converted to lactic acid, and, since metabolic waste products cannot be removed without a bloodstream, the lactic acid accumulates in the muscle. Typically there is a burst of muscular activity at the time of death due to trauma of the brain and spinal cord. Thus measured levels of creatine phosphate obtained within 5 minutes after death are usually depressed from those values found with biopsies or by nuclear magnetic resonance measurements on living muscle. Although the muscular activity rapidly subsides within a few min-

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utes after death, the sarcoplasmic reticulum calcium pump and the cell membrane sodiumpotassium pump continue to function to move their respective ions against the concentration gradients. The glycolysis pathway is the only source for this ongoing requirement of ATP. However, because there is a fixed supply of glycogen at the time of death, glycolysis can only continue for some limited time period postmortem. Usually glycolysis ceases before all the glycogen is depleted. Although the reasons for this cessation are not completely understood, possibilities include (a) the low pH that develops may inactive one of the glycolytic enzymes and/or (b) the conversion of adenine nucleotides to inosine derivatives may halt the glycolytic flux. Some ATP is regenerated by the myokinase-catalyzed reaction of two moles of ADP to form one mole of ATP and one mole of AMP. The AMP is converted to IMP and ammonia by the enzyme AMP deaminase. A graphical example of postmortem changes in a variety of muscle properties is shown in Fig. 9. The time scale would be appropriate for pig muscle undergoing typical postmortem processing procedures (i.e., transfer of the dressed carcass to a 0–4°C chiller at 30 minutes after death). Note that the pH decline is fairly linear and is paralleled by the increase in lactic acid concentration. The latter often reaches values in excess of 100 mMolar. ATP concentrations remain fairy stable for the first couple of hours post-mortem and then begin a linear decline. The commencement of this decline coincides with the depletion of the creatine phosphate. Creatine phosphate is usually depleted before the pH reaches 6.0. The overall shapes of the curves and relationships between the different parameters shown are consistent between different animals and species. The major differences will be in the time axis. The approximately 6-hour time course shown for the pig would be more like 18 to 24 hours in beef, 6 to 12 hours in lamb, and less than 3 hours for poultry. These are average times for muscles left on the carcass after slaughter. It should be noted that there is

Figure 8 Stress activation of the glycolysis pathway.

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Figure 9 Chemical and physical changes in muscle postmortem. The pattern shown would be typical for pig muscle undergoing normal metabolism. Abbreviations: ATP - adenosine triphosphate; CP - creatine phosphate; LA - lactic acid; Ext - extensibility.

considerable variation in the cooling rate at different locations in the carcass. Thus the surface will cool more rapidly than the deeper portions of the muscle tissue. One would therefore expect that the deeper portions of the round and chuck would reach the ultimate pH and go into rigor mortis before muscle that is closer to the surface. Likewise, in muscles susceptible to cold shortening, those regions within a couple of centimeters of the surface that cool very rapidly may go into rigor earlier than muscle 5 to 10 centimeters below the surface. The size of the carcass, the amount of fat cover, the temperature of the chiller, and the air velocity will all have a profound effect on rate of postmortem metabolism and development of rigor mortis. In addition the use of electrical stimulation to speed postmortem glycogen and high energy metabolite depletion will also have a profound effect on the time course of muscle metabolism. 2. Protein Changes A number of postmortem changes in the muscle proteins have been identified. The myofibrillar proteins desmin, troponin T, titin, nebulin, and vinculin all become partially or completely degraded during the first week postmortem (6). Although the proteolytic enzymes responsible for this degradation have not been unequivocally identified, the patterns of fragments generated and the proteolytic susceptibility in vitro all suggest that the calpains are involved. Calpains are calcium-activated proteases originally described by Dayton and coworkers (16). Three different isoforms of the calpains have been identified in muscle—m-calpain, -calpain, and P94-calpain. Millimolar levels of calcium activate mcalpain, and -calpain requires only micromolar concentrations for activity. The recently described P94-calpain is slightly larger than the other two isoforms—its role in protein degradation is unknown since to date it has been not possible to isolate the enzyme from muscle in an enzymatically active form. The current hypothesis regarding postmortem protein degradation suggests that the calcium in the sarcoplasmic reticulum leaks into the cytosol and activates the calpains after the muscle ATP is depleted (26). A number of pieces of evidence support this hypothesis. First, soaking muscle strips in calcium solutions or in-

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jecting muscle with calcium results in increased proteolytic degradation. Second, animals such as the callipyge lamb that have higher muscle levels of calpastatin (the natural inhibitor of the calpains) have lower rates of postmortem protein degradation (20). Third, the proteins that are degraded by the calpains in the test tube are the same ones that are broken down in postmortem muscle. The calpains are maximally active near pH 7, so they would be expected to have much lower activity at the usual ultimate pH of around 5.5. The activity of -calpain declines rapidly post mortem; m-calpain is more stable (5). Calpastatin activity also declines after death, so it remains unclear which of these components are most important in controlling postmortem protein degradation. B. Physical Alterations 1. Rigor Mortis The major physical change that occurs in postmortem muscle is the development of rigor mortis. The term comes form Latin and means the stiffness of death. The time for rigor mortis development can be estimated by alternately loading and unloading a muscle strip and recording the changes over time (3,12). Such a trace is illustrated in the upper part of Fig. 10. The extensibility remains relatively constant for some time postmortem (Fig. 10 bottom); this period is called the “delay” phase (5). Subsequently the extensibility declines rapidly during the “onset” phase. Finally, the muscle reaches a stage where there is no further decline in extensibility, and this is referred to as the “completion” of rigor. The time course of rigor mortis is linked to metabolite changes in the muscle. The completion of rigor corresponds to the point where the ATP has been depleted (Fig. 9). The onset period appears to start when the ATP levels begin to decline (note that the time courses shown in Fig. 2-9 and 2-10 are not for the same species or muscle). The loss of ex-

Figure 10 Rigor measurement and pattern. The upper trace demonstrates the pattern of weight addition and removal and the resulting changes in muscle length immediately post mortem. The lower trace shows the changes in extensibility from soon after death until the muscle is in full rigor. The pattern shown was obtained from a rabbit that had been anaesthetized before death. (Modified from Ref. 5, used by permission.)

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tensibility is due to the firm attachment of the myosin heads to actin. In the normal contraction cycle, ATP is required to dissociate these two proteins and allow the filaments to slide over one another. However, when there is no ATP present, the two proteins become firmly linked together and no longer allow muscle shortening or extension. The process is somewhat complicated by the fact that not all fibers in a muscle strip deplete their ATP at the same time because of biological variation and the difference in fiber types. The pattern shown in Fig. 9 bottom was produced from an animal that had been anaesthetized at the time of death. Animals slaughtered under commercial conditions will typically have a much shorter delay phase or even the absence of delay. The time course of rigor mortis is extended two- to threefold at 10°C versus 37°C (34). 2. Shortening Unrestrained muscle may also shorten as it goes into rigor mortis. This shortening can be as high as 25–30% of the initial length when the muscle is maintained at 37°C (31). Minimum shortening occurs at temperatures near 15°C. Similarly, if muscle is held isotonic, it will develop force during the onset of rigor mortis. The force developed is much weaker than that produced by living muscle during contraction (estimated to be less than 5%) (5), but is sufficient to significantly change the sarcomere length and the tenderness properties of muscles still attached to the carcass. Force begins to develop at the beginning of the onset phase of rigor (42). This force may be developing due to a rise in cytoplasmic calcium as the ATP nears depletion or because of the activation of contraction due to binding of myosin heads to actin (9). C. Unusual Patterns of Postmortem Metabolism 1. Thaw Rigor The term thaw rigor is somewhat of a misnomer. The name refers to the shortening that occurs when muscle is rapidly frozen pre-rigor and then subsequently thawed. Muscle that has been treated in this way shortens markedly (as much as 70–80%) and loses large of amounts of liquid (more than 25% of the initial weight) as drip (36). The mechanism of this phenomenon is believed to be the disruption of the sarcoplasmic reticulum due to ice crystal formation followed by the release of calcium upon thawing (27). The calcium then activates contraction since there is ATP remaining in the region of the myofibrils. The degree of shortening depends largely on the size of the muscle piece frozen and thawed. As the surface regions thaw, the inner parts provide a rigid scaffold to prevent shortening. The ATP is used up rapidly after thawing, so the outer portions will go into rigor before the core regions are thawed. Thus the degree of shortening may be minimal on a large muscle piece that has been frozen and thawed pre-rigor. 2. Cold Shortening The usual dependence of postmortem metabolism rate on temperature does not hold in certain cases. The muscles that rely primarily on oxidative metabolism may undergo a slow but significant shortening if excised and held at temperatures below 10°C [see Locker (36) for review]. Locker and Hagyard (31) termed this phenomenon “cold shortening.” The muscle length can decline as much as 50% in unrestrained muscle (35). Cold shortening is a slow process; the time course may be minutes to an hour and depends on the cooling rate. The shortening occurs before there is any reduction in muscle ATP levels (39). This shortening can also occur on the carcass, particularly with muscles not placed under stretch when sus-

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pended from the rail (35). However, the force is weak; Bendall (5) estimates that it is only about 2% to 3% of the maximum produced in a muscle tetanus. Cold shortening is believed to be caused by a gradual rise in the cytosolic calcium level by release from either mitochondria or the sarcoplasmic reticulum (the calcium pump operates more slowly at low temperature). The slightly elevated calcium causes a weak contractile response and the muscle shortens. The weak activation of the myofibrillar ATPase activity also increases the glycolysis rate compared to that at 5°C (5). Cold shortening is usually only a significant problem in beef and lamb. The rapid cooling that occurs with high efficiency chillers exacerbates the problem. Cold shortening leads to an increase in meat toughness (35), so improvements in processing efficiency and food safety are offset by deleterious effects on meat quality. 3. Pale, Soft, Exudative Condition An unusual postmortem phenomenon [first described in pigs by Ludvigsen (32)] is one in which the muscle becomes pale in color, develops a soft texture, and exudes large amounts of liquid. The post-mortem metabolic rate is vastly increased, with ATP depletion, completion of rigor mortis, and pH values as low as 5.3 occurring within 10 to 15 minutes after death (Fig. 11) (11). The low pH that develops while the muscle temperature is still high leads to a denaturation of some of the muscle proteins, notably myosin. This reduces the water-holding activity of the muscle and results in excess drip loss. The rapid postmortem glycolysis is often caused by a mutation in the ryanodine receptor (the calcium release channel) at the interface between the sarcoplasmic reticulum and T tubules (19). The mutation of the ryanodine receptor’s arginine, at position 615 on the molecule, to cysteine results in the protein leaking calcium even when there is no nerve signal for contraction. The elevated cytosolic calcium postmortem causes activation of the myofibrillar ATPase and the resulting acceleration of glycolysis. Attempts to remove animals carrying the mutation are in progress using genetic tests, but not all animals that develop the PSE condition can be identified by this procedure. It may be that there are other mutations in the ryanodine receptor that cannot be picked up by the current test. Such is the case with ryanodine receptor mutations in humans where at least 21 different mutation sites have now been described (8). The incidence of the PSE condition in the United States is approximately 15% (14).

Figure 11 Variation in postmortem pH decline in pigs. The pH decline pattern is closely related to the resulting muscle color. (From Ref. 10, used by permission.)

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Attempts to change animal handling and postmortem processing procedures to reduce the incidence and severity of PSE development have been partially successful. Gentle handling and allowing pigs a rest period before slaughter reduces the incidence of PSE (45). Rapid cooling post mortem, such as briefly immersing a carcass in liquid nitrogen (7), markedly reduces the PSE problem; however, this procedure has not been adopted commercially. Recently PSE has been prevented by early postmortem injection of sodium bicarbonate (25). The bicarbonate appears to slow the rate of pH decline as well as to elevate the ultimate muscle pH. It is now clear that a condition related to PSE also occurs in chicken and turkey muscle (see references 2 and 43 for reviews). With some turkeys the ATP and postmortem pH declines are accelerated, and the resulting muscle has a reduced cooked yield (40). There is also a precipitation of phosphorylase and a reduction in myosin solubility in muscles that have undergone this rapid postmortem glycolysis. The incidence is greater as the result of stress due to hot weather. Whether this rapid glycolysis condition in poultry muscle is related to a mutation in the ryanodine receptor remains to be determined. 4. Dark, Firm, Dry Condition The DFD condition in pigs often results from the same ryanodine receptor mutation that causes PSE. In DFD conditions, the glycogen has been largely depleted before death and lactic acid therefore does not accumulate in the muscle. The time to rigor mortis completion is very short and the resulting ultimate pH is high (it may be greater than 6.5). The meat is dark in color and has a firm texture. The surface is dry and sticky to the touch. Such meat has excellent properties for use in processed meat products because of its high water binding activity. Its unusually dark color may deter purchase at the retail meat case. However, because it only occurs in animals that may alternatively become PSE, conditions that reduce its incidence are usually sought. 5. Dark Cutter The dark cutter condition occurs in beef muscle having a high ultimate pH [see Tarrant (44) for review]. Like the DFD condition in pigs, dark cutters result from the antemortem depletion of glycogen. However, this depletion is not due to a genetic condition but rather appear to result from a stress response (28). The incidence of the dark cutting is often high in bulls (as much as 15%) since they tend to fight. Dark cutting can be reduced be avoiding mixing unfamiliar animals prior to slaughter. Meat from dark cutters may cause difficulty in retailing, but it is a superior product for use in processing because of its high pH and resulting improved water-holding ability. 6. RN Phenotype in Pigs A genetic condition in some pigs results in a different form of altered postmortem metabolism (38). The RN (Napole gene) condition is common in the Hampshire breed of pigs. It is characterized by a significant increase in the glycogen levels in the muscle of the live animal and an ultimate pH that is lower than normal (i.e., 5.3–5.4 instead of 5.5). The rate of postmortem pH decline is normal. However, the lower ultimate pH results in a greater drip loss and a slightly paler color. Animals with this condition can be determined using estimates of the “glycolytic potential” (GP). The GP equals 2([glycogen glucose units]  [glucose]  [glucose 6-phosphate])  [lactate]. Since the glycolytic potential is the sum of the glycogen and its major postmortem breakdown products, it should give similar results whether determined on biopsy samples or from muscle at any time point post-

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mortem. The differences in glycolytic potential are large; a recent study found values of 135 for normal homozygotes and 214 for heterozygotes carrying the RN allele (33). Use of a cutoff value of 180 would have completely segregated the two genetic groups. Muscle from pigs carrying the RN allele often is more tender than that from noncarriers (17). The nature of the genetic alteration causing these large changes in muscle glycogen storage has not been determined to date.* IV. SUMMARY Muscle tissue is complex structurally and is nonhomogeneous in its protein composition and metabolic emphasis. The rate and extent of metabolic changes postmortem have important effects on the color of meat, its texture, and its usefulness for inclusion in processed meat products. In addition there are profound differences in the rates of postmortem metabolism between muscles from different species. For example, electrical stimulation may be beneficial in speeding postmortem metabolism in beef and therefore prevent cold shortening, but acceleration of metabolism in pigs may lead to greater incidence of pale, soft, exudative meat. Thus, an understanding of the biochemical and physical processes that muscle undergoes postmortem is necessary for optimum design of slaughter and meat processing procedures and the use of this tissue for food.

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Bagshaw, C.R. Muscle Contraction. Chapman & Hall, London, 1993. Barbut, S. Problem of pale soft exudative meat in broiler chickens. Br Poult Sci 38:355–358, 1997. Bate-Smith, E.C. and J.R. Bendall. Factors determining the time course of rigor mortis. J Physiol 110:47–65, 1949. Bendall, J.R. The shortening of rabbit muscles during rigor mortis: relation to the breakdown of adenosine triphosphate and creatine phosphate and to muscular contraction. J Physiol 114:71–88, 1951. Bendall, J.R. Postmortem changes in muscle. In: Bourne, G.H. (ed.) The Structure and Function of Muscle, 2nd ed. Volume II, pp 243–309. Academic Press, New York, 1973. Boehm, M.L., T.L. Kendall, V.F. Thompson, and D.E. Goll. Changes in the calpains and calpastatin during postmortem storage of bovine muscle. J Anim Sci 76:2415–34, 1998. Borchert, L.L., and E.J. Briskey. Prevention of pale, soft, exudative porcine muscle through partial freezing in liquid nitrogen post-mortem. J Food Sci 29:203–209, 1964. Brandt, A., L. Schleithoff, K. Jurkat-Rott, W. Klingler, C. Baur, and F. Lehmann-Horn. Screening of the ryanodine receptor gene in 105 malignant hyperthermia families: novel mutations and concordance with the in vitro contracture test. Hum Mol Genet 8:2055–2062, 1999. Bremel, R.D., and A. Weber. Cooperation within actin filament in vertebrate skeletal muscle. Nature New Biol 238:97–101, 1972. Briskey, E.J. Etiological status and associated studies of pale, soft, exudative porcine musculature. Adv Food Res 13:89–168, 1964. Briskey, E.J., L.L. Kastenschmidt, J.C. Forrest, G.R. Beecher, M.D. Judge, R.G. Cassens, and W.G. Hoekstra. Biochemical aspects of post-mortem changes in porcine muscle. J Agr Food Chem 14:201–206, 1966.

* Note added in proof: The mutation for the RN condition has recently been identified. See Ref. 47.

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Briskey, E.J., R.N. Sayre, and R.G. Cassens. Development and application of an apparatus for continuous measurement of muscle extensibility and elasticity before and during rigor mortis. J Food Sci 27:560–566, 1962. Brooks, G.A. Mammalian fuel utilization during sustained exercise. Comp Biochem Physiol B Biochem Mol Biol. 120:89–107, 1998. Cannon, J.E., J.B. Morgan, F.K. McKeith, G.C. Smith, S. Sonka, J. Heavener, and D.L. Meeker. Pork chain quality audit survey: Quantification of pork quality characteristics. J Muscle Foods 7:29–44. Cassens, R.G., and C.C. Cooper. Red and white muscle. Adv Food Res 19:1–74, 1971. Dayton, W.R., D.E. Goll, M.G. Zeece, R.M. Robson, and W.J. Reville. A Ca2-activated protease possibly involved in myofibrillar protein turnover. Purification from porcine muscle. Biochemistry 15:2150–2158, 1976. Enfalt, A.C., K. Lundstrom, A. Karlsson, and I. Hansson. Estimated frequency of the RN-allele in Swedish Hampshire pigs and comparison of glycolytic potential, carcass composition, and technological meat quality among Swedish Hampshire, Landrace, and Yorkshire pigs. J Anim Sci 75:2924–2935, 1997. Engel, A.G. and Franzini-Armstrong, C. (eds.). Myology, 2nd ed. McGraw-Hill, New York, 1994. Fujii, J., K. Otsu, F. Zorzato, S. DeLeon, V.K. Khanna, J.E. Weiler, P.J. O’Brien, and D.H. MacLennan. Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia. Science 253:448–451, 1991. Geesink, G.H., and M. Koohmaraie. Postmortem proteolysis and calpain/calpastatin activity in callipyge and normal lamb Biceps Femoris during extended postmortem storage. J Animal Sci 77:1490–1501, 1999. Greaser, M.L. Conversion of muscle to meat. In: P.J. Bechtel (ed.) Muscle as Food. pp. 37–102, Academic Press, San Diego, CA, 1986. Greaser, M.L., and A.M. Pearson. Flesh foods and their analogues. In: A.J. Rosenthal (ed.) Food Texture: Perception and Measurement, pp 228–258. Aspen Publishers, Inc., Gaithersburg, MD, 1999. Hochachka, P.W. Cross-species studies of glycolytic function. Adv. Exp. Med. Biol. 474:219–229, 1999. Huxley, H. The double array of filaments in cross-striated muscle. J Biophys Biochem Cytol 3:631–647, 1957. Kauffman, R.G., R.L.J.M. van Laack, R.L. Russell, E. Pospiech, C.A. Cornelius, C.E. Suckow, and M.L. Greaser. Can pale, soft, exudative pork be prevented by postmortem sodium bicarbonate injection? J Anim Sci 76:3010–3015, 1998. Koohmaraie, M. Biochemical factors regulating the toughening and tenderization of meat. Meat Sci 43:193–201, 1996. Kushmerick, M.J., and R.E. Davies. The role of phosphate compounds in thaw contraction and the mechanism of thaw rigor. Biochim Biophys Acta 153:279–287, 1968. Lawrie, R.A. Physiological stress in relation to dark-cutting beef. J Sci Food Agric 9:721–727, 1958. Lawrie, R.A. Conversion of muscle to meat: biochemistry. In: D.E. Johnston, M.K. Knight, D.A. Ledward (eds.) The Chemistry of Muscle-based Foods, pp 43–61, Royal Society of Chemistry, Cambridge, UK, 1992. Locker, R.H. Cold-induced toughness of meat. Adv Meat Res 1:1–44. AVI Publishing Company, Westport, CT, 1985. Locker, R.H., and C.J. Hagyard. A cold shortening effect in beef muscles. J Sci Food Agric 14:787–793, 1963. Ludvigsen, J. Muscular degeneration in pigs. Int. Vet. Cong., Stockholm, Sweden, 1:602, 1953. Lundstrom, K., A.C. Enfalt, E. Tornberg, and H. Agerhem. Sensory and technological meat quality in carriers and non-carriers of the RN-allele in Hampshire crosses and in purebred Yorkshire pigs. Meat Sci 48:115–124, 1998.

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20.

21. 22.

23. 24. 25.

26. 27. 28. 29.

30. 31. 32. 33.

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Postmortem Muscle Chemistry 34. 35. 36. 37. 38. 39.

40.

41. 42. 43. 44.

45. 46. 47.

37

Marsh, B.B. Rigor mortis in beef. J Sci Food Agric 5:70–75, 1954. Marsh, B.B. and N.G. Leet. Studies on meat tenderness. III. The effects of cold shortening on tenderness. J Food Sci 31:450–459, 1966. Marsh, B.B., and J.F. Thompson. Rigor mortis and thaw rigor in lambs. J Sci Food Agric. 9:417–424, 1958. Matthews, G.G. Cellular Physiology of Nerve and Muscle. Blackwell Science Inc; Oxford, UK, 1998. Naveau, J. Contribution á l’étude du déterminisme génétique de la qualité de viande porcine Héritabilité du Rendement Technologique Napole. J Rech Porc Fr 18:265–276, 1986. Newbold, R.P. Changes associated with rigor mortis. In: E.J. Briskey, R.G. Cassens, and J.C. Trautman (eds.) The Physiology and Biochemistry of Muscle as a Food, pp 213–224. University of Wisconsin Press, Madison, WI, 1966. Pietrzak, M., M.L. Greaser, and A.A. Sosnicki. Effect of rapid rigor mortis processes on protein functionality in pectoralis major muscle of domestic turkeys. J Anim Sci 75:2106–2116, 1997. Richter, E.A., B. Kiens, H. Galbo, and B. Saltin (eds.) Skeletal Muscle Metabolism in Exercise and Diabetes, Plenum Press, New York, 1998. Schmidt, G.R., R.G. Cassens, and E.J. Briskey. Development of an isotonic-isometric rigorometer. J Food Sci 33:239–241, 1968. Sosnicki, A.A., M.L. Greaser, M. Pietrzak, E. Pospiech, and V. Sante. PSE-like syndrome in breast muscle of domestic turkeys: a review. J Muscle Foods 9:13–23, 1998. Tarrant, P.V. The occurrence, causes and economic consequences of dark-cutting in beef—A survey of current information. In: D.E. Hood and P.V. Tarrant (eds.) The Problem of Dark Cutting in Beef. pp 3–34. Martinus Nijhoff, Hague, Netherlands, 1998. Van der Wal, P.G., B. Engel, and B. Husegge. Causes for variation in pork quality. Meat Sci 46:319–327, 1997. Xiong, Y.L., C.-T. Ho, and F. Shahidi (eds.) Quality Attributes of Muscle Foods. Kluwer Academic Publishers, Dordrecht, the Netherlands, 1999. Milan, D., J.-T. Jeon, C. Looft, V. Amarger, A. Robic, M. Thelander, C. Rogel-Gaillard, S. Paul, N. Iannucelli, L. Rask, H. Ronne, K. Lundström, N. Reinsch, J. Gellin, E. Kalm, P. LeRoy, P. Chardon, and L. Andersson. A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle. Science 288:1248–1251, 2000.

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3 Meat Color OWEN A. YOUNG and JOHN WEST MIRINZ Centre AgResearch, Hamilton, New Zealand

I. INTRODUCTION II.

THE NATURE OF COLOR

III.

PIGMENTS IN LEAN MEAT

IV.

THE CHEMISTRY OF MYOGLOBIN COLOR A. Reactions of Myoglobin with Oxygen B. Effect of pH and Temperature on Autoxidation C. Reactions of Myoglobin with NO and CO D. Metmyoglobin Reductase

V.

VI. VII.

THE FUNDAMENTALS OF RAW MEAT COLOR A. Main Factors Affecting Raw Meat Color B. Chronology of Meat Color from Slaughter to Display MEASUREMENT OF MEAT COLOR APPLIED ASPECTS OF COLOR IN MEAT A. Species, Animal Age, and Muscle Type B. Effect of Vitamins on Color Stability C. Effect of Processing Conditions D. Effect of Packaging, Temperature, Storage, and Display Conditions E. Effect of Abnormal Rigor Conditions on Meat Color F. Effect of Ionizing Radiation G. Cooked Meat Color

VIII. FAT COLOR IX.

CONCLUDING REMARKS REFERENCES

I. INTRODUCTION The appearance of meat is important to retail shoppers. Appearance includes size; shape; the relative quantities and distribution of lean tissue, fat and bone; and color. Each of these factors is evaluated, either consciously or unconsciously, as buyers examine meat on retail display. Customers know that bone is inedible and fat is less nutritious than lean meat. Less

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obvious is the learned need to inspect meat to compensate for variability in the product or to protect against deliberate misrepresentation. In the meat industry’s present state, few consumers will buy meat sight unseen, which contrasts sharply with their willingness to buy manufactured products where consistency is tightly controlled. Consumers consider that the color of lean tissue is a particularly important indicator of meat quality. Red meat tends to turn brown with time when exposed to air. Meat also tends to spoil with time due to microbiological activity, so the browning of meat with time is a rough indicator of spoilage. However, it is important to realize that the two events— browning and spoilage—are largely independent, and with modern hygiene control, meat exposed to air will become brown long before spoilage develops. Moreover, as is discussed in Chapter 13, meat tends to become more tender the longer its is stored, so the modern preoccupation with “freshness” in foods can be detrimental to the eating quality of meat. Nonetheless, the connection between browning and spoilage persists, so meat color remains of prime importance to meat industries throughout the world. This chapter explores meat and fat color and how it is affected by species, breed, diet, age, processing, storage, and display. II. THE NATURE OF COLOR The color of an object is the perception of the spatial patterns of different wavelengths of light that emanate from that object. When light strikes an object, each wavelength contributing to that light suffers three fates to varying degrees: some passes through, some is absorbed, and some is reflected. The fraction of each wavelength reflected by an object is fundamentally important to its color. If blue and green wavelengths pass through the object and/or are absorbed more than red wavelengths, the object will reflect more red light and so appear red to the brain. The spatial patterns refer to the place from where the wavelengths are reflected (e.g., deep within an object or on the surface), and to the manner of reflectance—scattered like light from frosted glass or spectral like light from a mirror. However, the fundamental color of an object, whether shiny, matte, or translucent, depends on the absorbance of light by pigments in that object. III. PIGMENTS IN LEAN MEAT Unlike many fish species, the meat of land mammals—particularly if undomesticated—is characteristically red. The pigment responsible for most of this color is the protein myoglobin. This protein consists of a globular protein of about 153 amino acids (about 17,000 g/mole), housing a porphyrin ring structure held in a pocket of the protein (Fig. 1a). At the ring’s core is a cavity large enough to hold a transition metal ion. Porphyrin-like structures occur widely in nature. In the case of green plants, the metal is magnesium, which generates the green pigment chlorophyll, whereas in myoglobin the metal is iron. The combination of iron and porphyrin is called heme. Myoglobin is chemically very similar to the blood protein hemoglobin, which also contains iron bound in porphyrin. Muscle contains some hemoglobin, but its hemoglobin content is much lower than that of myoglobin. This hemoglobin is restricted to the fine vascular supply that permeates the muscle. The drip that leaks from raw steaks and other meat cuts is not blood as is commonly believed, but mainly myoglobin dissolved in the muscle cell exudate (Hunt and Hedrick, 1977).

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Meat Color

A

41

B

Figure 1 (a) A schematic view of myoglobin showing heme (a porphyrin ring plus iron) nestled in the globin, the protein part of the molecule. (b) A closeup of heme showing iron (Fe2) coordinated to four nitrogens of the porphyrin ring (incompletely drawn) plus a histidine residue in the globin protein. The sixth site is available for oxygen binding.

Because myoglobin is the dominant pigment in muscle, measurements of iron concentration, myoglobin concentration, and color are all strongly correlated. If one muscle appears redder than another, it very likely contains more myoglobin and, thus, more iron. The porphyrin ring structure held in the confines of the myoglobin protein accounts for four of the six coordination sites available on the iron atom (Fig. 1b). These four sites are the nitrogen atoms of the porphyrin’s pyrrole groups. A fifth coordination site is a strategically placed histidine molecule resident in the globular protein. The sixth coordination site is available for binding oxygen or other small molecule that qualifies. Binding at the sixth site is largely responsible for the various colors of meat, mainly red, but also purple, brown, and other colors. In the live animal, myoglobin functions to temporarily bind oxygen, bridging the gap between oxygen bound to hemoglobin in blood, and chemically reduced oxygen bound to hydrogen (as water) produced by respiration in mitochondria. The concentration of myoglobin differs from species to species (Table 1). Whales and other marine mammals have very high concentrations of myoglobin, which allows them to remain underwater for extended periods. Selective breeding for enhanced muscle growth (hypertrophy) has been applied to many domestic species, but among the meat animals, pigs, cattle and chickens have been modified the most. However, the increase in the quantity of total muscle protein has not been matched by a similar increase in the quantity of myoglobin, with the net result that myoglobin concentration is lower in highly selected breeds particularly for pigs. Within an animal, different muscles often have different concentrations of myoglobin, generally reflecting their role in the animal. Muscles involved in sustained repetitive action, like breathing (diaphragma), contain higher concentrations of myoglobin than muscles used less often (Table 2). The lateral line muscle of fish, which is used for sustained, low-power movement, is very much redder than the muscle groups used for short bursts of high-power movement. The pectoralis muscle of chicken, used for power takeoffs, is paler than leg muscles.

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Table 1 Myoglobin in the Muscles of Several Species or Breeds (Mature Animals) Approximate Concn. (mg/g)

Species Cattle (breed unstated) a Sheep (Dorset)b Pig (Hampshire) Wild pig

Reference

2–5 3–7 3–6 Higher than for domestic pigs 0.1–5 0.5–1 4.4–5.2 60 50–72 80

Chicken Tuna c Human a Whale (Hyperoodon rostratus) Dolphins Seal (Phoca vitulina)

Hunt and Hedrick (1977) Ledward and Shorthose (1971) Topel et al. (1966) Rahelic and Puac (1980) Nishida and Nishida (1985) Brown (1962) Möller and Sylvén (1981) Scholander (1940) Dolar et al. (1999) Robinson (1939)

a

For a range of muscles. Longissimus. c Light muscle. b

Muscles low in myoglobin are generally tailored to glycolysis (Chapter 2) as a means of generating ATP, whereas muscles rich in myoglobin generate ATP through oxidative metabolism. It therefore follows that redder muscles are also richer in mitochondria and, as part of the oxidative machinery, richer in cytochromes. As the name suggests, cytochromes are colored, again due to iron bound in porphyrin. However, cytochromes are present in much lower concentrations than myoglobin (Akeson et al., 1960; Rennere and Labas, 1984). Thus, the color of meat is dominated by the color reactions of myoglobin.

Table 2 Concentration of Myoglobin (mg/g) Within Muscles of Different Species Species Muscle Longissimus Psoas major Gluteus medius Semimembranosus (outer) Semimembranosus (inner) Semitendinosus (outer) Semitendinosus (inner) Biceps femoris Rectus femoris Diaphragma

Cattle a,b

Pig (Hampshire)c

Tunad

3.48 3.71 4.11 3.91 3.56 2.97 1.95

2.94 6.37

Light meat Lateral line muscle

4.05

Chicken e

5.06 5.66

Pectoralis Vastus lateralis Vastus intermedius Biceps femoris Rectus femoris Gizzard

~7

a

Hunt and Hedrick (1977). Renerre and Labas (1984). c Topel et al. (1966). d Brown (1962). e Nishida and Nishida (1985). b

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0.7 20

~0.1 2.8 5.0 0.7 2.5 19

Meat Color

43

IV. THE CHEMISTRY OF MYOGLOBIN COLOR A. Reactions of Myoglobin with Oxygen In its role as an oxygen store, the sixth coordination site on the iron in myoglobin’s porphyrin ring reversibly binds molecular oxygen (Fig. 1). When bound, the color of myoglobin changes from a purple-red to a bright red (Reaction 1). Myoglobin  O2 → Oxymyoglobin (Purple-red) (Bright red)

(Reaction 1)

For oxymyoglobin in isolation or in meat, this binding causes reflectance around 470 nm (blue wavelengths) to be reduced, and reflectance at the red end of the spectrum (600 nm) to be increased (Fig. 2). Meat color changes from purple-red to bright red. Reaction 1 can be rewritten as: Mb2  O2 → Mb2O2

(Reaction 2)

to emphasize the point that the iron atom at the business end of the myoglobin molecule in these two forms is in the 2 (ferrous) oxidation state. The sixth coordination site of myoglobin (or hemoglobin for that matter) is not specific for just oxygen. However, there are some important constraints in the formation of stable complexes with other molecules. First, the cleft that holds molecules in myoglobin is small (Takano, 1977)—only small molecules need apply. Second, only molecules that can

Figure 2 Comparative reflectance spectra for solutions of myoglobin (sometimes called deoxymyoglobin), oxymyoglobin, and metmyoglobin. These reflectance spectra are perceived as purple-red, bright red, and brown, respectively. The color change from myoglobin to oxymyoglobin is achieved by a reduction in blue wavelengths and an increase in red wavelengths. Reflectance spectra of meat have flatter peaks, but the main features remain.

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induce a low energy state can form stable complexes. Although many small compounds can fulfil this role, producing a range of colored compounds (Dymicky et al., 1975), only three ligands are usually important in the context of meat: oxygen (O2), nitric oxide (NO), and carbon monoxide (CO). The exact details of how the electrons redistribute themselves in the complexes with the three relevant molecules are unimportant here, except that the molecule donates two electrons to the complex while the iron ‘back-donates’ electrons to form extra stabilizing bonds (Giddings, 1977). These extra bonds develop only when iron is in the ferrous (Fe2) oxidation state. In the ferric state (Fe3), the higher charge of the iron binds electrons more tightly and prevents this important back-donation of electrons (Livingstone and Brown, 1981). For this reason, myoglobin in the ferric state—called metmyoglobin—cannot bind oxygen but binds a molecule of water instead. Metmyoglobin’s inability to bind oxygen is crucially important because metmyoglobin slowly forms from oxymyoglobin, both in live muscle and in meat. In the case of the live animal, metmyoglobin formation must be avoided because metmyoglobin is useless as a oxygen store. In the case of meat, metmyoglobin formation must be avoided because this pigment is brown (Fig. 2) and not the attractive bright red that consumers value. The reaction of oxymyoglobin to form metmyoglobin is termed autoxidation because it occurs with oxymyoglobin as the sole reacting species. Compared to the rate at which myoglobin binds oxygen to form oxymyoglobin, a matter of seconds, the rate of autoxidation is measured in hours. Although autoxidation is a comparatively slow reaction, it is still commercially important because meat on retail display in air or in an oxygen-enriched atmosphere must maintain a bright-red color for several days. Before describing the kinetics of browning in meat on retail display, it is useful to further explore the kinetics of oxymyoglobin autoxidation, because much meat color can be explained in terms of autoxidation chemistry. Figure 3 shows the relationship between the partial pressure of oxygen and the first order rate constants for the autoxidation of oxymyoglobin to metmyoglobin (George and Stratmann, 1952). The higher the rate constant, the faster the reaction. After a steep rise from zero in the complete absence of oxygen, the rate constant peaks at an oxygen partial pressure of around 1.5 mm of mercury, then declines to level out at around 40 mm. (On this scale 760 mm is one atmosphere of pure oxygen.) It turns out that the partial pressure of 1.5 mm corresponds to the concentration required to exactly half-saturate the myoglobin with oxygen (50% oxymyoglobin). This concentration is within the range normally found in live muscle, where oxygen reversibly binds to or is released from myoglobin, depending on the muscle’s activity. Thus the tendency to form metmyoglobin— which cannot bind oxygen—is greatest in the very working range of myoglobin in the living muscle. Autoxidation appears to be an unavoidable side reaction of the main event, mediating oxygen transfer between hemoglobin and mitochondria. Not surprisingly, an enzyme, metmyoglobin reductase, is active in muscle (Livingston et al., 1985) to reduce metmyoglobin (Fe3 ) back to the oxygen-binding form, myoglobin (Fe2). Metmyoglobin reductase is discussed shortly. The detailed mechanism of autoxidation is still debated, but recent research indicates that oxymyoglobin decomposes into ferrimyoglobin (Fe3 state) and superoxide radical (O 2 ) as a first step. The latter is capable of starting a series of damaging oxidative reactions in the cell, including damage to the remaining oxymyoglobin. Brown and Mebine (1969) showed that in solution, 0.75 mole of oxygen is evolved for each mole of myoglobin

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45

Figure 3 The rate of autoxidation as affected by the partial pressure of oxygen (where 760 mm is atmospheric pressure). Autoxidation occurs most rapidly at 1.5 mm, which is the point at where myoglobin is exactly half saturated with oxygen. (From George and Stratmann, 1952.) oxidized. Their reaction scheme accounts for the evolution of oxygen and predicts that acid conditions (low pH) will promote the reaction: H  Mb2O2 → Mb3  0.5H2O  0.75O2 (Oxymyoglobin) (Metmyoglobin)

(Reaction 4)

B. Effect of pH and Temperature on Autoxidation Figure 4 plots the rate constant of metmyoglobin formation as a function of pH in the range relevant to meat and meat products. The rate is high around pH 5.5 and decreases markedly as the pH increases to around 6.0 then slowly levels out. This result is consistent with Reaction 4 and suggests that autoxidation will proceed at different rates in meat of different pH values. As might be expected, the higher the temperature the higher the autoxidation rate. Brown and Mebine (1969) reported that a decrease in temperature from 22° to 2°C resulted in a 40-fold decrease in the rate of autoxidation, whatever the pH. C. Reactions of Myoglobin with NO and CO The complexes, nitric oxide myoglobin (Mb2NO) and carboxymyoglobin (Mb2CO) maintain myoglobin in the ferrous form. The former compound, which is also bright red, is the basis of cured meat technology, and this subject is examined in detail in Chapter 20. However, it is useful to briefly examine Mb2NO in the context of myoglobin stability. Although NO dissociates from the myoglobin complex far more slowly than does oxygen, Mb2NO is still considered unstable. This is because in meats, oxygen is often present in

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much higher concentrations than NO, which is usually added by way of traces of sodium nitrite during conventional salting. Excess oxygen can displace NO, which in turn can be lost to through oxidation to nitrate. In curing technology, the loss of NO is prevented by cooking, which generates a stable pink compound, nitrosylhemochrome, which is responsible for the characteristic pink color of cured meats. Carbon monoxide (CO) has a stronger affinity for myoglobin (and hemoglobin) than oxygen. When pale meats like pork are cooked in a gas oven, pinking is sometimes evident on the outer layers of the meat (Maga, 1994). Combustion generates small quantities of CO or NO that bind to the myoglobin, forming stable pink compounds that are visible on a pale background. (The very strong affinity of CO for heme means that death from carbon monoxide poisoning is quick, even when the concentration of CO in air is low. Victims turn pink, and are sometimes described as good looking corpses.) D. Metmyoglobin Reductase In the live animal, concentrations of metmyoglobin in muscle are kept very low due to reductase activity in the muscle tissue (Hagler et al., 1979; Livingston et al., 1985). Metmyoglobin reductase activity is located predominantly on the mitochondrial surface (Arihara et al., 1995). The enzyme requires the reductant NADH, and cytochrome b5 as an electron transfer mediator. The pH activity profile shows a peak around pH 6.5, falling steeply as pH falls (Fig. 5). The enzyme does not lose activity in stored meat and is unaffected by oxygen (Echevarne et al., 1990). Therefore it will be active both in the oxygenated surface layer of meat or deeper in anaerobic tissue, provided NADH is also present. Each of these properties is important in understanding the color behavior of meat on display.

Figure 4 The effect of pH on the rate of autoxidation to metmyoglobin. (From Brown and Mebine 1969.)

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47

Figure 5 The effect of pH on relative metmyoglobin reductase activity. (From Hagler et al., 1979.) V. THE FUNDAMENTALS OF RAW MEAT COLOR A. Main Factors Affecting Raw Meat Color The color relations of a piece of meat exposed to air are more complicated than for pure myoglobin in solution in the presence of dissolved oxygen. There are several reasons for this, including: 1.

Muscle as meat continues to respire after slaughter, so molecular oxygen continues to be reduced by NADH at the end of the mitochondrial electron transport chain. This respiratory system competes with myoglobin for the oxygen that diffuses into the meat from the atmosphere. (Diffusion from the cut surface of meat contrasts with the situation in the live animal, where the vascular system supplies oxygen to all parts of the muscle.) However, oxygen consumption decreases with time postmortem (Lanari and Cassens, 1991), so the balance between metabolic oxygen consumption and oxygen binding by myoglobin changes with time. 2. The enzyme metmyoglobin reductase remains active in meat and providing the reductant NADH is present, metmyoglobin can be reduced to the ferrous form (myoglobin) capable of binding oxygen to produce oxymyoglobin. 3. Meat represents muscle in a degradative state. A number of reactions occur that generate free radicals, molecular species with a single unpaired electron (Freeman and Crapo, 1982). Free radicals are highly reactive with tissue components—particularly unsaturated fats—resulting in a host of degradative events. A key free radical in tissues, including meat, is superoxide (O 2 ). This species is generated as a normal by-product of electron transport in mitochondria, ac-

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Young and West

counting for about 2% of mitochondrial oxygen consumption. In the living cell, the combined actions of the enzymes superoxide dismutase and catalase within mitochondria convert superoxide to molecular oxygen, preventing damage to the cytosol by superoxide. Because molecular oxygen is not a free radical, it can do no damage. In muscle as meat, however, the membranes that maintain the integrity of mitochondria—and indeed the entire muscle cell—become degraded allowing free radical species to damage cellular components. These events accelerate metmyoglobin formation. How these three main factors interact and determine the color of meat at various times after slaughter is now examined. B. Chronology of Meat Color from Slaughter to Display At slaughter, the vascular system ceases to deliver oxygen to muscle, and within minutes the muscle becomes anoxic. Using the human heart muscle as an analogy, anoxia in skeletal muscles can be viewed as a “muscle attack.” The oxymyoglobin present in muscle at the time of slaughter becomes depleted of oxygen that is consumed in mitochondria to sustain respiration. The muscle takes on the purple-red hue of (deoxy) myoglobin. At this time the muscle pH is still close to 7.0, the normal pH in live, rested muscle. If a thin piece of muscle is excised at this time and held up to the light, it is not only dark red but also somewhat translucent due to light passing through. In the absence of oxygen, glycolysis is accelerated in muscle, generating lactate as the end product and hydrogen ions from the associated cycling of ATP and ADP (see Chapters 2). The pH falls and if sufficient glycogen is present, a final (ultimate) pH of about 5.5 is attained. This is the normal final pH expected for muscles of an unstressed well-fed animal. As the pH falls towards 5.5, the myofibrillar proteins—principally actin and myosin—approach their isoelectric points and in that state they bind less water, creating gaps between the myofibrils. This creates steps in refractive indices (Offer et al., 1989), which bend light as it crosses from one medium to another (Fig. 6), resulting in light scattering and increased reflectivity. The degree of scatter increases with the size of the gap and

Figure 6 Schematic of light scattering when myofibrils—normally in contact and hydrated in prerigor muscle (left)—shrink when muscle is in rigor at normal pH (right). As light (arrows) passes from one refractive medium, myofibrils, to another, the gaps between myofibrils, it is scattered. Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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Figure 7 Kinetics of blooming at a freshly cut surface of meat either freshly in rigor or after chilled storage in the absence of air for 2 weeks. Blooming was judged by chroma, the intensity of color. The lower curve is a power curve and the upper an exponential. (Adapted from Young et al., 1999.) the extent to which proteins are denatured. For muscle in rigor at a normal pH, translucency is much reduced, and muscle takes on the familiar appearance of meat, particularly as atmospheric oxygen contacts exposed meat surfaces and turns them red according to Reaction 1. In the meat trade, the meat is said to “bloom” and is the color consumers associate with freshness and high quality. The kinetics of blooming in freshly exposed meat surfaces are shown in Fig. 7. The axis labeled chroma represents the intensity of color, which is a good indicator of blooming in meat freshly exposed to the air. Consider first the curve for meat freshly in rigor. Over a period of 10 or more hours, the surface color develops as oxygen diffuses into the meat and forms oxymyoglobin. This occurs only at the surface, usually within the top few millimeters. It usually occurs no deeper than this because although the solubility of oxygen in meat is higher than that required to fully saturate myoglobin (Fig. 3), the rate of oxygen diffusion into meat is less than the rate at which the mitochondria consume oxygen. If the meat had no respiratory activity, oxygen would gradually diffuse to the core of the meat cut, turning the entire meat mass bright-red (Cornforth and Egbert, 1985). This does not happen. The net result of oxygen diffusion and consumption is a steeply decreasing gradient of oxygen concentration from the air to the interior of the meat cut. This gradient is illustrated in Fig. 8, upper. If meat is stored for some time, say 2 weeks, and a new surface is exposed to air, the kinetics of blooming are different from those of meat freshly in rigor (Fig. 7). Blooming not only occurs more rapidly but also is more obvious (higher chroma). This is because the respiratory activity of meat is reduced during storage and oxygen is not consumed so rapidly. When this meat is fully bloomed (after 4 hours), the oxymyoglobin layer is therefore thicker, typical of chill-stored meat on display under an oxygen-permeable wrap in a supermarket (Fig. 8, lower). Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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Young and West

Figure 8 Schematic of oxygen concentration and color of meat in air, bloomed freshly in rigor (upper curve) and bloomed after storage (lower curve). If the meat remains on display in the meat case, it will eventually turn brown and become unsaleable. As described earlier, the brown pigment metmyoglobin is responsible for this color change, and where this pigment first appears in a meat cut on display (Fig. 8, lower) can be predicted from the biochemistry of its formation. Metmyoglobin slowly forms wherever oxygen penetrates, but provided NADH is present and metmyogloin reductase is active, metmyoglobin is reduced back to the oxygen-binding form, myoglobin. In the degradative, oxidative environment of muscle as meat on display, NADH eventually becomes depleted, and the rate of metmyoglobin formation begins to exceed the rate of its reduction to myoglobin. Because the rate constant Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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51

for metmyoglobin formation is greatest at low oxygen concentrations (Fig. 3), it is unsurprising that metmyoglobin is first evident at the interface between the outer oxymyoglobin layer and the inner bulk of anoxic meat where deoxymyoglobin prevails (Fig. 8 lower). The three different colors can often be seen if meat is cut with sharp blade at right angles to the exposed surface. As time on display increases, the brown layer migration occurs both inward and outward until the surface of the steak is completely brown. Migration toward the center of the meat occurs because as the oxygen-consuming activity of the meat declines, the concentration of oxygen rises ultimately leading to metmyoglobin formation. Superimposed on these events is the cellular damage caused by free radicals. With continuing exposure to oxygen, free radical formation is accelerated, further contributing to irreversible browning. Implicit in the above model of color in meat and its systematic study is the ability to measure meat color. Methods to do this are now described.

VI. MEASUREMENT OF MEAT COLOR Meat color is measured for many reasons, including grading, matching customer specifications, measuring consumer response to color, measuring color changes, and determining the causes of discoloration. Meat mainly reflects light in a diffuse way from the surface. However, there is some spectral reflectance from the glossy surface of wet meat, and because meat is partially translucent, a portion of the incident light is transmitted below the surface and reflected internally. When the reflected color of meat is assessed or measured, samples must be sufficiently thick to ensure that no light is reflected from the background. Above all, the measurement of meat color demands a systematic approach to data collection, whether color is scored by a sensory panel (subjective assessment) or measured by an instrument (objective assessment). In the case of panel assessment, the usual techniques of discrimination are used—triangle tests, intensity, preference. Any of these tests is complicated by the heterogeneity of meat and the environment in which the assessments are carried out. For instance, panelists scoring the color acceptability of lean meat can be unconsciously influenced by the color of the associated fat. Recommendations for visual appraisal of meat are given by the American Meat Science Association (AMSA, 1991). Objective (instrumental) measurements are based on three-dimensional “color space” parameters and are usually performed with reflectance colorimeters. A recent reference method for meat color measurement (Honikel, 1998) specifies the L*, a*, b* color scale (CIE, 1986). L* is a measure of lightness where 0 equals black and 100 equals white (Fig. 9). Values of a* indicate redness and greenness, and b* values indicate yellowness and blueness. Hue angle, which defines the color, is arctangent (b*/a*) determined by rotation about the a* and b* axes. Chroma, a measure of color intensity, is calculated as 2 兹苶 (a苶*苶 苶b苶*2苶), the length of the thick arrow in Fig. 9. Several other color spaces are in com苶 mon use, including the older Hunter L, a, b color space and the Yxy space. Honikel’s (1998) reference method also defines procedures for preparing muscle samples, the light source, geometry of illumination and viewing, observer angle, and other parameters that must be specified to ensure the data are reliable and can be compared with data from other sources. Not least are the number of replicates and the sampling strategy, given that browning often appears in patches. Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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Figure 9 In L* a* b* color space, the tip of the thick arrow is defined by lightness (70 on a scale of 0 to 100), redness (26 on a scale of 60 to 60), and yellowness (15). The hue is arctangent 2 15/26 (
30°) and the chroma, or intensity, is the length of the thick line, 兹(1 苶5苶苶 苶苶26苶2苶), as if it were  projected flat on the circle.

Proportions of the three major myoglobin pigments in whole meat can be calculated from spectra obtained with a reflectance spectrophotometer. The calculations depend on wavelengths known as isobestic points, where the extinction coefficients for two or three of the pigments are the same. Although not a perfect isobestic point, 525 nm has been used for all three pigments, and 572 nm for the reduced (Fe2) pigments (Fig. 2). Stewart et al. (1965), van den Oord and Wesdorp (1971), and Krzywicki (1979) have each devised equations that yield values on proportions of the three major myoglobin pigments. The main value of instrumental color measurements lies in the ability to produce objective and reproducible values at different times and locations. Also, instrumental color measurements can be taken where panel assessments cannot, and the use of instruments is cheaper. However, instrumental data must ultimately be correlated with visual judgments. Color measurements indicating the proportion of meat pigments, usually metmyoglobin, have often been correlated with analytical panel and consumer scores. For example, Hood and Riordan (1973) found that the proportion of sales of discolored meat was inversely related to the percentage of metmyoglobin. The proportion of sales fell as the percentage of metmyoglobin increased over the range 5 to 33%. Color space parameters have been correlated with all manner of subjective scales. Scales used to grade meat often relate to the paleness or darkness of meat or to chroma. Several studies of vitamin E effects on discoloration of beef during retail display indicate that a* values and hue angle are good indicators of discoloration. As discoloration progresses,

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a* values fall and hue angles increase. As well, color intensity (chroma) falls. The meat becomes dull. Existing instrumental methods of measuring meat color rely on average measurements. However, the color of meat cuts is usually not even, due to marbling and uneven discoloration. Future instruments are likely to use digital cameras linked to a computer that will analyze the color attributes of each pixel of the image, permitting a more detailed view of meat color. In experiments described in the next section, objective color measurements have been fundamental to studies on the effects of animal factors, processing factors, and subsequent meat handling to retail sale on meat appearance. VII.

APPLIED ASPECTS OF COLOR IN MEAT

A. Species, Animal Age, and Muscle Type The concentration of myoglobin in muscle varies not only with species, breeds, and individual muscles (Tables 1 and 2), but also with age (Fig. 10). Concomitantly, the oxidative activity due to mitochondria also increases with animal age. With higher concentrations of myoglobin, the potential for bright red meat is higher. However, opposing this effect is the oxygen-consuming activity of muscle, which tends to lower the concentration of oxygen in the meat, so raising the potential for metmyoglobin formation (Fig. 3). The tendency for meat to brown is very marked in venison, and in meat from marine mammals like seal and whale. The myoglobin concentration in these meats is high—particularly in seals and whales where the need to store oxygen as oxymyoglobin is important

Figure 10 Iron concentration, an indicator of myoglobin concentration, in beef semimembranosus muscle at different ages. Concentration increases irrespective of breed. (Redrawn from Boccard et al., 1982.)

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in deep dives. At first sight, one would predict that these meats would bloom strongly, but the blooming tendency is offset by the high oxygen consumption rates of the associated mitochondria, which are abundant in these meats. Even within a species different muscles differ in their color stability, and attempts have been made to explain differences in terms of myoglobin concentration, metmyoglobin reductase activity and mitochondrial activity. Ledward (1985) proposed that high metmyoglobin reductase activity was the most important factor in preventing meat discoloration. However, more recent research indicates that the opposite is true. Echevarne et al. (1990) and Lanari and Cassens (1991) found that individual muscles and breeds having lower color stability had high levels of metmyoglobin reductase activity. These breeds and muscles also had a high oxygen consumption rate (Table 3). The inverse relationship between color stability and metmyoglobin reductase activity might be explained in the context of muscle in the live animal. Highly oxidative muscles contain high concentrations of myoglobin and have a greater need for metmyoglobin reductase to maintain myoglobin in the active ferrous state. High oxygen consumption rates generate more free radicals in mitochondria. In the live animal these are routinely metabolized before damage is caused, but in muscle as meat, the mechanisms to eliminate free radicals progressively fail as membrane integrity is lost with time after slaughter. Free radicals promote metmyoglobin formation. B. Effect of Vitamins on Color Stability Supplementation of livestock diets with the vitamin E (-tocopherol) results in a reduced oxidation of lipids in the meat from animals fed those diets (Krukovsky et al., 1949; Webb et al., 1972). This vitamin, which is fat soluble and occurs in the membranes that ramify all cells, not only helps prevent lipid oxidation but also retards metmyoglobin formation in meat exposed to air so improving color stability. Vitamin E is not synthesized in animal cells; instead it must be obtained from plants through the diet. Green plant tissue contains much higher concentrations of vitamin E than mature or senescent tissue (Brown, 1953). Concentrations in grains are particularly low. Meat from unsupplemented grain-finished cattle has a vitamin E content of around 0.5 g /g of lean meat whereas a typical content in equivalent pasture-fed animals is 5 g/g (West et al., 1997).

Table 3 Relative Metmyoglobin Reductase Activities and Oxygen Consumption Rates of Two Muscles from Two Breeds Color stability in Breed and muscle Holstein Gluteus medius Longissimus Crossbreed Gluteus medius Longissimus

Oxygen consumption rate

Metmyoglobin reductase activity

100 a 57

100 a 71

75 32

69 35

Breed

Muscle

Lower Lower Higher Higher

a

Values are relative to gluteus medius in Holstein. Source: From Lanari and Cassens (1991).

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Figure 11 The relationship between vitamin E concentration in beef longissimus and hue angle after 7 days on display. (From Liu et al., 1996.)

Vitamin E supplementation increases the display life of meat from grain-finished animals. The results of Liu et al. (1996) are typical for cattle. Supplementation at 0.5 g per day per bovine for 126 days doubled the display life for a number of muscles as meat. These researchers also measured the vitamin E concentration in the meat on display and found that hue angle increased markedly below about 3 g/g of meat (Fig. 11), which appears to be a threshold concentration ensuring a good display life. The retardation of browning by vitamin E is probably linked to an inhibition of lipid oxidation. Products of lipid oxidation are more water soluble than the parent compounds and can enter the cytoplasm to react with oxymyoglobin and accelerate metmyoglobin accumulation (Schaefer et al., 1995). Viewed another way, membranes tend to remain intact when vitamin E is present, minimizing the metabolic chaos that ensues when normally compartmentalized cellular components are allowed to mix. Lanari et al. (1996) proposed a reaction sequence to explain the data in Fig. 12. Cattle were supplemented with vitamin E at two levels, then the time course of meat browning due to metmyoglobin was tracked. After a rather flat induction period, metmyoglobin formation accelerated. The length of the induction period was increased by higher vitamin E supplementation (Fig. 12). These workers proposed the following reaction sequence: autoxidation produces metmyoglobin and superoxide radical from oxymyloglobin; superoxide generates lipid free radicals that oxidize another oxymyloglobin; as a free radical scavenger, vitamin E is an alternative substrate to oxymyloglobin and so slows metmyoglobin accumulation; after the vitamin E is consumed, metmyoglobin formation proceeds uninhibited. They concluded that differences in color stability between muscles depend on the combined effects of vitamin E content and the susceptibility of pigments and lipids to oxidation. For color-labile muscles, the induction period is shorter.

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Figure 12 Kinetics of metmyoglobin formation in beef on display, from cattle supplemented with vitamin E at two levels. (From Lanari et al., 1996.) Addition of vitamin E is much more effective in stabilizing meat color when it is incorporated through a dietary pathway than when it is added postmortem to ground meat (Mitsumoto et al., 1993). This indicates that incorporation of vitamin E into cellular membranes is a critical step in its role as an antioxidant. If browned meat is sprayed with a dilute solution of ascorbic acid (vitamin C) or sodium ascorbate, metmyoglobin is rapidly reduced to myoglobin. In the presence of air, the bloomed appearance is restored. This effect is due to the reducing power of this vitamin. Vitamin C occurs naturally in muscle, and in the living cell, it regenerates vitamin E, suggesting it plays an integral role in maintaining the color stability of meat. Unlike vitamin E, the concentration of vitamin C in muscle is tightly controlled, at around 7 g per g. (This concentration is much lower than in fruits and vegetables, and vitamin C in meat is not a significant dietary source.) If an animal is orally supplemented with vitamin C, the concentration in blood rises rapidly then falls quickly to the homeostatically controlled concentration, making improvement of color stability through diet supplementation difficult. Hood (1975) intravenously injected cattle with massive doses of vitamin C immediately before slaughter and found that meat color stability was enhanced. The effect was more pronounced in color-labile than in color-stable muscles. In contrast to vitamin E, vitamin C is water soluble and is very effective in retarding discoloration when added to whole-tissue meat as a spray (Okayama et al., 1987) or as an adjunct during grinding (Mitsumoto et al., 1991). In the latter study, vitamins C and E were individually effective at slowing metmyogloin formation, whereas when applied simultaneously, they completely inhibited metmyogloin formation over 7 days (Fig. 13). In the same experiment, fat oxidation was negligible where vitamins C and E were applied together, again suggesting a close link between fat oxidation and metmyoglobin formation.

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Recent experiments in the authors’ laboratory have explored the fate of naturally occurring (endogenous) vitamins C and E in the bloomed layer of ground meat compared with ground meat stored anoxically (Fig. 14). In the bloomed layer (oxygen present) the vitamin C concentration fell to zero in 3 days but persisted for at least 7 days in the absence of oxygen. Vitamin E was more resilient. In this experiment, the bloomed layer browned completely after 4 days, suggesting a significant role for endogenous vitamin C in the normal kinetics of meat browning. Vitamins C and E are not the only antioxidants in meat that are derived from diet and may contribute to color stability. Other antioxidants abundant in green leaf tissue include polyphenolic flavanols and ubiquinols. C. Effect of Processing Conditions Processing conditions of carcasses have major effects on meat quality, particularly tenderness (see Chapter 15). However, processing also affects other qualities such as color. The rate of postmortem pH fall in pig muscle is typically double that in ruminant muscle. As a result, pig muscle is subjected to relatively low pH values at relatively high temperatures. Under these conditions, myofibrillar proteins tend to denature, and in this state are more reflective and so the meat appears lighter. (A parallel condition is seen in fish, where biochemically unstable fish proteins denature when exposed to an acidic sushi marinade even at chill temperatures.) Because porcine muscles contain significant concentrations of myoglobin, the net color attained on rapid pH fall when muscles are warm is light red, a color most consumers consider attractive for pork. Myofibrillar proteins are not the only proteins affected by the combination of low pH and high temperature. Proteins in membranes, including mitochondrial membranes, and

Figure 13 Effects of added vitamins on metmyoglobin formation in ground beef. (From Mitsumoto et al., 1991.)

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Figure 14 Fate of vitamins C and E in bloomed and anoxic layers of ground beef at 4°C in the dark. Thin layers of ground beef were exposed top and bottom to an atmosphere of pure oxygen or pure nitrogen.

probably many other proteins are damaged by this combination. Membranes become leaky, leading to the cellular chaos that helps generate free radicals which in turn promote metmyoglobin formation. Also, where these damaged proteins are enzymes, loss of activity often results. If mitochondrial enzymes are inactivated, oxygen consumption falls, with significant effects on color. Ledward (1985) explored the color effects of different pH/temperature /time regimes during processing by using electrical stimulation to accelerate glycolysis, with its attendant rapid fall in pH (Chapter 13), and water baths to control temperature. A semimembranosus muscle (stimulated) that entered rigor at 41°C developed metmyoglobin on display at the same initial rate as the contralateral muscle that entered rigor at 1°C (Fig. 15). However, after extended display, metmyoglobin formation stabilized in the muscle that entered rigor at low temperature but not in the other. This effect of high temperature and low pH during rigor attainment leading to increased metmyoglobin formation applied generally to the several muscles studied. This study also described the importance of muscle location on a carcass for color stability. In superficial muscles such as the longissimus, which cool rapidly on the carcass, stimulation will have a minimal effect on color stability. In deep muscles like the semimembranosus, which cool slowly during chilling, low pH values and high temperatures often coexist, leading to more exudative and paler meat. Slow cooling often leads to excess drip and sometimes to “two-toning” in muscles, where slower cooling inner parts of a muscle are paler than outer parts. In Ledward’s (1985) experiments, the temperatures examined were extreme, but even a narrower temperature range can affect meat color (Young et al., 1999). The effects are more subtle in a narrower temperature range but can be important where meat is stored chilled for long periods before display, as in the international chilled meats trade.

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D. Effect of Packaging, Temperature, Storage, and Display Conditions The packaging of meat to protect it against microbial contamination, and low temperature storage to slow microbial growth are relatively recent innovations in the history of the meat trade. These technologies have made interstate and international trade in meats possible, and have significant effects on meat color as perceived and evaluated by the final consumer. For each case the effects can often be explained in terms of the biochemical models discussed earlier. 1. Frozen Meat When meat freezes, the formation of ice crystals within what was once a uniformly hydrated product changes its optical properties. The meat becomes less reflective, so frozen meat looks darker than chilled. On exposure to air, frozen meat blooms or maintains the blooming existing at the time of freezing. If bloomed frozen meat remains exposed to air, blooming is maintained for a time but is eventually lost. The color of the frozen meat becomes dark red-brown that is a combination of low light reflection, surface drying, and metmyoglobin formation. Historically, much frozen meat in international trade was stored and transported exposed to air, producing a very unattractive product. Frozen meat may be cut and thawed before display. After thawing, a freshly exposed surface of meat will bloom, but the meat remains less reflective than meat that was never frozen and so appears darker and less attractive than chilled meat (Jeremiah, 1981a). More-

Figure 15 Metmyoglobin formation in semimembranosus muscles that entered rigor at high or low temperatures. The meat was displayed at 1°C. (Adapted from Ledward, 1985.)

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over, the longer meat is stored frozen, the shorter is its display life on thawing (Jeremiah, 1981b; Moore, 1990). The formation of ice crystals in meat causes structural damage in muscle cells. Membranes are broken and cellular components that are normally kept apart are allowed to mix. As well, because ice crystals exclude salts, the ionic strength of the remaining unfrozen water increases. Particularly in the presence of oxygen, these events lead to the formation of free radical species that accelerate tissue degradation and oxidation, which is linked to color deterioration. 2. Chilled Meat Meat freezes at 1.5°C, and meat stored at any temperature between this and ambient can be described as chilled. The temperature/time history between these limits has a significant effect on bloomed color stability, which in turn translates to the display life before meat turns brown. Figure 16 compares the effects of storage at three temperatures on the acceptability of pork color on aerobic display. Clearly, the cooler the meat the better. The longer meat is stored, the greater the tendency for metmyoglobin to form. Figure 17 shows how the color or quality of venison on display is reduced after long-term storage. Display conditions are also critically important for color. Obviously, blooming occurs only in the presence of oxygen. The most common method of display in serve-yourself meat cabinets comprises a cut of raw meat on a polysytrene tray and drip pad, with an overwrap of oxygen-permeable film to protect the cut against contamination and drying. Oxygen diffuses freely through the film so the meat blooms. Another technology—which is common in U.K. markets—employs oxygen-enriched atmospheres to obtain a thicker

Figure 16 Effect of storage temperature on color acceptability during retail display at 7°C. (Adapted from Jeremiah and Gibson, 1997.)

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Figure 17 Color quality score of venison on retail display after storage in vacuum packs for up to 18 weeks. (Adapted from Seman et al., 1988.)

layer of blooming. This technique works because at a high oxygen partial pressure oxygen diffusion dominates over oxygen consumption, so the red layer is thicker. By maintaining the metmyoglobin layer deeper in the meat, oxygen enriched atmospheres delay the time before metmyoglobin appears. However, given time, a thick metmyoglobin layer eventually becomes visible. At the other atmospheric extreme, meat displayed in a clear plastic vacuum pack will not become brown because no oxymyoglobin is present to be oxidized. Display life under vacuum is limited only by microbiological spoilage and flavor deterioration with time. For red meats on retail display, the worst environment lies between the two extremes of exposure to air (or oxygen) and a vacuum that totally excludes air. Plastics that are slightly oxygen permeable allow enough oxygen to enter the pack to satisfy the oxygen consumption demands of mitochondria but not enough to bloom the meat to any degree. These conditions favor metmyoglobin formation (Fig. 3). Clear or opaque vacuum packaging may offer an alternative to meat marketers where the maintenance of bloomed color on display is difficult. In the case of venison, which has high concentrations of myoglobin and mitochondria, the bloomed appearance does not last long. The problem is exacerbated when chilled venison is stored for weeks or even months before display, as is the case in international trade (Seman et al., 1988). If customers expected venison to appear purple-red at retail display rather than bright red, or if the meat were not visible at all, bloomed display life would not be an issue. However, educating consumers to abandon their long-standing habit of judging meat by its redness presents a major marketing challenge. The temperature of meat on display has a major effect on bloomed color stability, due to the marked increase in autoxidation with increasing temperature (Brown and

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Mebine, 1969). This effect is compounded by the increased consumption of oxygen by residual repiratory enzymes in meat as temperature increases, so lowering oxygen concentration. Provided meat is sold soon after slaughter, browning does not usually occur. However, when meat is stored before display, as is common in the international meat trade, retail display temperatures become very important. Huge gains in display life can be made when display temperatures are close to the freezing point of meat. Another reason to maintain low display temperatures is to minimize the growth of spoilage organisms. Despite these needs, temperatures around 0°C are not commonly achieved in supermarket displays. Temperatures inside retail display cabinets vary widely, frequently up to 7°C. The spoilage bacteria that grow on the surface of meat are sometimes responsible for generating green pigments through the generation of H2S or H2O2 from bacterial metabolism. These compounds react with the porphyrin ring structure to break one or more double bonds, markedly altering the color of the heme. This can be demonstrated by immersing meat in a H2O2 solution. The role of different bacteria in producing unusual colors is described in Chapter 19. 3. Light During retail display the pack is exposed to light, whether natural through windows, or by incandescent or fluorescent lights in display cabinets. The perceived color of meat varies with the spectral profile of the incident light. Specific profiles are used to emphasize the quality sought by consumers, often by enhancing the red end of the spectrum so reflected red wavelengths strike the eye. However, light also promotes metmyoglobin formation through photochemical autoxidation. Shorter wavelengths, particularly in the ultraviolet range, are the most damaging. At pH 5.4 and 0°C, the relative rates of autoxidation are 1, 10, and 4700 at 546, 366, and 254 nm respectively (Bertelsen and Skibsted, 1987). Photochemical autoxidation is only slightly temperature dependent, whereas thermal autoxidation—the type normally considered in the context of browning—is greatly increased with increasing temperature above 0°C (Brown and Mebine, 1969; Andersen et al., 1989). Thus for frozen meat, light-induced discoloration exceeds thermally induced discoloration, whereas in chilled meat the reverse is true. Nonetheless, fluorescent display lighting does promote discoloration of chilled meat. This is exemplified in Table 4 for three qualities of pork. The effect of light on the nitrosylhemochrome, which is responsible for the pink color of cooked cured meats, is discussed in Chapter 20.

Table 4 Effect of Cool White Fluorescent Light on Percent Oxymyoglobin at the Surface of Pork of Three Qualities During Storage Over 7 Daysa Meat quality Light condition

Dark-cutting

Normal

PSE

Dark Light

58 45

52 44

51 42

a

Means pooled over 7 days. PSE
Pale, soft, exudative.

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Figure 18 Changes in metmyoglobin content on the surface of pork of three rigor conditions during display for 7 days. (From Zhu and Brewer, 1998.)

E. Effect of Abnormal Rigor Conditions on Meat Color The abnormal appearance of pale, soft, exudative pork (PSE pork) and of dark-cutting meat can be explained in biochemical and biophysical terms. PSE pork is pale for several reasons. First, the rapid fall in muscle pH while the muscles are warm causes denaturation of muscle proteins that increase reflectivity beyond the normal reflectivity of myofibrillar proteins in rigor (Fig. 6). Second, the excessive drip takes myoglobin with it. Finally, PSE conditions are more common in pig breeds whose muscles have a low myoglobin concentration, and thus are predisposed to lighter colored meat. Dark-cutting meat is dark red for roughly the opposite reasons. Relatively undenatured proteins are unreflective, so the meat appears darker due to absorption of light. The water binding capacity of dark-cutting meat is high, so no myoglobin is lost through drip. Finally, the oxygen consumption rate is high (see below) so surface blooming is poor, as explained earlier. The comparative rates of metmyoglobin formation in the three contrasting rigor conditions (dark-cutting, normal, and PSE) are well described by Zhu and Brewer (1998). The pH values of the meats were 6.16, 5.52, and 5.44. Figure 18 shows the kinetics of metmyoglobin accumulation during display, increasing in the order dark-cutting, normal, PSE. Biochemical events contributing to this difference include the effect of pH on autoxidation rate (higher at low pH values, Fig. 4), the activity of metmyoglobin reductase (higher in dark-cutting meat, Fig. 5), and the relative oxygen consumption rate of the muscle (Fig. 19). Higher pH, as occurs in dark-cutting meat, favors oxygen consumption and at first sight might be expected to promote metmyoglobin formation by lowering oxygen concentrations in meat and generating free radicals. Insofar as it affects autoxidation rate and re-

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Figure 19 Oxygen consumption rate in pork of three rigor conditions during display for 7 days. The rate is percent of oxymyoglobin converted to (deoxy)myoglobin in 10 minutes. (Adapted from Zhu and Brewer, 1998.)

ductase activity, pH appears more important than oxygen consumption in determining the relative color stability in the three rigor conditions. F. Effect of Ionizing Radiation Ionizing radiation is used to reduce or eliminate microbiological contamination in foods. The radiation is generated by radioactive isotopes ( -radiation from 60Co), x-rays, or beams of high-energy electrons. Whatever the source, the radiation produces free radicals and electrons as primary species that decay to produce charged and neutral free radicals. The use of ionizing radiation to reduce contamination by pathogenic and spoilage organisms is generally regarded as safe up to a limit. Meat has been an obvious target for preservation. However, the generation of free radicals in any food is likely to cause some adverse effects, particularly where oxygen-containing free radicals formed in a high-moisture food like meat can react with fats and proteins. Free radicals accelerate the onset of rancidity. However, the concern here is color. Irradiation of meats often causes an increase in redness, although the degree is dose- and meat species–dependent (Millar, 1994). Poultry and pork tend to redden more than beef and lamb. The reddening is particularly evident in the absence of oxygen, indicating that oxymyoglobin is not the pigment responsible. Carbon monoxide is produced in irradiated meats (Furuta et al., 1992), so the increase in redness is probably caused by formation of carboxymyoglobin (Millar, 1994), a stable pink pigment discussed earlier in the context of myoglobin chemistry. In the presence of oxygen, the surface of irradiated meats tends to brown due to metmyoglobin formation, but even in vacuum packs, beef goes brown (Nanke et al., 1998). In contrast, similarly treated pork and turkey remain red. The reason for these species differences is unknown, but the

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color of meat in any situation will represent a balance between the four pigments (deoxy, oxy, met, carboxy) and possibly others not yet identified. G. Cooked Meat Color On cooking, meat tends to lighten in color and also tends to turn a brown-gray hue. The lightening is due to an increased reflection of light arising from light scattering by denatured proteins (an advanced case of the effects in Fig. 6). The loss of chroma and change in hue arise from changes to myoglobin. The compound largely responsible for the browngray hue is globin hemichrome(s). In this type of pigment, the iron, in the Fe3 state, is still held in the porphyrin ring structure (Fig. 1b), but the heme no longer binds to the protein in same way as it did in myoglobin, oxymyoglobin, or metmyoglobin. This is because the globin (the protein part of myoglobin) has been denatured due to heat. The formal coordination between the iron atom and the strategically placed histidine (Fig. 1b) may be replaced by links to other nitrogenous bases on the globin (Giddings, 1977) or on other proteins present in cooked meat (Ledward, 1971). Another compound involved in cooked meat color is globin hemochrome, in which the iron is in Fe2 state. Its color is typically dull red, the exact hue depending on which nitrogenous base is coordinated to the iron. The balance between hemochromes and hemichromes is affected by the state of the meat before cooking (van Laack et al., 1996; Warren et al., 1996). Generally, the more metmyoglobin that was present in the raw state, the more rapidly meat turns brown-gray, indicating that the oxidative state of meat affects the color obtained on cooking. Ground meat, which is significantly exposed to oxygen during grinding, browns more readily than whole tissue meat on cooking. Other factors affecting cooked color include species, animal maturity, muscle type, freeze/thaw, the presence of denaturing agents, and meat pH. As examples, Geileskey et al. (1998) showed that at 60°C, browning occurred twice as rapidly in lamb as in beef, and twice as rapidly in longissimus as in shoulder muscles (where pH was the same). Lytras et al. (1999) showed that myoglobin denatured at twice the rate at 70°C when 2% salt was added. Myoglobin is increasingly stable toward pH 7, accounting for the frequently uncooked appearance of higher pH meat when it is heated to normal cooking temperatures. The color of cooked meat and meat products is a poor guide to the internal cooking temperature attained. The importance of this is twofold. First, in catering systems, where meats are cooked to a standard internal temperature, the color of portions can vary, particularly where the pH is variable. This is commercially unacceptable. Second, pathogens such as Escherichia coli O157 are inactivated when meat is cooked to a minimum of 70°C for 2 minutes. In many cases, ground lamb or beef that has browned on cooking will have achieved that temperature, but not in all cases. VIII. FAT COLOR White or cream-white intermuscular and marbling fat is the market ideal in the United States and other sophisticated markets. However, this is not always achievable. Fat can be colored by carotenoid pigments from the animal’s feed and heme pigments from blood or drip. Fat from pasture-fed animals always contains a mixture of yellow/orange carotenoid pigments, mainly -carotene (Yang et al., 1992). Concentrations of carotene in fat vary between breeds and with animal management practices. Yellow fat is more

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common in animals from some dairy breeds and becomes more obvious through increased pigment concentration where subcutaneous fat levels decrease when feed is inadequate. Yellow fat is not a problem when animals are fed on grain. When pasture-fed animals are finished on grain, at least four weeks are required to reduce the carotene in fat to acceptable levels (Forrest, 1991). In countries where beef animals are finished almost exclusively on grain, some consumers incorrectly but not unreasonably equate yellow fat, from pasture finishing, with a disease condition. Yellow discoloration is much most apparent in hot fat on freshly slaughtered animals. As the fat cools and crystallizes, it appears progressively paler. This is because warm fat is more translucent than cold fat. Any measurements of fat color should take this into account. Fat on freshly slaughtered animals must be at least 12 mm thick to avoid background interference in color readings. Fatty tissues can be come discolored with blood due to bruising or, during processing, if capillaries that pass through the subcutaneous fat break. At various times after slaughter, fat tissues can be also discolored by pigments from drip. Fatty tissue absorbs this drip remarkably rapidly by migration between fat cells, resulting in entirely pink fat tissue. Oxidation of the heme to metheme can then produce an unslightly grey discoloration of the fat. Bell et al. (1996) reported that this discoloration was the limiting factor in the retail display life of steaks prepared from chill-stored vacuum and carbon dioxide–packed sub-primal beef cuts. However, this discoloration can be retarded by spraying the fat with a dilute solution of sodium ascorbate before packing (West, J. unpublished data). IX. CONCLUDING REMARKS The future importance of raw meat color as a perceived indicator of meat quality will be determined by changes in food marketing, which in recent years has moved markedly toward prepared meals in the U.S. and other affluent markets. For this mode of meat presentation, cooked meat color is more important, although that color is usually overlaid with colors from spices, sauces, and other meal ingredients. However, raw meat will continue to be sold to meet the needs of those consumers who, for economic, religious, or culinary reasons, prefer to buy meat raw and cook it themselves. For these consumers, particularly the gourmets, color will remain an important criterion for purchase, unless consistency can be controlled to the point that a customer is willing to buy meat as a branded item sight unseen. That will require a major change to the mindset that says if it looks good it will eat well. As is explained in other chapters that belief is not necessarily true. REFERENCES AMSA. American Meat Science Association committee on guidelines for meat color evaluation. Proc. 44th Ann. Recipr. Meat Conf. Kansas State University. pp 2–17, 1991. Andersen, H.J., G. Bertelsen, L., and L.H. Skibsted. Colour stability of minced beef. Ultraviolet barrier in packaging material reduces light-induced discoloration of frozen products during display Meat Sci 25:155–159, 1989. Akeson, A., G.V. Ehrenstein, G. Hevesy, and H. Theorell. Life span of myoglobin. Arch Biochem Biophys 91:310–318, 1960. Arihara, K., R.G. Cassens, M.L. Greaser, J.B. Luchansky, and P.E. Mozdziak. Localization of metmyoglobin-reducing enzyme (NADH-cytochrome b5 reductase) system components in bovine skeletal muscle. Meat Sci. 39:205–212, 1995.

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Bell, R.G., N. Penney, and S.M. Moorhead. The retail life of steaks prepared from chill stored vacuum and carbon dioxide-packed sub-primal beef packs. Meat Sci 42:165–178, 1996. Bertelsen, G., and L.H. Skibsted. Photooxidation of oxymyoglobin. Wavelength dependence of quantum yields in relation to light discoloration of meat. Meat Sci 19:243–251, 1987. Boccard, R.L., D.E. Cronjé, and M.C. Smit. Pigment content (myoglobin) in muscles of Afrikaner and Friesland bulls and steers of different ages. In: Meat Symposium. The vital role of science and technology in the meat industry. Animal and Dairy Science Research Institute, Pretoria, South Africa, 1982. Brown, F. The tocopherol content of farm feeding-stuffs. J Sci Food Agric 4:161–165, 1953. Brown, W.D. The concentration of myoglobin and hemoglobin in tuna flesh. J Food Sci 27:26–28, 1962. Brown, W.D., and L.B. Mebine. Autoxidation of oxymyoglobins. J Biol Chem 244: 6696–6701, 1969. CIE. Colorimetry 2nd ed. Commission International de l’Eclairage, Publication CIE 15.2. Vienna, 1986. Cornforth, D.P., and W.R. Egbert. Effect of rotenone and pH on the color of pre-rigor meat. J. Food Sci. 50:34–44, 1985. Dolar, M.L.L., P. Suarez, P.J. Poganis, and G.L. Kooyman. Myoglobin in pelagic small cetaceans. J. Exp. Biol. 202:227–236, 1999. Dymicky, M., J.B. Fox, and A.E. Wasserman. Color formation in cooked model and meat systems with organic and inorganic compounds J. Food Sci 40:306–309, 1975. Echevarne, C., M. Renerre, and R. Labas. Metmyoglobin reductase activity in bovine muscles. Meat Sci 27:161–172, 1990. Forrest, R.J. Effect of high concentrate feeding on the carcass quality and fat coloration of grassreared steers. Can J Anim Sci 61:575–580, 1981. Freeman, B.A., and J.D. Crapo. Biology of disease. Free radicals and tissue injury. Lab Invest 47:412–426, 1982. Furuta, M., T. Dohmaru, T. Katayama, H. Toratoni, and A. Takeda. Detection of irradiated frozen meat and poultry using carbon monoxide as a probe. J Agric Food Chem 40:1099–1100, 1992. Geileskey, A., R.D. King, D. Corte, P. Pinto, and D.A. Ledward. The kinetics of cooked meat haemoprotein formation in meat and model systems. Meat Sci 48:189–199, 1998. George, P., and C.J. Stratmann. The oxidation of myoglobin to metmyoglobin by oxygen. 2. The relation between the first order rate constant and the partial pressure of oxygen. Biochem J 51:418–425, 1952. Giddings, G.C. The basis of color in muscle foods. CRC Crit. Rev Food Sci Nutr 9:81–114, 1977. Hagler, L., R.I. Copppes, and R.H. Herman. Metmyoglobin reductase. Identification and purification of a reduced nicotinamide adenine dinucleotide-dependent enzyme from bovine heart which reduces metmyoglobin. J Biol Chem 254:6505–6514, 1979. Honikel, K.O. Reference methods for the assessment of physical characteristics of meat. Meat Sci. 49:447–457, 1998. Hood, D.E. Pre-slaughter injection of sodium ascorbate as a method of inhibiting metmyoglobin formation in fresh beef. J Sci Food Agric 26:85–90, 1975. Hood, D.E., and E.B. Riordan. Discolouration in pre-packaged beef: measurement by reflectance spectrophotometry and shopper discrimination. J Food Technol 8:333–343, 1973. Hunt, M.C., and H.B. Hedrick. Profile of fiber types and related properties of five bovine muscles. J Food Sci 42:513–517, 1977. Jeremiah, L.E. The effects of frozen storage and thawing on the retail acceptability of ham steaks and bacon slices. J Food Qual 5:43–58, 1981a. Jeremiah, L.E. 1981b. The effects of frozen storage and protective storage wrap on the retail case-life of pork loin chops. J. Food Qual. 5:311–326. Jeremiah, L.E., and L.L. Gibson The influence of storage and display conditions on the retail properties and case-life of display-ready pork loin roasts. Meat Sci 47:17–27, 1997.

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Krukovsky, V.N., J.K. Loosli, and F. Whiting. The influence of tocopherols and cod liver oil on the stability of milk. J Dairy Sci 32:196–201, 1949. Krzywicki, K. Assessment of relative content of myoglobin, oxymyoglobin and metmyoglobin at the surface of beef. Meat Sci 3:1–10, 1979. Lanari, M.C., and R.G. Cassens. Mitochondrial activity and beef muscle color stability. J. Food Sci 56:1476–1479, 1991. Lanari, M.C., D.M. Schaefer, Q. Liu, and R.G. Cassens. Kinetics of pigment oxidation from steers supplemented with vitamin E. J Food Sci 61:884–889, 1996. Ledward, D.A. On the nature of cooked meat hemoprotein. J Food Sci 36:883–888, 1971. Ledward, D.A. Post-slaughter influences on the formation of metmyoglobin in beef muscles. Meat Sci 15:149–171, 1985. Ledward, D.A., and W.R. Shorthose. A note on the haem pigment concentration of lamb as influenced by age and sex. Anim Prod 13:193–195, 1971. Liu, Q, K.K. Scheller, S.C. Arp, D.M. Schaefer, and M. Frigg. Color coordinates for assessment of dietary vitamin E effects on beef color stability. J. Anim. Sci 74:106–126, 1996. Livingston, D.J., and W.D. Brown. The chemistry of myoglobin and its reactions. Food Technol 35:244–252, 1981. Livingston, D.J., S.J. McLachlan, G.N. La Mar, and W.D. Brown. Myoglobin:cytochrome b5 interactions and the kinetic mechanism of metmyoglobin reductase. J. Biol. Chem. 260:15699– 15707, 1985. Lytras, G.N., A. Geileskey, R.D. King, and D.A. Ledward. Effect of muscle type, salt and pH on cooked meat haemoprotein formation in lamb and beef. Meat Sci 52:189–194, 1999. Maga, J.A. Pink discoloration in cooked white meat. Food Rev Int 10:273–286, 1994. Millar, S.J. The effect of ionising radiation on the appearance of meat. Ph.D. Thesis. The Queen’s University of Belfast, Northern Ireland, 1994. Mitsumoto, M., R.N. Arnold, D.M. Schaefer, and R.G. Cassens. Dietary versus postmortem supplementation of vitamin E on pigment and lipid stability in ground beef. J. Anim. Sci. 71:1812–1816, 1993. Mitsumoto, M., C. Faustman, R.G. Cassens, R.N. Arnold, D.M. Schaefer, and K.K. Scheller. Vitamins E and C improve pigment and lipid stability in ground beef. J Food Sci 56:194–197, 1991. Möller, P, and C. Sylvén. Myoglobin in human skeletal muscle. Scand. J Clin Lab Invest 41:479–482, 1981. Moore, V. Increase in retail display of frozen lamb chops with increased loin storage time before cutting into chops. Meat Sci 28:251–258, 1990. Nanke, K.E., J.G. Sebranek, and D.G. Olson. Color characteristics of irradiated vacuum-packaged pork, beef, and turkey. J Food Sci 63:1001–1006, 1998. Nishida, J., and T. Nishida. Relationship between the concentration of myoglobin and parvalbumin in various types of muscle tissues form chickens. Br Poult Sci 26:105–115, 1985. Offer, G., P. Knight, R. Jeacocke, R. Almond, T. Cousins, J. Elsey, N. Parsons, A. Sharp, R. Starr, and P. Purslow. Food Microstructure 8:151–170, 1989. Okayama, T., T. Imai, and M. Yamanoue. Effect of ascorbic acid and alpha-tocopherol in storage stability of beef steaks. Meat Sci 21:267–273, 1987. Rahelic, S., and S. Puac. Fibre types in longissimus dorsi from wild and highly selected pig breeds. Meat Sci 5:439–450, 1980. Renerre, M., and R. Labas. Variability between muscles and between animals of the color stability of beef meats. Sciences des Aliments 4:567–584, 1984. Robinson, D. The muscle hemoglobin of seals as an oxygen store in diving. Science 90:276–277, 1939. Schaefer, D.M., Q. Liu, C. Faustman, and M.C. Yin. Supranutritional administration of vitamins E and C improves antioxidative status of beef. J Nutr 125:1792S–1798s, 1995. Scholander, P.F. Experimental investigations on the respiratory function in diving mammals and birds. Hvalradets Skrift 22:1–131, 1940.

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Seman, D.L., K.R. Drew, P.A. Clarken, and R.P. Littlejohn. Influence of packaging method and length of chilled storage on microflora, tenderness and colour stability of venison loins. Meat Sci 22, 267–282, 1988. Stewart, M.R., M.W. Zipser, and B.M. Watts. The use of reflectance spectrophotometry for the assay of raw meat pigments. J Food Sci 30:464–469, 1965. Takano, T. Structure of myoglobin refined at 2.0 Å resolution. II. Structure of deoxymyoglobin from sperm whale. J Mol Biol 110:569–584, 1977. Topel, D.G., R.A. Merkel, D.L. Mackintosh, and J.L. Hall. Variation of some physical and biochemical properties within and among selected porcine muscles. J Anim Sci 25:277–282, 1966. van den Oord, A.H.A., and J.J. Wesdorp. Analysis of pigments in intact beef samples. J Food Sci 6:1–13, 1971. van Laacke, R.L.J.M., B.W. Berry, and M.B. Solomon. Effects of precooking conditions on color of cooked beef patties. J Food Prot 59:976–983, 1996. Warren, K.E., M.C. Hunt, and D.H. Kropf. Myoglobin oxidative state affects internal cooked color development in ground beef patties. J Food Sci 61:513–519, 1996. Webb, J.E., C.C. Brunson, and J.D. Yates. Effects of feeding antioxidants on rancidity development in pre-cooked, frozen broiler parts. Poult Sci 51:1601–1605, 1972. West, J., O.A. Young, M.P. Agnew, and T. Knight. Levels of -tocopherol in beef from New Zealand pastures. 43rd Int Congr Meat Sci Technol, Auckland, 1997. pp 350–351. Yang, A., T.W. Larsen, and R.K. Tume. Carotenoid and retinol concentrations in serum, adipose tissue and liver and carotenoid transport in sheep, goats and cattle. Australian J Agric Res 43:1809–1817, 1992. Young, O.A., A. Priolo, N.J. Simmons, and J. West. Effects of rigor attainment temperature on meat blooming and colour on display. Meat Sci 52:47–56, 1999. Zhu, L.G., and M.S. Brewer. Metmyoglobin reducing capacity of fresh normal, PSE, and DFD pork during retail display. J Food Sci 63:390–393, 1998.

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4 Flavors of Meat Products TZOU-CHI HUANG National Pingtung University of Science and Technology, Pingtung, Taiwan CHI-TANG HO Rutgers University, New Brunswick, New Jersey

I. INTRODUCTION II.

PRECURSORS FOR MEAT AROMA FORMATION A. Inosine Monophosphate B. Thiamin C. Alliin and Deoxyalliin D. Strecker Aldehyde E. 1-Pyrroline F. Fatty Aldehydes G. Carbohydrate Degradation Products

III. PATHWAYS FOR THE FORMATION OF SOME MEAT AROMA VOLATILES A. Acetaldehyde-Derived Meat Aroma B. Dicarbonyl-Derived Meat Aroma C. Fatty Aldehyde–Derived Meat Aroma D. Cysteamine-Derived Thiazoles and Thiazolidine E. 1-Pyrroline–Derived 1,3,5-dithiazine and Pyrroline F. Furan-Derived Meat Aroma G. 2.4-Decadienal–Derived Thiophene, Thiapyran, Pyridine, and Dithiazines IV. FACTORS CONTROLLING MEAT AROMA FORMATION A. Effect of Cooking Conditions on Meat Aroma Formation B. Effect of Buffer on Aroma Generation C. Effect of Water Content on Aroma Generation D. Role of Lipids in Meat Aroma Formation E. Effect of Fermentation on Meat Flavor REFERENCES

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I. INTRODUCTION Characteristic meat flavor is a product of the volatile and nonvolatile compounds. Volatile compounds identified in meat flavor include hydrocarbons, alcohols, carbonyls, carboxylic acids, esters, lactones, ethers, sulphur-containing compounds as well as different classes of heterocyclic compounds, namely furans, pyridines, pyrazines, oxazoles, thiazoles and thiophenes (Table 1). II. PRECURSORS FOR MEAT AROMA FORMATION Raw meat is a rich reservoir of nonvolatile precursors of cooked meat flavor. Components of meat flavor formed by the thermal breakdown of fats, proteins, and carbohydrates are considered as the major precursors for thermally generated meat aroma. Model systems composed of D-glucose and L-cysteine have long been used to study the thermal generation of nitrogen and sulfur-containing flavor compounds (2,3). An early attempt to synthesize meat flavor involved the heating of either cysteine, plus some additional amino acids or hydrolyzed protein, with pentose or hexose (4). Reaction of an aqueous solution of cystine with thiamin, glutamate and ascorbic acid produces a complex mixture of compounds with an overall flavor resembling that of roasted meat. (5). A. Inosine Monophosphate The role of inosine monophosphate (IMP) as a precursor of meat flavor has been examined by heating muscle with and without added IMP. A number of thiols and disulfides containing furan groups were isolated from the meat systems, with much larger amounts

Table 1 Classification of Volatile Compounds Found in Meat Number of compounds Compounds Hydrocarbons Aldehydes Ketones Alcohols Phenols Carboxylic acids Esters Lactones Furans Pyridines Pyrazines Other nitrogen compounds Surur compounds Halogenated compounds Miscellaneous Total

Pork (uncured)

Pork (cured)

Mutton

Chicken

Beef

45 35 38 24 9 5 20 2 29 5 36 24

4 29 12 9 1 20 9 — 5 — — 3

26 41 23 11 3 46 5 14 6 16 15 8

71 73 31 28 4 9 7 2 13 10 21 33

123 66 59 61 3 20 33 33 40 10 48 37

31 4 7 314

31 1 11 135

12 — — 226

33 6 6 347

126 6 16 681

Source: Adapted from Ref. 1.

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formed in the meat containing IMP. The amounts of these sulfur compounds were also higher in meat systems in which the pH had been reduced by the addition of acid (6). Effects of addition of meat flavor precursors inosine-5-monophosphate, to cooked beef on formation of volatile flavour compounds were studied. Concentration of sulfur-substituted furans, mainly those containing the 2-methyl-3-furyl moiety, were significantly increased by the presence of flavor precursors, particularly 5-IMP. B. Thiamin Thiamin has been recognized as the precursor for the formation of meat aroma compounds, methyl-3-furanthiol and bis-(2-methyl-3-furyl)disulfide. Thiamin degradation generates a series of sulfur-containing meaty compounds; hydrogen sulfide is an important precursor that can react with furanones to give an intense meat flavor. The acid degradation of thiamin leads to the formation of 4-amino-5-(hydroxymethyl)-2-methylpyrimidine and 5-(2hydroxyethyl)-4-methylthiazole. The latter is supposed to be a key substance for the generation of series processed meat flavors. On the other hand, the formation of meaty flavor compounds is favored by the thermal degradation of thiamin in a slightly akaline pH value (7). Some key intermediates, hydrogen sulfide and 3-mercapto-5-hydroxy-2-pentanone, were formed. Further degradation of the latter can generate additional intermediates, 3,5dimercapto-2-pentanone, 3,5-dihydroxy-2-pentanone, 3-hydroxy-5-mercapto-2-pentanone, and by a keto-enol tautomerization of 3,5-dihydroxy-2-pentanone, 1,4-dihydroxy3-pentanone, 1-mercapto-4-hydroxy-3-pentanone, 1-hydroxy-4-mercapto-3-pentanone, and 1,4-dimercapto-3-pentanone (8). The intermediates 3-mercapto-5-hydroxy-2-pentanone and 5-dimercapto-2-pentanone are important precursors for aroma compound generation. C. Alliin and Deoxyalliin Alliin and deoxyalliin are two important nonvolatile flavor precursors of garlic. The isolate from the interaction of alliin and glucose possessed a very good roasted meaty character, the isolate from the interaction of deoxyalliin and glucose possessed a slightly roasted meaty flavor with garlic character. Thiazoles, especially 2-acetylthiazole, were found to be the predominant volatile interaction products of alliin and glucose (9), whereas pyrazines, especially 2,5-dimethyl-, methyl-, and trimethylpyrazine were found to be the predominant volatile interaction products of deoxyalliin and glucose. Some thiazoles together with some pyrazines, thiophenes, ketones, furans, and cyclic sulfur-containing compounds were thought to contribute to the roasted meat-like flavor in the model systems. D. Strecker Aldehyde High temperature treatment of meat such as roasting or grilling may lead to the pyrolysis of peptides and amino acids. When foods are roasted at temperatures higher than 100° to approximately 200°C, pyrolysis may participate in aroma formation taking place on the surface of the foods (10). The pyrolytic products frequently include ammonia, carbon dioxide, amine, hydrocarbon, nitriles, and carbonyl compounds. The compounds formed on pyrolyzing a peptide depended on the sequence of amino acids. As suggested by Dwivedi, this could explain the formation of volatile compounds in meat not obtained on heating model systems of single amino acids (11). Degradation of glutathione releases hydrogen sulfide rapidly when heated (12), but ammonia is released relatively slowly, producing hetero-

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cyclic compounds containing nitrogen (13). Ammonia released from amino acids, peptides, and proteins may impart numerous reactions leading to the formation of meat aromas. Different amino acids liberate free ammonia at different rate. The effects of an oil medium on the release of free ammonia from each of five amino acids (glycine, L-aspartic acid, L-asparagine, L-glutamic acid, L-glutamine) and the formation of pentylpyridines were studied. Among the five amino acids, only asparagine and glutamine generated ammonia readily by deamidation of amide side chains at 180°C under oil conditions, even though both of them produced less ammonia than under aqueous conditions (14). Strecker degradation is one of the most important reactions associated with the generation of the precursors for thermal formation of meat aroma and observed by Strecker (15). It involves the oxidative deamination and decarboxylation of an amino acid in the presence of a dicarbonyl compound. This leads to the formation of a Strecker aldehyde, containing one fewer carbon atoms than the original amino acid, and an -aminoketone (16). Ammonia may also be produced from amino acid by the Strecker degradation. A number of -dicarbonyls or vinylogous dicarbonyls as well as reducing sugar (glucose) are able to degrade -amino acids via Strecker degradation (17). Mechanism of the Strecker degradation of 1-phenylalanine initiated by 2-oxopropanal was proposed in honor of Strecker (18). Several Strecker aldehydes are well-known cooked beef components, e.g., acetaldehyde (sharp, penetrating, fruity), 2-methylpropanal (penetrating, green), 3-methylbutanal (malty, green) 2-methylbutanal (etheral, bitter, almond, green) and phenyl, and may derive from alanine, valine, leucine, isoleucine and phenylalanine respectively. The Strecker degradation of methionine is another source of the sulfur-containing intermediate, methional. Methanethiol and 2-propenal are produced (19). Besides the thermal degradation of thiamin, the amino acid cysteine is a well-known precursor of meat flavor. Hydrogen sulfide, mercaptoacetaldehyde, and acetaldehyde were identified as common products when cysteine was boiled with various carbonyl compounds. Among them, acetaldehyde is known to be formed from the hydrolysis or Strecker degradation of cysteine (10). In addition to cysteine, sulfur containing amino acid cystine and glutathione will liberate hydrogen sulfide, which is a major reactant for meaty aroma generation (20). The amino group in the free cysteine molecule is accessible to dicarbonyl compound and Strecker degradation is possible (21). E. 1-Pyrroline 1-pyrroline was identified as effective intermediates in generating the roast-smelling food odorant 2-acetyltetrahydropyridine and 2-acetyl-1-pyrroline (22). The oxidation of proline with periodate may lead to the formation of 1-pyrroline (23). Heating L-proline with various reducing sugars leads to the formation of 1-pyrroline as well (24). Strecker degradation of proline and/or arginine and/or ornithine may produce Strecker aldehyde, 4-aminobutanal, which is an equilibrium compound of 1-pyrroline (25). Biologically, in yeast (26) and Bacillus cereus (27), 1-pyrroline has been characterized as an important intermediate for the formation of 2-acetyl-1-pyrroline. 1-Pyrroline is proposed to be derived enzymatically through decarboxylation of proline and reduction of glutamic acid. In addition to yeast and bacteria, 1-pyrroline and 2-acetyl-1-pyrroline were identified in taro (28) volatiles, indicating that 1-pyrroline may be synthesized in vivo as well. Flavor contribution and formation of the intense roast-smelling odorants 2-propionyl-1-pyrroline (PP) and 2-propionyltetrahydropyridine (PTHP) in Maillard-type reac-

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tions have been proposed (29). A formation pathway for both odorants is created from the same intermediate, 1-pyrroline, when reacted with either 2-oxobutanal (yielding PP) or 1hydroxy-2-butanone (yielding PTHP). F. Fatty Aldehydes Three main mechanisms for the formation of volatile reactive carbonyls are discussed: (a) Strecker degradation of amino acids; (b) non-enzymatic oxidation of fatty acids and (c) retro-aldol condensation. The identification of many heterocyclic compounds from reactions between aldehydes and products from Maillard reactions indicates that these compounds could be formed during the cooking of meat. Some of the aldehydes investigated must be considered Strecker degradation products of amino acids mentioned above. However, other studies have shown that aldehydes from lipid oxidation, such as pentanal, hexanal, 2-hexenal, 2-decanal and 2,4-decadienal can as readily enter these reactions as can those of shorter chain length (30). Degradation of Lipid. Lipid oxidation is one of the major causes of quality deterioration in raw and cooked meat products (31). Processes involved in meat product processing, which include cutting, grinding, mixing, and cooking, can enhance the degradation of polysaturated fatty acid hydroperoxides into secondary products. During cooking, thermal and oxidative degradation of depot triglyceride and tissue phospholipids occur simultaneously. In the absence of oxygen, lipids thermally degrade through dehydration, decarboxylation, hydrolysis, dehydrogenation, and carbon-carbon cleavage. Through hydrolysis, free fatty acids are released (32). The procedure of direct heating the meat sample under acidic (pH 3 or lower) conditions enhances the degradation of the existing lipid hydroperoxides and generates additional degradation products. Of the volatiles produced by lipid oxidation, aldehydes are the most significant flavor compounds. The accepted mechanism of lipid oxidation involves hydroperoxide formation, followed by the production of fragments containing various fuctional group (33). Autoxidation is a free radical chain reaction and is initiated by the extraction of a hydrogen atom from the methylene carbon of an unsaturated fatty acid. For fatty acid with two or more double bonds in a non-conjugated system, the release of the methylene hydrogen is stabilized by delocalization of the free radical over five carbons. Reaction of oxygen with the free radical generates peroxy radicals, and the reaction is then propagated by formation and decomposition of hydroperoxides. Aldehyde can be produced by scission of the lipid molecules on either side of the radical. The products formed by these scission reactions depend on the fatty acids present, the hydroperoxide isomers formed, and the stability of the decomposition products. Temperature, time of heating, and degree of autoxidation are variables that affect thermal oxidation (34). Aldehydes are the major components identified in volatiles of cooked meat. Octanal, nonanal, and 2-undecenal are oxidation products of oleic acid, and hexanal, 2-nonenal, and 2,4-decadienal are major volatile oxidation products of linoleic acid (35). 1. 2,4-Decadienal The autoxidation of linoleic acid creates 9- and 13-hydroperoxides. Cleavage of 13-hydroperoxide leads to hexanal and the breakdown of 9-hydroperoxide produces 2,4-decadienal (36). Subsequent retro-aldolization of 2,4-decadienal will produce 2-octenal and hexanal (37). 2,4-decadienal is known to be one of the most important flavor contributors to deep-fat-fried foods (38).

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Phospholipid fraction has been identified as primary substrates in development of oxidative deterioration in muscle foods (39,40). Phospholipids are essential structural components of all cells and contain a much higher proportion of unsaturated fatty acids than the triglycerides, including significant amounts of arachidonic acid (20:4). Arachidonic acid constitutes greater than 50% of the polyunsaturated fatty acids in meat phospholipids. Autoxidation of arachidonic acid may have a role in the development of off-flavors in meat. Various aldehydes, ketones, aldehyde esters, hydrocarbons, and alcohols were identified. The major products included hexanal, methyl 5-oxopentanoate, pentane, methyl butanoate, and 2,4-decadienal, which could be important to off-odor development in oxidized food systems containing arachidonate (41). Reactions involving phospholipids contribute considerably to the flavor and aroma of cooked meat. After 1 hour at 132°C, ethanolamine-containing phospholipids rapidly lost most of their component aldehydes, but not their fatty acids, whereas choline-containing phospholipids lost their aldehydes much more slowly. In both cases, the presence of ribose or glycine had little effect on the loss of aldehydes. In the meat itself, after heating at 132°C, there was a 90% loss of aldehydes, which was similar for both the ethanolamine- and choline-containing phospholipids (42). 2. Short-Chain Aldehydes Short-chain aldehydes such as acetaldehyde, butanal, and hexanal, probably derived from the decomposition of 2,4-decadienal. Under aqueous conditions 2,4-decadienal undergoes , -double-bond hydration and retro-aldol condensation to give hexanal and acetaldehyde (37). Hexanal can then condense with acetaldehyde, suggested to be involved in the formation of 3,5-dimethyl-1,2,4-trithiolane or 5,6-dihydro-2,4,6-trimethyl-4H-1,3.5-dithiazine. Benzaladehyde is considered a typical thermal degradation product of 2,4-decadienal (43). The effect of adding glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE) to the decomposition of linoleic (18:2) acid was evaluated at 70°C to study effects of phospholipids on off-flavor production during lipid oxidation in cooked meat products by monitoring the production of low mol. wt. headspace volatiles. Hexanal was the most prominent volatile produced in all treatments. Volatile production rate in the 18:2  GPE treatment was generally greater as compared to the 18:2  GPC and 18:2 treatments (44). Water-mediated retrol-aldol degradation of , -unsaturated carbonyls appear to be significant as a means to thermally generate flavor-active carbonyl. The reaction cascade proceeds rapidly from the conjugated carbonyl through its hydration and subsequent fragmentation (45). Retrol-aldol degradation of (E,Z)-2,6-nonaldienal to (Z)-4-heptenal and acetaldehyde, and (E,Z)-2,4-decadienal to 2-octenal hexanal and acetaldehyde, have been demonstrated to be thermally driven at neutral pH. G. Carbohydrate Degradation Products During thermal treatment of carbohydrates, several dicarbonyls are formed (17). The condensation of the carbonyl group of the reducing sugar with the amino compound gives a glycosylamine. Subsequently, this rearranges and dehydrates, via deoxyosone, to various sugar dehydration and degradation products such as furfural and furanone derivatives. When a ketose is involved instead of an aldose sugar, a ketosylamine is formed that undergoes a Heyns rearrangement to form a 2-amino-2-deoxyaldose (Heyns product). At temperature above 100°C, 1-amino-1-deoxy-2-ketoses undergo 2,3-enolization to give a 1-

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amino-2,3-enediol from which an amine is eliminated to form a methyl-2,3-dicarbonyl intermediate (46). Yayalan has recently proposed an alternative mechanism, e.g. direct dehydrations from cyclic forms of the Amadori compounds to account for the many products observed in model systems and in foods (47). 1,2-enolization of the Amadori product will result, after dehydration and deamination, in the formation of a 3-deoxyosone. The latter compound can be formed by a similar pathway from the Heyns product. The 3-deoxyosone is readily converted to the corresponding furfural from a pentose, and 5-methylfurfural from a hexose. Furan derivatives are also formed by the further dehydration of the 1methyl-2,3-dicarbonyl compounds, yielding 4-hydroxy-5-methyl-3(2H)-furanone or the 2,5-dimethyl homologue, from the pentose and hexose sugars, respectively. Ribose is one of the main sugars in muscle. It is usually associated with phosphate and exists as ribose phosphate. In meat, it has been proposed that ribose phosphates, from ribonucleotide, is the principal precursor of furan (48). Dephosphorylation and dehydration of ribose phosphate form the important intermediate, 4-hydroxy-5-methyl-3(2H)-furanone (HMF), which readily reacts with hydrogen sulfide (49). 4-hydroxy-5-methyl-3(2H)-furanone and the structurally related 2,5-dimethyl-3(2H)-furanone (HMF) have been isolated from natural beef broth (50). These compounds are considered to be involved in the formation of meaty flavors through their reaction with either hydrogen sulfide or sulfur-containing amino acids. A method is described for producing a pure, food-grade 2,5-dimethyl4-hydroxy-2,3-dihydrofuran-3-one by heating a 6-deoxyhexose in the presence of an amino acid in an aqueous medium at pH 4-8. The hydroxyfuranone thus obtained is suitable for flavoring meat products (51). In addition to the ring-type furan derivatives, a number of hydroxycarbonyl and dicarbony fragmentation compounds can also be formed, e.g. glyoxal, glycolaldehyde, acetaldehyde, glyceraldehyde, pyruvaldehyde, hydroxyacetone, dihydroxyacetone, diacetyl, acetoin and hydroxydiacetyl. 2-Oxobutanal was shown to be formed in high yields (29 mol %) by reacting acetaldehyde and glycolaldehyde, two well-known degradation products of carbohydrates (52). Recently, the reaction between 4-hydroxy-5-methyl-3(2H)-furanone and cysteine or hydrogen sulfide was reinvestigated. Formation of 2,3-pentanedione was proposed. The key steps are the acid hydrolysis of 4-hydroxy-5-methyl-3(2H)-furanone to yield 1-deoxypentatosone followed by the reduction and acid-catalyzed dehydration of the latter. Retroaldolization of the 1-deoxypentatosone, produced from the hydrolysis of 4-hydroxy-5-methyl-3(2H)-furanone, can yield 2-oxopropanal (pyruvaldehyde) and, subsequently, hydroxyacetone by reduction. Aldol condensation of the latter with acetaldehyde, followed by dehydration, would yield 2,3-pentanedione. The formation of diacetyl from 4hydroxy-5-methyl-3(2H)-furanone could follow pathways similar to those involved in the formation of 2,3-pentanedione. The fragmentation of the carbohydrate chains of the Amadori and Heyns products or the rearranged 1- and 3-deoxyketone proceeds by a series of retroaldolization reaction. These reactions can lead to a series of -dicarbonyl compounds such as 2-oxopropanal, 2,3-butanedione, 1,2-ethandial, and hydroxycarbonyl compounds such as 1-hydroxy-2-propanone, 2-hydroxyethanal, 2,4-dihydroxypropanal, and 1,3-dihydroxy-2-propanone (51). The loss of one carbon to give diacetyl occurs via retroaldol condensation with the elimination of formic acid. In the presence of cysteine the formation of 3-mercaptobutan-2-one requires aldol condensation between acetaldehyde and pyruvaldehyde followed by elimination of formic acid by retroaldol condensation. The resulting hydroxybutanone readily reacts with hydrogen sulfide to yield 3-mercaptobutan-2one. In the formation of mercaptoalkanones, it has been proposed that -dicarbonyls are reduced to hydroxyakanones before substitution by hydrogen sulfide (53).

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Mercaptoalkanone are known to form readily by the reaction of -dicarbonyl compounds with hydrogen sulfide (54). The formation of -dicarbonyls from 4-hydroxy-5methyl-3(2H)-furanone is a key step in the formation of mercaptoalkanones, such as 3-mercaptopentan-2-one and 2-mercaptopentan-3-one. Three alkanediones, 2,3-pentanedione, 2,4-pentadione, and 3,4-hexadione were identified in the reaction mixtures containing cysteine. Three interesting meat aroma precursors, 3-mercaptobutan-2-one, 3-mercaptopentan-2-one, and 2-mercaptopentan-3-one, which had been reported previously in cysteineribose model systems, were detected in those systems containing hydrogen sulfide. III. PATHWAYS FOR THE FORMATION OF SOME MEAT AROMA VOLATILES Heterocyclic compounds have been identified as important volatile components of many foods. The odor strength and complexity of these compounds makes them desirable as flavoring ingredients. Heterocyclic compounds are primarily formed through nonenzymatic browning reactions. A. Acetaldehyde-Derived Meat Aroma Acetaldehyde is involved in many reactions, explaining the formation of reportedly meaty 1-(methylthio)ethanethiol (55), 3,5-dimethyl-1,2,4-trithiolane (56), trithioacetaldehyde (57), thialdine (56), 2,4-dimethylthiazole (58), 2,4-dimethyl-5-ethylthiazole (59), 2,4,5trimethyl-3-thiazoline (60), 2,4,5-trimethyl-3-oxazole (61) and 2,4,5-trimethyl-3-oxazoline (62). 3,5-Dimethyl-1,2,4-trithiolane was first identified by Chang and co-workers (63) in the volatiles of boiled beef and has been found in the volatiles of cooked chicken (64). It is reported to have roasted and onion-like flavors (65). In addition to 3,5-dimethyl-1,2,4trithiolane, 3,5-diisobutyl-1,2,4-trithiolane were identified in the volatiles isolated from fried chicken flavors (66). These trithiolanes possess roasted, roasted-nut, crisp bacon-like and pork rind-like aromas. 3,5-Diisobutyl-1,2,4-trithiolane has been produced in a model system containing isovaleraldehyde, ammonia, and hydrogen sulfide (67). Instead of acetaldehyde, isovaleraldehyde is involved in the reaction leading to the formation of 3,5-diisobutyl-1,2,4-trithiolane. Isovaleraldehyde arises via Strecker degradation of leucine. Ammonia and hydrogen sulfide may arise via thermal degradation of amino acids and cysteine or cystine. Several cyclic sulfur-containing compounds have been identified in meat flavor. Thialdines are sulfur and nitrogen-containing heterocyclic flavor components produced nonenzymatically during thermal processing (e.g., by cooking, frying, boiling, roasting, baking etc., of foodstuffs). Their origin in food flavors lies in a series of complex reactions in which lipids and amino acids play a dominant role (68). Cyclic dithia compounds, especially those containing a keto group, are believed to contribute to meat flavor (69). A number of cyclic dithia compounds impart fatty, spicy, roasted, mushroom or sulfury notes to overall meat flavor. Possible formation mechanisms of thialdines have already been described (70). At high temperature approximating roasting conditions (i.e., Shigematsu condition), the products formed reflect the fact that amino acid pyrolysis (rather than Strecker degradation) is an important reaction (71). Cysteine is primarily transformed into six products, namely mercaptoacetaldehyde, actaldehyde, cysteamine, ethane-1,2-dithiol, hydrogen sulfide, and

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ammonia (72). When cysteine was degraded at 180°C, representing frying temperature, both 3,5-diisobutyl-1,2,4-trithiolane and 2,4,6-trimethylperhydro-1,3,5-dithiazine (thialdine) were identified as major products (73). A similar mechanism for the formation of thiadiazines and dithiazines was proposed (74). Fatty aldehydes, hydrogen sulfide, and ammonia are often constituents of food flavor that are converted into mixtures of alkyl-substituted thialdines by heating, 2,4,6trimethylperhydro-1,3,5-dithiazine (thialdine), the main representative of this class of compounds, was described as early as 1847. The first identification of the trimethyl derivative in a food product was described in heated pork. Thialdine was isolated and identified from pressure-cooked beef, cooked beef flavor, roasted lamb fat and cooked mutton (68). In the presence of ammonia, dithiazines and thiadiazines are formed. One dithiazine (i.e., thialdine) has been reported in heated beef aroma (55). Model reactions using aldehydes and hydrogen sulfide lead to the formation of another group of interesting sulfur compounds. At atmospheric pressure, acetaldehyde and hydrogen sulfide give mainly cyclic trimers, eg. dioxathianes, oxadithianes, and trithiane (75). 2,4,6-trimethyl-1,3,5-trithiane has been found in cooked beef aroma (68). 1,3,5Trithiane has also been identified in model reactions of fatty aldehydes, hydrogen sulfide, thiol, and ammonia (70) and from furfural, hydrogen sulfide, ammonia reaction mixture as well (76). However, reaction of acetaldehyde and excess hydrogen sulfide in a closed vessel give rise via bis-(1-mercaptoethyl) sulfide as intermediate to some compounds, namely 3,5-dimethyl-1,2,4-trithiolane and diethyldisulfide. In 2,4,6-trimethyl-1,3,5-dioxathiane, 2,4,6-trimethyl-1,3,5-oxadithianes, 2,4,6trimethyl-1,3,5-trithiane, 2,4,6-trimethyl-1,3,5-thialdine, 2,4,6-trimethyl-1,3,5-thiadiazine and 3,5-dimethyl-1,2,4-trithiolane, acetaldehyde provides the carbon skeleton on the ring structure (Fig. 1) B. Dicarbonyl-Derived Meat Aroma An extensive and systematic study of similar reactions, but using -dicarbonyls in addition to aldehydes, hydrogen sulfide, and ammonia, have been conducted (68). Various combinations were studied, the -dicarbonyls being 2,3-butanedione, 2,3-pentadione, or pyruvaldehyde; the aldehydes used were acetaldehyde or propanal. All reaction mixture gave relatively similar products, e.g., thiazoles, thiazolines, oxazoles, oxazolines, isothiazoles, thialdine, dithiolenes, trithianes, and mercaptoalkenes. Aldehydes formed by Strecker degradation of amino acids in the presence of -diketones react with ammonium sulfide in the presence or in the absence of acetoin (3-hydroxy2-butanone) giving a variety of oxygen, sulfur, and/or nitrogen-containing heterocyclic compounds (77). Sugars are the major suppliers of carbonyls and amino acids for nitrogen. Thiazole and oxazoles may be formed from related mechanisms, involving the formation of an intermediate imine from the reaction of an aldehyde with ammonia. Reaction of the imine with a carbonyl produces an oxazoline that may undergo oxidation to the corresponding oxazole. If hydrogen sulfide is present, it may react with the dicarbonyl to give an -mercatoketone which then yields thiazoline and thiazole from reaction with the imine. Oxazoles and oxazolines, which are oxygen and nitrogen-containing heterocyclics, have been identified in many kinds of heated foods and have significant sensory contribution (78). They possess potent sensory quality at very low concentration described as green, sweet, and nutty aroma and have been identified in soy sauce (79), and cooked beef (60). 2,4,5-Trimethyl-3-oxazoline was first found in the flavor of meat as early as 1968 (63).

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Figure 1 Formation mechanism for acetaldehyde-derived meat aroma.

2,4,5-Trimethyl-3-oxazoline was reported later to be the major compound in boiled beef (80), canned beef stew (81). 2,4,5-Trimethyl-3-oxazoline was isolated and characterized from a model reaction of ammonia, acetaldehyde, and 3-hydroxy-2-butanone. The condensation between the amino group of 3-amino-2-butanone and the keto group of 3-hydroxy-2-butanone leads to the formation of a Schiff base. The Schiff base may rearrange to form 2-(1-hydroxyethyl)-2,4,5-trimethyl-3-oxazoline and then 2,4,5trimethyl-3-oxazoline. A similar 2,4,5-trimethyl-3-oxazoline formation mechanism has been proposed by Piloty and Baltes (82). They found that oxazoles, pyrazines, pyrroles, and pyridines were formed in the heated model systems of amino acids and 2,3-butanedione (Fig. 2). Thiazoles are a class of compounds possessing a five-membered ring with sulfur and nitrogen in the 1 and 3 position respectively. A number of thiazole derivatives were found in cooked meat (83). 2-Acetyl-2-thiazoline has been found in beef broth (84). Thiazoles are considered one of the main constituents which give a meaty flavor. Alkylthiazole identified Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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in meat products arises from a Maillard-type reaction between amino acids and sugars or sugar-derived carbonyl compounds. An alternative formation pathway has been reported (36). Four alkylthiazoles, namely 4-methylthiazole, 4,5-dimethylthiazole, 4-methyl-5ethylthiazole and 4-methyl-5-vinylthiazole, identified in pork may be the thermal degradation products of thiamin. Several thiazoles with C4-C8 n-alkyl substituents in the 2-position have been reported in roast beef (85) and fried chicken (64). Other alkylthiazoles with longer 2-alkyl substituents (C13-C15) were found in the volatiles of heated beef and chicken with the highest concentrations in beef heart muscle (86). Aliphatic aldehydes from lipid oxidation are the likely sources of the long n-alkyl groups in these compounds. Recently, a large number of alkyl-3-thiazolines have been isolated from cooked beef (87). Most of the thiazolines contained C5-C9 n-alkyl substituents in the 2-position. The most likely routes to the 3-thiazolines and thiazoles are from -hydroxyketones or -diones (-dicarbonyls), hydrogen sulfide, ammonia, and aldehydes (Fig. 2). Lipid oxidation provides long chain aldehydes.

Figure 2 Formation of thiazoles, thiazolines, oxazoles, oxazolines and pyrazines by the reaction of aldehydes with dicarbonyls, hydrogen sulfide, and ammonia.

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Formation of 3-thiazolines from an -hydroxyketone involves the substitution of the hydroxyl group with an thiol group. This is followed by nucleophilic attack by the sulfur at the carbon atom of an imine intermediate formed by the reaction between ammonia and an aldehyde. Subsequent ring closure, with the elimination of water, gives the 3-thiazolines. Oxidation of the thiazoline results in the formation of the corresponding thiazole (87). A slightly different mechanism could occur with the alkane--diones. The initial step requires nucleophilic addition of thiol group to one of the carbonyl groups. This intermediate could be reduced to a mercaptoketone, or it could react with an imine intermediate, leading to a thiazole (Fig. 2). Pyrazines are very important constituents of meat aromas. Most of them contain a methyl group as a substituent that demonstrates the outstanding role of diacetyl, pyruvic aldehyde and corresponding sugar decomposition products. Other important substitutes are ethyl-, propynyl-, vinyl-, allyl-, and propenyl groups. In the simple Maillard reaction mixture, the alkylpyrazines were quantitatively major products when amino acid was the nitrogen source (88). Volatile aroma compounds from beef steaks, shallow fried without oil, were identified. Pleasant flavor qualities were much stronger for the 280°C extract than for the 300°C extract; this was mainly due to the combination of 2-ethyl-3,5-dimethylpyrazine and 2-propyl-3-methylpyrazine (89). At least 22 pyrazines were identified in the flavors of fried chicken and roasted chicken (90). The alkylpyrazines accounted for almost 80% of the total headspace volatiles of well-done grilled pork (91). Pyrazine formation was studied as a model for carbohydrate fragmentation in the Maillard reaction (92). Thus 1-13C-glucose, 2-13C-glucose and 1-13C-fructose were reacted with asparagine in 1,2-propanediol, and the volatile products isolated by steam distillation and extraction. The product mixture consisted mainly of dimethyl-, monomethyl, and, to a lesser extent, trimethylpyrazines. The 13C-incorporation in the pyrazines obtained from all three labeled hexoses was in agreement with retro-aldolization of the intermediate deoxyglucosones as the main cleavage mechanism. Both 1-and 3-deoxyglucosone appear to play an approximately equal and important role in the formation of the methylated pyrazines. Alkylpyrazines were formed from a reaction of the degraded nitrogenous substances, NH3, RNH2 from proteins, peptides, amino acids and phospholipids, and -dicarbonyl compounds in food (93). Formation pathways for alkylpyrazines have been proposed by numerous researchers (94). An important route to alkylpyrazines is from -aminoketones, which are formed in Strecker degradation or from the reaction of -dicarbonyls with ammonia. Condensation of two aminoketone molecules yields a dihydropyrazine that oxidized to the pyrazine (Fig. 2). C. Fatty Aldehyde–Derived Meat Aroma 1. Pyrazines Long n-alkyl substituted pyrazines, butyl- and pentyl-pyrazines are found in meat (95). Two pentyl-substituted and one butyl-substituted pyrazines are also observed in grilled pork. Propylpyrazine may be formed from the interaction product of lipids, proteins, and carbohydrates following a similar mechanism proposed by Chiu et al. (96). The combination of these alkylpyrazines may lead to the formation of the characteristic fried meaty aroma of Chinese fried pork bundle (97). Recent studies on the interaction between the Maillard reaction and lipid degradation leading to the formation of desirable flavor compounds of foods are also discussed (95).

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The effect of long-chain aldehydes on the formation of long-chain alkyl-substituted pyrazines was investigated in model systems of acetol and ammonium acetate with the addition of pentanal or hexanal. When the systems were reacted at 100°C for 4 hours, 2.5dimethyl-3-pentylpyrazine and 2,6-dimethyl-3-pentylpyrazine were formed in the model system with added pentanal, and the corresponding hexylpyrazines were formed in the hexanal system. Formation pathways are proposed for some of these pyrazines (96). The formation of higher carbon number substituted pyrazines is hypothesized to be due to the intervention of aldehydes. The aldehydes, originating from lipid degradation, could facilitate addition to the metastable dihydropyrazine compound, formed by the condensation of two molecules of aminoketones (98). The latter is a product of the Strecker degradation of amino acids with -dicarbonyl compounds. Figure 2 shows the mechanism for the formation of long-chain alkyl-substituted pyrazine. 2. Pyridines 2-Isobutyl-3,5-diisopropylpyridine was identified in fried chicken and has a roasted cocoalike aroma (66). The formation of pyridines involves the reaction of aldehyde and ammonia at high temperature and is known as the Chichibabin condensation (99). As a matter of fact, the investigation of the condensation of aldehydes, ketones, , -unsaturated carbonyl compounds with ammonia to form substituted pyridines can be dated back to 1950 (100). In a continuation of the above studies, the same authors showed that when glycine and propanal were heated at 180°C, 3,5-dimethyl-2-ethylpyridine, 3,5-dimethylpyridine were formed immediately (101). D. Cysteamine-Derived Thiazoles and Thiazolidine An alternative pathway for thiazole formation derived from the condensation between cysteamine and aldehyde or 2,3-butanedione has been proposed. Cysteamine, which is a decarboxylated derivative of cysteine, produced a series of alkylthiazolidines (102). Aroma compounds thiazolidine and thiazine, were reported by different research groups from the same model system, cysteamine and 2,3-butanedione. Thiazolidines generally possess a characteristic popcorn flavor (103). 2-acetyl-2-methylthiazolidine was first characterized by Umano et al. (104) from the head space of a heated D-glucose/L-cysteine model system. The yield of 2-acetyl-2-methylthiazolidine is less than 0.01% (GC peak area). They hypothesized that the reaction between cysteamine, the decarboxylated cysteine, and 2,3-butanedione, a glucose degradation product, may lead to the formation of 2-acetyl-2methylthiazolidine. Under the experimental conditions described, a standard mixture composed of formaldehyde, actaldehyde, propionaldehyde, butyaldehyde, valeraldehyde, and hexanal were reacted with cysteamine as described. Thiazolidine, 2-methylthiazolidine, 2-ethylthiazolidine, 2-propylthiazolidine, 2-butylthiazolidine, and 2-pentylthiazolidine were formed (105). A mechanism was proposed to elucidate the formation of a thiazole and thiazolidine, in aldehyde or 2,3-butanedione/cysteamine model systems (Fig. 3). Buffering dramatically promotes thiazolidine formation from aldehyde or 2,3-butanedione and cysteamine. Phosphate tends to stabilize the carbocation formed, and this may lead to completion of the cyclization by attacking the amino nitrogen on the activated carbon. Protic solvent, by removing the water molecule, further enhances thiazolidine formation. Redox reaction catalyzed by phosphate ions results in the conversion of thiazolidine to the corresponding thiazoline through hydride transfer (106).

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Figure 3 Formation of thiazoles, thiazolines, and thiazine by the reaction of aldehydes with cysteamine.

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E. 1-Pyrroline-Derived 1,3,5-Dithiazine and Pyrroline A formation pathway has been proposed for both odorants from the same intermediate, 1pyrroline, when reacted with either 2-oxobutanal (yielding 2-acetyl-1-pyrroline) (107) or acetaldehyde and hydrogen sulfide (yielding pyrrolidino(1,2-e)-4H-2,4-dimethyl-1,3,5dithiazine) (108). 2-Oxobutanal was shown to be formed in high yields (29 mol %) by reacting acetaldehyde and glycolaldehyde, two well-known degradation products of carbohydrates. Pyrrolidino(1,2-d)-4H-2,4-dimethyl-1,3,5-dithiazine possesses an extremely low odor threshold. 1-Pyrroline and compound pyrrolidino(1,2-d)-4H-2,4-dimethyl-1,3,5dithiazine were produced at the same time during heating and were increased by longer heating. It is postulated that compound pyrrolidino(1,2-d)-4H-2,4-dimethyl-1,3,5-dithiazine was formed secondarily from 1-pyrroline with acetaldehyde and hydrogen sulfide in the shellfish during heating (108). The reaction between 2-oxopropanal and proline was suggested earlier (109) as a key step in the formation of 2-acetyltetrahydropyridine. The reaction of 1-pyrroline with 2-oxopropanal was previously postulated to be a key step in the generation of 2-acetyl-1-pyrroline (Fig. 4). The production of volatile compounds in the reaction of the lipid peroxidation product (E) 4,5-epoxy(E)2-heptenal with lysine or bovine serum albumin was studied in order to characterize alternative mechanisms for production of volatile pyrrole derivatives in foods (110). A mixture of the lipid and the amino acid or the protein was stirred overnight at 37°C and the volatiles formed were collected using a Tenax TA trap. Several aldehydes, ketones, alkylfurans, alkyl- and acyl-pyrroles, and alkylpyridines were identified by GC/MS. Formation of pyrrole derivatives in these reactions suggested the existence of alternative mechanisms for production of these flavor-associated compounds, previously considered to be formed mainly via Maillard reactions. This new route does not need the presence of sugars, and might contribute significantly to volatile pyrrole production in foods under conditions unfavorable for classic Maillard reactions (110).

Figure 4 Formation of acylpyrole and bicyclic-1,3-dithizine by the reaction of aldehydes and 1pyrroline.

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In addition to 2,4,6-trimethylperhydro-1,3,5-thiadiazine, other alkyl-substituted dithiazines, including 2-ethyl-4,6-dimethyl,4-ethyl-2,6-dimethyl,2-propyl-4,6-dimethyl-, 2-butyl-4,6-dimethyl-,2-pentyl-4,6-dimethyl- and 4-petyl-2,6-dimethyl-1,3,5-dithiazines, were also found in chicken and beef (68) flavor. These alkyl-substituted dithiazines were identified in flavor of pork as well. The higher homologue of 3,5-dimethyl-1,2,4-trithiolane, 3,5-diisobutyl-1,2,4-trithiolane, was identified in the volatiles isolated from fried chicken flavor (66). F. Furan-Derived Meat Aroma Sulfur-containing furans and thiophenes and related disulfides are known to possess strong meat-like aromas and exceptionally low odor threshold values. Such compounds have been found in model Maillard reaction systems and in cooked meat where they are considered to contribute to the characteristic aroma (111). Possible precursors of these compounds in meat are pentose sugars and cysteine. One of the main sources of pentoses in meat is the ribonucleotide, inosine-5-monophosphate (IMP), which accumulates in meat during postmortem glycolysis (111). Furans or thiophenes substituted on position 3 by a thiol, sulfide, or disulfide group are considered as key compounds to meat flavor with savory and meaty aromas and very low thresthold values (112). An essential structural requirement for meaty aroma is a 5- or 6-membered ring, which is more or less planar and substituted with an enol, a thiol, and a methyl group adjacent to the thiol (Fig. 5). Meat, such as beef, mutton, pork, or poultry, treated with a raw soy sauce, retains the flavor of soy sauce and the well-balanced enzyme activities (proteinase, collagenase, elastase) acquired during brewing. The treatment eliminates the powdery (roughened) surface and other disadvantages attributable to conventional tenderizing agents. The raw soy sauce acts intensively on the connective tissue of the meat, and to a lesser extent on myofibrils, producing meat with good flavor and tenderness (113) 2-Methyl-3-furanthiol has been identified as the most important flavor compound contributing to the meaty perception of chicken broth (114) and as a character impact compound in the aroma of cooked beef (115). Another compound structurally related to 2-methyl-3-furanthiol and identified as a primary odorant in chicken broth is 2-furfurylthiol (114). This compound possesses a threshold of 5 ppt and aroma qualities such as roasted and sulphury. Such compounds are thermally generated from the reaction of furfural and cysteine (116). Aroma concentrates from cooked and roasted beef, pork, and chicken were isolated and reported to contain aliphatic, cyclic, and heterocyclic S-containing components. Constituents identified included several iso- and anteiso-methyl-branched long-chain aliphatic aldehydes primarily found in beef aroma as well as an homologous series of alkyl-substituted cyclopentene-1-carbaldehydes in chicken flavor (117). Reaction products formed from the aqueous mixture (1:1:1:1 molar ratio) of cysteine, xylose, thiamin, and ascorbic acid heated at 120°C for 5 hours in a laboratory autoclave were isolated and characterized (118). Compounds were identified as 6-methylbicyclo[4.3.0]-2,5-dithia-7-oxanonene-3, 2-(2-furyl)-1,3-dithiol, cis- and trans-2-(2-furyl)-4acetyl-1,3-oxathiane, 4-(2-methyl-3-furylthio)-4-(2-furyl)-3-thiabutanal, 4,6-bis-(2-furyl)3,5-dithiahexanal, and 4-(3-thienylthio)-4-(2-furyl)-3-thiabutanal. Spectral data and odor descriptions are given and possible pathways for formation of these compounds are postulated (118).

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Figure 5 Formation from ribonucleotides of 4-hydroxy-5-methyl-3(2H)-furone, and its reactin with hydrogen sulfide.

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1. Thiophene, Thiophenone, Thiirane Thiophenes are responsible for the mild sulfurous odor of cooked meat (83). Four reactions were carried out to compare the sulfur-containing compounds formed via Maillard reaction/Strecker degradation of cysteine with furaneol and via the participation of hydrogen sulfide in the thermal degradation of furaneol. GC-MS analysis showed that certain sulfurcontaining compounds, such as 2.5-dimethylthiophene 2,5-dimethyl-4-hydroxy-3(2H)thiophenone, and 3,5-dimethyl-1,2,4-trithiolane were found in four reactions, whereas thiirane and 2-methylthiophene were found only in the Strecker degradation of cysteine and furaneol. Furthermore, this study showed that more sulfur-containing compounds were formed in the participation of hydrogen sulfide than in the Maillard reaction/Strecker degradation of glutathione and even cysteine, indicating that the availability of hydrogen sulfide in the reaction may be the limiting factor in the amount and the type of sulfur-containing compounds formed in the reactions. The amino group of the cysteine residue in glutathione is peptide bonded and cannot participate in Strecker degradation with a dicarbonyl compound. Therefore, the reaction mechanisms involved in the reaction between cysteine and furaneol would be different from those in the reaction between glutathione and furaneol. Thiophenes are responsible for the mild sulfurous odor of cooked meat (83). Several thiophenes were identified in grilled pork. Alkyl-substituted dithiazines have been found in the flavors of beef and pork (68). Three compounds, 2-methyl-3-furanthiol, 2-methyl-3-thiophenethiol and 3-thiophenethiol, were recently found in the 4-hydroxy-5-methyl-3(2H)-furanone/cysteine reaction mixture (119). 2-Methyl-3-furanthiol and 2-methyl-3-thiophenethiol had both been found in cysteine-ribose model systems (120). They were also reported by van der Ouweland and peer (51) to be major products of the reaction between mmf and hydrogen sulfide. These authors also reported that the di- and tetrahydro derivatives of 2-methyl-3-furanthiol and 2-methyl-3-thiophenethiol were also found. Pathway for the formation of 2-methyl-3furanthiol involves the initial reduction of hmf to give a hydroxyhydrofuranone, which undergoes acid-catalyzed dehydration to 2-methyl-3-hydroxyfuran. Ketonization of this, followed by the addition of hydrogen sulfide and the elimination of a molecule of water, gives the thiol 2-methyl-3-furanthiol. The substitution of the oxygen ring in 2-methyl-3-furanthiol by a sulfur atom via ring opening was suggested previously (49). 2-Methyl-3-thiophenethiol was suggested to form via a route similar to that proposed above for the furanthiol. If the intermediate 2-methyl-3 (2H)-furanone or 2-methyl-3(2H)-thiophenone were reduced to the corresponding hydroxy derivatives prior to substitution of the MOH by MSH, 4,5-dihydro-2-methyl-3-furanthiol and the corresponding thiophenethiol would result. G. 2,4-Decadienal–Derived Thiophene, Thiapyran, Pyridine, and Dithiazines A large number of long-chain alkyl-substituted heterocyclic compounds, including thiophenes, pyridines, thiazoles and other sulfur-containing compounds were detected in the interaction systems. The occurrence of such compounds in cooked meat products has been reviewed (121). These compounds are O-, N- and S-, heterocycles containing n-alkyl substituents (C5-C15). The alkyl groups usually derived from aliphatic aldehydes, obtained from lipid oxidation. The reaction pathway for the formation of 2-pentylpyridine, 2hexylthiophene, and 2-pentylthiapyran from 2,4-decadienal has been proposed (Fig. 6). The reaction of 2,4-decadienal with ammonia is a likely route to 2-pentylpyridine. Related

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Figure 6 Formation of 2,4-decadienal–derived thiophene, thiapyran, pyridine, and dithiozines by the reaction of aldehydes.

reactions between dienals and hydrogen sulfide may be responsible for the formation of 2alkylthiophene with C4-C8 alkyl substituents, which have been reported in pressure-cooked beef. 2-pentylpyridine is commonly found in the volatiles of cooked meat. A number of other alkylpyridines have been reported in lamb fat (122). Recently, the mechanism for the formation of 2-pentylpyridine in the reaction of 2,4-decadienal with either cysteine or glutathione ( -glu-cys-gly) in an aqueous solution at high temperature (180°C) was studied (123). The release of free ammonia from amino acids, especially L-asparagine and L-glutamine, leads to the formation of pentylpyridines as reported by Kim and Ho, 1998 (124). When each of the amino acids was reacted with 2,4-decadienal under oil conditions, an increased amount of 2-pentylpyridine could be produced from asparagine and glutamine compared to aqueous conditions. However, only a small amount of 3-pentylpyridine was formed in the asparagine and glutamine that produced a large amount of 2-pentylpyridine under oil conditions. When each of the five amino acids was reacted with 2,4-decadienal under oil conditions an increased amount of 2-pentylpyridine could be produced from asparagine and glutamine compared to aqueous conditions. Specifically, larger than 10 times the amount of 2-pentylpyridine was found in the reaction of glutamine and 2,4-decadienal under oil conditions than under aqueous conditions, despite the fact that both asparagine and glutamine could liberate less ammonia in oil systems (124) 2-Alkylthiophenes with C4-

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C8 alkyl substituents, which derived from the interaction between dienals and hydrogen sulfide, have been reported in pressure-cooked beef (125). IV. FACTORS CONTROLLING MEAT AROMA FORMATION A. Effect of Cooking Conditions on Meat Aroma Formation A wide range of temperature conditions exist during normal cooking of meat the center of a rare steak may only reach 50°C, the center of roast meat may attain 70°–80°C, and the outside of grilled or roast meat will be subjected to much higher temperatures and localized dehydration of the surface will occur. On the other hand, in stewing the meat remains at a temperature of 100°C, in the presence of excess water, for several hours (54). The large numbers of heterocyclic compounds reported in the aroma volatiles are associated with roasted, grilled, or pressure-cooked meat rather than boiled meat where the temperature does not exceed 100°C (91). In pressure-cooked pork liver at 163°C, quantitatively over 70% of the total amount of volatiles were furans and pyrazines (126); in boiled pork, the volatiles were dominated by aliphatic aldehydes and alcohols with only very small quantities of heterocyclic compounds. The effects of cooking conditions on the formation of volatile heterocyclic compounds in pork has been compared (91). Well-done grilled pork contained 66 heterocyclic compounds including pyrazines thiazoles, thiophenes, furans, and pyrroles. Pork cooked under less severe roasting or boiling conditions contained considerably fewer heterocyclic compounds. The alkylpyrazines accounted for almost 80% of the total headspace volatiles of well-done grilled pork (91). It has been well accepted that alkylpyrazines are formed from a reaction of the degraded nitrogenous substances, ammonia, primary amine from proteins, peptides, amino acids and phospholipids, and -dicarbonyl compounds in food (83). Like alkylpyrazines, alkylthiazoles identified in pork arise from a Maillard-type reaction between amino acids and sugars. It has been demonstrated that the requirement for the formation of these compounds is severe heating. The mode of cooking may also affect the flavor formation via wet versus dry cooking. From the mechanistic point of view, cooking conditions affect the generation of aroma compounds significantly. Volatile compounds formed from the reaction of 3-hydroxy-2-butanone/ammonium acetate at 25°, 55°, and 85 °C were investigated. Six compounds were characterized. Among the volatile compounds identified, an interesting intermediate compound, 2-(1-hydroxyethyl)-2,4,5-trimethyl-3-oxazoline, was found. The formation pathway of these volatile compounds was proposed (127). In these model systems, 2-(1-hydroxyethyl)-2,4,5-trimethyl-3-oxazoline was formed at reaction temperatures below 25°C. Tetramethylpyrazine was the major component when the reaction temperature was higher than 85°C. On the other hand, tetramethylpyrazine was the major component when the reaction temperature was higher than 85°C. The amounts of 2-(1-hydroxyethyl)2,4,5-trimethyl-3-oxazoline and tetramethylpyrazine increased linearly with increased heating time at 55°C. The reaction between 3-hydroxy-2-butanone and ammonium sulfide was studied (128). In addition to oxazole thiazole and thiazoline were formed in the presence of sulfide. Four well-known compounds, 2,4,5-trimethyl-3-oxazoline, 2,4,5-trimethyl-3-oxazole, 2,4,5-trimethyl-3-thiazoline and 2,4,5-trimethyl-3-thiazole were identified. On the other hand, tetramethylpyrazine was the major product with a reaction temperature higher than 100°C. In a related study, volatile components in maize flour, extruded under different con-

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ditions obtained by varying product temperature (120°, 150°, or 180°C), moisture level (14%, 18%, or 22%), and residence time (35s or 60s), were identified and evaluated (129). Increasing the product temperature, reducing the moisture level, or prolonging the residence times generally increased the numbers and quantities of Maillard-derived compounds, such as pyrazines, pyrroles, furans, and sulfur-containing heterocycles. In lowtemperature (120°C) and high-moisture (22%) extrusions, the main volatiles were compounds associated with lipid degradation, with few compounds derived from the Maillard reaction. Increasing the temperature and reducing the moisture level to 18% gave rise to the formation of some pyrazines and thiophenones. A marked increase in quantities of 2-furfural, 2-furanmethanol, and alkylpyrazines occurred in the extrusions at 180°C and 14% moisture level. Under these conditions, other nitrogen- and sulfur-containing heterocyclic compounds were also generated (129). B. Effect of Buffer on Aroma Generation 1. Schiff Base Formation in Meat Aroma Generation The first steps for the Maillard reaction involve the addition of a carbonyl group of the open-chain form of a reducing sugar to the primary amino group of an amino acid, peptide, or other compound. The elimination of water gives a Schiff base that cyclizes to give the corresponding N-substituted aldosylamine (130). An important route to alkylpyrazines is from -aminoketones, which are formed in Strecker degradation or from the reaction of dicarbonyls with ammonia. Condensation of two aminoketone molecules yields a dihydropyrazine that oxidizes to pyrazine (131). The condensation between cysteamine and aldehyde or 2,3-butanedione leads to the formation of cysteamine-derived thiazoles and thiazolidines (105,106). The key step is the formation of a Schiff base. 2. Effect of Phosphate on Schiff Base Formation Buffer dramatically promotes Schiff base formation from amino and aldehyde groups. Phosphate-mediated flavor compound formation has captured the interest of food processors who are trying to use this technique to obtain a better quality flavor monomer. With regard to the enhancing effect of phosphate buffer on Schiff base formation. Schwimmer and Olcott (132) have found a 12-fold increase in the brown color (420 nm) formed in a glycine/hexose model system in a phosphate buffer (0.33 M, pH 6.5) as compared with that in water. Sauders and Jevis (133) have reinvestigated the role of buffer salts in non-enzymic browning and confirmed the findings of Schwimmer and Olcott (132). Mori and Manning (134) have performed a large-scale synthesis of a Schiff base from AlaHis and glyceraldehyde in a potassium phosphate buffer at pH 7, and the significant bifunctional catalytic effect of phosphate on the Schiff base formation in diketopiperazine from aspartame has recently been reported by Bell and Weetzel 10. Recently, phosphate buffer (pH 5.0) was used as a reaction medium for the formation of an intense roasty, popcorn-like odorant, 5-acetyl-2,3-dihydro-1,4-thiazine (135). Phosphate buffer (pH 5.7) has also been utilized in the reaction system of L-cysteine and D-ribose to produce meat-like aroma compounds (86). The phosphate buffer system served as either a proton donor or acceptor, which catalyzed the Schiff formation. Extra phosphate may also have catalyzed the dehydrogenation reaction. This was due to the buffer effect of the phosphate system. A sufficient amount of phosphate ions serves both as a proton donor and acceptor.

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The proton from hydrogen phosphate protonated the carbonyl carbon of 3-hydroxy2-butanone, which facilitated the formation of an ionic intermediate. Subsequent rearrangement of this intermediate was catalyzed by phosphate and led to the formation of an aminoketone. Condensation of two molecules of the aminoketone and subsequent dehydrogenation produced one molecule of TMP. Redox reaction catalyzed by phosphate ions results in the conversion of thiazolidine to the corresponding thiazoline through hydride transfer (106). The intermediate of TMP formation was characterized as tetramethyldihdyropyrazine(TMP) using gas chromatography–mass spectrometry. A 15N-labeled ammonium acetate/3-hydroxy-2-butanone model system was used to confirm the incorporation of a nitrogen atom in the molecule of tetramethyldihydropyrazine. C. Effect of Water Content on Aroma Generation Numerous meat flavor compounds were formed in interaction between amino acids, the pentose sugar, and phospholipids (136, 137). The volatile profile of the Maillard reaction formed in a system with excess water differs greatly from those of low-moisture systems. Cysteine/ribose model reactions carried out in aqueous solutions with the concentrations of the reactants similar to those found in boiled meat were compared with that performed in the absence of water or with a relatively low moisture content (5:1, water:solids) performed at 185°C, which is similar to the condition on the surface of grilled meat (138). In aqueous systems, the major volatile components were thiols such as 2-methyl-3-furanthiol, 2-furanmethanethiol, 2-thiolphenethiol, 2-methyl-3-thiolphenethiol, 2-mercapto-3butanone, and two mercaptopentanones (139). However, in the corresponding dry or lowmoisture systems, 3,5-dimethyl-1,2,4-trithiolane, 3-methyl-1,2,4-trithane, 3,6-dimethyl1,2,4,5-tetrathiane, 1-(2-furannymethyl)-1H-pyrrole, and 5-ethyl-4-methylthiazole were the major components (138). In the presence of large amounts of water, furn- and thiophenethiols, mercaptoketones, and other oxygenated sulfur heterocylics, such as dithiolanones, are formed from the action of hydrogen sulfide on ribose-derived Maillard reaction products (140). On the other hand, trithiolane, trithiane, and tetrathiane can be formed from the interaction of acetaldehyde, hydrogen sulfide and mercaptoacetaldehyde, all of which are produced by the thermal degradation of cysteine. At low moisture levels the thermal degradation of cysteine is much more prominent than reactions of ribose with hydrogen sulphide, which requires relatively high water content. The addition of phospholipids to the dry and low-moisture system produced only small amounts of Maillard/lipid interaction products, and the only significant lipid oxidation products were a series of 2-alkanones. Under aqueous conditions, large quantities of Maillard/lipid interaction products, including 2-pentylthiophene, 2-hexylthiophene, 2pentylthiapyran, and 2-pentylpyridine, were produced together with large amounts of lipid oxidation products such as pentylfuran, alkanals, and alkanols. The quantity of volatiles generated from the reaction of 2,4-decadienal with cysteine or glutathione were almost the same. However, it has been shown that cysteine degradation in aqueous medium produces four times as many volatiles as glutathione (141). Methanol was used as a reaction medium for the Schiff base formation between amines and glucose by Rosen et al. (142) as early as 1958, and a similar solvent effect has been observed by Herraiz and his coworkers (143). The protic solvent attracted the water molecule, which led to completion of the Schiff base formation. The mechanism proposed in this paper is directly applicable to nearly all reactions involving a Schiff base formation.

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It has been demonstrated that the requirement for the formation of these compounds is severe heating. An investigation of the volatile components formed from the interaction of cysteine, xylose, and tributyrin at 200°C led to the formation of furans, or furanones, pyrroles, pyridines, thiophenes, thiazoles, and a number of other heterocyclic compounds containing sulfur or sulfur and nitrogen. The low moisture content of the reaction mixture and the relatively high temperature of reaction (200 to 220°C) were the key factors in the formation of a large number of thiazoles (144). However, acetylthiazole was present in large amounts in the boiled pork, suggesting that a different mechanism is involved in its production. D. Role of Lipids in Meat Aroma Formation The role of the lipid fractions of meat, both adipose tissue and structural phospholipids, has been the subject of a number of studies. Aqueous extracts of beef, pork, and lamb had similar aromas when heated, and when fats were heated, they yielded the species characteristic aromas. Interesting triangle taste tests reveal that the addition of 10% fat (beef or pork) enabled the panel to distinguish the lean meats more easily (145). The addition of pork fat to either lean beef or pork resulted in a substantial increase in hexanal but only small changes in most other volatiles (146). It was suggested that lipids provide volatile compounds that give the characteristic flavors of different species, and the lean is responsible for a basic meaty flavor common to all species (147,148). It is generally accepted that meaty notes come mainly from such sulfur-containing compounds as 2-methyl-3-furanthiol and bis-(2-methyl-3-furyl)disulfide, which are generated from water-soluble precursors in lean meat (149). Phospholipids rather than triglycerides were important in determing meat flavor (150). In mammalian tissue, structural phospholipids contain significant amounts of certain polyunsaturated fatty acids, in particular arachidonic (20:4), docosapentaenoic (22:5), and docosahexaenoic (22:6). Foods cooked in lard usually have better flavor than when cooked in vegetable oils. Pork fat was dry-rendered or wet-rendered to obtain crude lard. These two different lards were then refined, resulting in dry-rendered lard and wet-rendered lard (151). Results of sensory evaluation showed that crude dry-rendered lard had the strongest flavor intensity among the four lards. Crude dry-rendered lard also contained the highest amounts of trace components (phospholipids and amino acids) and flavor-contributing volatiles after heating at 190°C. These results seemed to indicate that wet-rendering and refining of lard could reduce the trace components that might be important in producing lard flavor (151). E. Effect of Fermentation on Meat Flavor Generation of volatile compounds (via chemical or enzymic oxidation of unsaturated fatty acids and further interactions with proteins, peptides and amino acids, or Strecker degradations) during dry curing of meat products has been extensively discussed Biochemical mechanisms involved in development of dry cured hams and other meat products including proteolysis (caused by calpains and cathepsins) and its effects on flavor; lipolysis by various enzymes at different stages of processing; and control of enzymic activities, e.g., by controlling relative humidity(RH), temperature, and salting levels, has been discussed as well (152). Numerous reports have indicated that carbonyl components make a significant contribution to the flavor of cured meat (153–155). 2,3-Octanedione and 2,4-decadienal were found in pork; 3-hexenal, 3-methylhexanal, 2-heptenal, octanal, 2-octenal, and decanal were found in chicken; and 3,3-dimethylhaxanal were uniquely identified in beef.

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The long processing of the dry-cured hams (12 months) provides ample time for the occurrence of lipolytic and oxidative degradation of unsaturated fatty acids. Aldehydes have been shown to be the major oxidative decomposition components. It is suggested that 2,4decadienal may be derived from linoleic acid (156). Unsaturated aldehyde, 2,4-decadienal and 2-undecenal may undergo further oxidation to shorter-chain aldehydes during the long process (157). In addition to fatty aldehydes, branched aldehydes such as 2-methylpropanal, 2methylbutanal and 3-methylbutanal arise from Strecker degradation of the amino acids valine, isoleucine and leucine, respectively (158). The intense proteolytic activity produced during the dry-curing process results in an increased concentration of free amino acid. Though these carbonyl components were detected in the cured meat flavor concentrate, they were absent in the cooked uncured meat. Two pyrazines, methylpyrazine and 2,6-dimethylpyrazine were found in the headspace volatiles of Serrano dry-cured ham. The temperature used during the dry-curing process is not as high as in cooking, therefore fewer pyrazines are found in the cured meat (159). 2-Oxopropanal is proposed to be the precursor of 2,6-dimethylpyrazine (160). In addition, the chemical reaction during the curing process—lactic acid fermentation—deserves special interest. Potential and actual effects of lactic acid bacteria or Micrococcaceae on catabolism of carbohydrates, proteins and amino acids, and lipids and fatty acids in fermented meat products (such as hams and sausages), and their influence on flavor development, have been discussed, covering carbohydrate catabolism, proteolysis, amino acid catabolism, lipolysis, fatty acids oxidation, and ester production. (161). One of the investigations indicated that hexanal, a major lipid oxidation product, was found to be present in uncured meat at a concentration of 12.66 /0.08 mg /kg, but only 0.03 mg / kg was present in the cured product. Also, the concentration of other carbonyl compounds was higher in uncured pork, whereas these compounds were either present in reduced amounts or not detectable in the cured meat (162). It is reasonable to postulate that aldehyde precursors for aroma generation may occur during the curing process.

Figure 7 Formation of acetaldehyde, 2, 3-butanedione, and 3-hydroxy-2-butanone.

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Lactic acid bacteria produce significant amounts of acetaldehyde, 2,3-butanedione, 2,3-pentanedione and 3-hydroxy-2-butanone. Formation of acetaldehyde, 2,3-butanedione, and 3-hydroxy-2-butanone is shown in Fig. 7 (163). Sterile pork loin tissue has been inoculated with Lactobacillus plantarum and Lactobacillus fermentum, placed in sterile sample bottles, purged with CO2, and stored for up to 24 days at 3°C. L. plantarum grew more rapidly than L. fermentum under these conditions. Profiles of volatile compounds were similar for inoculated and sterile pork loin tissue (164). It is expected that during the thermal treatment of the cured meat products, all these precursors may participate in the reactions leading to the formation of various heterocyclic meat aroma compounds following the pathways described previously. REFERENCES 1. 2. 3. 4. 5.

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5 Analytical Methods OWEN A. YOUNG, DEBORAH A. FROST, JOHN WEST, and TERRY J. BRAGGINS MIRINZ Centre AgResearch, Hamilton, New Zealand

I. INTRODUCTION II. STATISTICAL ISSUES IN ANALYTICAL METHODS A. Sources of Error B. Replication C. Calibration D. Costs of Error Minimization E. Sampling III. SAMPLE PREPARATION A. Equipment and Methods of Comminution IV. OVERVIEW OF ANALYTICAL METHODS V. MOISTURE VI. PROTEIN A. Protein Determination by Nitrogen B. Amino Acid Composition VII. FAT A. Crude Fat B. Total Fat after Acid Hydrolysis C. Total Lipids by Chlorinated Solvents D. Fatty Acid Profile VIII. ASH A. Minerals IX. CARBOHYDRATE X. VITAMINS XI. CHOLESTEROL XII. COMMON TESTS FOR MEAT PRODUCTS XIII. NONINVASIVE PHYSICAL METHODS

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XIV. TESTS COMMONLY USED IN MEAT RESEARCH XV. SUBSTITUTION, SPECIES AND ANIMAL IDENTIFICATION, AND IRRADIATION A. Substitution and Species Identification B. Identification of Individual Animals C. Detection of Irradiated Meat XVI. LABORATORY MANAGEMENT A. Costing Analyses, Calculating and Reporting Results B. Quality Systems XVII. THE FUTURE OF ANALYTICAL METHODS FOR MEAT REFERENCES

I. INTRODUCTION As we enter the millennium, it is difficult to buy packaged food that does not display information about energy, fat, protein, salt, or many other food components. Even when the product is unprocessed, such as fresh fruits, vegetables, raw meat or fish, advertising often makes substantial claims about the product’s composition—rich in this or low in that. In earlier times, cheeses were simply cheeses bought on price and flavor, and rump steak was rump steak bought on similar criteria. In recent history several factors have conspired to create an awareness of food composition that pervades all affluent societies. The factors include a major decrease in the cost of food production, reduced physical activity, and medical advances that have increased life spans. A significant outcome in affluent societies has been an increase in obesity and associated diseases, particularly in the United States. As a consequence, the composition of foods has become a national obsession. In the case of meat and meat products, issues of composition are particularly important. On the one hand, most people regard meat as an important part of their diet and their overall enjoyment of food. On the other hand, the public is bombarded with real and purported facts about the effects of total fat, saturated fat, cholesterol, and residues in meat on health concerns such as obesity, heart disease, and cancer. Faced with defending meat from dietary concerns and scares, the meat industry could be forgiven for wishing that composition analysis and labeling laws would simply go away. This will not happen. Instead, there are constant if not increasing demands on the industry for composition data to satisfy the demand of food manufacturers, consumers, and bureaucracies. The main components of interest in terms of the analysis of meat and meat products are moisture, protein, fat and fatty acids, cholesterol, iron, sodium, and calculated values such as energy. Another less obvious component is qualitative rather than quantitative— species of origin. Several major religions have strict dietary laws relating to meat origin. For example, pork is forbidden under Islamic and Judaic law. Even without religious edicts, many societies and groups within societies reject certain species as meat animals whereas other cultures eat meat from these species without question. Horsemeat is not a big seller in the United States, but is a normal butchery item in France. Consumers like to know what species they are eating. In this respect, a fraudulent kangaroo and horsemeat substitution scandal with Australian export beef in 1980 threatened that country’s entire meat export trade. Species identification is important for another reason: trade barriers are often based on quotas by species. This chapter summarizes the analytical methods used to measure the composition

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of meats and meat products. An analytical method is basically a laboratory recipe to qualify or quantify some chemical compound or chemical class of interest. At the root of quantitative analysis are issues of statistics that should be grasped before the recipes are followed. II. STATISTICAL ISSUES IN ANALYTICAL METHODS A. Sources of Error Each step in an analytical procedure introduces error. Some errors are random and some are systematic. In the case of weighing for instance, a random error would be the variation in weight recorded for repeated measurements of the same subsample, perhaps arising from the balance’s circuitry or vibration in the balance bench. A systematic error might be a consistent overestimate of the weight. Both types of error can and do occur at the same time. The classic target shooting example illustrates this well (Fig. 1). Random error is the scatter of hits (formally termed precision), whereas systematic error is the averaged distance of the hits from the bullseye (accuracy). The degree of precision and accuracy required will govern the effort applied to minimizing error. Replication and calibration respectively tackle the two sorts of error. B. Replication Random errors are overcome by replication so that a meaningful average can be obtained. The greater the error, the more replication is required to allow for the error. For any ana-

Figure 1 The target example of precision and accuracy.

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lytical procedure, performing replicated measures at each analytical step highlights those where the most random error occurs. These are the steps that should be replicated in the method’s application if higher precision is demanded. C. Calibration Systematic errors are minimized by calibration against reference standards. For example, the accuracy of laboratory balances should be routinely calibrated with a certified series of weights. Calibration is a basic activity of quality management systems. D. Costs of Error Minimization Replication and routine calibration both cost money, and a balance must be struck between the cost of better precision and accuracy and the needs of the client. In many cases, the client will be blissfully ignorant of the distinction between precision and accuracy, but the importance of both in respect of cost should be made known, particularly where analysis is ongoing and the outcome is commercially important. Experience has shown that the greatest source of random error is the variability inherent in the raw meat itself. The ways of minimizing this problem are now examined. E. Sampling If an analyst wanted to determine the relative proportions of fat, protein, and moisture (water) in a one ton container of meat pieces, analyzing the entire ton would give perfect answers (from calibrated methods) but would destroy all the meat. Therefore a sample is taken. If the sample were small, say 50 g, and taken from a single site in the container, the answer would almost certainly bear no relation to the true proportions in the entire ton; a piece of fat or lean might easily dominate the sample causing completely misleading results. If the sample size were increased, say to 1 kg, the chances of obtaining a more representative answer would increase. Further improvements would be obtained with a 10 kg sample and so on. Clearly, the sample size is important in the quality of the determination. More information about the one ton of meat could be obtained by taking a number of samples, say 20 replicates of 50 g, from randomly chosen sites throughout the carton. The proportions of fat, protein, and water would vary from replicate to replicate, but an average of the 20 results would give a fairly accurate result. The result would certainly be more reliable than that from the single 1 kg sample at a single site, which could be particularly fatty or lean. Moreover, the spread of values around the average would give the analyst an idea of how variable the meat was from site to site. Sample size and sample replication are fundamental issues facing food analysts who on the one hand are required to provide determinations with maximum certainty but on the other are expected to charge a minimum for the service. This tension between uncertainty and cost pervades all analysis and pivots on the relative heterogeneity of the food in question. In the case of a stirred wine vat, a single 5 ml sample (~5 g) is sufficient to give an acceptably reliable answer for many analytes. As a solid derived from complex organisms comprising many tissue types, meat is inherently heterogeneous, requiring a more thoughtful approach to sampling. The formal mathematics of sampling are covered in statistical texts and are beyond the scope of this chapter. In situations where routine testing of somewhat defined meat and meat products is undertaken, sampling size and replication should be formally examined to Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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minimize expense but at the same time provide an adequate answer. In other cases, the analyst is faced with ill-defined meats and products about which judgments must be made and made quickly. Experience helps to resolve the conflict between certainty and cost. As a general rule, the larger the sample size and the number of replicates, the more certain the determination but the higher the cost.

III. SAMPLE PREPARATION The whole of each 50 g sample (replicate) from the meat carton discussed above could be analyzed for fat, protein, and moisture. However, some preliminary steps are normally required before the analysis can be performed. In any analysis the equipment must be scaled to match the sample size. Because analysis of 50 g samples often requires large glassware items and large solvent volumes, which increase costs and hazards to workers, the original 50 g sample is normally subsampled to obtain a smaller mass to analyze. The original sample is likely to be heterogeneous, however, so it is usually comminuted before subsampling. This reduction in tissue particle size also makes extraction of analytes more efficient due to increased surface area, and also more predictable if all particles are of similar size. Nearly all analytical procedures for meat require a preliminary comminution step. In cases where a homogeneous meat product is to be analyzed, comminution may not be necessary. For example, a 2 g sample of emulsion sausage would be representative of the entire sausage. Nonetheless, comminution is often required to reduce particle size. A. Equipment and Methods of Comminution Whatever technique is chosen, it is critically important that comminution does not damage or otherwise change the nature of the analytes to be measured. The most common cause of this is blunt equipment. Not only can blunt equipment cause variable comminution and analyte damage, but it also makes comminution much more difficult. The need for sharp equipment cannot be overemphasized. Working parts should be also clean and dry, and metal should be free from rust. The standard butcher knife is the most basic tool and much size reduction and tissue randomization can be accomplished quickly and easily. The standard laboratory grinder (or mincer) is essential equipment in an analytical laboratory for meat. The cutting plates typically have a diameter around 70 mm, and hole sizes ranging from 2.5 to 4 mm. So-called kidney plates are often included in the grinding assembly to aid comminution of large meat pieces. Where possible, the meat or meat product should be well chilled or even lightly frozen before grinding. Grinders work best when cool, as even the sharpest grinder generates heat. Bowlchoppers have rapidly rotating knives that cut meat to the required consistency, governed by the time the bowlchopper is allowed to run. As with grinders, damaging temperature increases can occur. The finer the comminution, the greater the heating. Liquid nitrogen can be used judiciously to control temperature where this is important. Where an analyte is prone to oxidation from exposure to oxygen and heat, tissue is comminuted as a solid in an excess of liquid nitrogen. Typically the tissue is pulverized by mortar and pestle while the nitrogen simultaneously displaces oxygen, cools, and embrittles the sample. Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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When meat tissue is extracted with a solvent, aqueous or otherwise, the analyst can choose from a wide range of proprietary blenders. Traditional blenders by Sorvall and Waring have been joined by rotor/stator shearing elements sourced mainly from Europe that operate in excess of 20,000 revolutions per minute. IV. OVERVIEW OF ANALYTICAL METHODS Four components of meat dominate analytical methods: moisture, protein, fat, and ash, which is a collective term for the minerals present in meat. Protein, fat, and minerals are fundamentally important in human nutrition, and methods to determine them have existed for decades. As knowledge of food chemistry has grown in the last century, methods have been developed to determine the specific amino acids, fatty acids, and individual elements that are components of the protein, fat, and ash. Similarly, methods have been developed for vitamins in meat and—in response to perceived or real health concerns—for cholesterol, a component of cell membranes. Methods have also been developed for other analytes common in meat products, and, in recent decades, methods to identify species of origin and even animal of origin. Whether analysis is at its simplest level (for example, moisture determination of ground meat) or is more esoteric (for example, determination of geometric isomers of unsaturated fatty acids), the mission is fundamentally the same. The analyte of interest must be isolated or otherwise distinguished from all other analytes and measured. These steps must be performed in a reproducible way such that repeated measures of the analyte in identical replicates of the substance will always yield a result within set limits. This goal is achieved with standard methods that have been road tested and refined over many decades. The Official Methods of Analysis of AOAC International provides statistically verified methods for meat and for foods (AOAC, 1999). In the sections that follow, many of these methods are referenced by AOAC number, but the details are omitted. Instead, the principles that underlie the methods are explored. A variety of non-AOAC methods are also discussed, spanning noninvasive physical methods, meat quality attributes, and species identification. Finally, aspects of analytical laboratory management are examined. V. MOISTURE Moisture constitutes about 70 to 77% of raw muscle. The moisture content falls as the fat content of the meat increases, and it can be markedly lower in processed meats, such as sausages, that contain additional ingredients. In many processed products, reliable control of moisture content is important for preservation because a low moisture content lowers water activity (see later) and helps inhibit microbial growth. Measurement of meat moisture content is not a complicated test but as with all analyses, care must be taken to ensure accurate sample weighing. Moisture content, or more accurately volatile matter content, is determined by weight difference before and after removing the water from the samples—usually by drying. AOAC Method 950.46 dries a weighed sample in a forced air, convection, or vacuum oven between 95 and 125°C for 2 to 16 hours or until the sample attains constant weight, depending on the drying method used. Alternative drying methods include infrared, ultraviolet light, or microwave ovens. Before reweighing, the dried sample must be cooled in a dry air environment, such as a desiccator, to prevent uptake of moisture from room air. Dried meat

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samples, particularly samples of meat and bone meal, are hygroscopic and readily absorb moisture from the air. This can have a significant influence on the accuracy of the test, especially when measuring samples with a low moisture content, where sample weight differences can be small. Quick and effective moisture loss is aided by carefully mixing the samples with acidwashed sand to increase the surface area of the sample and prevent clumping of the meat that might trap moisture during the drying step. Care must also be taken to prevent excess weight loss due to oxidation of fat in the sample. For high fat samples rich in oxidation-prone fatty acids, drying in a vacuum oven at 70°C is useful for minimizing loss. More rapid moisture determination can be accomplished by noninvasive physical methods to be discussed later. These methods, which extend to other meat analytes, need careful calibration with standard chemical methods that are the ultimate arbiter. VI. PROTEIN As a generic molecular species, protein is a linear polymer of different amounts of 20 amino acids. The possible combinations and permutations of the linear sequence of amino acids are immense, but from a dietary perspective only the total mass of protein in a unit weight of food and the amino acid profile—the relative proportions of the amino acids—are important. (Sequence is usually not important because protein is hydrolyzed in the digestive system to amino acids, which are used to build proteins specific to the consuming organism.) The amino acid profile is important because some amino acids cannot be synthesized by humans and must be obtained from diet. Meat is rich in the so-called essential amino acids—lysine, leucine, isoleucine, and sulfur-containing amino acids—and in this sense meat is a high-quality protein. In the middle of the nineteenth century, the discovery of amino acids and their effects on growth and wellbeing led to the development of routine methods for determining amino acids as well as total protein. A. Protein Determination by Nitrogen Nitrogen is present in all amino acids, so most methods for determining protein measure the quantity of nitrogen present in the meat and use a multiplication factor to calculate the quantity of protein. However, the relationship between nitrogen and protein depends on the amino acid composition: the percent weight of nitrogen in individual amino acids ranges from 8.6 for tyrosine to 35.9 for arginine. Fortunately, relatively constant proportions of myosin and actin dominate meat protein, and together contain approximately 16% nitrogen. Therefore a multiplication factor of 6.25 is commonly used for raw (or cooked) meat. The presence of collagen in meat’s connective tissue complicates the calculation because the correct factor for collagen is about 5.36, reflecting its higher proportion of nitrogen compared with other meat proteins. In meat containing high proportions of connective tissue, lower multiplication factors should be used (Benedict, 1987). The situation becomes even more complicated in meat products, which frequently contain soy or milk protein. However, as long as the correct nitrogen-protein conversion factor is applied, protein determination by nitrogen measurement is considered the most accurate and reliable method currently available.

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The most common procedure for the determining of meat nitrogen is the Kjeldahl method, named after its inventor. Here the protein is digested at high temperature with concentrated sulfuric acid, sodium sulfate, and a metal catalyst, to convert nitrogenous substances in the meat to ammonium salts. Addition of concentrated alkali then converts the ammonium salts to free ammonia that is distilled with steam and collected in either hydrochloric acid (AOAC 928.08) or dilute boric acid solution (AOAC 981.10) containing suitable colored pH indicators. In the boric acid procedure, the ammonia increases the pH and is titrated with acid to the original pH. In the hydrochloric acid procedure, excess hydrochloric acid is back-titrated to neutral with sodium hydroxide solution. The ammonia can alternatively be measured as colored ammoniacal complexes. To increase sample throughput in a busy laboratory, semiautomated Kjeldahl equipment is available to analyze for protein in batches of samples (AOAC 977.14). An alternative method for determining protein nitrogen employs combustion. This releases the nitrogen into pure oxygen, which changes its thermal conductivity (AOAC 992.15). This combustion method lends itself well to automation. However, careful comminution to yield homogeneous tissue is very important for this method, as typically a very small sample is analyzed. As for Kjeldahl nitrogen, a conversion factor is used to relate nitrogen to protein. The fat and moisture content of raw meats can be used to estimate the protein content accurately enough for quality control purposes. This estimate works because the percent content of fat, protein, moisture, and ash in meat should sum to 100. The ash content is low, about 1%, and sufficiently constant not to influence the estimation. B. Amino Acid Composition In proteins, the 20 amino acids are joined by peptide bonds that must be hydrolyzed (broken) before the individual amino acids can be measured. Hydrolysis is accomplished by refluxing the meat sample with concentrated hydrochloric acid (6M) for up to 24 hours. This aggressive treatment causes partial or complete loss of some labile amino acids, notably tryptophan, but most are resilient. (Special hydrolysis methods are used to recover labile amino acids.) An aliquot of the hydrolysate is evaporated to dryness, and the residue is dissolved in the buffer required for subsequent liquid chromatographic analysis. In the original chromatographic technology, the amino acids were separated from each other by ion exchange and after they emerged from the ion exchange column they were visualized colorimetrically with the classic ninhydrin reagent (AOAC 994.12). In more modern methods, fluorescent complexes are prepared before chromatography. In these methods, separation is accomplished with reverse-phase columns largely on the basis of polarity and pH. Hydroxyproline deserves special mention as it is an indicator amino acid of collagen. The collagen content of meat and meat products is often of particular interest to food processors because it alters batter gelation properties, and in Germany the levels of collagen in meat products are governed by food laws. After acid hydrolysis, hydroxyproline is measured by a colorimetric reaction (Stegemann and Stalder, 1967), and related to collagen by a conversion factor, typically 7.14, that reflects the fact that collagen contains about 14% hydroxyproline. VII.

FAT

Total fat and the fatty acid profile of fat are probably the most topical of all nutritional components in foods. This interest is engendered by obesity in Western societies and the assoCopyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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ciated heart disease and diabetes. Obesity is mainly caused by excess energy from overeating. On a weight basis, fats have the highest energy content of all the food components, and the trivial name for obesity—fat—reinforces the mental association between fat in foods and obesity. Of all fats in the human diet, those from animal foods have attracted the most adverse publicity because their fatty acid profile is usually high in saturated fatty acids (Chapter 1) and because of the statistical association of animal fats with dietary cholesterol. Fat measurements are usually made on the food product as sold to the consumer, but in the product’s preparation the manufacturer is obviously concerned with the fat characteristics of the ingredients. Thus the routine measurement of fat in a heterogeneous and naturally variable ingredient like raw meat is important. Fats as recognized by the general public are a subclass of what are technically called lipids, but fat is often used interchangeably with lipid. Although several classification systems have been proposed—usually based on the solubility of lipids in organic solvents— there is no agreed international definition of the term lipid. For nutritional purposes, however, total fat can be defined as the whole body of fatty acid–containing substances. Other organic compounds such as sterols, terpenes, and waxes that are dissolved by organic solvents are not considered fats, but if present in a food these compounds can be extracted along with the fat and contribute to the measured weight. Meat fats are mainly tri-, di-, and monoacylglycerols but also include some phospholipids, cholesterol esters, and free fatty acids. Triacylglcerols are the dominant form of storage lipid in animals (typically 95% by weight). This class of compounds comprises a glycerol backbone with three fatty acids esterified to the three alcohol groups of the glycerol. Mono- and diacylglcerols are similar to triacylglcerols, whereas phospholipids are diacylglycerols with the third glycerol alcohol group esterified to a phosphate derivative. The particular method used for determining fat defines the fat content. For instance, crude fat is defined as the petroleum ether* or diethyl ether-extractable portion. Extraction with a mixture of chloroform and methanol, on the other hand, will extract crude fat, plus other more polar compounds such as phospholipids. Solvent polarity is a prime determinant of fat extraction, but other factors also influence extraction. These include acid treatments to make the fat easier to extract, water washing to remove carbohydrate, extraction time, particle size, moisture content, and subtleties of the sample matrix. The subtleties of sample matrix are illustrated by this example: if emulsion sausages are made from prerigor meat rather than rigor meat, fat in the resulting cooked meat gel is less accessible to the extracting solvent.† Significant underestimates of fat by crude fat methods are common with emulsion sausages made with prerigor meat. One way of overcoming these problems is by a vigorous pretreatment such as acid hydrolysis. Outlines of some fat methods are now presented. A. Crude Fat Crude fat, also known as the ether extract or the free lipid content, is the traditional measure of fat in meat and meat products. The most common method of extraction usually employs a Soxhlet apparatus—another inventor’s name. * Petroleum ether is not strictly an ether, but rather a mixture of alkanes, typically hexanes, of different boiling points. Diethyl ether is also a suitable solvent for crude fat determination but presents greater safety risks. † The reasons relate to the relative salt solubility of muscle proteins in pre- and postrigor muscle.

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A comminuted meat sample is weighed into a Soxhlet thimble (either cellulose or glass fiber) and mixed with dry acid-washed sand. The sand increases the surface area of the meat, allowing better penetration of the solvent. The sample is dried, and the fat is extracted by continuously refluxing solvent through the sample. The extraction rate varies with the solvent and is fast for diethyl ether and slower for higher-boiling-point solvents such as hexanes. Extraction can take as long as 16 hours. The solvent containing the extracted fat is evaporated, and the fatty residue is dried to a constant weight. The fat content is calculated as a percentage of the original wet sample weight. Meat products containing water-soluble carbohydrates should be washed with water prior to the solvent extraction because the presence of carbohydrates may lead to erroneously high fat values due to their extraction. Several semiautomated systems are now available to speed the extraction. More recently, extraction methods incorporating high pressures and temperatures have been commercialized. Supercritical fluids, usually carbon dioxide, can also be used as the extracting solvent under high pressure. Standard methods for the extraction of crude fat include AOAC Methods 960.39 and 991.36 and ISO 1444–1973. B. Total Fat after Acid Hydrolysis Some sample matrices resist the removal of fat with petroleum ether. Acid hydrolysis is a useful way of making the fat more available for Soxhlet extraction (ISO 1443–1973; British Standard 4401, 1970). The name “total fat” reflects this improvement. Digestion with hydrochloric acid liberates the fat by hydrolyzing proteins and carbohydrates. The mixture is filtered, washed with water to remove acid and other water-soluble material, and then dried. A conventional Soxhlet extraction recovers the fat. C. Total Lipids by Chlorinated Solvents Mixtures of chloroform (or methylene chloride) and methanol extract lipids more thoroughly than other solvents. Chlorform:methanol mixtures are also gentle in the sense that lipids extracted at room temperature by this mixture undergo little chemical change, making this mixture the best starting point for detailed lipid analysis. Because the mixture is a good solvent for phospholipids, the following methods are particularly useful for tissues such as liver, kidney, and brain that are rich in phospholipids. However, chlorinated solvents are not suitable for very high fat samples where underestimates are common. In the Folch method, the comminuted meat or meat product is mixed in a defined ratio with chloroform:methanol themselves in a set ratio, 2:1. Two phases result, a solid phase largely comprising protein, and a liquid phase containing all lipid and some water-soluble components. After filtration to recover the liquid phase, water is added to resolve it into two phases, a denser chloroform-rich phase that contains the lipid and a lighter aqueous phase. The former is recovered, the solvent evaporated, and the lipid determined by weight (Christie, 1984). AOAC 983.23 employs the same principle as the Folch method, but employs a rather different procedure. Maxwell et al. (1980) introduced another method whereby meat is ground with the drying agent anhydrous sodium sulfate. The mixture is packed in a column from which the lipids are eluted by methylene chloride:methanol as a single water-free phase. Chlorinated hydrocarbons present a significant environmental risk and lipid determination with chloroform and related chemicals should be used only where essential.

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D. Fatty Acid Profile In the context of meat and meat products, the main aim of fatty acid analysis is determination of different fatty acids in the range from about 12 carbon atoms to 22. (Fatty acids with 16 and 18 carbon atoms dominate fats in meat.) Beyond the length of the fatty acid’s alkyl chain backbone, the degree of unsaturation (the number of double bonds) is of particular interest, as is the positions of these bonds in the alkyl chain, so defining isomers. Drilling deeper still, each double bond can be classified as to whether it adopts a cis or trans geometric orientation, another level of isomer complexity. At varying depths of detail, this information, termed the fatty acid profile, is of interest to food manufacturers, nutritional and medical experts, bureaucrats, and consumers. In many cases, data are required to satisfy law, whether that law is based on sound scientific data or not. The profile is typically expressed as either the weight of the individual fatty acids per unit weight of the food, or as percentage by weight of the total fatty acids reported. The fatty acid profile is determined by gas chromatography. The fatty acids within the extracted fats are first converted to methyl or sometimes butyl esters. As alkyl esters the fatty acids are relatively nonpolar and volatile, and they can be resolved to high precision on chromatographic columns. The eluting esters are identified by their retention times and quantified by comparison with authentic ester standards. The choice of chromatographic column and chromatographic conditions is governed by what the analyst is looking for, because one column and one set of conditions will not resolve all fatty acids and their isomers. The details of fatty acid analysis, from fatty acid methylation techniques through to chromatography, are not discussed here. Readers are referred to Christie (1989), and to House (1997), who describes a method for the gas chromatographic determination of total, saturated and monounsaturated fats in foods that is aimed at satisfying U.S. requirements for food labeling. VIII. ASH The ash content of raw meat is low and relatively constant but can vary greatly in processed meat products and rendered meat and bone meal. Ash is the inorganic residue that remains after combustion at high temperature (500 to 600°C) in the presence of air. Analytical methods vary in the charring procedure and combustion temperature. Temperatures above 600°C cause some minerals to be lost by volatilization. Some methods also recommend the addition of reagents, such as magnesium acetate, to speed combustion. Care must be taken during drying, charring, and combustion, as it is easy to lose some of the sample. Unintentional, rapid ignition during charring may cause small particles of sample to be blown out of the crucible. Similar particulate loss can occur when the crucible is being removed from the furnace after combustion, as the ash is very light and susceptible to air currents. The use of a crucible lid during ashing and handling is recommended. A. Minerals Minerals in meats and meat products is a collective term for nutrient elements often quoted in food composition tables. Depending on the method of isolation, the elements may include sulfur, which is retained by acid digestion but is prone to loss as volatile sulfur oxides upon ashing. Thus, ash and minerals are not synonymous but are related terms. Ash from raw lean beef contains (in descending order of abundance) ionic forms of

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potassium (about 360 mg/100 g), phosphorus, sodium, magnesium, calcium, zinc, iron (around 3 mg/100 g), and other trace minerals (Anderson and Hoke, 1990). Although the color of meat is dominated by iron in the form of the heme protein myoglobin, iron is one of the lesser minerals in meat. Nonetheless, its measurement is very important, as heme iron is the most readily absorbed source of iron in the human diet. The iron content in meat can be measured spectroscopically after colored complexes are formed. However, inductively coupled plasma atomic spectroscopy procedures similar to that described in AOAC 990.08 are now more common and convenient for this and all other minerals. IX. CARBOHYDRATE Raw meats contain negligible carbohydrate. However, in processed meat products, carbohydrate content is an important measure of nonprotein additions such as cereals, starches, and gums. Carbohydrate content is commonly estimated by difference: [100 percent (percent weight of fat  protein  moisture  ash)]. If more detailed qualitative and quantitative data are required for added carbohydrates, tests specific for the various classes of carbohydrate would have to be applied. X. VITAMINS Meat is a significant dietary source of some B group vitamins. Vitamins B1 (thiamine), B2 (riboflavin), niacin (plus niacinamide), and B6 (pyridoxine, pyridoxal plus pyridoxamine) can be measured in a single hydrolysate obtained by acid digestion and multienzyme hydrolysis of meat samples. Automated chemical procedures similar to those in AOAC 942.23, 981.15, and 975.41 can be used to determine the first three vitamins, and vitamin B6 can be determined by paired-ion high pressure liquid chromatography (Gregory and Feldstein, 1985). Vitamin B12 (cyanocobalamin) can be determined by the radioassay method of Millar et al. (1984). Meat is not a significant dietary source of vitamin E, but this vitamin is important for color maintenance in raw meat. Vitamin E (tocopherol) supplementation of cattle feed is now common practice. In the method of Phalzgraf et al. (1995), meat is saponified with potassium hydroxide in a methanolic solution. The tocopherol is extracted into an organic solvent, resolved by liquid chromatography, and detected by fluorescence. XI. CHOLESTEROL Historically, the etiology of arterial disease was attributed to the intake of cholesterol from foods such as meat and eggs, and this belief spawned analytical methods that demonstrated which foods were “unhealthy” and which were “healthy.” Although other factors such as overall diet are more important in the etiology, the analytical methods are still used to satisfy labeling legislation and to keep the chlolesterol bogeyman alive for commercial reasons. In meat, cholesterol is determined by direct treatment of samples with potassium hydroxide in alcoholic solution followed by extraction of cholesterol into a nonpolar solvents, typically hexanes. The hydroxide hydrolyzes fats to the potassium salts of fatty acids (soaps), which are insoluble in the solvent (Kovacs et al., 1979). The plant sterol stigmasterol, which does not occur in meat, is added as an internal standard before determination

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of cholesterol by gas chromatography. This procedure does not require derivatization of the extract prior to chromatography. If cholestane is used as the internal standard, derivatization is required to prevent contaminants from interfering with the standard peak. AOAC 994.10, which uses toluene as the extracting solvent, also requires derivatization of the extract and is, moreover, an unpleasant procedure. XII.

COMMON TESTS FOR MEAT PRODUCTS

The salts contained in meat products include common salt (NaC1), phosphate(s), and nitrite and nitrate, each imparting useful properties to the food. The concentrations of these additions are often measured for legislative reasons stemming from real or perceived health concerns, and for quality control purposes. This section outlines the methods in common use, and also describes three other common tests applied to meat products: water activity, gel characteristics, and fat oxidation. Whereas sodium in (unprocessed) raw meats is measured by methods described under Minerals (above), added salt is usually measured as the chloride ion through its reaction with silver ion added as silver nitrate to extracts of meat products. Two methods are in common use. The Volhurd back-titration method measures excess silver ions by two reactions resulting in a colored ferrithiocyanate complex. The now more favored direct titration method employs a silver ion–specific electrode. For both methods a standard salt solution is used for calibration, which assumes that the chloride’s counter ion is sodium. This is true for most meat products, but in some products a proportion of potassium chloride (KC1) is used in place of NaC1 for dietary purposes. If the analyst is not aware of this substitution, the concentration of sodium ion will always be overestimated. Simple and complex phosphates are added to meat products for a number of purposes. The counter ion is either sodium or potassium and the various phosphates used contain different quantities of water as water of crystallization. In view of this complexity, it is common to express phosphates as either elemental P or phosphorus pentoxide (P2O5). In the most common analytical method, meat products are ashed, then dissolved in acid solution where the phosphorus is present as simple phosphate rather than the polyphosphates common in meat products. The phosphate is reacted with an acidic solution of molybdic and vanadic acids to yield a colored complex that is measured colorimetrically. The calibration salt, usually KH2PO4, represents a known weight of P2 O5. As discussed in Chapter 20, nitrite, and indirectly nitrate, is used in meat as a curing agent, where nitric oxide forms a stable pink complex with the heme of myoglobin. For determination, extracted nitrite forms a diazonium salt with the primary amines sulfanilamide or sulfanilic acid. This salt in turn is coupled to an aromatic amine to form a (colored) azo dye. Nitrate is unreactive in this system. Of the several methods available to determine nitrate, the most common is chemical reduction of nitrate to nitrite with cadmium metal followed by determination of nitrite. Both anions are often present in the same meat product, so extracted nitrite is determined first, then nitrate is reduced to nitrite, which is remeasured, and the original quantity of nitrate is calculated by difference. Ascorbate or its isomer erythorbate, which is frequently included in meat products, can interfere with the determination of nitrite. Various protocols have been described to minimize this chemical’s effect (Fox et al., 1984). The basis of reporting the concentration of nitrite/nitrate depends on convention and food laws, and care must be taken when comparing data from different sources. Water activity (aw) is a measure of how much of the water in a product is “free,” referring to the water that is not chemically or physically bound. Rephrased, the aw of a

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product is the water content that makes itself noticeable externally. (Moisture content, by contrast, refers to all water evaporable at high temperature in an oven.) Products with absolutely no free water have an aw of 0.00, pure water has an activity of 1.00. The aw of a saturated salt solution (NaC1) is around 0.75. The ingredients present in meat products, such as salt, sugars, and phosphates, result in a lower water activity for the product than for raw meat (aw approx.
0.97). For many products a reduced aw is important for their preservation because microorganisms will not grow below certain aw levels (Troller, 1980). Water activity is formally defined as the ratio of water vapor pressure above the sample to the water vapor pressure of pure water at the same temperature. It is equal to the relative humidity of air in equilibration with the product. In one technology to measure aw, a progressively cooled mirror is employed to determine the dew point of air above the sample. At the temperature where moisture condenses, the mirror’s reflectance changes and this is monitored photoelectrically. Simultaneously, infrared thermometry pinpoints the sample temperature. These data are used to calculate aw. The temperature range of interest is between approximately 5° and 40°C, the range that shelf-stable foods might experience during storage. For the purpose of preservation, determining the aw is more useful than determining the moisture content. Besides, water activity determinations take a matter of minutes whereas conventional moisture determinations take many hours. Heating of meat and meat products causes the muscle proteins, principally myosin, to denature, so converting those proteins from the sol to the gel state. This latter state is most familiar to the consumer as texture of cooked sausage, which has characteristics perceived by the consumer like hardness, springiness, fracturability, and others, characteristics that are immensely important in defining the quality of the sausage. Two fundamental rheological properties that are well correlated with sensory panels scoring for these characteristics are stress and strain to the point of fracture. Stress has units of force per unit area whereas strain is unitless, being expressed as the increase in deformation as the gel is deformed by the stress. Of the several methods to obtain these data from meat sausage gels, the torsion test is particularly useful (Hamann, 1983): cylindrical gels about 30 mm long and 19 mm in diameter are formed with a lathe into a capstan shape, 10 mm in diameter at the midpoint. The capstan’s ends are then counter-rotated at a defined speed to the point of fracture, which always occurs near the midpoint. Stress and strain to fracture are calculated from the torque and angular displacement. The fracture data are often highly variable even from very homogeneous sausage gels, so many replicates need to be tested to get meaningful results. In meat products, spoilage arising from fat oxidation usually occurs before microbial spoilage. Spoilage from fat oxidation is manifest as the rancid odors and flavors of aldehydes, short chain fatty acids and other compounds that are generated from peroxidation of unsaturated bonds in fatty acids. Of the very many oxidation compounds generated, malondialdehyde and some others have the useful property of forming a pink complex with 2thiobarbituric acid that can be measured with a spectrophotometer. One common indicator of fat oxidation, the TBARS test, exploits this property. In the original execution of the test (Tarladgis et al., 1960), a sample of meat product is blended with water, acidified, then distilled to volatilize the oxidation products of which malondialdehyde is the most abundant thiobarbituric acid reactive substance. An aliquot of the distillate is reacted with the acid, and the light absorbance at a suitable wavelength is related to the concentration of the reactive substances by a calibration curve based on malondialdehyde. Other variations of the

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TBARS test do not require distillation but employ various methods to isolate the colored complex for measurement. XIII. NONINVASIVE PHYSICAL METHODS Three technologies with potential for noninvasive measurement of meat composition are the Neugat system, dual-energy x-ray absorptiometry (DEXA), and near-infrared (NIR) spectroscopy. The Neugat system uses coincident low intensity beams of neutrons and gamma rays to determine the composition of many materials. The interaction of the radiation with the material causes scattering (neutrons) or absorption (gamma rays), in both cases reducing the intensities of neutrons and gamma rays reaching the detector. After calibration, the intensity of the gamma radiation and the ratio of neutrons to gamma rays at detection are used to determine the ratio of any two constituent components of the material, provided all other components remain constant. The quantity of radiation is minute. In a typical application, the total exposure during measurement is equivalent to one week of normal background radiation. The Neugat system has been successfully used to determine the amount of water in wood chips, grain, coal, and dairy products, the amount of alcohol in beer and so forth; and, importantly, the proportion of fat in meat (Bartle, 1995). A commercial Neugat system can measure the fat content of manufacturing meat at better than 1% accuracy at a rate of 15 tons per hour (Loeffen et al., 1997). DEXA is an advance on the X-ray attenuation method for determination of fat in ground meat (Chapter 1). DEXA is used in diagnostic medicine as a tool for detecting osteoporosis, and in airport security for detection of weapons and explosives in baggage. It relies on the differential absorption of two energy bands of x-radiation by the material under test. As with the Neugat system, changes in the intensity of the radiations reaching the detector can be used to determine the ratio of any two components of the material. Medical DEXA scanners have been tested for determination of lean, fat, and bone in ovine carcasses, with an accuracy for fat content similar to that of Neugat (Clarke et al., 1999). While this work is largely experimental, the imaging capacity of x-ray systems offers potential for analysis of the composition and spatial location of meat both on and off the carcass. NIR methods are based on the absorption of defined infrared wavelengths of light by the target material. These wavelengths can penetrate many materials opaque to the eye, and this property is exploited to gain compositional information. In its simplest form, a near-infrared light source shines on a sample and the reflected light is collected. The difference between the intensity of source and reflected light is measured at each of many wavelengths, and collectively represents the difference spectrum due to the material. The difference spectrum is correlated with each chemically measured analyte (e.g., fat, moisture content). In practice, only some of the many available wavelengths of light are used for each correlation because others only add to “noise.” Sophisticated mathematical procedures are used to sort out the useful wavelengths. Although application in an industrial situation is far from straightforward, NIR devices offer considerable advantages over wet chemical tests: NIR is largely noninvasive, analytes that can be determined in a single spectral collection are limited only by the analyte correlations available, the test results are available within minutes at most, little labor is required, and NIR systems lend themselves to automated testing. As always, though, wet

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chemical tests remain the cornerstone of analysis against which NIR methods—and other noninvasive methods—must be calibrated, in many cases frequently. XIV. TESTS COMMONLY USED IN MEAT RESEARCH In the value chain from farm to retail, some of the most important measures are those described elsewhere in this chapter; principally, moisture, protein, fat, and fatty acid profile. Although these measures are important to the professional researcher as well, other attributes of meat are also routinely evaluated. These include color, pH, drip, water-holding capacity, tenderness, protein solubility and emulsifying ability. Measurement of color is described in Chapter 3. The pH of meat has very important effects on meat color, microbial stability, and eating quality, as described in Chapter 12 and others. pH is usually measured with a probe-style electrode that, after calibration, is inserted into a meat cut to obtain a reading. In another technique, a 1 gram sample is excised from the meat and homogenized in 10 ml of water. The pH is then measured in the slurry. Method details are well described in meat science research papers and recommended procedures have recently been described by Honikel (1998). Measurement of fluid loss (termed drip or purge) is important at all points in the value chain. No trader wants to buy meat that will suffer a significant loss in weight during their ownership. Ideally, fluid should not be released from meat until it is eaten, but the reality is that from the time of slaughter, fluid is progressively lost. The pattern of loss from slaughter to consumption depends on species, genetic predisposition to rapid glycolysis, meat ultimate pH, processing variables, and time. Drip is measured by recording the loss in weight of a suspended meat sample (80 to 100 g) during a period of holding, say 24 hours, at a controlled chill temperature (Honikel, 1998). The measurement of water-holding capacity is a variation of drip measurement where force is applied to extract the fluid. (The word “water” is not an accurate descriptor but is in common usage.) Kauffman et al. (1986) compared a number of methods. In one, fluid loss is generated by centrifugation. In another popular method, a meat sample is placed on a filter paper disk and squeezed between two perspex plates. The area dampened by the exudate and the area occupied by the squashed meat are used to calculate the water holding capacity. Tenderness is probably the most researched quality attribute of meat from domestic animals. In outline, tenderness can be measured by a number of compressive, shearing, and tensile methods after meat has been cooked in a standard way to a standard temperature and cut to a standard size with defined grain orientation. In common with most measurements on meat, replication is required to provide statistically valid data. Some reference methods for tenderness are described by Claus (1995), American Meat Science Association (1995), and Honikel (1998). Protein solubility, also known as protein extractability, is often used as a measure of muscle protein denaturation that can occur on frozen storage, for example. Denatured proteins are generally less soluble than the native forms. There is no standard method for measuring protein solubility but many methods follow that of Helander (1957). Low concentrations of KCl dissolve sarcoplasmic proteins principally, while high concentrations dissolve that fraction plus myofibrillar proteins. Extracted proteins are measured by the Kjeldahl method (see earlier). The ability of muscle proteins to emulsify fat in comminuted meat products is an important quality. The higher the emulsification ability the better. In the method of Swift et

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al. (1961), vegetable oil is progressively introduced to a salt-extracted solution of meat proteins continuously blended at high speed. When the oil-in-water emulsion can hold no more oil, the emulsion collapses to a water-in-oil state with a marked drop in viscosity. The quantity of oil required to reach this point is the measure of emulsifying capacity. Several variations of the method have been devised, including the emulsion activity index by proposed by Pearce and Kinsella (1978). XV. SUBSTITUTION, SPECIES AND ANIMAL IDENTIFICATION, AND IRRADIATION A. Substitution and Species Identification Barramundi is a particularly popular fish caught and bought in Australia. Folklore has it that much more barramundi is bought than caught. For high-value foods such as fish and meat, species substitution for illicit financial gain is probably as old as commerce itself. Substitution is relatively easy because raw muscle tissue from different species is often similar in appearance, and once incorporated in a comminuted product, identification based on appearance is completely lost. At another level, substitution of declared or implied meat product ingredients with cheaper ingredients is very common. These cheaper ingredients are either from the species declared (typically offals, blood, connective tissue), or are non-meat ingredients. Sometimes these practices break no laws but in the longer term can condemn the meat product to a lower price category, so defeating the intent to make more money. In many countries, however, meat products such as sausages have zero tolerance for certain offals, and minimum/maximum limits for fat, lean meat, collagen, salt, added carbohydrates, and substitute proteins from plant or dairy sources. These ingredients in meat products are therefore often monitored. Ingredient substitution with non-meat products can also generate religious concerns: the simultaneous consumption of dairy and meat proteins is prohibited for Jews who observe strict dietary laws (Kashruth). Religious strictures, perceived or real health concerns, and cultural likes and dislikes are the main drivers of species identification for consumer protection. All methods to determine species are based on biochemistry in one form or another because qualitative and quantitative biochemical traits set all individuals apart from one another. Methods to determine species have kept pace with biochemical technology. Prior to the 1990s, the main methods of species identification were based on the properties of proteins (Barai et al., 1992). In these techniques, proteins are extracted from the meat or meat products with solvents and subjected to electrophoretic or immunological analysis. Polyacrylamide gel electrophoresis is the most common electrophoretic method in use, where migration of proteins in response to an electric field applied across a flat gel depends on their net charge. Protein bands in the gel are visualized by staining and the pattern generated by the different migrations is often characteristic of different species. A variation on gel electrophoresis is isoelectric focusing, in which proteins migrate in a pH gradient on a gel to the point where the net charge on each protein is zero. Subtle differences in charge for a given protein due to species are often easy to detect in a defined pH range. Typical immunological methods rely on diffusing the extracted proteins of interest against antibodies to proteins from a range of different animal or plant species. A precipitin band indicates recognition of antigen (say, one protein of a given species) for its corresponding antibody that was previously induced in animals or culture cells, and purified for

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the purpose. In gels, the diffusion rates of individual extracted proteins and antibodies are frequently different, so precipitin bands form in different regions of the gel sheet, generating a pattern characteristic of a particular mix of antigens that might be present in a sausage (say, a beef muscle protein, a chicken muscle protein, and casein from cow’s milk). The well-known ELISA technique is a refinement of the basic immunological method, where an enzyme—usually a peroxidase—is covalently linked to the antibody. The enzyme amplifies the precipitin complex by generating many more chemical equivalents of (colored) reaction product than are inherent in the precipitin complex. The ELISA technique is sometimes used with fluorescent markers, which are detectable in minute quantities. However, very high sensitivity can be a problem rather than a benefit, as minute contamination by foreign antigens can be detected. Quantitation is difficult, so minute traces of chicken, for example, on equipment also used to make beef sausages could lead to false accusations of species substitution. A major problem with these protein-based methods is that they do not work well with cooked meats and meat products. On heating, proteins tend to become insoluble, which means they are difficult to extract and analyze on gels. Moreover, immunological methods are based on shape recognition, and the native shape of proteins is lost when they are heated to cooking temperatures. One approach to this has been to dissolve the cooked proteins in concentrated urea, followed by a renaturation step by dialysis. Other methods of detecting substitution include amino acid and peptide composition, particularly for the rarer amino acids such as 3-methylhistidine (Johnson et al., 1986; Plowman and Close, 1988), and fatty acid profile in storage fats. For fatty acids, indicators and specific markers of species include the saturation to unsaturation ratio, a fatty acid unique to pork (Sawaya et al., 1990), and branched chain fatty acids common in goat and sheep but insignificant in other species (Wong et al., 1975). However, the methodologies to determine these defining amino acid and fatty acid profiles are complex and the results are usually equivocal. Absolutely unequivocal identification can, however, be determined from the nucleic acid profile, by technologies that can extend to the level of the individual animal. The 1990s have seen the rapid development of DNA-based methods for species identification. DNA is a remarkably stable chemical, able to withstand boiling and freezing, although if samples are not preserved, by drying or freezing for example, tissue enzymes or bacteria can degrade DNA very quickly. DNA can be chemically extracted for analysis from most tissues, including skin, muscle, blood, and even bone. Along with DNA’s fundamental specificity, its stability makes it attractive for identification purposes. For instance, even meat products cooked to high temperatures yield satisfactory qualities and quantities of DNA for species identification. Contemporary DNA methods to identify species analyze “high copy number” DNA such as the small loop of DNA associated with the mitochondria. An animal cell typically contains hundreds of mitochondria, so mitochondrial DNA is present in large numbers of copies compared with the DNA from the nucleus. This is true even where the cell is multinucleate as in the case of a muscle fiber. Where only low quantities of DNA can be extracted, the simple but elegant technique of polymerase chain reaction (PCR) can be used to replicate the DNA to a large numbers of copies. Certain sections of mitochondrial DNA, such as the cytochrome b gene, have been analyzed to yield a unique DNA sequence for virtually every vertebrate species. Comparison of the test sample DNA sequence to a database of DNA sequences provides unequivocal identification of tissue from species as diverse as whales, fish, frogs, deer, and marsu-

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pials (Bartlett and Davidson, 1992). However, sequencing is technically demanding and requires the expertise of a dedicated laboratory. For rapid analysis of common meat species, DNA probes can be employed. The probe is a short fragment of DNA (amplified to high copy number) that is relatively or absolutely specific for a given species. In one typical test procedure, DNA is extracted from the meat product, purified, denatured into the two complementary strands, and immobilized on a nylon membrane. The DNA probe is added, hybridizing with the complementary DNA if it is present. Binding is visualized with the aid of radioisotopes, or with colorimetric or fluorescent markers. Probes can have a higher or lower specificity depending on the application. A probe prepared to unequivocally distinguish chicken from turkey may be useless at distinguishing beef from bison. The challenge is to prepare probes that are specific enough for the job in hand. A good example of a contemporary DNA technique is described by Matsunaga et al. (1999). In this example, species-specific DNA probes based on the cytochrome b genes were prepared for five meat species. Each probe had a different number of base pairs and thus a different and characteristic migration in an electrophoretic gel. Comparison with the migration of DNA extracted from cooked meats (up to 120°C) permitted species identification. B. Identification of Individual Animals In recent years there have been major food safety scares, many connected to meat and meat products. Traceability is becoming a major issue for retailers. One of the key assurances a retailer can give is that the meat came from a named source with certain defined standards. In order to provide this assurance, a paper or electronic trail is needed, tracing product from source to retail. DNA analysis offers an alternative approach to conventional trails, which are obviously prone to fraud. The uniqueness of each individual is in its DNA. With the exception of clones and identical twins, no two sheep, cattle or pigs are identical in every respect. DNA tracing relies on the ability to read the unique DNA code, or genetic signature, that belongs to an individual animal. The entire nuclear DNA code is too large and complex to be useful, so DNA testing involves the use of small sections of DNA, called DNA markers. The combination of several markers provides a DNA profile, or DNA “fingerprint” that is analogous to a retail barcode unique to a retail product. This DNA profile can be obtained directly from a live animal (virtually any tissue) or any products derived from the animal. The profile does not change with the animal’s age or nutritional status or with season. Current application of DNA tracing in the meat industry involves taking a small tissue sample from every animal processed in a packing plant and storing this tissue in a DNA bank. To verify the origin of a piece of meat at retail, its DNA profile is compared with the profiles of shortlisted samples from the bank to obtain an identifying match. The key to such systems are technologies that allow rapid sampling of carcasses and storage of the samples for easy retrieval. The easiTrace® system, a product of the authors’ company, is an example of the successful integration of this tissue sampling, storage, and trace-back from retail. C. Detection of Irradiated Meat -Irradiation is a proven way of reducing microbial contamination of foods while causing minimal chemical change to the tissue compared with most other preservation methods. For

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reasons linked to the horror of nuclear weapons and accidents, there is widespread public concern about irradiation. Although much of the counter argument is emotional, consumers in democracies maintain they have a right to know if their food has been irradiated, and in many cases this belief has been translated into law. In turn, such laws have spawned tests designed to detect irradiation. For any food an unequivocal test would be the demonstration of a unique irradiation-generated (radiolytic) compound(s). This has proved difficult because the changes wrought in food due to irradiation are mediated by free radical mechanisms that occur naturally in foods, although usually more slowly. Meat contains about 80% water, and a legal dose of -radiation will generate about 5 mmole of free radicals per kilogram of food (Swallow, 1988). These include superoxide and hydroxyl radicals. These primary radicals are highly reactive and in the aqueous environment of meat combine with fats and proteins to generate secondary radicals and downstream degradation products. Few if any degradation products are unique radiolytic products. Moreover, as detection limits drop due to improvements in analytical techniques, compounds once thought to be unique to irradiated foods are found to be present naturally. In solid material such as bone, some free radicals last for weeks rather than fractions of a second, and this longevity has been exploited with physical methods such as electron spin resonance. Detection of irradiated bone is well established (Scotter et al., 1990), and there is even enough bone in mechanically separated meat to detect irradiation (Gray and Stevenson, 1989). Stable free radicals in solids can also be detected by luminescence. In the aqueous environment of meat, where free radicals are short-lived, analysis of volatile hydrocarbons has been useful in the detection of irradiation. Radiolysis of fatty acids generates unusually high quantities of alkenes. Linoleic acid, for instance, gives rise to heptadecadiene and hexadecatriene (Kavalam and Nawar, 1969). In blind tests on irradiated chicken, beef, and pork, Nawar et al. (1990) successfully identified irradiated samples through hydrocarbons analysis. Another family of fat oxidation products, the cyclobutanones, have been proposed as unique markers of irradiation (Stevenson, 1991). Several methods based on DNA have also been proposed. Modified bases such as thymidine glycol are strong indicators of irradiation (Debble et al., 1990). Of several biological methods, the direct epifluorescent filter technique to aerobic plate count ratio (DEFT/APC) is the most useful. The basis of this method is as follows: irradiation does not reduce the fluorescence due to acridine orange binding to dead and live microbes isolated (by filtration) from meat, but the aerobic plate count due to live microbes is markedly reduced through irradiation. A high ratio therefore indicates irradiation (Betts et al., 1988; Betts, 1991). XVI. LABORATORY MANAGEMENT A. Costing Analyses, Calculating and Reporting Results Estimating costs is often difficult as each step in an analytical method has inherent error in costs of labor and materials. Moreover, costs are usually sensitive to the number of samples to be analyzed. In developing a useful costing system, a typical starting point would be a brief description of each of the steps for a particular analysis. The steps usually extend from sample receipt to presentation of the results (and invoicing if required), and must include early and late steps such as handling samples on receipt and cleaning up. The latter is frequently overlooked. Factors affecting each of these steps and their interactions need to be specified. This written description is then translated

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into simple equations for a spreadsheet, which is then tested under a number of what-if scenarios. However, the spreadsheet’s utility must ultimately be tested with real samples in quantities that test the limits of the calculations. When the analyst is satisfied with the spreadsheet, most cells are protected so that the critical parts of the calculation cannot be accidentally changed, causing under- or overcharging. As a component of laboratory information management systems, spreadsheets are also used to calculate results. Again key cells are protected. Auditing techniques are sometimes applied to the outputs of formulas such that if a result is outside a defined limit, the analyst is alerted. It is fundamentally important to present analytical results in a form that the client understands and finds useful. In most cases the value reported is some fraction (e.g., percent, parts per million, mole per kg) of the sample as received, although this should not be assumed. The basis of presentation should be established at the outset. For many clients the word “average” is readily understood, but not so its synonym “mean.” Whereas standard deviations are sometimes required for legal purposes, the expression often has no meaning to meat industry personnel. As an alternative, the spread of values can be shown by quoting the highest and lowest value, or by expressions that state what fraction of values lie between x and y on either side of the average z. The format of reports should be examined for clarity and readability. Above all, the key to good reporting is understanding the client’s needs. B. Quality Systems When the analyst, and later the client, views a set of results that confirms preconceptions, analyst and client satisfaction are guaranteed. However, when results are at variance with preconceptions, problems arise unless the laboratory has a quality system in place. A quality system gives confidence that the results—no matter how unexpected or unwelcome— are valid within normal experimental limits. A quality system is a formal way of minimizing random and systematic error arising from equipment and procedures. To cite an example, a quality system for the determination of moisture might require that the temperature of the drying oven was always within a specified range. Every day, week, or other period (depending on the anticipated variability and the importance of the determination) the temperature of the oven is measured with a thermometer whose precision and accuracy can be traced through periodic audit to a national standard. The oven temperatures are recorded and monitored for trends. Likewise, the laboratory balances are calibrated routinely, usually by a certified agency with its own quality system. In quality systems, it is important that precision and accuracy are both controlled. When the former is controlled but not the latter, the analyst could be justifiably accused of producing junk data, but happily consistent junk (Fig. 1, upper left quadrant). Quality systems are tailored to the needs of the laboratory and its clients. The most basic of quality systems demands at least routine calibration of balances. In many cases, only some methods are fully monitored. However, detailed systems are sometimes needed that demand fully calibrated glassware and interlaboratory comparisons. As with all systems, a sensible trade-off between benefit and cost must be struck. A common criticism of quality systems is that they are time and paper wasters. This appears true when things are going smoothly, but when results are unusual the analyst can look to the checks and balances of a quality system and sign the reports with confidence.

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XVII. THE FUTURE OF ANALYTICAL METHODS FOR MEAT Measurement is fundamental to any technology, and in the case of meat science applications, the measurement of protein and fat, and in turn, amino acids and fatty acids, has been a cornerstone. In terms of human nutrition these are the macronutrients of meat, and the need for their future analysis is assured. Methods for their determination will evolve with an emphasis on increased speed and decreased cost, but without loss of precision or accuracy. As for the micronutrients of meat, such as minerals and vitamins, some will be of enduring interest while others, such as rare fatty acids and cholesterol, may cease to be of analytical interest as the concerns of societies move to fresh issues or obsessions. If and when irradiation of meat products becomes common, interest in its detection may wane. In contrast, guarantee of origin through traceability and guarantee of species are likely to receive increased attention as nucleic acid-based methods improve. Enduring concerns based on religion, and current societal concerns for animal welfare, environmental damage, food safety, and what we eat in general, will be powerful drivers of these nucleic acid technologies. REFERENCES American Meat Science Association. 1995. Research guidelines for cookery, sensory evaluation, and instrumental tenderness measurements of fresh meat. AMSA and National Live Stock and Meat Board, Chicago. Anderson, B.A., and I.M. Hoke. 1990. Composition of foods: beef products, Agriculture Handbook 8–13, United States Department of Agriculture, Human Nutrition and Information Service. AOAC Methods. In: Official Methods of Analysis (16th Ed.). 1999. AOAC International, Gaithersburg, Maryland. Barai, B.K., R.R. Nayak, R.S. Singhal, and P.R. Kulkarni. 1992. Approaches to the detection of meat adulteration. Trends. Food Sci. Technol. 3:69–72. Bartle, C.M. 1995. Features of the measurement of fat in meat products using the neutron/gamma transmission method. Appl. Rad. Isotopes 46:741–750. Bartlett, S.E., and W.S. Davidson. 1992. FINS (Forensically Informative Nucleotide Sequencing): A procedure for identifying the animal origin of biological specimens. Bio Techniques 12:408–411. Benedict, R.C. 1987. Determination of nitrogen and protein content of meat and meat products. J. Assoc. Off. Anal. Chem. 70:69–74. Betts, R.P. 1991. The detection of irradiated foods using the direct epifluorescent filter technique. In: Potential New Methods of Detection of Irradiated Food. Report EUR 13331, pp 86–90. Commission of the European Communities, Luxembourg. Betts, R.P., L. Farr, P. Bankes, and M.F. Stringer. 1988. The detection of irradiated foods using the direct epifluorescent filter technique. J. Appl. Bacteriol. 64:329–335. British Standard 4401. 1970. British standard methods of test for meat and meat products. Part 4: Determination of total fat content BS 4401. H.M. Stationery Office, London. Christie, W.W. 1984. Extraction and hydrolysis of lipids and some reactions of their fatty acid components. In: H.K. Mangold (Ed.) CRC Handbook of Chromatography: Lipids. Vol. 1, CRC Press, Boca Raton, Florida. Christie, W.W. 1989. Gas Chromatography and Lipids. A Practical Guide. The Oily Press, Ayr, Scotland. Clarke, R.D., A.H. Kirton, C.M. Bartle, and P.M. Dobbie. 1999. Application of dual-energy X-ray absorptiometry for ovine carcass evaluation. Proc. N. Z. Soc. Anim. Prod. 59:272–274. Claus, J.R. 1995. Method for the objective measurement of meat product texture. In: 48th Annual Reciprocal Meat Conference, pp. 96–101, San Antonio. Am. Meat Sci. Assoc. and National Live Stock and Meat Board.

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Debble, D.J., A.W. Jabir, B.J. Parsons, C.J. Smith, and P.A. Wheatley. 1990. Changes in DNA as a possible means of detecting irradiated food. In: Food Irradiation and the Chemist. Roy. Soc. Chem. Special Publication 86, pp 57–59. London. Fox, J.B., R.C. Doerr, and R. Gates. 1984. Effects of residual ascorbate on determination of nitrite in commercial cured meat products. J. Assoc. Off. Anal. Chem. 67:692–697. Gray, R., and M.H. Stevenson. 1989. The effect of post-irradiation cooking on the ESR signal in irradiated chicken drumsticks. Int. J. Food Sci. Technol. 24:447–450. Gregory, J.F., and Feldstein, D. 1985. Determination of vitamin B6 in foods and other biological materials by paired-ion high-performance liquid chromatography. J. Agric. Food Chem. 33:359–363. Hamann, D.D. 1983. Structural failure in solid foods. In: M. Peleg and E.B. Bagley (Eds.) Physical Properties of Foods. pp. 351–383. AVI Publishing Co., Connecticut. Helander, E. 1957. Muscle-protein determination. Acta Physiol. Scand. 41, suppl. 141:1–99. Honikel, K.O. 1998. Reference methods for the assessment of physical characteristics of meat. Meat Sci. 49:447–457. House, S.D. 1997. Determination of total, saturated, and monounsaturated fats in foodstuffs by hydrolytic extraction and gas chromatographic quantitation: collaborative study. J. Assoc. Off. Anal. Chem. Int. 80:555–563. ISO 1443-1973. Meat and meat products—determination of total fat content. International Organization for Standardization, Geneva. ISO 1444-1973. Meat and meat products—determination of free fat content. International Organization for Standardization, Geneva. Johnson, S.K., W.J.P. White, and R.A. Lawrie. 1986. Observations on the 3-methylhistidine content of bovine, ovine, and porcine muscles. Meat Sci. 18, 235–239. Kauffman, R.G., G. Eikelenbloom, P.G. van der Wal, B. Engel, and M. Zaar. 1986. A comparison of methods to estimate water-holding capacity in post-rigor porcine muscle. Meat Sci. 18:307–322. Kavalam, J.P., and W.W. Nawar. 1969. Effects of ionizing radiation on some vegetable oils. J. Am. Oil. Chem. Soc. 46:387–390. Kovacs, M.I.P., W.E. Anderson, and R.G. Ackman. 1979. A simple method for the determination of cholesterol and some plant steroids in fishery-based food products. J. Food Sci. 44:1299–1301. Loeffen, M.P.F., R.D. Clarke, P. Petch, C.M. Bartle, and R. Kelly. 1997. On-line measurement of the chemical lean content of manufacturing meat. Proc. 43rd Int. Congr. Meat Sci. Techol., Auckland, New Zealand, 238–239. Matsunaga, T., K. Chikuni, R. Tanabe, S. Muroya, K. Shibata, J. Yamada, and Y. Shinmura. 1999. A quick and simple method for the identification of meat species and meat products by PCR assay. Meat Sci. 51:143–148. Maxwell, R.J., W.N. Marmer, M.P. Zubillaga, and G.A. Dalickas. 1980. Determination of total fat in meat and meat products by a rapid, dry column method. J. Assoc. Off. Anal. Chem. 63:600–603. Millar, K., A.T. Albyt, and G.C. Bond. 1984. Measurement of vitamin B12 in the livers of sheep and cattle and an investigation of factors influencing serum vitamin B12 levels in sheep. N.Z. Vet. J. 32:65–70. Nawar, W.W., Z.R. Zhu, and Y.J. Yoo. 1990. Radiolytic products of lipids as markers for the detection of irradiated meats. In: Food Irradiation and the Chemist. Roy. Soc. Chem. Special Publication 86, pp 13–24. London. Pearce, K.N., and J.E. Kinsella 1978. Emulsifying capacity of proteins: evaluation of a turbidimetric technique. J. Agric. Food Chem. 26:716–723. Phalzgraf, A., H. Stenhart, and M. Frigg. 1995. Rapid determination of -tocopherol in muscle and adipose tissues of pork. Z. Lebensm. Unter Forsch. 200:190–193. Plowman, J.E., and E.A. Close. 1988. An evaluation of a method to differentiate the species of origin of meats on the basis of the contents of anserine, balenine and carnosine in skeletal muscle. J. Sci. Food Agric. 45:69–78.

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Sawaya, W.N., T. Saeed, M. Mameesh, E. El-Rayes, A. Husain, S. Ali, and H. Abdul Rahman. 1990. Detection of pork in processed meat: experimental comparison of methodology. Food Chem. 37:201–219. Scotter, S.L., P. Holley, and R. Wood. 1990. Co-operative trial of methods of analysis to detect irradiation treatment of chicken samples: initial trial. Int. J. Food Sci. Technol. 25:512–518. Stegemann, H., and K. Stalder. 1967. Determination of hydroxyproline. Clin. Chim. Acta 18:267–273. Stevenson, M.H. 1991. The detection of irradiated foods using chemical methods of analysis. In: Potential New Methods of Detection of Irradiated Food. Report EUR 13331, pp 171–176. Commission of the European Communities, Luxembourg. Swallow, A.J. 1988. Can we tell if our food has been irradiated? Chem. Brit. 24:102–105. Swift, C.E., C. Lockett, and A.J. Fryar. 1961. Comminuted meat emulsions—the capacity of meats for emulsifying fat. Food Technol. 15:480–482. Tarladgis, B.G., B.M. Watts, M.T. Younathan, and L. Dugan. 1960. A distillation method for the quantitative determination of malonaldehyde in rancid foods. J. Am. Oil Chem. Soc. 37:44–48. Troller, J.A. 1980. Influence of water activity on microorganisms in foods. Food Technol. May: 76–82. Wong, E., C.B. Johnson, and L.N. Nixon. 1975. The contribution of 4-methyloctanoic (hircinoic) acid to mutton and goat meat flavour. N.Z. J. Agric. Res. 18:261–266.

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6 Meat Biotechnology M. B. SOLOMON U.S. Department of Agriculture, Beltsville, Maryland

I. INTRODUCTION II. GENETIC SELECTION AND MANAGEMENT STRATEGIES III. GROWTH-PROMOTING AGENTS A. Appetite Stimulants B. Antibacterial Growth Promoters C. Rumen Modifiers D. Anabolic Steroids and Related Substances E. Endogenous Somatotropin F. Exogenous Somatotropin G. Growth Hormone Releasing Factor H. Somatostatin I. Beta-Adrenergic Agonists IV. TRANSGENIC ANIMALS A. Swine with Growth-Related Transgenes V. CONCLUSIONS REFERENCES

I. INTRODUCTION Biotechnology is the implementation of biological techniques to produce or modify products and to manipulate cell genome and function. Use of science for the improvement of muscle foods has involved natural selection of dominant traits, selection of preferred traits by cross-breeding, the use of endogenous and exogenous growth factors, and ultimately gene manipulation and cloning to produce desirable changes in meat/carcass quality and yield. Until recently, improvements in the quality of meat products that reached the marketplace were largely the result of postharvest technologies. Extensive postharvest efforts have been implemented to improve or to control tenderness, flavor, and juiciness. Tender-

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ness, flavor, and juiciness are the sensory attributes that make meat products palatable and are often the attributes that consumers consider when making their selection to purchase meat products. Consumers not only have been interested in the quality (palatability) but also have been concerned with the nutritional value, safety, and wholesomeness of the meat they consume. The public has been inundated with warnings about the health risks of consuming certain types or classes of foods (in particular, the fat profile). Consumers became more health and weight conscious in the 1980s, desiring fewer calories in their diet. In fact, this decade was considered the “decade of nutrition.” However, present trends suggest that there is less concern among consumers about many of the substances previously viewed as harmful. There is also less concern about calories. Consumers appear to prefer traditional and familiar food—foods they have always eaten. New technologies (e.g., biotechnology) and alternative production methods appear to hold great promise for improving the quality and yield attributes of animal products. A wide range of biotechnology strategies for altering the balance between lean and adipose tissue growth and deposition in meat-producing animals are available. These include genetic selection and management (production) strategies. More recently, the confirmation of the growth-promoting and nutrient repartitioning effects of somatotropin, somatomedin, -adrenergic agonists, immunization of animals against target circulating hormones or releasing factors, myostatin mutations, polar overdominance (callipyge mutation), and gene manipulation techniques have given rise to a technological revolution for altering growth and development in meat producing animals. II. GENETIC SELECTION AND MANAGEMENT STRATEGIES The main genetic alteration during the past 40 years has been to decrease carcass fatness and increase lean tissue deposition. These alterations have been via growth rate and mature size of meat animals, particularly through genotypic and sex manipulation. There have been numerous papers on the effects of breed and sex condition on carcass composition and meat quality, and therefore this will not reviewed in this chapter. III. GROWTH-PROMOTING AGENTS Growth-promoting agents are substances that enhance growth rate of animals without being used to provide nutrients for growth such as nutrient partitioning agents. Growth-promoting agents are anabolic; that is, they produce more body tissues and thereby result in more rapid animal growth. Growth-promoting agents cause changes in carcass composition, mature weight, and efficiency of growth. Many substances qualify as “growth-promoting agents” despite their varied origin and chemical nature. Examples of growth promoting agents are the following: antibacterial agents (e.g., antibiotics), rumen modifiers (e.g., monensin, lasalocid), steroids (e.g., testosterone, estrogen), -adrenergic agonists (e.g., clenbuterol) and somatotropin (growth hormone). Growth-promoting agents influence growth in three ways: (a) by stimulating feed intake (appetite stimulants) and thereby increasing the supply of nutrients available for growth, (b) by altering the efficiency of the digestive process, resulting in improved supply and/or balance of nutrients derived per unit of feed consumed (antibacterial agents, rumen additives, i.e., ionophores), and (c) by altering the manner in which the animals utilize or partition absorbed nutrients for specific growth processes.

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The effects of various growth-promoting agents on improving efficiency of growth, changing nutrient requirements (thereby increasing lean meat yield), and concomitantly decreasing fat have been thoroughly reviewed (1–9). It is not the intent of this chapter to review all the plethora of literature on growth and development but instead to discuss the implications these biotechnological interventions have on muscle and meat quality. A. Appetite Stimulants Many growth-promoting agents influence voluntary feed intake; however, responses in voluntary intake are generally small and inconsistent. Stimulating appetite (intake) could result in improved growth rate and the production of edible protein (lean tissue). Scientists are investigating the use of drugs to control appetite. If successful substances are developed, it may be possible to enhance meat production in the future by increasing or decreasing an animal’s appetite. B. Antibacterial Growth Promoters A wide variety of antibiotics (antibacterial agents) enhance growth in primarily nonruminant animals. Some common antibiotics that are used to remedy clinical infections are successful at promoting growth. Included in these common antibiotics are penicillins, tetracyclines, bacitracin, avoparcin, and virginiamycin. These antibiotics are active against gram-positive bacteria and, in most instances, are not retained in the tissues of the animal. As much as a 20% improvement in growth rate and feed efficiency have been observed with the administration of antibacterial agents to nonruminants. C. Rumen Modifiers A special class of antibiotics whose principal site of action is the reticulorumen of ruminants has been investigated. These include monensin (Rumensin, Romensin) and lasalocid (Bovatec), which are classified as ionophores. Rumen additives are generally administered to growing ruminant animals. The type of diet fed to the animal receiving rumen modifiers has an effect on the outcome. Voluntary intake is depressed when rumen modifiers are included in concentrate diets, whereas little depression in intake is observed when included in forage-based diets. Improvements in growth rate and productivity are generally less than 10%. The mode of action of rumen modifiers is poorly understood but is thought to be a result of altering the metabolism (digestive process) of rumen microflora. Rumen modifiers act against gram-positive bacteria in the rumen, causing a shift in patterns of volatile fatty acid production, improved digestive efficiency, reduced bloat and reduced production of methane and hydrogen. In a study using lambs reported by Solomon et al. (10) and Fluharty et al. (11) on the effects of energy source and ionophore (lasalocid) supplementation on carcass characteristics, lipid composition and meat sensory properties suggested that ionophore supplementation had no significant effect on either carcass/meat quality or yield characteristics (10, 11). Diet (forage vs concentrate) offered more opportunities for manipulating carcass composition without jeopardizing meat quality than the use of ionophore supplementation. D. Anabolic Steroids and Related Substances The importance of steroids such as androgens and estrogens in regulating growth is evident from distinct differences between male and females in growth and composition. Castration

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Table 1 Steroid-Related Growth-Promoting Agents Class Natural estrogens Estrogen analogues

Natural androgens Androgen analogues Natural progestagens Progestagen analogues Combined products

Active agent(s)

Trade name

Estradiol-17B Diethylstilbesterol Hexestrol Zeranol (resorcyclic acid lactone) Testosterone Trenbolone acetate Progesterone Melengestrol acetate Trenbolone acetate  estradiol Testosterone  estradiol Testosterone propionate  estradiol benzoate Progesterone  estradiol Progesterone  estradiol benzoate Zeranol  trenbolone acetate

Compudose® DES Ralgro® Finaplix® MGA Revalor® Implix BF® Synovex-H® Implix BF® Synovex-S® Forplix®

of males, which removes their primary source of androgens (testosterone), is the oldest method of manipulating growth by non-nutritional mechanisms. A number of steroid-related growth-promoting agents that are either available for commercial use or have been extensively researched exist. These are listed in Table 1. These agents are most effective in castrated males or females (especially in ruminants). Naturally occurring and synthetic estrogens and androgens have been used to improve efficiency of growth and carcass composition of meat animal for more than 50 years. Steroid-related growth-promoting agents generally increase live weight gain and improve carcass lean-to-fat ratios in ruminants but are not as effective in swine. In fact, many of the growth-promoting agents are not approved for use in growing swine in the United States. Their mode of action is unclear but many have a direct effect on muscle cells. A review of the biosynthesis and metabolism of the naturally occurring estrogens and androgens has been published (12). The literature on growth-performance responses to anabolic steroids indicates great variability, ranging from no response in feedlot bulls (13) to a 70% increase in average daily gain in heifers (14). The efficacy of anabolic steroid implants is summarized in several reviews (12, 15–18). In a recent study (19) looking at the effects of different growth-promoting implants on muscle morphology in finishing steers, these researchers found Revalor implants to be most effective, followed by Ralgro implants, in increasing lean mass through muscle fiber hypertrophy. Synovex implant had the least pronounced capacity for muscle growth enhancement. Other studies (see exogenous somatotropin section) have compared combining anabolic steroids with somatotropin administration in cattle. E. Endogenous Somatotropin A group of peptide hormones, i.e., somatotropin (ST), growth hormone-releasing factor (GRF), somatostatin, insulin-like growth factor–I (IGF-I), insulin, and thyrotropic hormone, work in harmony to regulate and coordinate the metabolic pathways responsible for tissue formation and development. Even though relationships between growth and circulating levels of some of these peptide hormones have often produced conflicting results, the majority of data indicates that the genetic capacity for growth is related to increased circulating levels of somatotropin and IGF-I in livestock.

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The anterior pituitary secretes three hormones (ST, prolactin and thyroid stimulating hormone) that influence growth and carcass composition. Somatotropin, often called growth hormone (GH), is the most notable, with commercial growth-promoting potential. Somatotropin is a small, single-chain polypeptide, made up of 191 amino acids, secreted by the pars distalis of the pituitary’s adenohypophysis. The structure of somatotropin varies among species. Release of ST is stimulated by growth hormone releasing factor (GRF or GHRH) produced in the hypothalamus (20). Understanding how to control the production of these hormones within the meat animal is a long-term goal of scientists. F. Exogenous Somatotropin 1. Porcine Somatotropin There is a growing database supporting the use of pituitary-derived porcine somatotropin (pST) as an agent to improve efficiency of growth and carcass composition in swine. Turman and Andrews (21) and Machlin (22) were the first to demonstrate that daily exogenous administration (injection) of highly purified pST dramatically altered nutrient use, resulting in improved growth rate and feed conversion of growing-finishing pigs. Pigs injected with pST had less (35%) fat and more (8%) protein. However, their original observations were of little practical significance because purification of porcine ST from pituitary glands was not economical. A single dose required 25–100 pituitary glands. More recently, the development of recombinant deoxyribonucleic acid (DNA) technology has provided a mechanism for large-scale production of somatotropin. The gene for ST protein is inserted into a laboratory strain of Escherichia coli, which can be grown on a large scale and from which ST can be purified and concentrated for use (23). There is also a growing database supporting the use of recombinantly derived pST (rpST). No significant differences between the effectiveness of pST and rpST have been observed. With greater emphasis on lean tissue deposition and less lipid, the optimal genetic potential for protein deposition of an animal is a very important concept in that this potential, or ceiling, defines the protein requirement of the animal. In defining the optimal/genetic potential for protein deposition, ST is used as a tool to maximize genetic potential for protein accretion. Administration of pST to growing pigs elicits a pleiotropic response that results in altered nutrient partitioning. In studies with growing pigs, significant improvements of 40% in average daily gain and 30% in feed conversions can be achieved by administration of pST. Research has also shown that the effect of pST is enhanced by good management and nutritional practices (24–26). Furthermore, a 60% reduction in carcass fat and a 70% increase in carcass protein content can be attained. The magnitude of response has varied in the various studies performed since the initial classical studies (21,22). Different interpretations in response have been attributed to differences in experimental designs. These include initial and final weight of pigs, length of study, genotype, sex, dose of ST, nutritional conditions, and time of injection. However, despite these differences in design, it is quite apparent that pST or rpST increases average daily gain by as much as 40%, decreases carcass fat deposition by as much as 60%, and concomitantly increases carcass protein (lean) accretion by 70%. Daily injections of pST has been the method of choice for pST administration. Porcine ST must be administered by injection because it is a protein and would be inactivated by digestive enzymes if given orally. The response from pST administration is not related to the site of injection nor to the depth of injection. Recently, researchers (27, 28) in-

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vestigated the effects of daily pST administration compared with injecting larger doses of pST over an extended period of time. Frequency of administration influenced the magnitude of responsiveness to pST treatment. Results indicated that optimal benefit would be realized by a delivery system that mimicked a daily surge of pST. Mechanisms of pST action have been reviewed and discussed in numerous reviews (several listed above). Clearly, pST affects many metabolic pathways that influence the flow of nutrients among various tissues of the body. Many have concluded that the mechanism by which pST decreases fat content in pigs is via the inhibition of lipogenesis (Table 2). These changes in metabolism and cell proliferation lead to the alteration of carcass composition via the reduction of nutrients normally destined to be deposited as lipid to other tissues. Studies have demonstrated that regardless of the stage of growth, pigs respond to exogenous pST at all ages. However, rapidly growing animals do not seem to benefit from exogenous pST as much as do finishing animals with respect to fat alterations. This is not surprising, because pigs that are growing rapidly and are more efficient are not producing much fat. a. Meat Tenderness. Administration of pST represents a technology with a promise not only for improving production efficiency but also as a means for packers and retailers to offer leaner pork products. A multitude of pST studies have been performed in evaluating the flexibility of pST technology in conjunction with other variables (e.g., management and environmental conditions) to alter carcass and muscle characteristics of morphology as well as meat palatability. These studies typically found that administration of pST to barrows reduced tenderness (increased shear force) by as much as 39% in the longissimus (LM) muscle and 15% in the semimembranosus (SM) when compared with controls. Although the reason for the increase in shear values representing reduced tenderness remains unclear, Solomon et al. (29) proposed that it may involve an alteration of the muscle composition and/or the cold shortening phenomena of muscle. Solomon et al. (30) found that pST administration to boars (Table 3) and gilts lowered shear force of the LM (13.9% and 17.1%, respectively), thus improving muscle tenderness. Klindt et al. (31) reported that extended administration of pST for 18 weeks to barrows resulted in reduced tenderness and Table 2 Biological Effects of Somatotropin on Adipose and Skeletal Muscle Tissue Tissue

Effect

Physiological process affected

Adipose tissue

↓a ↓ ↓ ↓ ↓ ↑b ↑ ↑

Skeletal muscle (growth)

↓ ↑ ↑

Glucose uptake Glucose oxidation Lipid synthesis Lipogenic enzyme activity Insulin stimulation of glucose metabolism Basal lipolysis Catecholamine-stimulated lipolysis Ability of insulin to inhibit lipolysis Insulin binding unaffected Somatotropin binding unaffected Protein degradation Protein synthesis Satellite cell proliferation

a b

↓
Decreases ↑
Increases

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Fiber Characteristics for Transgenic, pST-treated Fed Pigs Treatmentsb Item Carcass Total lipid, g/100 g Cholesterol, mg/100 g Lean Total lipid, g/100 g Cholesterol, mg/100 g Longissimus muscle 10th rib back fat, mm Area, cm2 Shear force, kg/1.3 cm Fiber type, % SO FOG FG Fiber area, m2 SO FOG FG Giant fiber Number Area, m2

T-Control

T-bST

T-oST

Significance pST

pST-Control

SEM

T

pST

27.00 68.71

4.49 77.18

4.82 67.87

18.64 68.48

25.18 70.72

1.7 2.5

* *

* NS

2.89 48.64

1.38 55.58

.96 49.33

2.33 50.13

3.21 45.38

.5 1.5

* NS

* NS

24.8 33.91 3.32

2.2 32.37 3.46

2.4 33.17 3.88

13.2 42.61 4.83

18.7 33.29 5.61

1.5 3.1 .5

* NS NS

* * *

12.4 20.0 67.6

7.2 24.2 68.6

13.3 22.9 63.8

14.0 24.5 61.5

12.5 21.0 66.5

3.9 2.4 2.8

* * NS

NS NS NS

* * *

* * *

3053 3669 4359

2694 1979 2749

3166 2180 4356

0

0

0

3713 3121 4795

3311 2785 4344

3.3 7147

7248

226 225 407

Meat Biotechnology

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Table 3 Comparison a of Total Carcass and Lean Tissue Lipid and Cholesterol Content, Longissimus Muscle Area, Shear Force and

.3

133

NS indicates not significant (P  .05); *
P  05. a Wet weight basis. b T-control
control boars for transgenics; T-bST
transgenic (boars) with bovine somatotropin gene; T-oST
transgenic (boars) with ovine somatotropin gene; pST
exogenously treated boars with porcine somatotropin.

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juiciness, but this was not observed with a 6-week pST administration period. It is difficult to determine whether these tenderness differences found in barrows will be perceived by the consumer; however, it should be noted that most of the shear force values reported were within the shear values associated with normal pork products. Research is needed to determine whether consumers and/or trained sensory panels can detect tenderness differences or other possible problems with pST-treated pork. In a study (32), time post mortem of sampling muscle from pST-treated barrows for subsequent shear force analysis had a significant effect on tenderness. Differences in shear force tenderness between pST and control pigs were virtually eliminated when loin chops were removed from the carcass and frozen within 1.5 hours post mortem compared with controls (frozen 5 days post mortem). Some of the inconsistencies reported in the literature for shear force and tenderness as a result of pST administration may be a result of inconsistencies in the time that the meat sample is removed and subsequently frozen. Minimal observable differences in processing yields, color retention, or composition of products from control and pST-treated pigs have been observed. From the wealth of literature, it appears that pST affects carcass composition and not quality (other than possibly tenderness). However, pale, soft, exudative (PSE) muscle has been observed in a couple of pST studies (discussed in pST Muscle Morphology section). b. Muscle Morphology. The consensus is that pST exerts a hypertrophic response on carcass muscles, which can be seen at both the cellular level (fiber types) and with the naked eye (carcass conformation and loin-eye area). Most of the research demonstrates that muscle fiber area increased in size with the use of pST. However, a rate-limiting factor in the hypertrophic response of muscles to pST administration was the level of dietary protein used (32) in combination with pST administration. This confirms that the beneficial effects of pST is dependent on good management and nutritional practices. Porcine ST administration has little effect on the (percentage) distribution of muscle fiber types (Table 3). Solomon et al. (33, 34) reported that alterations in muscle fiber type populations are often associated with differences in physiological maturation rates of the animals studied. Sorensen et al. (35) observed that genotypes with relatively large muscle fibers are less responsive to pST treatment than genotypes with relatively small muscle fibers; other investigators (29, 30, 32, 36, 37) observed hypertrophied (giant) fibers in muscles from pST-treated pigs (Table 3). Whether the giant fiber anomalies occurred through increased activity associated with compensatory (flux) adaptations or from fibers undergoing degenerative changes has yet to be determined. The occurrence of giant muscle fibers has been associated with stress-susceptible pigs, which exhibit pale, soft, exudative (PSE) muscle. In two pST studies (30, 37), pST-treated pigs exhibited PSE muscle (30% and 62% incidence, respectively), which is much higher than the 12% that is reported as normal occurrence by packers. One explanation offered for the increased incidence of PSE in these studies was a seasonal effect. The experiments were conducted during the summer with average temperatures ranging above 35°C for the duration of the experiments. Perhaps pST administration in conjunction with elevated environmental temperatures may induce the PSE syndrome. However, one cannot discount the possibility that the occurrence of PSE in these two studies and the absence of PSE in other studies could be due to genetic differences between pigs used in the different studies. Aalhus et al. (38) did not observe an increase in the proportion of giant fibers or an interactive effect with the halothane genotype. They indicated that the effects of pST appear to be muscle and gender specific, which may be the result of differences in maturity and rates of growth at the time of pST administration. Although they observed minor reductions in the muscle color (paler) and increased drip as

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well as higher shear values, these quality differences were not always significant nor did they result in PSE meat. Ono et al. (39) reported on the effects of pST administration to barrows on muscles located within different regions of the body (i.e., locomotive vs postural muscles). Based on the analysis of changes of the percentage of muscle fiber types from 20 to 90 kg body weight, they suggested that the fiber type transformation from small slow-twitch oxidative (SO) to large fast-twitch glycolytic (FOG) fibers plays an important role in muscle size enlargement as animals grow. Treatment with pST showed different effects on muscle fiber growth among the different fiber types and different locations of muscles in the body, suggesting some relationship between pST and muscle function and metabolism and/or muscle maturation. Ji et al. (40) found that the enhanced muscle growth achieved by pST was not associated with altered expression of the p94 or -actin gene (key genes relative to muscle growth), or with an increase in the abundance of any calpastatin transcription product. c. Carcass Composition. Lipid composition studies have demonstrated that the lipid content from pST-treated pigs was as much as 27% less in the lean tissue and 23% less in the subcutaneous fat compared to controls. Carcasses (Fig. 1) from pST-treated pigs contained 22% less saturated fatty acids (SFA), 26% less monounsaturated fatty acids (MUFA), and no difference in polyunsaturated fatty acids (PUFA) compared with controls (41). The administration of pST resulted in lean tissue (Fig. 2) containing as much as 40% less SFA, 37% less MUFA and no difference in PUFA compared to controls. The subcutaneous fat from pST-treated pigs contained 33% less SFA, 24% less MUFA, and 9% less PUFA than controls. Cholesterol content in the subcutaneous fat from pST pigs was 12% higher than from control pigs. Cholesterol content of intramuscular fat was similar. These results indicate that significant reductions in total lipid and all three classes of fatty acids can be achieved using pST. This represents a favorable change in regard to human dietary guidelines. Few differences in fatty acid profiles of the intramuscular fat extracted from cooked pork rib chops as a result of pST administration have been observed (42). Choles-

Figure 1 Relative fatty acid composition for carcasses from transgenic and pST administered pigs.

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Figure 2 Relative fatty acid composition for lean from transgenic and pST administered pigs. terol content of cooked chops from pigs receiving pST in their study were greater than controls. Differences in cholesterol values may have resulted from less concentration of the cholesterol during cooking (heating). Lonergan et al. (43) reported that pST treatment did not alter the overall fatty acid saturation in subcutaneous or perirenal fat depots, but resulted in a greater reduction of the more saturated middle and inner layers of subcutaneous fat at the 10th rib location. As a result, the more unsaturated outer layer of subcutaneous fat was present in a greater proportion in pST-treated pigs. Oksbjerg et al. (44) found a change to more polyunsaturated fatty acids and less saturated fatty acids in the backfat of pST treated gilts. The decrease in total SFA and MUFA, and virtual no change in PUFA, support the conclusion that the mechanism by which pST decreases carcass fat content in pigs is by the inhibition of lipogenesis. A decrease in fat synthesis would lead to a decrease in the production of SFA and MUFA with little effect (change) in the amount of PUFA. The majority of PUFA in pig tissues are the result of dietary fatty acids linoleic and linolenic, and are not synthesized. However, the possibility of an increased turnover of storage lipids, at the level of triacylglycerol synthesis or hydrolysis, exists (45). Use of pST is similar to cattle implants and anabolic agents used to enhance growth in that all cause increases in growth and more efficient utilization of feed. However, they are different in chemical structure. Porcine ST is a protein that is not active orally and is readily digested like any protein. Since pST is a protein and is broken down in the gastrointestinal tract, human ingestion of pST would present no dangers because digestive processes would inactivate the protein and provide no residues. Digestion would break the protein down into its component amino acids, making it available for normal metabolic processes. This has been one of the concerns for the acceptance of the use of pST in pigs. Caperna et al. (46) reported that daily pST treatment to growing barrows enhanced collagen deposition in the skin, head, and viscera, whereas non-collagen protein deposition and collagen maturation were enhanced in the carcass tissues.

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2. Bovine and Ovine Somatotropin There is evidence that exogenous bovine and ovine ST improves efficiency of growth and lean-to-fat ratio in ruminant animals. The database is much less extensive than it is for swine. Bovine somatotropin has been found to increase gain and protein deposition in feedlot steers, but reports in the literature suggest the greatest effect on steers is in the noncarcass fraction (47,48). An additive effect of estrogen and somatotropin treatment on weight gain and protein deposition of steers has been suggested (49,50). Rumsey et al. (51) found that rbST (Somavubove) and the estrogenic growth promoter Synovex-S independently increased growth and protein deposition in young beef steers. The combined treatments consistently demonstrated an additive effect on growth rate, carcass growth, protein deposition, and the energy efficiency of protein deposition. The combination of these two growth-promoting agents was also effective in some muscles beyond the response obtained with either treatment alone (52,53). The overall muscle growth effects were greater when the two growth-promoting agents were combined than when they were administered singularly. Similar results were observed (54) using rbST (Posilac) and Revalor-S (a trenbolone acetate and estrogen implant). Vann et al. (55) observed that rbST alone administered to creep-fed beef calves increased muscle mass but did not affect satellite cell number or concentration of myosin light chain-1f mRNA. The increased muscling appeared to be the result of a greater distribution of FG fibers, which possess larger cross-sectional areas than the other fibers (56). G. Growth Hormone Releasing Factor Somatocrinin, often called growth hormone releasing factor (GRF) or growth hormone releasing hormone (GHRH), is a peptide hormone belonging to the glucagon family of the gut. Effects on growth of the administration or manipulation of GHRH are likely to be due to direct effects on the secretion of ST. Exogenous GRF administration increases concentrations of ST in serum of meat animals (57). Long-term administration of GRF has been shown to stimulate growth in rats and in humans by increasing both the secretion and synthesis of pituitary ST (58). H. Somatostatin Somatostatin, another hormone produced by the hypothalamus, acts directly on the adenohypophysis of the pituitary gland to inhibit ST release (20). Endogenous ST secretion could be enhanced by GRF agonists or somatostatin antagonists. Enkephalins, also hypothalamic peptides, stimulate ST release, and it is likely that enkephalin agonists could also be used to enhance endogenous secretion (59). Although somatostatin was originally purified from the hypothalamus, it is now recognized that many cells and tissues throughout the body secrete this peptide. One of these networks releases somatostatin to affect ST release (60). Several studies [reviewed (61)] used somatostatin antibodies to attempt to neutralize circulating levels of somatostatin in blood and promote growth by increasing blood ST concentrations. Although this technique appeared to be an attractive possibility for manipulating growth, immunization against somatostatin did not consistently stimulate whole body growth. Lack of a response may result from the multitude of sources (cells and tissues) that secrete somatostatin. Immunization against somatostatin has led to other strategies based on immunization techniques that may neutralize or amplify hormonal signals via receptors for hormones.

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Passive (62) and active (63) immunization against somatostatin markedly increased serum concentrations of ST. As a result of the increased concentration of ST, researchers set out to determine if immunization against somatostatin would improve growth in carcass lean:fat ratio. As much as a 22% improvement in ratio of gain and 13% improvement in efficiency of gain in lambs was reported (64). However, no significant effect on carcass composition was realized. This would be expected if the effect were mediated via ST or ST receptors (65). I. Beta-Adrenergic Agonists The discovery of -adrenergic agonists, which are chemical analogues of epinephrine, norepinephrine, and catecholamines, is a promising development in growth-promotion applications in animals. Included in this class of compounds are the following: clenbuterol, cimeterol, ractopamine, L-644,969 and L-640,033, and isoproterenol. -adrenergic agonists are orally active and thus may be administered in the feed. Many are chemically stable and extremely potent, making successful development of implants for cattle, sheep, and swine likely. -agonists improved live weight gain (15%) and feed conversion efficiency (15%) in meat-producing animals in research trials. Carcass protein (muscle) content is substantially increased (25%) while carcass fat is decreased (30%). These changes were observed in intact and castrated males and females. Unlike the effects of anabolic steroids, the effects of -agonists were not dependent on sex of the animal. They represent an altered pattern of metabolism such that nutrients are directed or partitioned away from adipose (fat) tissue and directed toward lean (muscle) tissue. For this reason, the term “nutrient repartitioning agents” is commonly applied to adrenergic agonists. Mechanisms (Table 4) by which -agonists influence growth have been reviewed (66–68). Changes in carcass lean content is primarily the result of hypertrophy (increased cell size), rather than hyperplasia (increased cell number). -agonists appear to exert their effects on skeletal muscle by reducing degradation rate without altering the rate of protein Table 4 Biological Effects of -Adrenergic Agonists on Adipose and Skeletal Muscle Tissue Tissue Adipose tissue

Effect

Physiological process affected

↓a

Glucose uptake Glucose oxidation unaffected Lipid synthesis Lipogenic enzyme activity Insulin stimulation of glucose metabolism Basal lipolysis Catecholamine-stimulated lipolysis Insulin inhibits stimulation of lipolysis Insulin binding unaffected Somatotropin binding unaffected Protein degradation Protein synthesis Satellite cell proliferation

↓ ↓ ?b ↑c ↑ ↑

Skeletal muscle (growth)

↓ ↑ ↑

↓
decreases. ?
questionable. c ↑
increases. a b

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synthesis. Muscle tissue is continually being degraded and resynthesized in animals. Nitrogen retention in skeletal muscle is increased by -agonists yet decreased in skin/hide and visceral organs. This decrease in nitrogen retention of non-carcass components, together with reduced fat deposition, accounts for the increased carcass yield in animals treated with

-agonists. Hypertrophy of cross-sectional area of skeletal muscle fiber types is typical of all red-meat-producing animals treated with -agonists. Stimulating increased rates of lipolysis (fat breakdown) and decreased rates of lipogenesis (fat synthesis) reflect decreased carcass fat deposition (69). Additionally, the supply of energy available for fat synthesis may be reduced in treated animals, both because an increased proportion of dietary energy is used for protein synthesis and because the -agonists elicit a general increase in metabolic rate. Growth-promoting -agonists were found to reduce plasma levels of insulin and somatomedin-C, but did not elevate plasma ST levels. This suggests the concept that growthpromoting -agonists work directly through skeletal muscle cell receptors and not indirectly through the elevation of plasma ST or insulin concentrations (69). A critical factor in the usefulness of -agonists is likely to be the degree to which their effects are retained after withdrawal, because a recommended withdrawal period is likely to be required. Growth rates decline following withdrawal. Effects on the carcass are not as rapid but the advantages are not sustained after prolonged periods of withdrawal. 1. Porcine In the majority of studies, daily gain was not significantly increased by feeding of -agonists. In fact, some studies showed a depression in growth rate. The repartitioning effects of -agonists appear to increase with dose rate in pigs and result in a decrease in carcass fat. Minimal differences according to sex (gilt vs. barrow vs. boar) have been observed. Minor increases in meat toughness have been reported for pigs treated with -agonists. In a recent review, Warriss (70) concluded that -agonists fed to pigs do not promote pale, soft, exudative (PSE) meat but might lead to a greater propensity for dark, firm, dry meat. -agonists appear to have no effect on water-holding capacity and ultimate muscle pH. Ruminants appear to be more responsive than swine to -agonists. 2. Bovine and Ovine Response to treatment with -agonists has been studied in bulls, steers, and heifers as well as rams, wethers, and ewes in different trials. Responses have ranged from no response to an improvement of 48% in growth rate and feed conversion. Beef carcasses treated with agonists during the finishing phase contain less fat and more lean than controls. -agonists are very effective in repartitioning utilizable energy away from fat deposition and toward protein accretion in rams, wethers and ewes. Ruminants seem to be quite susceptible to meat toughening when treated with -agonists. Hamby et al. (71) were among the first to report that toughening of meat (lamb) occurs in -agonist–treated animals. Shear force in the longissimus muscle increased 114% in treated lambs. Although treated lambs had less carcass fat, Hamby et al. (71) concluded that some factor other than cold-shortening was involved in the 114% increase in toughness as a result of a low r2 value for linear regression of shear force on neutral lipid content. The hypothesis that reduced tenderness is due to reduced protein degradation has been considered (68). Collagen and its respective cross-linking is a major determinant in the texture (tenderness) of cooked meat (72). If -agonists cause an increase in growth by reducing protein degradation, this would allow the collagen molecules more time to cross-link, (72).

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The increase in cross-linking would enhance the toughness independently of postmortem proteolytic degradation. Beermann et al. (73) demonstrated a significant (as much as 70%) reduction of M calcium dependent proteinase (CDP) activity in skeletal muscle of lambs fed a -agonist for 3 or 6 weeks. This reduction was synonymous with a dose-dependent increase in shear-force values of treated lambs.

IV. TRANSGENIC ANIMALS Development of recombinant DNA technology has enabled scientists to isolate single genes, analyze and modify their nucleotide structures, make copies of these isolated genes, and transfer copies into the genomes of livestock species. Such direct manipulation of genetic composition is referred to as genetic engineering, and the term transgenic animal denotes an animal whose genome contains recombinant DNA. The dramatic achievements in molecular biology during the past decade and the development of micromanipulation for early-stage embryos provided the combined capabilities for introducing cloned genes into the mouse genome in 1980 (74,75). The transfer of genes was immediately recognized as an important scientific achievement. However, the subsequent creation of a “super” mouse by the transfer of a rat somatotropin gene provided the convincing evidence that demonstrated the potential offered by gene transfer (76). At the Ohio University (Athens), transgenic mice carrying a modified version of the bovine somatotropin gene that originally created the “super” mouse were found to produce “mini” mice (approximately half the size as the controls). Modifying a somatotropin gene and incorporating it into mouse DNA that in turn prevents stimulating growth lends itself as a powerful tool for probing the hormone’s function. This suggests that somatotropin does more than promote growth. The first U.S. patent for a transgenic animal, a mouse expressing a foreign oncogene created by Harvard University researchers, was issued in 1988. The issue of that patent triggered intense criticism from animal rights activists, ethicists, and environmentalists. As a result, the government did not issue any further patents. Environmentalists and others argue that there are dangers in releasing transgenic animals into the environment, as well as ethical issues that have not been fully explored. Scientists had been struggling with practical problems involved in transferring genes from one animal to another. Scientists inserting genes from a variety of different species, for example the pig, have encountered significant problems. One unresolved major difficulty is the inability to insert genes precisely into animals’ DNA, the building block of genetics. Instead, the gene is inserted into the nucleus of a cell with the hope that the gene lands in an appropriate location. In addition, many pigs develop severe health problems, e.g., ulcers, pneumonia, arthritis, cardiomegaly, dermatitis, and renal disease. The successful use of genetic engineering to enhance carcass composition and the efficiency of meat production in livestock depends on many factors. These include identification, isolation, and modification of useful genes or groups of genes that influence meat quality and quantity. Control of the time and level of expression of the inserted genes in transgenic animals so that their health status is either improved or not diminished affects the successful insertion of these genes into the genome. The progress and methods of transferring genes in farm animals was published in the 1995 ACS Symposium Series—Genetically Modified Foods: Safety Issues (77). Recently, Pursel et al. (78) reported on the successful expression of insulin-like growth factor–I (IGF-I) in skeletal muscle of transgenic swine. The general health of the IGF-I transgenic and control pigs did not differ in physical appearance, be-

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havior, ability to tolerate summertime heat stress, or reproductive capacity. These types of problems were observed for the transgenic pigs expressing bovine growth hormone (79). In April 1997, a Scottish scientist, Ian Wilmut, successfully cloned a fully grown sheep. Named Dolly, the sheep represented the first time DNA from a mammal had been used to produce an exact genetic replica. The Roslin Institute research team in Edinburgh took DNA from single mammary cells from a 6-year-old ewe, inserted it into fertilized eggs, then implanted the eggs in 13 ewes, one of which became pregnant and gave birth to the cloned lamb, Dolly. This method proved that mature cells other than reproductive cells contain DNA capable of programming regeneration of an entire animal. In August of 1997, a Wisconsin-based company, Deforest, successfully cloned a Holstein bull calf. In June of 1999, researchers at the University of Connecticut announced the birth of a cloned calf from an adult farm animal using cells from the ear of the adult, not the reproductive organs. Similarly, scientists in Hawaii announced the successful cloning of mice using tail cells from adult males, again the successful exhibition of cloning without using reproductive organs/cells. With regards to the cloning of farm animals, most of the research focuses on state-of-the-art science and cutting-edge methodologies, technical improvements, and current progress toward producing transgenic animals for medical and agricultural applications. To date, the effects of cloning farm animals on carcass and meat composition and quality have not been investigated. A tremendous amount of variation in carcass components, such as muscle development, fat content distribution, tenderness, and flavor, exists among and within breeds of each species. Animal breeders have successfully utilized selection from this genetic variation to improve farm animals for many years. Unfortunately, the quantitative genetic approach has yielded few clues regarding the fundamental genetic changes that accompanied the selection of animals for superior carcass attributes. Few single genes have been identified that have major effects on carcass composition. A national effort to map the genes of meat animals is under way. In cattle, the double-muscle gene is responsible for muscle hyperplasia and hypertrophy and for enhanced lean tissue deposition. In sheep, the callipyge mutated gene is responsible for muscle hypertrophy and enhanced lean tissue accretion. In pigs, the halothane sensitivity gene (Hal) is associated with increased yield of lean meat and porcine stress syndrome. Pigs homozygous for “Hal” are susceptible to stress and have a high incidence of pale, soft, exudative (PSE) meat. These genes offer considerable potential for investigation of carcass composition in meat-producing animals. However, except for the Hal gene, which has been identified as a single mutation in the ryanidine receptor gene, the specific product of each gene remains to be identified. A. Swine with Growth-Related Transgenes A number of transgenic pigs containing various ST transgenes have been raised (80). Production of excess ST in transgenic animals caused multiple physiological affects but did not result in “giantism” as was expected based on the earlier production of “super” mice as described (76). However, transgenic pigs that have excess ST levels exhibited numerous unique carcass traits. Reduced carcass fat, alteration of muscle fiber profiles, thickening of the skin, enlargement of bones, and redistribution of major carcass components occurred in transgenic pigs. Some of these effects are similar to those observed after daily injections of pST; others are considerably different (Table 4). Possibly, these differences are the consequence of continual presence of excess ST in the transgenics whereas injections of pST provide a daily pulse of excess ST.

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1. Carcass Composition and Meat Tenderness The first transgenic animals for which carcass and meat quality was evaluated were the TbST pigs at the USDA-ARS-Beltsville, MD, research facility. Carcass fat was dramatically reduced (Fig. 3) in transgenic pigs that expressed a bST transgene at five different live weights (81). This difference in fat became greater among transgenic and non-transgenic littermates as the pigs approached market weight. Total cholesterol content of ground carcass tissue of bST transgenic pigs was not different from sibling control pigs at any of the designated weights. However, as body weight increased from 14 to 92 kg, the cholesterol content decreased for both groups of pigs. Analysis of fatty acids showed that carcasses of bST transgenic pigs consistently contained less total (expressed as g/100 g tissue) SFA than sibling control pigs at each body weight. These differences in SFA were primarily a result of reductions in palmitic, stearic and myristic acid. Both myristic and palmitic acids have been reported to be hyperlipidemic and hypercholesterolemic in humans. Consumption of hypercholesteremic fatty acids by humans has come under attack by health professionals in the United States. Carcasses from bST transgenic pigs contained less total MUFA and PUFA fatty acids than sibling control pigs. Similar observations (Fig. 2) were found for ToST (transgenic pigs with an ovine somatotropin gene). Carcasses and lean tissue from transgenic pigs (Figs. 1 and 2) had near the optimum ratio of 1:1:1 for SFA:MUFA:PUFA, as recommended (82). When carcasses were separated into the four primal (pork) cuts, the hams of the bST transgenic pigs were significantly larger and the loins were significantly smaller than those of the sibling control pigs (83). The intramuscular fat for each primal cut (lean portion only) showed large differences between bST transgenic pigs and the controls. In spite of these dramatic reductions of fat throughout all primal cuts, evaluation of tenderness by shearforce determination indicated there were no significant differences between the two groups of pigs for the longissimus (loin) muscle (Table 3). Although somatotropin is considered the primary growth-promoting hormone in mammals, many of its effects are thought to be mediated by insulin-like growth factor-I (IGF-I). Transgenic pigs have been produced using a skeletal -actin regulatory sequence to direct expression of an IGF-I gene specifically in skeletal muscle (78). The underlying

Figure 3 Carcass lipid accretion in control and transgenic pigs from 14 to 92 kg.

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rationale was to initiate a paracrine response with IGF-I to enhance muscle development without altering the general physiology that might occur from an endocrine response. Founder T pigs were mated to non-T pigs to produce G1 transgenic and sibling control progeny. In comparison to sibling controls, T females and intact males had less carcass fat (9.9% and 8.1%, respectively), and more muscle (8.6% and 3.6%, respectively) (78). In a follow-up study, using barrows and gilts (84), IGF-I transgene pigs had larger (34%) loin eye areas and heavier (range 9–24%) muscle weights of five major muscles of the carcass (84). Neither average daily gain nor feed efficiency differed for T and control pigs. T and control pigs did not differ in general appearance, and no gross abnormalities, pathologies, or health-related problems were encountered as was observed for transgenic pigs with somatotropin genes. Thus, enhancing IGF-I specifically in skeletal muscle had a positive effect on carcass composition of swine. Shear force values for control pigs was 6.52 kg and 6.42 kg for IGF-I T pigs (84). 2. Muscle Morphology Morphological evaluation (Table 3) of bST transgenic-pig skeletal muscles revealed bST transgenic pigs had fewer SO fibers and more FOG fibers than control pigs. The population of FG was similar for the transgenic and control pigs; however, the classical porcine fiber arrangement with SO fibers grouped in clusters surrounded by FOG and FG fibers was less evident in the transgenic muscle (85). Morphological fiber profiles for T-bST pigs resembled that of bovine muscle rather than porcine muscle. Hypertrophied (giant) fibers, which were identified in pST-injected pigs, were not observed in bST transgenic pigs (Table 3). The shift in the percentage of SO fibers to FOG fibers in the bST transgenic pigs has not been identified in pigs that have received daily injections of pST. Muscle fiber growth patterns in bST and oST transgenic pigs differ markedly from that seen in muscle of pigs injected daily with pST. All three fiber types are enlarged in pST-treated pigs, whereas in bST transgenic pigs, only SO fibers appear to hypertrophy during growth compared to controls. In the T-oST pigs, both the SO and FG fibers hypertrophy similar to controls during growth, whereas the FOG fibers remain much smaller than controls (Table 3). No giant fibers were observed in muscle tissue from bST transgenic pigs. Even though bST transgenic pigs were highly stress sensitive, there were no signs of pale, soft, exudative meat. The IGF-I transgene pigs from the Pursel et al. (78) study as reported in Bee et al. (86) exhibited an increase in FG fibers and a decrease in FOG fibers. All fibers increased in size, with the hypertrophic response being greatest for the SO fibers, followed by the FOG and FG fibers. The IGF-I transgene pigs from the Eastridge et al. (84) study showed that there was no difference in fiber type percentages between the T pigs and controls, but that the increase in muscle mass was due to an increase in muscle fiber area (hypertrophy) for all three fiber types. Bee et al. (86) also reported that IGF-I transgene expression altered the distribution of slow and fast isomyosin forms. 3. Bovine and Ovine with Growth-Related Transgenes To date, producing transgenic cattle by microinjection of DNA into pronuclei is inefficient and extremely costly, in large part due to the cost of maintaining numerous pregnancies to term. Many pregnancies result in non-transgenic progeny. The success rate in both bovine and ovine is significantly less than that for swine. No carcass data are available for transgenic bovine or ovine. The cloning technology described by Wilmut in 1997 has introduced

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the successful cloning of cattle and sheep, however, these research programs have not looked at carcass data as well. V. CONCLUSIONS The potential for manipulation of growth and composition of farm animals has never been greater than at present because of the wide array of strategies for altering the balance between lean and fat. Recent discoveries of repartitioning effects of somatotropin, select adrenergic agonists, as well as the variety of growth-promoting agents, and gene manipulation techniques offer a wide range of strategies. Although progress is being made, much more needs to be accomplished. Eating quality and safety must not be sacrificed as leaner animals are developed. We are still a long way from fully understanding the integrated mechanisms resulting from manipulation of growth and carcass composition and possible effects on meat quality (either positive or negative) as a result of the techniques described in this chapter. REFERENCES 1. 2. 3.

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RC Vann, TG Althen, WK Smith, JJ Veenhuizen, SB Smith. Recombinant bovine somatotropin (rbST) administration to creep-fed beef calves increases muscle mass but does not affect satellite cell number or concentration of myosin light chain-1f mRNA. J Anim Sci 76:1371–1379, 1998. RC Vann, TG Althen, MB Solomon, JS Eastridge, EW Paroczay, JJ Veenhuizen. Recombinant bovine somatotropin in (rbST) increases size and proportion of fast-glycolytic muscle fibers in semitendinosus muscle of creep-fed steers. J Anim Sci 79:108–114, 2000. WM Moseley, LF Krabill, AR Friedman, RF Olsen. Administration of synthetic human pancreatic growth hormone-releasing factor for five days sustains raised serum concentrations of growth hormone in steers. J Endocrinology 104:433–439, 1985. RG Clark, G Chambers, J Lewin, ICAF Robinson. Automated repetitive microsampling of blood: growth hormone profiles in conscious male rats. J Endocrinology 111:27–35, 1986. EM Convey. Strategies to increase meat yield and reduce fat/cholesterol. In: Proceedings of 6th Biennial Symp. American Academy Vet. Pharm. and Therapeutics on Animal Drugs and Food Safety, 1988, pp 27–32. GS Tannenbaum. Physiological role of somatostatin in regulation of pulsatile growth hormone secretion. In: Advances in Experimental Medicine and Biology, Vol. 188, Somatostatin, eds. YC Patel and GS Tannenbaum, New York: Plenum Publishing Corp., 1985, pp 229–259. GSG Spencer. Hormonal manipulation of animal production by immunoneutralization. In: Control and Manipulation of Animal Growth, ed. PJ Buttery, DB Lindsay, NB Haynes. London: Butterworths, 1986, pp 279–291. W Vale, C Riveir, M Brown. Regulatory peptides of the hypothalamus. Annual Review Physiology 39:473–527, 1977. MA Varner, SL Davis, JJ Reeves. Temporal serum concentrations of growth hormone, thyrotropin, insulin, and glucagon in sheep immunized against somatostatin. Endocrinology 106:1027–1032, 1980. GSC Spencer, GJ Garssen, IE Hart. A novel approach to growth promotion using auto-immunization against somatostatin. I. Effects on growth and hormone levels in lambs. Livestock Production Sci 10:25–37, 1983. GSC Spencer, GJ Garssen, PL Bergstrom. A novel approach to growth promotion using autoimmunization against somatostatin. II. Effects on appetite, carcass composition and food utilization in lambs. Livestock Production Sci 10:469–477, 1983. JP Hanrahan, JF Quirke, W Bomann, P Allen, JC McEwan, JM Fitzsimons, J Kotzian, JF Roche. -agonists and their effects on growth and carcass quality. In: Recent Advances in Animal Nutrition, eds. W Haresing, DJA Cole. London:Butterworths, 1986, pp 125–138. PEV Williams. The use of -agonists as a means of altering body composition in livestock species. Nutr Abstr Reviews 57B:453–464, 1987. A Moloney, P Allen, R Joseph, V Tarrant. Influence of beta-adrenergic agonists and similar compounds on growth. In: Growth Regulation in Farm Animals. Adv. in Meat Research, Vol. 7, eds. AM Pearson, TR Dutson. New York: Elsevier Appl Sci, 1991, pp 455–513. LA Muir. Effects of beta-adrenergic agonists on growth and carcass characteristics of animals. In: Designing Foods, National Res Council, Washington, DC, 1988, pp 184–193. PD Warriss. The influence of -adrenergic agonists and exogenous growth hormone on lean meat quality. Proceedings Brit. Soc. Anim. Prod. winter meeting, 1989, p 52. PL Hamby, JR Stouffer, SB Smith. Muscle metabolism and real-time ultrasound measurement of muscle and subcutaneous adipose tissue growth in lambs fed diets containing a -agonist. J Anim Sci 63:1410–1421, 1986. AJ Bailey. Connective tissue and meat quality. Proceedings of 33rd International Congress of Meat Sci and Technology, 1987, p 152. DH Beermann, SY Wang, G Armbruster, HW Dickson, EL Rickes, JG Larson. Influences of beta-agonist L-665,871 and electrical stimulation on postmortem muscle metabolism and tenderness in lambs. Proceedings of 42nd Annual Reciprocal Meat Conf. 42:54, 1989.

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Solomon RL Brinster, HY Chen, ME Trumbauer, MK Yagle, RD Palmiter. Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proceedings of National Academy Sci USA 82:4438–4444, 1985. RD Palmiter, RL Brinster. Germ-line transformation of mice. Annu. Rev. Genet. 20:465–478, 1986. RD Palmiter, RL Brinster, RE Hammer, ME Trumbauer, MG Rosenfeld, NC Birnberg, RM Evans. Dramatic growth of mice that develop from eggs microinjected with metallothioneingrowth hormone fusion genes. Nature 300:611, 1982. KH Engel, GR Takeoka, R Teranishi. Genetically Modified Foods: Safety Issues. ACS Symp. Series #605, 1995. VG Pursel, RJ Wall, AD Mitchell, TH Elsasser, MB Solomon, ME Coleman, F DeMayo, RJ Schwartz. Expression of insulin-like growth factor-I in skeletal muscle of transgenic swine. In: Transgenic Animals in Agriculture, eds JD Murray, GB Anderson, AM Oberbauer, MM McGloughlin, CAB International. 1999, pp 131–144. VG Pursel, CA Pinkert, KF Miller, DJ Bolt, RG Campbell, RD Palmiter, RL Brinster, RE Hammer. Genetic engineering of livestock. Science (Wash DC) 244:1281–1288, 1989. VG Pursel, CE Rexroad Jr. Status of research with transgenic farm animals. J Anim Sci 71(Suppl. 3):10–19, 1993. MB Solomon, VG Pursel, EW Paroczay, DJ Bolt, RL Brinster, RD Palmiter. Lipid composition of carcass tissue from transgenic pigs expressing a bovine growth hormone gene. J Anim Sci 72:1242–1246, 1994. National Research Council. Designing Foods. Animal Product Options in the Market Place. Washington, DC: National Academy Press, 1988. MB Solomon, VG Pursel. Partitioning of carcass components of transgenic pigs. Proceedings of 40th International Congress of Meat Sci and Technology, S-VII.01, 1994, pp 11–17. JS Eastridge, MB Solomon, VG Pursel, AD Mitchell. Response of porcine skeletal muscle enhanced by an IGF-I transgene. Proceedings Annual Reciprocal Meat Conference, 1999, p 125. MB Solomon, NC Steele, TJ Caperna, VG Pursel. A further look at the effects of growth hormone on morphological muscle characteristics in pigs. Proceedings of 37 th International Congress of Meat Sci and Technology (1), 1991, pp 497–501. G Bee, MB Solomon, VG Pursel. Expression of an IGF-I transgene on skeletal muscle morphology in swine. FASEB J 11:A415, 2405, 1997.

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7 Microbiology of Meats DOUGLAS L. MARSHALL and M. FARID A. BAL’A Mississippi State University, Mississippi State, Mississippi

I. INTRODUCTION II. MEAT CONTAMINATION AND DECONTAMINATION III. MEAT AS A SUBSTRATE FOR MICROBIAL PROLIFERATION IV. MICROBIOLOGY OF FERMENTED AND CURED MEATS V. MEAT-ASSOCIATED FUNGI VI. MEAT-ASSOCIATED PARASITES VII. MICROBIAL MODELING VIII. SUMMARY ACKNOWLEDGMENTS REFERENCES

I. INTRODUCTION Contamination of sterile animal muscle used as food is a direct consequence of slaughtering and dressing of animal carcasses. Wide ranges of microorganisms from different sources are introduced onto moist muscle surfaces that are rich in nutrients. It is argued that only a small portion (10%) of these microorganisms is capable of survival and proliferation during storage, distribution, and retail sales of meats. Additionally, an even a smaller portion will eventually predominate and cause spoilage. Survival and proliferation of microorganisms deposited on meat surfaces depends on their ability to withstand processing and storage conditions and to utilize available nutrients in the muscle through assimilation or proteolysis of complex molecules into readily utilizable substrates. Over the years, efforts to preserve meats have focused on retarding microbial growth or killing selected contaminants by applying chemical, physical, or biological treatments whose net outcome should slow or prevent the growth of spoilers or allow harmless fermentative microorganisms to predominate. In either case, successful treatments extend product shelf life and allow its delivery from farm/processor to remote consumption areas.

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Microflora of meat products available to consumers at the retail level is a reflection of the environment in which they were processed and the conditions under which they were stored. From a numerical standpoint, using the per capita meat consumption of 88 kilograms (2,109) and an average microbial load of 10,000 colony forming units (CFU) per gram, a family of four annually brings into their home nearly 1 billion microorganisms associated with raw meat products. The nature and composition of viable microorganisms associated with consumer health risks and associated economic impact on the meat industry vary with the nature of the animal, processing, packaging, storage, and handling conditions. Meats can acquire a large variety of pathogenic and spoilage microorganisms during primary and further processing (Table 1). Pathogens can include Clostridium perfringens, Staphylococcus aureus, Salmonella spp., pathogenic Escherichia coli, Campylobacter spp., Yersinia enterocolitica, Listeria monocytogenes, and Aeromonas hydrophila (44,49,112). Meat pathogens can cause self-limiting human enteric diseases or systemic and fatal infections of the immunocompromised, the elderly, and the young. Spoilage of meats is largely dependent on initial microbiological quality and subsequent storage conditions. Pseudomonas spp. predominate in chilled air–stored meats, (38) Enterobacteriaceae in temperature-abused meats, (83) lactic acid bacteria and Micrococcaceae in meats packaged with preservatives (82,88) and Brochothrix thermosphacta in vacuum- and modified atmosphere–packaged products (118). Gill (42) reviewed the potential sources of meat contamination during slaughter and butchering of food animals. Animal health, hide, viscera, feces, oral microflora, and carcass handling are all potential sources of cross-contamination of sterile muscle during dressing operations. Ultimately, the microbiological quality of dressed carcasses relies a great deal on the skill level of operators during dressing operations, in particular skinning and evisceration, more so than on physical facilities or the types of stock slaughtered (87). Several decontamination approaches have been proposed to enhance the microbiological quality and safety of dressed carcasses (24). Treatments with organic acids (30), hot water (56), steam pasteurization, and steam carcass vacuuming (122) have been implemented in some processing plants. Bacterial species vary widely in their susceptibility to decontamination treatments, with 2 to 3 log population reductions possible (24,26). Determining the microbiological quality of dressed animal carcasses requires obtaining samples for laboratory analysis. Classical sampling methods have included excising a meat surface portion (16,80) or swabbing a defined surface area delimited by a template (57) to determine microbial loads per unit area. Obtained samples are either suspended and homogenized in a diluent and bacterial counts determined directly by spread plate, pour plates, hydrophobic grid membrane filtration (HGMF), or indirectly by ATP bioluminescence or impedance measurements (51,58). According to Gill and Jones (51), HGMF can enhance detection sensitivity to 1 CFU/100 cm2. For routine microbiological monitoring of hygienic quality of carcasses, USDA-FSIS mandates combining samples obtained from three sites of a given carcass and determining E. coli counts (127). In any event, carcass sampling remains a technique and its usefulness in predicting microbiological quality or microbial profiles depends on obtaining a true representative sample in high volume operations (27,79,119). From a practical standpoint, it is impossible to monitor dressed carcasses for all potential pathogens and spoilage microorganisms they may harbor. This consideration limits meat microbiological monitoring to either total aerobic plate counts or to E. coli counts as an indicator of fecal contamination (127). The latter approach is currently favored in view that total aerobic plate counts bear no indication to the potential presence of pathogens (117). However, the use of E. coli counts, or other potential indicator organisms, shows that no single indicator organism is effective for all types of foods (14).

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Consumers view quality aspects of meats as being good taste, tender, juicy, fresh, lean, healthy, and nutritious. However, cross-cultural differences exist in what is designated as fresh meat (60). The butcher in most developed countries may no longer be the guarantor of meat freshness, quality, and safety because supermarket chains supplied from major processors dominate the meat processing and retailing business. This chapter will address the microbiology of meat products with emphasis placed on the roles that processing, storage, and retailing exert on the microflora. II. MEAT CONTAMINATION AND DECONTAMINATION The microbiological profile of meat products presented to consumers is the sum total of slaughtered animal health, conditions under which it was reared, quality of slaughtering, processing, packaging, and conditions under which the meat was stored. Table 1 lists the common genera of microorganisms found on fresh, processed, and vacuum-packaged meats. Gill (42) reviewed the potential sources of meat contamination during slaughtering and butchering of food animals. Animal health, hide, viscera, feces, oral microflora, and carcass handling are all potential sources of cross contamination of sterile muscle during dressing operations. With cattle and sheep, the major source of initial meat contamination is the animal hide or fleece (6,7,42,62). These sources are exposed to soil, feces, water, and oral microorganisms during animal rearing. It would seem logical that cleaning the hide or fleece before dressing should reduce the number of potential contaminants; however, several studies have failed to confirm this point (9,10,128). On the other hand, scalding treatments applied to pigs destroys gram-negative bacteria, leaving predominantly gram-positive bacteria as survivors (39). Subsequent pig carcass dehairing reintroduces gram-negative bacteria from accumulated detritus and contaminated recirculated water (12,44,49). Animal hides not only introduce spoilage bacteria such as Pseudomonas, Acinetobacter, and Moraxella but also may introduce potential pathogens such as C. perfringens, S. aureus, Salmonella spp., E. coli, Campylobacter spp., Y. enterocolitica, L. monocytogenes, and A. hydrophila (44,49,112). Foodborne pathogens of animal origin can cause human gastroenteritis, and in high-risk populations they can lead to systemic and sometimes fatal infections (8). Other potential risks of meat contamination involve the potential transfer of antibiotic-resistant microorganisms to dressed meats and their ability to subsequently transfer this resistance to other microorganisms in the human gut (102). Evisceration is another critical step where operator skill is required to avoid spilling fecal matter onto skinned carcasses. It has been shown that bacteria deposited on carcasses during eviscerating operations predominantly originate from the mouth and the anus (50). Ultimately, the microbiological quality of dressed carcasses heavily relies on the skill level of operators during skinning and evisceration, more so than on processing plant physical facilities or the type of animal slaughtered (87,90). Little information exists on the impact of misprocessing events on microbiological quality of dressed carcasses. Gill (42) advocated treating common mishaps, such as spilling of gut contents onto carcasses or contamination introduced by operator handling, as events that necessitate special treatment. Accidentally contaminated carcasses should be flagged and detained for extra trimming and washing to remove visible contamination. Gill and Jones (50) showed that carcass pasteurization combined with a modified dressing process that prevents contamination from animal mouth and viscera can produce dressed pig carcasses with average E. coli counts of 1 per carcass and total counts of 2 CFU/cm2. Enhancing the microbiological quality of

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Table 1 Genera of Microorganisms Commonly Found on Meats Type of meat

BACTERIA Achromobacter Acinetobacter Aeromonas Alcaligenes Alteromonas Bacillus Brochothrix Campylobacter Carnobacterium Citrobacter Clostridium Corynebacterium Enterobacter Enterococcus Escherichia Flavobacterium Hafnia Janthinobacterium Klebsiella Kluyvera Kocuria Kurthia Lactobacillus Lactococcus Leuconostoc Listeria Microbacterium Micrococcus Moraxella Paenibacillus Pantoea Pediococcus Proteus Providencia Pseudomonas Psychrobacter Salmonella Serratia Shewanella Staphylococcus Vibrio Weissella Yersinia

Gram Rxn

Fresh

                                          

X XX XX X X X X X X X X X X XX X X X X X X X X X X X X X XX X X X X X XX XX X X X X X X

Processed

Vacuum packaged

X X

X X

X X X

XX XX

X X X

X X XX

X X

X XX X X X X

X X XX X X X

X X

X

X X

X

X

X

X X X

X

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X X

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Table 1 Continued Type of meat Gram Rxn YEASTS Candida Cryptococcus Debaryomyces Hansenula Pichia Rhodotorula Saccharomyces Torulopsis Trichosporon MOLDS Acremonium Alternaria Aspergillus Aureobasidium Botrytis Chrysosporium Cladosporium Fusarium Geotrichum Monascus Monilia Mucor Neurospora Penicillium Rhizopus Scopulariopsis Sporotrichum Thamnidium

Fresh XX X X X X X

Processed

Vacuum packaged

X XX

X XX X X X X X

X

X XX X

X XX X XX X X XX X X XX XX XX

X X X X X XX X X X

X
known to occur, XX
most frequently isolated. Source: Dillon (25); Garcia-Lopez et al. (35); Jay (78).

dressed carcasses entails recognizing microbial hazards (12,91) and designing strategies to limit their spread to muscle foods during carcass dressing. Following dressing, carcasses are split and knife trimmed to remove visible contamination and bruised tissue (126). Studies of trimming practices have indicated that they are purely of aesthetic value and do not contribute to enhancing microbiological quality of dressed carcasses (42,43,57). Furthermore, trimming causes loss of meat and requires extra operator time and effort without necessarily improving microbiological quality (112). The requirement to meet pathogen reduction performance standards has prompted a number of studies in which antimicrobial intervention strategies have been compared. Washes with chemicals have been most advocated. Considerable literature exists on the antibacterial efficacy of dilute solutions of organic acids, hydrogen peroxide, chlorine, chlorine dioxide, and organic acid salts (63,93,108,110). Overall, such interventions can result in reduction of contaminants by 1 to 2 logs (24). In addition to benefiting meat safety, these

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Figure 1 Time required for spoilage development in frankfurters held at different refrigeration temperatures. (Data from Ref. 130.)

interventions can also improve meat quality. For example, a reduction in the numbers of psychrotrophs by 4 logs can greatly increase the refrigerated shelf life of frankfurters (Fig. 1). Studies that have addressed meatborne pathogens have shown varying susceptibilities to such treatments. Campylobacter jejuni and Y. enterocolitica are among the most susceptible pathogens to acid treatment (92,94), E. coli O157:H7 and L. monocytogenes are more resistant (13,108,120). Smulders and Greer (123) reviewed other prospects of organic acid decontamination and resultant adverse effects on muscle foods. Using organic acids as a decontaminant in abattoirs has been recommended (127); however, a primary concern to meat exporters is that European Union countries do not allow for product decontamination treatments with anything other than potable water (123). The use of hot water to decontaminate dressed carcasses has been studied with beef (15,23), sheep (28), pork (56), and buffalo (113). A typical outcome of carcass washing was the reduction of microbial loads by 2 logs with hot water treatment (80°C for 10 sec) (122). Another possible outcome of all wash operations is the uniform redistribution of contaminants from heavily soiled areas to the whole carcass (42). Hot water washes largely have not been adopted commercially in view that large volumes would be needed to uniformly heat a carcass surface. Moreover, economic reasons would impose recirculating hot treatment water, but sanitary concerns would not entertain such cost reductions (42). Alternatively, steam pasteurization has been advocated as a more viable option (19,29,97); however, to ensure adequate carcass surface heating, supra-atmospheric pressures are needed, which entails the need for specialized containment chambers (19). In commercial applications, steam is applied for 6.5 sec without appreciable sensory degradation and results in 2 log and 1 log reduction in E. coli and total aerobic counts, respectively (29,97,106). Steam pasteurization enriches growth of gram-positive bacteria on meats while reducing the more thermally susceptible gram-negative enteric pathogens (42). A less expensive alternative to steam pasteurization chambers involves the use of hot water or steam vacuum hand-held wands. Selected carcass areas can be treated by this method to remove visible contamination. A favorable report exists on the steam-vacuum approach to treat artificially contaminated meats (29). Use of this method in a commer-

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cial setting was advocated as an alternative to cleaning soiled carcass areas and to reduce the need for subsequent trimming (29). Following the dressing process, carcasses are transferred to chiller rooms. Rapid cooling before the development of rigor results in toughening of the muscle and subsequent loss of meat quality (125). On the other hand, slow cooling ensures rigor and allows for spoilage and pathogenic bacteria to proliferate (42). Commercial chilling of dressed carcasses use chill tunnels that rely on blasts of cold air (48) or spraying of carcasses with chilled water (55,59). It has been proposed that very fast carcass cooling can ensure muscle tenderness and microbiological quality (81); however, major capital spending would be required to implement fast cooling systems in abattoirs (42). Another consequence that accompanies air chilling is drying of the carcass surface by the reduction of surface moisture. Typically a 0.5 log microbial count reduction is observed as a consequence of surface drying (96). Alternative techniques have desiccated the carcass surface with dry heat (18). The major objection to carcass drying is the accompanying carcass weight loss (39). Accordingly, many abattoirs in North America have adopted intermittent spraying with water during the first hours of cooling to avoid weight loss (42). Gill (42) proposed an explanation for the observed reduction in Gram-negative and E. coli counts on spray-cooled carcasses. His twofold mechanism was that (a) spray cooling washed away surface bacterial loads and (b) surface freezing reduces gram-negative bacteria due to their greater susceptibility to freezing. Offal or organ meats are collected in bulk containers during carcass dressing. The geometry of their storage adversely affects subsequent chilling. Product on the periphery of storage containers cools faster than product in the center of the bulk container (42). Even in freezers of high cooling capacities, product at the centers of large containers will cool more slowly (5), thus providing greater opportunity for microbial proliferation. Offal meat pH is usually  6.0, which implies that the major hurdle in restraining microbial growth would be temperature and potential anaerobic conditions produced at the center of bulk containers (45,47). Enhancing the storage potential of offal can been achieved by vacuum or CO2 packaging to noticeably extend shelf life up to several weeks (46). However, efforts by the meat industry to ensure the microbiological quality of offal will remain proportional to their economic value on the market. Hot boning involves breaking down an animal carcass immediately after dressing as boxed manufacturing meats or for further processing into comminuted, cured, or cooked products. Boxing and stacking of hot boned meats in a cooler suffers similar limitations as the earlier described chilling of offal (111). However, temperature control is possible using ice or liquid carbon dioxide (42). Studies are needed to elucidate the effects of rigor development on the proliferation of microorganisms in hot-boned meats (42). Following chilling, carcasses are either broken down into primal cuts or transported to other processing plants for further processing. In either event, personnel hygiene and proper sanitation of equipment and work surfaces are prerequisites to ensure microbiological quality and safety of processed meat products. Visible cleanliness of equipment and work surfaces although advocated (127) may not be a true reflection of sanitary conditions unless coupled with microbiological or bioluminescent assays to ensure efficacy (34). Important sources of microbial contamination of processed meat products include detritus accumulated in inaccessible parts of equipment that subsequently contaminate products that come in touch with these surfaces (69,70). Under these circumstances, bacterial biofilms flourish due to repeated applications of water and the constant availability of nutrients from accumulated detritus. In cooled work environments, cold-tolerant (psychrotrophic) pathogens such as L. monocytogenes, Y. enterocolitica, and A. hydrophila can flourish

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(49,107). Ensuring the quality of processed meats entails making sure that biofilms do not persist day to day on work surfaces and equipment (131). Processed meats and primal cuts await their distribution in coolers. With raw meats, low storage temperature and low pH (5.5) due to rigor are the only two hurdles to slow bacterial proliferation. At all times, meat temperatures should not exceed 7°C to prevent pathogenic mesophiles from proliferating (11). Consequently, storage and transport of meat at chilled temperatures lower than 7°C is regarded as safe, although some pathogenic psychrotrophs can proliferate at this temperature (42,103). Limiting psychrotroph growth and further extending shelf life can be achieved by lowering the temperature of boxed meat to 1.5°C without obvious ice formation (54). At temperatures of 0°, 2°, and 5°C, chilled meat will have a storage life of 70%, 50%, and 30% of the storage life of meat stored at 1.5°C (42). Alternatively, meats can be frozen for extended periods of time; however, the microbiological concern for frozen meats requires that temperatures be maintained below 5°C, the minimum growth temperature of yeast and mold fungi (42,84,85). Although chill temperatures inhibit microbial growth, pathogens may survive for prolonged periods in meats even though they do not multiply. The microflora of meats available to consumers is the total sum of microorganisms acquired during processing of animal muscle food. Animal health, dressing skills, personnel hygiene, abattoir cleanliness, and adequate storage and holding temperature during distribution and retail influence the constitution and number of microorganisms present (17,72). It is unlikely that a single intervention can fully enhance quality and safety of meats (42). Rather, consumer education (1) coupled with an integrated approach, which ensures better understanding and optimization of each processing step (41), are more likely to enhance the future quality and safety of meats available to consumers. III. MEAT AS A SUBSTRATE FOR MICROBIAL PROLIFERATION Proliferation of microorganisms in meats is dependent on several factors, which include microflora composition, product temperature, previous product treatments, pH, available nutrients, oxidation-reduction potential, and the atmosphere surrounding the product. Many of these factors are not constant throughout the shelf life of a meat product. Understanding the influence of these factors on microbial growth and survival and the impact on meat spoilage has been greatly aided by the pioneering work of Ingram and Dainty (20,21,74,75). More recently, Nychas et al. (101) reviewed the subject of chemical changes associated with stored meats. Common defects of meats and associated bacteria are shown in Table 2. Microbial proliferation in meats occurs in the aqueous phase surrounding the product. This phase is rich in substrates readily utilizable by almost all microorganisms (98). Frozen (12°C) meats prevent the growth of contaminating microorganisms but allow for their abundant survival during storage. Spoilage of thawed meats is due to the number and type of microbes present before freezing and the time/temperature conditions of the product during thawing. Thawed meats are often more perishable than fresh meats because of the abundance of drip containing readily utilizable substrates for microbial metabolism. Although the concentrations of carbohydrates (primarily glucose and glycogen) are low in the aqueous phase in comparison to proteins, available concentrations are sufficient to support massive initial microbial proliferation (40,100). After glucose is depleted, microorganisms start using amino acids for energy and as a result produce volatile compounds that are responsible for spoilage odors (31,32,124). Many bacteria, including pseudomonads, produce ammonia during amino acid metabolism, which is a major cause of pH inCopyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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Table 2 Common Defects of Meats and Causal Bacteria Defect

Meat product

Slime

Meats

H2O2 greening

Meats

H2S greening H2S production Sulfide odor Cabbage odor Potato odor Putrefaction Bone taint Bone taint Pocket taint Internal taint Souring

Vacuum-packaged meats Cured meats Vacuum-packaged meats Bacon Ham Ham Whole meats Bacon Bacon Ham Ham

Bacteria Pseudomonas, Lactobacillus, Enterococcus, Weissella, Brochothrix Weisella, Leuconostoc, Enterococcus, Lactobacillus Shewanella Vibrio, Enterobacteriaceae Clostridium, Hafnia Providencia Burkholderia, Pseudomonas Enterobacteriaceae, Proteus Clostridium, Enterococcus Proteus, Vibrio Vibrio, Alcaligenes, Proteus Providencia Lactic acid bacteria, Enterococcus, Micrococcus, Bacillus, Clostridium

Source: Garcia-Lopez et al. (35); Gardner (37); Jay (78).

crease in spoiling meat products (101). Gram-negative bacteria predominate during aerobic spoilage and are generally responsible for the production of putrid and sulfury odors (35). The amino acids cystine, cysteine, and methionine are precursors for hydrogen sulfide, methylsulfide, and dimethylsulfide. Amino acid decarboxylation of lysine yields putrescine. Increasing putrescine levels correlate with increasing pseudomonad counts in meats. Decarboxylation of ornithine or arginine yields cadaverine. Increasing cadaverine levels correlate with increasing Enterobacteriaceae counts (22). It has been noted that pseudomonads preferentially deaminate amino acids, whereas Enterobacteriaceae preferentially decarboxylate (89). Bacteria, other than pseudomonads, responsible for malodorous volatile compounds include Shewanella (Alteromonas) putrefaciens, Proteus, Citrobacter, Hafnia, and Serratia (89). The expression of meat spoilage odors from the degradation of amino acids can be delayed by the addition of glucose to meats. The presence of glucose delays the utilization of amino acids by spoilage bacteria and their subsequent development of sensory spoilage characteristics (4,115). Lactate is another low molecular weight component utilized by meat microflora under both aerobic and anaerobic conditions. Lactate is generally utilized after glucose is depleted and can be used in similar strategies to retard spoilage (116). Indeed, according to Gill (40), as long as readily utilizable low molecular weight substrates are available, meat proteolysis is inhibited. Microbial metabolism in chilled air-stored meats is primarily oxidative (101). Aerobic gram-negative bacteria are the common cause of spoilage of meats stored at 4°C, with Pseudomonas species predominating. For example, P. fragi, P. fluorescens, and P. lundensis are the dominant species on beef, lamb, and pork (35). Acinetobacter, Psychrobacter, Moraxella, and psychrotrophic Enterobacteriaceae such as Hafnia alvei, Serratia liquefaciens, and Enterobacter agglomerans also occur but their numbers remain low relative to the dominant pseudomonads. Meats packaged under vacuum or modified atmospheres demonstrate a shift from a diverse flora to one that is predominated by lactic acid bacteria and Brochothrix thermoCopyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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Figure 2 Hypothetical development of off-odor and slime in meat due to psychrotrophic bacterial growth during storage under various atmospheres at 1°C. (Data from Refs. 3 and 95.)

sphacta (68). The impact of this shift in microflora is shown in Fig. 2. As illustrated, the time to develop off odors and slime is greatly extended when oxygen is removed from the headspace of packages. Dairy/cheesy odors of meat stored in gas mixtures with carbon dioxide are primarily due to diacetyl, acetoin, and alcohols produced by B. thermosphacta from glucose fermentation (21). End products of B. thermosphacta metabolism differ with the gaseous atmosphere composition. When oxygen tension is low (2 M oxygen), L-lactate, ethanol, and propanol are the main metabolic end products (101). Accordingly, ethanol and propanol could be used as spoilage indicators of meat stored under vacuum of modified atmosphere (98,101). Future prospects of meat substrate and bacterial metabolite studies could point the way to analytical techniques that could assess meat spoilage without resorting to time-consuming microbiological analysis. According to Jay (77), metabolite-based spoilage detection should ensure that (a) the spoilage indicator is not normally present in the food, (b) the indicator concentration should increase with storage time, and (c) the concentrations of the indicator should reflect the most predominant microorganisms and correlate with sensory quality. IV. MICROBIOLOGY OF FERMENTED AND CURED MEATS Comminuted raw meats fermented into various sausages requires the aid of salt, nitrate/nitrite, and desirable fermentative lactic acid–producing bacteria. When properly fermented, pathogens and spoilage bacteria will be eliminated or greatly reduced in numbers, which yields products with superior shelf-life attributes. In addition, desirable sensory properties are achieved. For example, cured red/pink color, robust flavor, and firm texture are unique to this product type (68). Growth and acid production of lactic acid-forming bacteria are promoted by pH below 6.0, water activity (aw) of 0.96 due to salt (2.5 to 3.0%), 100 ppm sodium nitrite, and 0.3% glucose. When stuffed into casings, the redox potential of the sausage is reduced,

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which further enhances activity of the lactic acid bacteria (68). Despite the wide variety of fermented meats around the world and various technical differences during processing, the bacteria that tend to dominate in naturally fermented sausages are usually Lactobacillus sake and Lactobacillus curvatus (61,73,114). Predominance of these two species is based on their ability to grow at reduced aw (0.91) and low temperature (4°C). Other psychrotrophic lactics (Carnobacterium spp., Leuconostoc spp. and Weissella spp.) are either less halotolerant (except Weissella halotolerans) or grow poorly below 7°C (Lactobacillus pentosus, Lactobacillus plantarum, Pediococcus acidilactici, and Pediococcus pentosaceus) (86). Many traditional fermentations use nitrate as the curing agent. As such, the presence of nitrate-reducing micrococci (Micrococcus varians, Staphylococcus carnosus, Staphylococcus xylosus, or Staphylococcus piscifermentans) is necessary to form nitrite, which is needed for proper quality (color) and safety (antibotulinal activity) (67,68,99). Because the reliability of natural fermentations is occasionally less than desired, most industrial processing of fermented sausages use domesticated starter culture bacteria. These starters are either single or mixed strains of homofermentative lactic acid bacteria. When nitrate is used, micrococci also are included (67,86). Commonly used lactics include L. sake, L. curvatus, L. plantarum, L. pentosus, and P. pentosaceus (P. cerevisiae) (33,66,86,129). Strains are selected primarily for their ability to rapidly acidify, accelerate ripening, and improve color intensity and stability at the fermentation temperature desired (Table 3) (64,65). Spoilage microflora in fresh sausages generally are similar to those found in ground meat. Type of meat, presence of preservatives, and storage temperature and atmosphere will determine the predominant microbes (35). Like fresh meats, products in air-permeable packaging will have pseudomonads predominating during low temperature storage and Enterobacteriaceae during higher temperature storage. Presence of these bacteria on fully cooked products is the result of post-heating contamination, usually during casing removal, slicing, and subsequent handling during packaging. Because of the facultatively anaerobic nature of most Enterobacteriaceae, they tend to predominate in vacuum-and modified atmosphere–packaged products stored at high temperatures (10°C) (35,104). However, this group competes poorly with lactic acid bacteria under proper chill storage conditions (12). Similarly, gram-negative bacteria usually will not spoil fermented sausages, dried meats, and canned meats (35). Bacteria that are resistant to salt and low aw, nitrite, and fermentation and ripening temperatures will be selected for in fermented sausage products. Many of the same microorganisms are found on cured unfermented products such as hams and bacon. For ex-

Table 3 Lactic Acid Bacteria Used as Starter Cultures for Fermented Sausages at Various Processing Temperatures Bacteria type

Process temperature (°C)

Bacterial species

Thermophiles

30–38

Mesophiles

20–25

Psychrotrophs

15–20

Pediococcus acidilactici Pediococcus cerevisiae Lactobacillus pentosus Lactobacillus plantarum Lactobacillus sake Lactobacillus curvatus

Source: Holzapfel (68).

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ample, Micrococcaceae, lactic acid bacteria (Carnobacterium and Leuconostoc), Vibrio, Enterobacteriaceae, and other gram-negative bacteria (Psychrobacter, Acinetobacter, and Proteus) are the major microbial groups found on cured products (12,35,37). Among the halophilic (salt loving) vibrios, Vibrio costicola is a common slime former (36). V. MEAT-ASSOCIATED FUNGI Carcasses aged at very low temperatures (5°C) can have surface defects caused by molds (Table 1). For example black, white, blue-green, and whisker spots may be evident (25). Cladosporium cladosporioides, Cladosporium herbarum, Penicillium hirsutum, and Aureobasidium pullulans were identified as causative agents of black spot (52,53); Chrysosporium pannorum and Acremonium sp. caused white spots (85). Blue-green spots were associated with Penicillium corylophilum and whisker spots were caused by Thamnidium elegans and Mucor racemosus (85). In addition to bacteria, several molds have been responsible for cured meat spoilage. Low aw and presence of oxygen selects for molds from the genera Aspergillus, Alternaria, Fusarium, Mucor, Rhizopus, Botrytis, and Penicillium (25,78). Some dry-cured products, such as European sausages, Italian salami, and country-cured hams, can support prolific growth of aspergilli and penicillia. There is some speculation that the characteristic flavors of these products are due in part to the presence of these fungi (25,78,105). Like many molds and bacteria, psychrotrophic yeasts are capable of growing on meats during refrigerated storage (Table 1). Most yeast-associated meat spoilage occurs when the product has been treated in such a manner to reduce the level and activity of contaminating bacteria. Such treatments usually include low pH by acidification or low aw by salting, drying, or freezing (25). In fresh meats, however, yeasts generally are unable to compete with bacteria because of their slower growth rates. As such, their numbers remain low in proportion to bacterial counts. Candida spp. are the predominate yeast isolated from raw meats (25,71). Spoilage caused by yeasts is typically related to slime formation on products such as dried sausages, wieners, cured hams, and salami. Debaryomyces spp. and Candida spp. are the predominate yeasts found in processed meats (25). VI. MEAT-ASSOCIATED PARASITES Several microscopic animal parasites may be harbored in meats (Table 4). Parasitic protozoa, flatworms, and roundworms associated with meat animals can be infectious to consumers. Unlike bacteria and fungi, the parasites do not grow in foods but are merely transported either intramuscularly or as surface contaminates. Surface contamination can come from contact with feces or more commonly from use of contaminated water supplies. Fortunately, most parasites are easily killed by proper cooking (80°C internal temperature) and handling of products. Long-term freezing (10°C for 30 days) or salting of meats also has been shown to inactivate many of the parasites (78). Among protozoa, the coccidian Toxoplasma gondii may cause toxoplasmosis in individuals consuming raw or undercooked meats from cattle, pigs, sheep, and goats (76,121). Sarcocystis hominis and Sarcocystis suihominis may be transmitted by consumption of raw beef and pork (78). Cryptosporidium parvum has been linked to ingestion of leftover beef tripe (78). The flatworm Fasciola hepatica is infrequently found in beef livers. Most other flat-

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Table 4 Parasites Commonly Associated with Meats Parasite

Genus

Meat

Protozoa

Toxoplasma Sarcocystis Cryptosporidium Fasciola Taenia Trichinella

Beef, pork, sheep, goat Beef Beef Beef liver Beef, pork Pork

Flatworms Tapeworms Roundworms Source: Jay (78).

worms are distributed in fish rather than meat animals (78). The common tapeworms Taenia saginata (Taeniarhynchus saginatus) and Taenia solium can cause mild human illness in consumers eating raw or undercooked beef and pork, respectively. Of more severity is the illness trichinosis, caused by consumption of raw or undercooked pork containing the roundworm Trichinella spiralis. This illness can occasionally be fatal (78). VII.

MICROBIAL MODELING

Predictive modeling of microbial growth and survival in meats has become an increasingly important tool in studying the behavior of spoilage and pathogenic microorganisms under different environmental conditions. Predicting the impact of intrinsic (nutrients, pH, salt, nitrite, etc.) and extrinsic (atmosphere, temperature) factors on microorganisms can help processors and regulators determine optimum conditions needed for enhanced quality and safety. Two computer-based modeling programs are available: Food Micromodel (Leatherhead, Surrey, UK) and Pathogen Modeling Program (USDA, Wyndmoor, PA). Example outputs from the USDA model are illustrated in Figs. 3 through 5. Figure 3 shows predicted growth potential of E. coli O157:H7 under specified atmosphere, temperature, pH, salt, and nitrite conditions. Figure 4 demonstrates the predicted amount of time needed to achieve a 3 log or greater reduction in the numbers of L. monocytogenes at pH 3.5, 16.9°C, and 0.5% NaCl. Figure 5 illustrates the predicted survival of S. typhimurium at 0°C with increasing gamma irradiation doses. VIII. SUMMARY The numbers and types of microorganisms found on meats are determined by the environment under which the animals were raised and processed, and the meat packaged and stored. With current technology it is nearly impossible to produce sterile meats without excessive thermal or irradiation processing. That said, proper animal husbandry, workplace sanitation, and processing will produce edible meats with acceptable microbial numbers and low or no human pathogens. However, it must be expected that raw meats will contain potential human pathogens. In addition, with the possible exception of canned meats, most processed meats will have a limited shelf life that is dictated by the quantity and kinds of spoilage microorganisms on the product. Meat processors have the expectation that farmers and feedlots will provide animals for slaughter that are safe for meat production. Like-

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Figure 3 Predicted aerobic growth of Escherichia coli O157:H7 using the USDA Pathogen Modeling Program.

Figure 4 Predicted low pH inactivation time needed for Listeria monocytogenes using the USDA Pathogen Modeling Program.

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Figure 5 Predicted irradiation inactivation curve for Salmonella typhimurium using the USDA Pathogen Modeling Program.

wise, consumers have the expectation that meat products available for consumption are safe and wholesome. Failure by the meat industry to meet these expectations frequently leads to microbial problems and ultimately decreased consumer demand. Therefore, understanding the quantity and nature of meatborne microorganisms remains a critical issue for long-term viability of the industry. ACKNOWLEDGMENTS The USDA Pathogen Modeling Program ver. 5.1 was developed by R. L. Buchanan, Ph.D., R. C. Whiting, Ph.D., and A. R. Pickard, Ph.D., at the Microbial Food Safety Research Unit of the USDA/ARS Eastern Regional Research Center in Wyndmoor, PA. REFERENCES 1. 2. 3. 4. 5. 6. 7.

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8 Meat Safety DANIEL Y. C. FUNG, MAHA N. HAJMEER, CURTIS L. KASTNER, JUSTIN J. KASTNER, JAMES L. MARSDEN, KAREN P. PENNER, RANDALL K. PHEBUS, J. SCOTT SMITH, and MARTHA A. VANIER Kansas State University, Manhattan, Kansas

I. INTRODUCTION A. Current Status of Meat Safety B. Meat Irradiation C. Dietary Supplements D. Genetic Modification E. Consumers’ Knowledge and Practices II. HISTORY OF MEAT INDUSTRY SAFETY A. Current Status of Meat Consumption B. Early Developments of Meat Safety C. Food Safety and Government Regulations III. MICROBIOLOGICAL HAZARDS ASSOCIATED WITH MEATS A. Meat Microbiology B. Microbiological Intervention Strategies C. Rapid Methods and Automation in Microbiology IV. CHEMICAL HAZARDS ASSOCIATED WITH MEATS A. Pesticide Residues B. Hormone Disruptors C. Antibiotic Residues D. Chemicals from Production or Processing V. PHYSICAL HAZARDS ASSOCIATED WITH MEATS: IDENTIFICATION AND CONTROL VI. CURRENT REGULATORY POLICIES AND INSPECTION A. Concepts of Hazard Analysis Critical Control Points (HACCP) B. Operational Steps in HACCP

* This material is based upon work supported by the Cooperative State Research, Education, and Extension Service, United States Department of Agriculture, under Agreement no. 93-34211-8362. Contribution no. 00-193-B from the Kansas-State Agricultural Experiment Station.

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Potential for Recall of Meat and Poultry Products USDA Policy on Recalls Processes of Conducting a Recall Imported Products: State vs. Federal Programs and Agencies Involved Definitions

VII. MEAT SAFETY IN THE FUTURE A. Food/Meat Safety and Research Needs B. Domestic and International Meat Safety in the Future and Meat Safety Standards VIII. SUMMARY REFERENCES

I. INTRODUCTION A. Current Status of Meat Safety The safety of food and meat is of major concern to consumers, processors, retailers, food service industry, government agencies, educational institutions, public health professionals, researchers, and the general public locally, regionally, nationally, and internationally. Meat safety during processing, packaging, transporting, storing, displaying, selling, cooking, serving, and eventually consumption ideally should be constantly under tight scrutiny by government officials, food processors, food handlers, food providers, and the consumers themselves. Although in developed countries food and meat usually are safe for consumption after proper preparation, many factors can lead to foodborne disease outbreaks. Some outbreaks can be mild and affect a small number of people, but others can be large and affect hundreds and thousands of people, resulting in serious short- and long-term consequences and even death. The purpose of this chapter is to examine some major issues related to meat safety. Although ascertaining the exact number of foodborne outbreaks in the world is impossible, the number may be in the hundreds of millions per year. In the United States, estimates have ranged from 1.4 million to 150 million cases per year (2). Todd (71) estimated that 12.6 million foodborne illness cases occurred per year, costing $8.4 billion. Bean and Griffin (9) reported that from 1973 to 1987, a total of 7,458 outbreaks with 237,545 cases occurred in the United States, of which 327 and 120 outbreaks were attributed to beef and poultry, respectively. A more recent report by Bean et al. (8) indicated that 2,423 outbreaks occurred and resulted in 77,373 cases from 1988 to 1992, with bacterial pathogens causing the largest percentage of outbreaks (79%) and cases (90%). Annual, national, direct and indirect costs (1993 dollars) were estimated to be $2.9 to $6.7 billion, respectively, for foodborne illnesses caused by Campylobacter jejuni or Campylobacter coli, Clostridium perfringens, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella (nontyphoid), and Staphylococcus aureus by the Centers for Disease Control and Prevention (15). The most recent estimates (September 16, 1999) by the CDC were 325,000 serious illnesses resulting in hospitalization, 76 million cases of gastrointestinal illnesses, and 5,000 deaths each year in the United States. This number of annual deaths was reduced from the 9,000 reported by CDC in previous years. Estimating the number of outbreaks and cases caused directly by meat products is difficult because sources for a large number of outbreaks were listed as “multiple vehicles” and “unknown”. For example, in 1992 the percentages of outbreaks by vehicle of transmission were beef (2.2%), chicken (1.7%); ham (0.5%); unknown meat (0.7%); turkey

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(0.7%), multiple vehicles (13.3%), and unknown vehicle (62.0%). However, there is no doubt that meat accounts for many foodborne disease outbreaks and cases annually. The most sensational outbreak related to meat was the Escherichia coli O157:H7 outbreak attributed to undercooked hamburger by a fast food chain in the Pacific Northwest in late 1992 and early 1993, which resulted in 501 cases, 151 hospitalizations, 45 cases of hemolytic uremic syndrome (HUS), and four deaths (3). This outbreak literally transformed U.S. consumers’ awareness of and concern about food safety issues and directly led to changes in government policy and regulation that resulted in improvement of safety in all areas of the food industry. This outbreak directly and indirectly stimulated the formation and active involvement in the public policy arena of consumer groups such as Safe Tables Our Priority (S.T.O.P.) and the Lois Joy Galler Foundation for Hemolytic Uremic Syndrome, Inc. Karen Penner, in a lecture entitled “Consumers and Food Safety” at the Excellence in Food Science X program held at Kansas State University on September 17, 1999, indicated that consumers’ fears about the food supply include pesticides, Salmonella, irradiation, biotechnology, growth hormones, E. coli O157:H7, lead, product tampering, and milk allergies. A survey by the Food Market Institute (FMI) (31) of consumers’ attitudes from 1994 to 1998 showed that confidence in food safety was lowest in 1994 (73%), peaked at 84% in 1996, and fell to 81% in 1998. The FMI also reported that about 70% to 75% of consumers rated product safety very important during this period. The report indicated where food safety problems occur, from highest to lowest: in processing, at restaurants, at home, during transportation, in markets, and on farms. The most important source of foodborne illness was mishandling of food, followed by “germs,” chicken, improper cooking, old food, beef, seafoods, mayonnaise, and fruit and vegetables. Telephone calls to the Kansas Department of Health and Education by consumers with questions about food safety issues increased from less than 500 in 1988 to 2000 in 1999 (Paige, S. personal communication, 1999). Current emerging issues related to consumers’ perceptions of food safety include irradiation, dietary supplements, genetically modified foods, and consumers’ practices. B. Meat Irradiation Meat irradiation appears to be imminent, especially with the use of electron beam technology rather than radioactive isotopes. The Titan Corporation Plant in Sioux City, Iowa, is on the verge of opening. The United States Department of Agriculture (USDA) has approved an irradiation level of 1.5 to 3.0 kGy for poultry (fresh or frozen), and proposed approval of maximum levels of 4.5 kGy and 7.0 kGy for fresh and frozen red meat, respectively. Internationally, 41 countries have clearances for commercial food irradiation, with meat and poultry clearances in 23 countries for different categories and different doses. Consumer acceptance of irradiated food is high. A report by the FMI and Grocery Manufacturers of America (32) showed that 60% of consumers were likely to buy irradiated meat in 1997 and 55% in 1998. John A. Fox (personal communication, 1999) reported that 80% of consumers surveyed are willing to purchase irradiated versus nonirradiated poultry, if the price is the same; 30% would pay a 10% premium; and 15% would pay a 20% premium. Thus, meat irradiation is moving in a positive mode with consumers at the present time. C. Dietary Supplements The Dietary Supplement and Health Education Act (DSHEA) of 1994 (an amended Food Additive Amendments of 1958) permits the use of vitamins, minerals, herbs or other botan-

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icals, amino acids, and other substances in foods. The safety issues involved include oversupplementation, use of botanicals not from plants typically used for food, use of hormone products, and the lack of purity of ingredients. Some of the problems include product recalls, deaths attributed to supplements containing ephedra, and reports of adverse effects. However, about 50% of the public use dietary supplements, as reported by Camire and Kantor (13). D. Genetic Modification According to Hoban (44), U.S. consumers’ acceptance of genetically modified food is as follows: 72% support agriculture biotechnologies, 90% support medical biotechnologies, 75% believe biotechnologies will result in personal family benefits, and about 66% would buy produce modified to taste better or remain fresher. Internationally, 61% of Europeans avoid products with modified ingredients. Market tests of cloned beef in Japan showed that the low price (50% lower than other beef) outweighed concerns for the new technology. Starting in April 2000 in Japan, tofu, corn snacks, and soy milk with genetically modified ingredients must have the proper labeling. Labeling of meat is under consideration. E. Consumers’ Knowledge and Practices The last hurdle in food safety is the consumer. Zhang et al. (79) identified some risky home consumption practices in Kansas: 26% of respondents to a survey canned their vegetables, 9% ate undercooked hamburger, 2% drank unpasteurized milk, and 56% consumed raw or undercooked eggs. In a national survey, Gravani et al. (41) reported that 92% of consumers were concerned about raw meat left out for more than 4 hours, 82% were concerned about cooked meat left out for more than 4 hours, 24% believed off-odor or flavor caused illness, 28% believed freezing kills harmful bacteria, and 17% did not wash their hands after handling raw poultry. Thus, consumers still need great deal more food safety education. Challenges in such education include recognizing current consumers as key players in food safety and informing them about emerging pathogens, about new technologies, and benefits and risks; stressing the need to change behavior as new knowledge is gained; and communicating to all that they are food safety educators, wherever they are in the food and nutrition system. Meat safety will be enhanced greatly by appropriate consumer education in all levels of society. II. HISTORY OF MEAT INDUSTRY SAFETY A. Current Status of Meat Consumption Meat is nutritious for humans and other living entities, such as microorganisms. Growth of undesirable microorganisms in meat and meat products may result in spoilage and foodborne illnesses. Thus, from ancient times, humans have devised ways to ensure the safety of meat mainly through religious practices. Meat is a major part of the human diet. Lupton and Cross (56) reported that U.S. per capita consumption of meat, poultry, and fish in 1990 was 191.5 lb, including 112.3 lb of red meat, 63.8 lb of poultry, and 15.4 lb of fish. Compared with similar data in 1965, the increases in consumption were 1113% for chicken, 146% for turkey, and 37% for fish, while consumption of red meat decreased by 10%. More recent data compiled by Weaber (77) in 1999 indicated that per capita consumption was 120.4 lb. for red meat and 96.7 lb.

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for chicken and turkey. Thus, consumption of red meat and poultry combined increased between 1990 and 1999 (176.1 lb. versus 217.1 lb.) Per capita spending on beef, pork, and chicken combined increased from $332.69 in 1986 to $425.57 in 1998; however, spending on beef decreased from 53.8% of the total in 1986 to 44.1% in 1998. There is no doubt that meat is the basis of a big industry in the United States and in many developed countries. For example, the volume of U.S. beef exports increased from about 170 million lb. in 1976 (valued at $110 million) to about 2,000 million lb in the late 1990s (valued at about $2.5 billion). Cattle sold by the 10 largest US companies in 1998 were worth $30 billion (77). With so much at stake, it is necessary to keep meat safe at all levels of production, processing, and sales. B. Early Developments of Meat Safety Upton Sinclair’s 1906 book (66) The Jungle is frequently labeled as the first public call to address meat safety in the United States. Although Sinclair’s book certainly did propel the tightening of U.S. food safety policy, public concern and governmental policies dealing with the safety of American food clearly were born much earlier. In fact, food safety issues were on the minds of pre-Colonial era pirates; the U.S. Army; President Abraham Lincoln; and, most noticeably, foreign importers of U.S. food products. During the 17th and early 18th centuries, pirates in the West Indies earned the name “buccaneers” for their characteristic practice of drying—or “boucaning”—beef. This practice enabled them to stock their ships with preserved, safe meat that they both consumed and sold, as reported by Price and Schweigert (65). Other preservation practices, which have beginnings dating back to 2000 B.C., have played a significant role in the history of the United States. For example, during the War of 1812, the U.S. Army purchased a disproportionate amount of its meat from New Englander Sam Wilson. Mr. Wilson, who was renowned for applying the basic food safety principles of using clean barrels and low temperature storage for his salted beef, stamped the letters “US,” for “United States,” on his barrels earmarked for sale to the Army. Interestingly, those in the army interpreted the letters to represent “Uncle Sam’s” meat, and over time, the origin of this term became obscure. Today, of course, the accepted connotation of “Uncle Sam” is the U.S. government itself (65). Formal U.S. food safety policy has its historical ties in the USDA. Indeed, if one is to comprehend the development of U.S. food safety policy, a familiarity with the history of this federal department is necessary. The USDA began as a sub-cabinet-level agency on May 15, 1862, when President Abraham Lincoln signed the enabling legislation for the department’s creation. Although President Lincoln had unsuccessfully pushed for cabinetlevel status for the department, he was nonetheless pleased that the United States now had a department to help enhance the productivity of the American farmer (45). This purpose— to make farmers more productive—was narrowly adhered to by the department during its initial years. Interestingly, however, other emphases, particularly regulation, within the department were hinted at early on in its history. Perhaps the most prophetic glimpse of a future expansion of the department’s activities came during President Lincoln’s Annual Message to Congress on December 1, 1862. Only 7 months after the inception of the agriculture department, Lincoln mentions that “some valuable tests in chemical science [are] now in progress in the laboratory” (4). Although it is certainly debatable for what purposes—to enhance productivity or food safety—such chemical tests were being conducted, the mere presence of this emphasis provided the groundwork for future food safety investigations in

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the department’s Bureau of Chemistry. In 1883, Harvey W. Wiley, the Bureau’s director, began to address public concerns regarding the widespread practice of selling colored lard as butter and other food adulteration atrocities (5). In addition to the chemical examination of foods, Wiley’s famous Poison Squad, a team of healthy young men who were fed adulterated food until their health deteriorated, was effective in arousing the public’s concern about food safety (5). One of the strongest influences on the development of U.S. food safety policy was foreign trade. In the late 1800s, European countries were beginning to voice their legitimate concerns about pleuropneumonia, tuberculosis, trichinae, and other animal diseases in U.S. livestock and meat exports (17). First among the international community to take action was England, which in 1879 required that all cattle imported from the United States be slaughtered within 10 days of importation in order to minimize the spread of pleuropneumonia. Italy and Hungary followed with trade restrictions on U.S. pork because of trichinae. Later, in 1880, Germany and Spain implemented bans on U.S. meat, as did France, Turkey, and Romania in 1881. Before the end of the 1880s, Greece and Denmark had joined the list of nations banning U.S. meat on the basis of food safety concerns (17). As a result of the significant shrinkage of willing international buyers, U.S. meat exports fell significantly. The country, having no animal or meat inspection systems in place, found itself with a huge credibility problem with food safety. This problem, felt most painfully by the meat packing and livestock industries, forced the U.S. government to inaugurate a service that would certify to foreign governments the healthfulness of American animals and the safety of its meat (17). The U.S. Congress, under great pressure from the meat packers and livestock producers, passed legislation in August 1890 to create an animal and meat inspection program (69). This piece of legislation actually was only a feeble attempt by the government to satisfy the packers and producers, and the country’s credibility problem persisted. Meanwhile, the USDA Bureau of Animal Industry, created in 1884 to conduct research on animal diseases, was priming itself for an expanded regulatory role. A marked enlargement of regulatory power was granted to the Bureau after the unable-to-export meat industry convinced Congress to establish an inspection program of real value. On March 3, 1891, the U.S. Congress added a truly substantive policy for inspecting animals and meat to the 1890 inspection law. Now the Bureau of Animal Industry clearly had both the power and budgetary authority to inspect and certify (to indicate passage of) U.S. animals and meat prior to exportation. The inspection and certification system affected offered-for-export salted pork and bacon, cattle, sheep, and swine (5). The first-ever inspection carried out as a result of the 1891 legislation occurred in New York City on May 12, 1891. The economic fruits of the legislation soon became apparent when, in September of 1891, Germany removed its restriction on U.S. pork. Later, Denmark, France, Italy, and Hungary followed by repealing their own trade bans. It was clear that the new meat and animal inspection policy had helped resuscitate exports by recapturing the respect of the international community (17). The 1890 Meat Inspection Act, after the 1891 modification and other amendments, also required inspection prior to the slaughter of cattle, sheep, and hogs that were bound for interstate trade. Postmortem inspection was to be implemented as well, but only at the discretion of the U.S. Secretary of Agriculture (17). Although it had helped reestablish international trade stability for meat and animals, the U.S. inspection program was still in its infancy and laden with problems. In 1894, Dr. D.E. Salmon, Chief of the Bureau of Animal

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Industry, complained that “The large number of abattoirs (slaughterhouses) which do an interstate trade has made it impossible up to the present time to extend the service sufficiently to include them all,” as recorded by the Department of Agriculture in 1894 (22). Although the USDA hailed the fact that only one in 5,000 cattle carried tuberculosis, it was concerned that because of meager Congressional appropriations, it was unable to inspect all meat and animals identified for interstate trade. Because of this lack of funding and the popularity of a congressionally mandated program to microscopically inspect pork (for export), officials within the USDA and the meat industry itself began to debate who should pay for inspection. Dr. Salmon at the Bureau of Animal Industry, writing on behalf of the Secretary of Agriculture, recommended the following in 1894: The Secretary of Agriculture recommends that the law providing for the inspection of export and interstate meat be so amended as to compel the owners of the meat inspected to pay the cost of the microscopic inspection. . . It is only equitable that those pay for the inspection who are directly pecuniarily benefited thereby. As the law exists today, any slaughtering establishment, no matter how insignificant, which declares it has or expects to have foreign trade in meats, has a legal right to demand governmental inspection and certification. It costs individuals nothing (22).

In 1899, the Department unsuccessfully appealed for an emergency appropriation to address anticipated needs in inspection (23). By 1905, U.S. meat inspection policy mandated both antemortem and postmortem inspections, and government and industry officials praised the program. Millions of dollars of annual foreign trade depended on the success of the meat inspection program (24), and this economic motivation fueled the program’s funding. This meat inspection program had more flaws, however. Because the policy applied only to products bound for interstate trade, a huge intrastate market of beef, pork, and lamb was not inspected. Similarly, condemned meat, although not permitted by the federal government for interstate trade, was subject only to state and municipal governance. Unfortunately, these local units of government usually were plagued by graft, corruption, and an increasingly powerful “Beef Trust” (66). At the end of the 19th century, this Beef Trust, or group of meat industry and government leaders that exploited the flaws of U.S. meat inspection policy while providing brutal labor conditions in order to achieve economic gains, was under scrutiny by the public. During the Spanish-American War, the U.S. Army had been supplied with rotten meat. Quickly, charges of graft in connection with this event were brought against members of industry and government by Theodore Roosevelt (40). The exposure of graft by the Beef Trust was best accomplished by Upton Sinclair, who, in 1904, wrote The Jungle. Although history books often label this work as a focused attack on food safety atrocities, it actually was fueled by a more broad motivation: socialism. Sinclair was hired by the socialist weekly The Appeal to Reason to investigate labor conditions in the Chicago Stockyards and provide a report. Sinclair, after spending 7 weeks amidst the brutalities of “Packingtown,” serially composed The Jungle week-by-week in The Appeal to Reason during 1904. He was unable to publish the book in its entirety until 1906. Although the primary purpose of The Jungle was to advocate socialism, Sinclair’s vivid descriptions of formaldehyde in milk, diseased meat, adulterated butter, tubercular pork, borax-coated and glycerine-filled sausage, and other public health atrocities gained the attention of the American public. Although President Theodore Roosevelt mocked Sin-

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clair for his socialist convictions, he used Sinclair’s work to push long-stalled legislation for meat inspection and pure food through Congress. These two 1906 measures, the Beveridge Amendment (now referred to as the Federal Meat Inspection Act or FMIA) and the Food and Drug Act, brought a new level of food safety assurance to the American public. In addition to this legislation, the United States was making major leaps in food safety technologies. In 1890, commercial pasteurization of milk was begun in the United States. In the same year, mechanical refrigeration for fruit storage was begun in Chicago (47). In 1910, the New York City Board of Health issued an order requiring the pasteurization of milk; this governmental mandate marked a significant moment in U.S. food safety policy. In 1939, the new Food, Drug, and Cosmetic Act became law, ushering in a new era of consumer-protection measures, while at the same time causing the Food and Drug Administration (FDA) to be removed from the Department of Agriculture despite objections from Secretary of Agriculture Henry Wallace (5). In 1958, the Food Additives Amendment was added to the Food, Drug, and Cosmetics Act. After World War II, poultry became a popular commodity, and in 1957, the federal Poultry Products Inspection Act (PPIA) was passed. This measure, like the FMIA, requires federal inspection for interstate commerce. In 1968, the FMIA and PPIA were broadened to mandate states to adopt inspection systems for their intrastate products that were “equal to” the federal inspection system. This mandate upset some states, but a provision was soon added to provide federal inspection to those states that did not have an adequate inspection program (5). The 1906 FMIA, the 1957 PPIA, and the 1958 Food, Drug, and Cosmetics Act still serve as the principal authorities for food safety policy in the United States. However, the face of the U.S. food supply has changed considerably over the past century, and new foodborne disease trends are developing. A food supply that is highly processed, shipped across the country, and imported from other countries has presented and will continue to present challenges to U.S. policy makers (33). A shift from a purely sight-smell-touch method of inspection to a prevention-based philosophy is being made in the USDA Food Safety and Inspection Service (FSIS) to address the microscopic pathogens that are causing 4,000 deaths and nine million cases of foodborne disease each year. The incorporation of Hazard Analysis Critical Control Point (HACCP) and other prevention-based policies is being implemented at present within both the USDA and the FDA. Consumers, scientists, and a significantly large portion of the food industry are embracing these new philosophies. Food safety policy in the United States continues to develop. Throughout its dynamic history, U.S. food safety policy has influenced, or been influenced by, our nation’s economic system, the voices of authors, and foreign governments. If history is any indication, future U.S. food safety policy issues will continue to be both intriguing and challenging (51). C. Food Safety and Government Regulations Consumer reaction to the 1993 outbreak of Escherichia coli O157:H7 forced a reassessment of our nation’s meat inspection system. In 1994, USDA Undersecretary for Food Safety, Michael Taylor, declared E. coli O157:H7 an adulterant in ground beef. The American Meat Institute and other trade organizations sued to block the implementation of this policy in federal court. A federal court judge upheld USDA policy, and a monitoring program for E. coli O157:H7 was instituted in 1995. Under this program, the USDA monitors samples of raw ground beef at retail stores and at grinding plants. Every positive sample results in a product recall.

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A recall at Hudson Foods in 1998 was the largest in U.S. history—25 million pounds—and resulted in dissolution of the company. After that incident, the USDA attempted to shift the responsibility for control of E. coli O157:H7 to the slaughter segment of the industry through Directive 10.010.1. Under this directive, grinding plants are exempted from USDA monitoring if their slaughter supplier employs a validated intervention technology and verifies its effectiveness through a routine microbiological testing program. In 1999, the four largest U.S. meat packers announced that they were in compliance with this directive. The focus of HACCP-based critical control points (CCPs) is to prevent, eliminate, or reduce hazards to an acceptable level. Steam pasteurization was introduced as a potential CCP for beef carcasses in 1995. In the same year, steam vacuuming was approved by the USDA for removal of physical defects from carcasses. A rule allowing for the irradiation pasteurization of ground beef and other meat products were implemented in February of 2000 by the USDA. The term pasteurization is defined by The American Heritage Dictionary as “The act or process of destroying most disease-producing microorganisms.” Technologies are under development to reduce bioload and allow for the pasteurization of meat and poultry products. Pasteurization can be achieved by chemical treatments (i.e., peracetic acid), heat (i.e., post-process pasteurization of processed meat), and irradiation with either gamma rays or an electron beam. By minimizing contamination, the irradiation pasteurization of raw meat and poultry products may be achieved at very low doses, thereby preventing undesirable quality changes. Measures to achieve bioload reduction in the live animal may include vaccines and/or the use of competitive exclusion. The objective of both the USDA and the meat industry is the elimination of pathogens from meat and poultry products. In 1995, the USDA proposed to make HACCP mandatory in all meat and poultry plants. Under this regulation, large plants were required to implement HACCP in January 1998 and small plants in January 1999. The final stages of HACCP implementation were complete in January 2000 with the inclusion of very small plants under USDA’s HACCP rule. The years following the 1993 Jack-in-the-Box outbreak will be remembered as a turning point in inspection and food safety, with the most significant achievements being the implementation of HACCP across the entire meat and poultry industry and the advent of pasteurization for raw meat and poultry products. III. MICROBIOLOGICAL HAZARDS ASSOCIATED WITH MEATS A. Meat Microbiology All living things interact with the environment that they inhabit. Therefore, the microbes on and in food animals are influenced by the surroundings in which they are reared or housed. A dirty environment with soil, mud, fecal materials, urine, stale water, insects, rodents, flies, and other animals will influence the microbial loads of the hair, hide, udder, skin, and exposed areas of the animal. Good sanitation of the environment will help reduce the microbes on the surface of the animal before transportation to slaughter facilities. The animal itself has an inherent microbial population in the gastrointestinal tract and organs. A healthy animal will have fewer pathogenic organisms, and a diseased animal will carry pathogens into the processing areas. During transportation, stress on the animals also will influence shedding of organisms into the transportation environment, for example, a truck or holding pen. In the processing area, the cleanliness of the facilities also influences the

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microbial load of the meat after slaughter and fabrication. Sticking, bleeding, and scalding operations will spread microorganisms onto and into various tissues. Fecal material from the surface of the animal or from the evisceration process will spread potential pathogens to the meat during fabrication. Contaminated water used to clean the processing facilities or wash the carcasses also can contribute to microbial contamination of the meat. Chilling, storing, aging, cutting, packing, transporting, distributing, handling, displaying, and selling of meat and meat products all can contribute to further contamination of the meat. The environment used for the preparations of raw meat for sale such as cutting, slicing, grounding, wrapping, and final presentation of the product to consumers also may add to the contamination level. Finally, at the cooking stage, improper time and temperature of food preparation may not render the meat safe for consumption. The entire “farm to table” chain can add to contamination of meat by microorganisms. Therefore food scientists, government agencies, and food processors all have responsibilities to design ways and means to minimize and eliminate these hazards and provide consumers with a safe product as detailed by Bourgeois et al. (10), ICMSF (46), Doyle et al. (26), Davies and Board (21). The contamination of carcasses and various meat cuts in terms of numbers and kinds of various spoilage bacteria, yeast, molds, pathogens, and emerging pathogens has been studied and reported extensively (48). The list of microorganisms found on meat and poultry is extensive. Frequently encountered genera include Pseudomonas, Bacillus, Brochothrix, Campylobacter, Clostridium, Escherichia, Enterobacter, Enterococcus, Streptococcus, Lactococcus, Lactobacillus, Listeria, Micrococcus, Staphylococcus, Pediococcus, Salmonella, Serratia, Yersinia, and other members of the family Enterobacteriaceae. Yeasts and molds found in meat products include Candida, Torulopsis, Saccharomyces, Rhodotorula, Mucor, Rhizopus, Penicillium, Geotrichum, and Aspergillus. The number of microorganisms on the surface of meat also varies greatly. Fung (35) developed a microbial scale to indicate spoilage potential of meat. A bacterial count on meat of 0 log to 2 log colony-forming units (CFU)/g is considered low. When the count reaches 3 log to 4 log CFU/g, it is considered intermediate. A count of 5 log to 6 log CFU/g is considered high. A count of 7 log CFU/g is considered the “Index of Spoilage,” because when the number reaches higher than 8 log CFU/g, the meat will have an odor, and at 9 log CFU/g, slime will appear. Most ground beef in supermarkets has 1 million bacteria per gram and will spoil within a week in home refrigerators. Excellent reviews on the subject were provided by Sofos (67) and Milner (57), who discussed the sources of contamination of red meat, poultry, and seafoods; types of contamination of red meat, poultry, seafoods and processed products; microbial effects on muscle foods; and control of microbial growth in muscle foods. A detailed presentation of important microbial groups in meats is provided by Douglas Marshall in the chapter on “Microbiology of Meats” in the volume. B. Microbiological Intervention Strategies A variety of intervention strategies have been used to reduce spoilage and pathogenic organisms on meat surfaces and meat products. Nutsch (61) reviewed these strategies in detail in a PhD. dissertation. The following are synopses of major intervention strategies: 1. Handling of Carcasses Hides and viscera were cited as significant sources of bacterial contamination during slaughter and processing. The slaughter environment such as walls, floors, air, and hands and garments of workers also were noted as potential sources of cross-contamination. Cor-

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rective measures include chilling carcasses as soon as possible, sanitizing knives between animals, minimizing contact between hide and skinned carcass surfaces, and general good environmental sanitation in the slaughter area. 2. Water Washing Many washing procedures have been tested with various combinations of temperature of water (35°C to 80°C), pressure (10 psi to 400 psi), contact time (4 sec to 36 sec), types of instruments, distance from the carcasses, and volume of water (e.g., 1.5 gallon, 1.5 L). Reductions of bacteria ranged from almost none to several logs, depending on the combination. The conclusion is that water washing has value in removing extraneous materials and reducing some microorganisms on the carcasses. 3. Hot Water Washing Here again, many combinations have been tested using various high temperatures such as 80°C, 85°C, or 96°C. Care must be taken to avoid discoloration of meat when using hightemperature washing. Reductions of bacteria again ranged from almost none to a 1 to 2 log CFU/cm2 reduction, depending on the time, temperature, pressure, and combinations thereof. On balance, hot water washing is more effective than cold water washing in reducing bacteria from carcasses. 4. Decontamination by Chlorine Incorporation of chlorine into water to wash carcasses has been investigated by many researchers. Chlorine levels used ranged from 20 ppm to 400 ppm, and the effectiveness is influenced by the temperature and pH of water. Reductions of microorganisms ranged from negligible to 2 log CFU/cm2. 5. Decontamination by Organic Acid Treatment A large body of research has been devoted to this form of decontamination. Acetic acid (1%, 2%, 4%, 5%) spray has been studied extensively. Reduction of bacteria seemed to be organism dependent. For example, in one study using 1% acetic acid, E. coli was reduced from 5 log CFU/cm2 to 2.2 log CFU/cm2, whereas Salmonella wentworth was reduced to 1.5 log CFU/cm2. Lactic acid at 2% or 3% also has been studied. Reduction was generally about 1 to 2 log CFU/cm2 after treatment. Some studies also combined acetic acid, lactic acid, and even propionic acid in the solution. In general, acid washes can reduce bacterial populations by about 2 log CFU/cm2 in optimum combinations. 6. Decontamination by Trimming Trimming has been used in commercial processing of meat to remove visible contaminants. Many trimming procedures for various types of tissues have been reported. Trimming is very effective in removing bacteria, because the organisms are removed physically from the area, and counts after trimming become very low. Reductions of 2 to 3 log CFU/cm2 have been reported for this procedure. Some studies also have combined washing and trimming. 7. Decontamination by Steam Pasteurization™ A commercial antimicrobial carcass intervention process called Steam Pasteurization (SPS™; Frigoscandia Food Process Systems, Bellevue, WA) is being used widely in the beef slaughter industry. This unit is a stainless steel tunnel encompassing the facility’s

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overhead rail system and is situated immediately prior to the point where carcasses enter the holding cooler (“hot box”). Carcasses enter the tunnel at normal line speeds and are exposed uniformly to saturated steam for 8–10 seconds, bringing the surface temperature up to 85–90°C. The second section of the unit applies a chilled water spray to quickly lower the surface temperature of carcasses and reduce adverse color effects. Nutsch et al. (62,63) found the system capable of reducing total aerobic bacterial counts on carcasses by approximately 1.5 log cycles from initial levels of approximately 2.5 log CFU/cm2. Coliformtype populations on carcasses were virtually eliminated. The SPS™ unit, as the last step in the slaughter phase, serves as a CCP in beef slaughter HACCP programs and is capable of continuously and automatically logging steam chamber temperature for all carcasses processed. 8. Decontamination by Steam Vacuuming Small, localized areas of visible contamination must be removed from carcasses prior to washing. These physical defects can be removed throughout the slaughter process by knife trimming and/or use of steam vacuuming. Steam vacuuming has become a standard practice in most slaughterhouses and uses a hand-held vacuum nozzle that is sanitized continuously by a steam spray. Visible contamination less than one inch in any dimension can be vacuumed from the carcass, thereby reducing yield loss from extensive trimming. Laboratory validation studies using artificially contaminated meat tissues have shown steam vacuuming to be effective in reducing microbial contamination on carcasses as reported by Dorsa et al. (25) and Phebus et al. (64). 9. Decontamination by Miscellaneous Methods Other forms of decontamination, including trisodium phosphate, ultraviolet radiation, postexsanguination dehairing, dry heat, ozone, and bacteriocins, have been used with various degrees of success. B. Rapid Methods and Automation in Microbiology Rapid methods and automation in microbiology are dynamic fields of study that address the utilization of microbiological, chemical, biochemical, biophysical, immunological, and serological methods for the study of improving isolation, early detection, characterization, and enumeration of microorganisms and their products in clinical, food, industrial, and environmental samples. In the past 15 years, food microbiologists have started to adapt rapid and automated methods in their laboratories. Fung (36–38) has provided detailed reviews on this topic. 1. Improvements in Sampling and Sample Preparation The Stomacher instrument developed by Tony Sharpe about 20 years ago has become a standard method for homogenizing food samples internationally. It involves putting food sample and diluents in a sterile bag and placing the bag in the Stomacher, which massages the food and dislodges the microbes into the diluent. Viable cell counts then can be made from the massaged sample. More recently, Tony Sharpe invented a new instrument named the Pulsifier, which can dislodge bacteria from food by pulsification in a bag. Fung et al. (39) evaluated the Pulsifier and found that it provided essentially the same bacterial counts as the Stomacher but with less food debris, which makes the sample better for subsequent microbiological manipulations such as ELISA tests or PRC tests.

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2. Alternative Methods for Viable Cell Count Procedure The conventional viable cell count method is time-consuming both in terms of operation and collection of data. Alternative methods that have been well tested in the past 10 years are the Spiral Plating, ISOGRID, 3M Petrifilm, and Redigel. All these methods have been shown to be acceptable in obtaining viable cell counts of foods and are less expensive when used routinely compared with the conventional method. 3. Instruments for Estimation of Microbial Population and Biomass The Bactometer, Malthus, and RABIT systems are used to measure impedance and/or conductance changes in food due to the growth of total microbes as well as target pathogens. Monitoring of adenosine triphosphate (ATP) to estimate microbial population and biomass has gained popularity in recent years. All living things have ATP; thus it can be used to estimate total counts in food and contamination in the environment. Currently, the trend is to monitor total ATP in the environment regardless of sources (e.g., from bacteria, yeast, mold, blood, or food particles) to ascertain cleanliness of the surfaces (i.e., hygiene monitoring). In this procedure, any amount of ATP beyond the background level will indicate contamination of the food preparation surfaces. Cleaning the surfaces properly will reduce the ATP level. 4. Miniaturized Microbiological Techniques Identification of microorganisms is an important part of quality assurance and control programs in the food industry. Miniaturized microbiological methods described by Fung (36) as well as API, Enterotube, Minitek, MicroID, IDS, and others are rapid and convenient for identifying large numbers of pathogens in clinical, food, and industrial samples. Vitek and Biolog are automated miniaturized systems that can identify clinical and environment isolates effectively. 5. Immunological Technologies Enzyme-linked immunoabsorbent assay (ELISA) tests have been very useful in the past 10 to 15 years in screening and diagnostic systems. Completely automated systems of ELISA tests such as the VIDAS system, Opus systems, Bio-tek, Detex, and others are now available. Another development is the rapid lateral migration of antigen-antibody complexes in test units for screening target organisms such as E. coli O157:H7, Salmonella, and Listeria, by kits such as VIP and Reveal. These kits provide negative or positive screening results in about 10 minutes after pre-enrichment of about 18 hr. Another exciting development in relation to immunology is immunomagnetic capture technology first developed by VICAM for Listeria and now popularized by Dynal for not only antibody-antigen reactions but also capturing of other target molecules on magnetic beads. After the capture, a powerful magnet is applied to the side of the test tube to separate these beads from the rest of the liquid matrix, thus greatly concentrating the target cells or molecules for further analysis. These methods can eliminate at least one day of detection time in many microbiological protocols. 6. Genetic-based Rapid Tests DNA and/or RNA probes have been used for more than 15 years in rapid detection of target pathogens such as Salmonella and Listeria. Polymerase chain reaction (PCR) for rapid amplification of target DNA has gained much attention recently as a rapid method for de-

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tecting target pathogens. Qualicon markets a BAX system for PCR reaction and also a Riboprinting system for characterizing subspecies of target pathogens such as E. coli O157:H7 and Salmonella. These types of genetic techniques certainly will become more important in the future. IV. CHEMICAL HAZARDS ASSOCIATED WITH MEATS A. Pesticide Residues Of all the food contaminants, pesticides probably have received the most interest worldwide. Most pesticides are acutely toxic to humans and animals; even ingestion of low levels over a long period of time can have adverse effects. Overall, very few, if any, foods are contaminated in the United States when pesticides are used according to the prescribed application guidelines. The regulation of pesticide use is handled differently by each country throughout the world, though there is a tendency toward a more unified approach. With the passage of the GATT treaty (General Agreement on Tariffs and Trade), we can expect to see a much broader approach to pesticide regulatory activities. The World Health Organization and the United Nations Environment Programme play major roles in evaluating and disseminating information on pesticide use and toxicity, as well as other types of toxic compounds. In the United States, pesticide regulation is under the auspices of three government agencies; the Environmental Protection Agency (EPA), the FDA, and the USDA-FSIS. The EPA is responsible for determining which pesticides are allowed in a particular food and what residue levels (if any) are acceptable. Pesticide residues for all foods are covered in Parts 150 to 189 of Title 40 of the Code of Federal Regulations (19). Title 40 has an alphabetical listing of approved chemicals (pesticides), a listing of approved food usages for each individual pesticide, and residual levels allowed in approved foods. In addition, it contains a listing of each commodity/food and pesticides allowed for that food. Both the FDA and USDA-FSIS (73) are responsible for monitoring pesticide residues in foods based on levels set by the EPA. The FSIS is responsible for meat and poultry products, and the FDA covers all other types of raw commodities and processed food products. Since 1995, the FDA has published on their Web site the results from yearly surveys of pesticide and chemical residues found in various food items, and that study should be consulted for specific details (30). For years, the USDA has published survey results of various chemical resides in animal products in what is known as the “Red Book.” Both the Red Book and the residue-sampling plan, known as the “Blue Book,” are available in hard copies and Web versions (74). Unfortunately, there is a lag of about 5 years between the sampling period and the date of publication, which limits its usefulness. In 1996, the Food Quality Protection Act (FQPA) became law and dramatically altered how pesticides are evaluated for human toxicity (76). The FQPA directed the US EPA to further evaluate pesticide risk for children, consider the cumulative effects of exposure to the pesticide and substances having a common mode of action, and consider the potential for endocrine-disrupting effects. In addition, the FQPA removed what has been called the “Delaney Paradox.” This was the situation in which the EPA could consider the benefit of a pesticide when approving it for use on raw food commodities, even if it was a weak carcinogen. However, if the food commodity was processed so that the levels were concentrated above the approved amounts for the original product, then the pesticide residue became a “food additive” and was governed by a different law, The Federal Food, Drug,

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and Cosmetic Act of 1954 (section 409). Under section 409, the Delaney clause prohibits the approval of any food additive that causes cancer in humans or animals. This section will discuss various pesticides, based on the organism that is to be eliminated or controlled. Particular attention will be focused on pesticide residues that continue to be associated with potential contamination of meat products. 1. Insecticides These are by far the most common chemicals used for pest control on both crops and animals. This group can be subdivided into categories based on chemical structure and mode of action. a. Carbamates. These pesticides can be either insecticides, herbicides, or fungicides and have in common a carbamic group in their structures. A variety of substitutions can occur around the carbamic group, which often will determine both the degree of toxicity and potential use. In recent years, carbamate residue has not been considered a problem in meat products. b. Organohalides (halogenated hydrocarbons). Strictly speaking, many classes of pesticides contain halogens, especially chlorine, and can be grouped in this category. The organohalide pesticides have a vast array of different structures but usually have at least one ring substituent, contain chlorine, and are extremely stable. Examples of this class include aldrin, chlordane, dieldrin, endrin, heptachlor, and DDT. This class of pesticides emerged around the time of World War II. The discovery was heralded as a major breakthrough in the control of various types of insects worldwide (20). The now-infamous DDT was introduced about that time and was critical in the control of malaria-bearing mosquitoes. It is probably the least toxic of the organohalides and was routinely applied directly to people, their living quarters, and water supplies. In the 1970s, it and similar pesticides were banned in the United States and most countries because of its bioaccumulation through the food chain, especially in predatory birds such as the bald eagle. The United Nations Environment Programme at its meeting held in Geneva in September 1999 addressed the worldwide control and production of these type of persistent organic pollutants (POPs) (72). Use of almost all of the POPs will be phased out with a few exceptions. There are still concerns that no current substitutes exist for DDT, which currently is produced only in China, India, Mexico, and Russia. It remains the most effective pesticide for use in the control of malaria-bearing mosquitoes and is considered essential in Africa and other tropical regions. The organohalides are neurotoxins and are noted especially for their persistence in the environment. Although many have been banned for years, they are still detected readily in the environment and fatty tissue of many animals, including humans. The major concerns with these pesticides are their potential teratogenicity (toxicity to fetuses), endocrine disruption, and carcinogenicity. c. Organophosphates (OPs). These represent a large class of organic compounds with a variety of uses as herbicides, fungicides, acaricides, and most notably insecticides. They were synthesized first in the 1800s, but the insecticidal properties were not discovered until the early 1900s. The German scientist Gerhard Schroder, who synthesized many of the early OPs, was instrumental in the practical synthesis steps for parathion, which is still in use today. The OPs can be divided into about 15 subgroups depending on the types of elements bound to the core phosphorus atom (16). True organophosphates contain the phosphate group with various ester linkages to organic substituents.

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The OPs are used on a wide variety of crops, grains, and food animals, such as poultry and cattle. As with carbamates, their toxicity and mode of action are associated with the irreversible inhibition of acetylcholinesterase. Because of fairly rapid breakdown, the OPs do not accumulate in fatty tissues or the environment (unlike organohalides). Many of the chlorinated OPs are strictly regulated, and, thus, many meat products are monitored by USDA-FSIS. d. Synthetic pyrethroids. The pyrethroids are synthetic insecticides modeled after pyrethrins, which are natural constituents of the flowers of certain chrysanthemums (60). Pyrethrins are the oldest known insecticides, with their use dating back to ancient China and the Middle Ages in the Persia region. Because the natural pyrethrins are not very stable when exposed to air and sunlight, most commercial pyrethrin-type insecticides are synthetic derivatives containing halogens, primarily chlorine and fluorine. The pyrethroids, like most other insecticides, are neurotoxic to insects. The major advantages of this group of insecticides are their low toxicity to humans and animals and their action against a wide variety of insects. However, they do cause a “burning” type of skin irritation, which may explain the origin of the name. The pyrethroids are approved for a wide variety of crops, including many fruits and vegetables (14). They also are used for pest control on pets and farm food animals. Pyrethroids have replaced many of the organohalides and organophosphates and are now found commonly in household insecticidal products. B. Hormone Disruptors 1. Polychlorinated Biphenyls (PCBs) The PCBs constitute a group of industrial chemicals that have good stability to chemical and thermal breakdown and are nonflammable. As with many of the organohalogen pesticides, they are considered POPs, and an effort is under way to bring about a worldwide ban. All PCBs typically contain two to nine chlorine atoms and two phenyl groups. Many different isomers exist because of the possible arrangement of the chlorine atoms around the phenyl rings. Prior to their production being halted in most countries in 1974, PCBs were used in electrical transformers, in electronic parts, and as flame retardants. Production was limited when toxicity was discovered. Until then, little effort was made to control disposal of waste containing PCBs. As a consequence, trace amounts are found in soil, water, and animals in various parts of the world. Other than a severe skin rash termed chloracne, exposure to high levels of PCBs has not caused any problems in adults. However, in two major epidemics in Japan in 1968 and Taiwan in 1978, people ingested rice cooking oils containing high levels of PCBs (1,000 ppm or greater) and developed a variety of conditions such as chloracne, liver disorders, fatigue, and nausea. Some smaller children exposed to the contaminated oils had delayed neurological and cognitive functioning (20). Based on this and other studies, the FDA designated PCBs as unavoidable environmental contaminants and set tolerances at 0.2 to 2.0 ppm for residues in many food products. 2. Polychlorinated Dibenzo-p-dioxins (PCDDs) Dioxins and dioxin-like compounds are environmental contaminants that are fat soluble and chemically stable. Dioxins originate from combustion of chlorine-containing organic compounds. Sources of exposure include industrial and municipal incinerators and com-

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bustion of leaded gasoline, diesel fuel, and wood. Dioxins also are by-products of chlorine bleaching of paper and pulp and are known to be present in the leachates from certain hazardous waste sites. They exhibit high toxicity and carcinogenicity in animal models and thus merit a considerable amount of concern for human public health. The dioxins are somewhat related to the PCBs and have been grouped with them by some research workers because of their hormone-mimicking properties. This group of chemicals contains two main categories of structurally similar, yet distinct, compounds: the polychlorinated dibenzo-dioxins and furans. The compounds can contain anywhere from 2 to 8 chlorine (or bromine) halogens, which give over 200 possible isomers or congeners. The compound 2,3,7,8 tetrachlorodibenzodioxin (abbreviated 2,3,7,8-TCDD or just TCDD) often is referred to in the lay press as dioxin. This is a misnomer, because many different types of dioxins exist. Although dioxins are present in the environment in very small amounts (parts per trillion), their known carcinogenicity and estrogen-like action are causes for concern. The estrogen-like activity is of special interest, because the potential to target many different genes can disrupt cell functions. Possible effects include disruption of the reproductive system in the developing fetus, immune system malfunction, and neurological disorders. Until recently, no major cases of food contamination with dioxins have occurred. That changed in the spring of 1999, when significant contamination was found in dairy products, eggs, chickens, baked goods, and some pork and beef products produced in Belgium. The contamination was so extensive that by June essentially all of these product were banned from worldwide trade including in the United States (see “Chemical Contaminants” at the U.S. FDA’s Web site (29). The original source of contamination appears to have been the addition of a technical mixture of PCB containing dioxins (formerly used as transformer oil) with an 80,000 kg batch of animal fat. The contaminated fat then was used to formulate 1.4 million kg of animal feed mix, which was distributed throughout Belgium and, in some cases, France and the Netherlands. Thus, many of the animals fed the feed produced in early 1999 showed levels of contamination 100 to 700 times higher than the U.S. legal limit of 1 ppt. Regulatory officials were able to identify the contaminated food items/products, and they were destroyed, but at great financial loss. The passage of the Food Quality Protection Act and amendments to the Safe Drinking Water Act in 1996 required the EPA to develop a screening and testing program for endocrine disruptors and to implement testing by August 1999. The agency has collected data and public comments on dioxin environmental contamination and appropriate testing procedures. It is important to note that dioxins are not the only endocrine disruptor–type compounds. Most of the restricted chlorinated hydrocarbons (chlordane, DDT, aldrin, heptachlor, and endrin) and PCBs also posses endocrine activity. Even though these compounds are no longer used (at least in Europe, the United States, and Canada), they still persist in the environment. The final report of the Endocrine Disruptor Screening and Testing Advisory Committee was made available on the EPA’s Web site in August 1998 (75). Estimates indicate that 50% to 90% of daily exposure to dioxins originates from food, primarily fish, meat, and dairy products, so efforts are directed at minimizing contamination from these sources. C. Antibiotic Residues A considerable number of drugs, including antibiotics, are regulated closely by the FDA (20). Antibiotics have a wide variety of toxic affects, including potential teratogenicity and

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mutagenicity. Some of them, especially in the penicillin family, can cause hypersensitivity reactions, which may be life-threatening in susceptible individuals. Because residue levels are seen only in food animals, the USDA-FSIS is responsible for monitoring them. In the past, antibiotics were used extensively to treat various animal illnesses. However, they are now used more selectively for therapeutic and disease-prevention purposes. Some antibiotics are fed to animals in subtherapeutic doses because they increase feed efficiency, i.e., enhance weight gain per amount of feed. All drugs are regulated so that only trace amounts are allowed in muscle foods, usually range below ppm (20). In many cases, the drugs must be withdrawn from the animals for a defined period before they are taken to market. Major issues have developed over the use of antibiotics in animal feed and/or treatments and how this practice contributes to the generation of antibiotic-resistant strains of bacteria. As noted in a recent review, the occurrence of antibiotic-resistant strains of pathogenic bacteria has become a worldwide problem in the treatment of human infectious diseases (54). Though the use of antibiotics in animals is not entirely to blame for resistance problems, a growing body of evidence suggests that use of subtherapeutical level of antibiotic drugs in feed is a major contributor. Thus, within the past several years, world regulatory agencies have moved to restrict antibiotic drugs used in treating human infections from use in both animal treatments and feed. In December of 1998, the European Union proposed a ban on the use of certain antibiotics as animal feed additives, and the U.S. FDA proposed new guidelines that severely restrict the use in animals of any antibiotics that are essential for treating bacterial infections in humans (28). D. Chemicals from Production or Processing 1. Heterocyclic Amines The toxic and mutagenic properties of heterocyclic amines were discovered by accident in the late 1970s by Takashi Sugionura and several of his coworkers at the National Cancer Center in Japan (70). They were evaluating the mutagenicity of cigarette smoke tars and decided to test foods that are commonly smoked, such as fish and meat. As expected, the foods did contain mutagenic activity. However, the application of smoke alone did not explain the large increase in mutagenicity observed with all the foods, indicating that some other type of compound was present. Further investigation showed that several different amino acids present on the surface of a cooked (browned or grilled) food are pyrolyzed into potentially carcinogenic substances. It is now known that tryptophan, phenylalanine, lysine, and glutamic acid each can yield several different types of mutagenic heterocyclic amines when exposed to the high temperatures of broiling. Physical variables such as temperature, time, and method of cooking significantly affect the mutagenic activity of cooked meat. Cooking temperature is the most important factor; a marked decrease in mutagenic activity is observed when meat is fried at lower temperatures. Moreover, the surface of well-done charcoal-broiled steaks contains much higher levels of heterocyclic amines than that of boiled beef. Recent data also have shown an apparent correlation in women between consumption of well-done meats (presumably containing higher HCA levels) and incidence of breast cancer (80). Several studies have suggested that these mutagens form in different types of meats. Hatch (42) has compiled an extensive list of heterocyclic amine levels in a variety of foods. Research on cooking temperatures suggests that the levels of heterocyclic amines vary con-

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siderably in a wide variety of processed meat products, according to Abdulkarim and Smith (1), and in restaurant-cooked meat products, according to Knize et al. (53). 2. Polycyclic Aromatic Hydrocarbons (PAHs) These are highly mutagenic and carcinogenic compounds that are pyrolytic products of burning fuel or organic compounds and are present in any type of smoke. They are found primarily in the environment, as a result of air pollution. However, PAHs have been found in a variety of foods, especially grilled, roasted, and smoked fish and meats (6). Significant levels are also present in grains, fruits, and vegetables. Charcoal-broiled and barbecued meats have some of the highest levels, about 30 to 40 times normal. Generation of PAHs occurs primarily by cooking or combustion at high temperatures and involves carbohydrates, peptides, and lipids. Lipid pyrolysis appears to cause the greatest production of PAHs in grilled products. The PAHs can enter the body by either ingestion or inhalation. Once absorbed, they are activated by liver enzymes to produce compounds that can interact with either proteins or DNA. The binding to DNA involves covalent bonding, which causes mutations and eventual carcinogenicity in some animal species. Though information is limited, PAHs are also thought to cause immunosuppression reactions in some animals. 3. Nitrosamines The N-nitrosamines are carcinogenic compounds formed from reactions between a secondary amine (amino acid) and nitrogen oxides and nitrous acid originating from nitrate or nitrite added to processed meat products. The reaction generally does not occur to a great extent unless high temperatures are applied; thus, levels of N-nitrosamines are low in most meat products. In the 1970s and early 1980s, extensive concern existed about potential nitrosamine exposure from eating processed meat products. Because of this, the use of nitrate and nitrite in curing meats was almost banned. Two major publications by the National Academy of Sciences provided insight on potential exposures to nitrosamines and nitrates and nitrites and the risk of cancer (58,59). Since then, nitrosamines have been found in a wide variety of foods, such as cheeses, beer, dried milk, dried fish, and mushrooms. Although the presence of nitrosamines in cured meat products still causes concern, the major regulatory thrust since the late 1970s has been on controlling the levels in bacon. Very specific regulations dictate maximum amounts of 120 ppm for sodium nitrite or 148 ppm for potassium nitrite, and 500 ppm for sodium erythorbate or sodium ascorbate in the product (18). The final nitrosamine contained in the cooked (fried) bacon is not allowed to be over 10 ppb, which is the level of detection. The only other cured meat products that have been scrutinized closely for nitrosamines are hams that had been smoked/cooked in rubber-containing elastic nets. Apparently, reactions at the surface and in the netting caused some nitrosamines to migrate to the surface of the product (27). Although nets containing rubber are still in use, efforts are under way to remove the precursors from the rubber. Because of the tighter process control on nitrites and nitrates added to various cured meat products, the debate over N-nitrosamines has subsided. As pointed out in the review by Cassens (14), the residual nitrite levels in cured meats have dropped dramatically from those reported in the 1970s. This indicates that cured meat products are not major sources of exposure to N-nitrosamines, as was once thought.

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V. PHYSICAL HAZARDS ASSOCIATED WITH MEATS: IDENTIFICATION AND CONTROL Physical hazards, when compared to biological and chemical hazards, may not be distributed as uniformly throughout the food product. Therefore, fewer individuals may be affected by a physical hazard event. Nonetheless, a HACCP plan must take into account physical hazards and their control (52). Katsuyama also noted a distinction between physical contaminants that cause physical injury and those that are aesthetically unpleasing. HACCP deals primarily with those physical contaminants that may cause injury. However, in some instances, control of filth adulteration, whether it results in a public health risk or not, comes under regulatory agency control. This certainly is true for international regulatory guidelines (55). The filth consideration is also a part of the U.S. regulatory approach, recognizing the goal of harmonization of international food safety standards. Physical hazards can result from incoming raw materials; poor personnel practices; and faulty processes, facilities, and equipment. The following list of examples of physical hazards was compiled from Katsuyama (52) and Boyle and Getty (11): Band-aids Bones/bone fragments Bullets/shot/BBs Carcass ID tags Cigarette butts Dirt, rocks Feathers Gasket materials Glass Grease Gum, wrappers Hair Hypodermic needles Insects Jewelry, buttons Metal Mold, mold mats Paint flakes Plastics Rodents/droppings Rubber Wood splinters Writing pen caps The control of these hazards begins with good manufacturing during preharvest management through further processing and handling before receipt by the ultimate consumer. Additional controls include, for example, carcass and product trimming, carcass washing, bone separators, metal detectors, magnets, x-ray devices, visual evaluation of incoming raw materials for defects, employee training, equipment and facility maintenance, and proper sanitation. However, complete control of the spectrum of potential physical hazards is impossible with current technology. One problem is that detection technology does not exist

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for certain contaminants—for example, vinyl gloves or pieces. The ability to be able to detect particulate contaminants regardless of the composition is needed. In the absence of that technology, Katsuyama (52) provided the following list of strategies to help prevent and control physical hazards in processing facilities: Complying with good manufacturing practice regulations Using appropriate specifications for ingredients and supplies Obtaining letters of guarantee from all suppliers Utilizing vendor certification Identifying types and sources of physical hazards Determining critical control points Installing equipment that can detect and/or remove physical hazards Monitoring the critical control points and documenting control performance Training employees Advances in the detection and control of physical hazards are needed and warrant increased research and development efforts. VI. CURRENT REGULATORY POLICIES AND INSPECTION A. Concepts of Hazard Analysis Critical Control Points (HACCP) HACCP is an acronym referring to Hazard Analysis and Critical Control Point system. The objectives of HACCP are to provide safe food for consumption and prevent chemical, physical, or biological hazards from occurring in food products. Originally, HACCP was developed jointly around 1959 by the Pillsbury Company, the National Aeronautics and Space Administration (NASA), and the United States Army Natick Research and Development Laboratories to assure safe foods for the U.S. space program (7,68). Later, HACCP was adopted voluntarily by several food companies in the United States as a preventive system to assure safe products and to reduce costs associated with unsafe food (e.g., recalls, lawsuits, or shutdowns). Following the Jack-in-the-Box E. coli outbreak in 1993, the USDA recommended that HACCP be mandatory in all meat and poultry plants. That recommendation was adopted, and on July 25, 1996, the Pathogen Reduction Final Rule mandating HACCP implementation was published. Also, several deadlines for implementation of HACCP were set: 1. 2. 3.

Jan. 26, 1998, in large meat and poultry plants, i.e., with 500 employees. Jan. 25, 1999, in smaller plants, i.e., with 10 or more employees but 500. Jan. 25, 2000, in very small plants, i.e., with 10 employees or having annual sales of $2.5 million.

HACCP has seven principles that need to be met: 1.

Conduct Hazard Analysis (HA): The HACCP team brainstorms to list and identify potential chemical, physical, or biological hazards during food production or preparation. Also, the team determines the significance of a hazard (e.g., low risk or high risk) and identifies preventive measures. 2. Identify Critical Control Points (CCPs): A CCP is a point in the process where a control can be applied and a potential food safety hazard prevented, eliminated, or reduced. A CCP decision tree can be used to help determine if a point in the process is a CCP or a Control Point (CP; any point at which a hazard can be con-

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3.

4. 5.

6.

7.

trolled). Once a CCP has been identified, a method for its control needs to be determined. Establish Critical Limits (CLs): Critical limits are needed for preventive measures associated with each CCP. They serve as boundaries for CCPs and help indicate when a deviation from the acceptable level has occurred. Establish CCP Monitoring Procedures: Monitoring procedures are necessary to adjust the process and maintain control during production or food preparation. Establish Corrective Actions (CAs): When monitoring indicates deviations, CAs are implemented to adjust for the deviation. In case of noncompliance, it is important to address the cause of the deviation, how the problem was corrected, and disposition of the product. Also, records of CA should be maintained (i.e., in case of deviations). Establish Verification Procedures: These can be conducted by the HACCP team or by outside consultants. The goal is to show that the CCPs, CLs, and the HACCP system as a whole are working. Establish Recordkeeping Procedures: Records on HACCP team, product description and intended use of product, flow diagram of process(es), CCPs, type of hazards, preventive measures, CLs, monitoring procedures, CAs, and verification procedures should be kept in an accessible location. This is important is cases where deviation(s) occurred.

For a HACCP system to be incorporated successfully into a food process, all of the principles must be implemented carefully for the specific process. Because the HACCP team has the responsibility of developing a successful HACCP, one of the first steps involves careful identification of that team or individual(s) who will serve as the lead HACCP person/people for your establishment. According to the USDA’s HACCP regulation, individual(s) developing HACCP plans must have successfully completed a course of instruction in the application of the seven HACCP principles to meat or poultry processing. Therefore, it is important to make sure that the individual(s) completes the required training. A list of introductory HACCP courses can be obtained from North American Meat Processors (NAMP) or by calling the International HACCP Alliance (409) 862-2036. After completion of the HACCP training, the individual(s) should have a working knowledge of the process required to develop and implement a HACCP program. The individual(s) also should have a HACCP reference book and handouts from the training course that should help them move forward. HACCP-trained individual(s) then should identify the people needed on the HACCP team. After the HACCP team members are identified, the next step involves gathering documents and materials that are needed to adequately develop the HACCP plan. This includes a copy of the actual HACCP regulation (Final Rule) and the technical amendments and issue papers related to the regulation. These documents can be obtained from the NAMP office, by downloading from the Internet, or by contacting USDA. The following Web sites also provide information that may be useful: http://ifse.tamu.edu/haccpall.html (International HACCP Alliance) http://www.usda.gov/agency/fsis/homepage.htm (USDA-FSIS) B. Operational Steps in HACCP It is understood that each establishment operates slightly differently from the next, even if they are producing the same products. Therefore, each HACCP team may not need to use

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the same type of information. The items listed below are fairly basic, and each team can add or delete material from the list as needed. The main thing is that the identified material should be available to the HACCP team members as they begin to develop the HACCP plan. 1. Written sanitation standard operating procedures (SSOPs) manual or document and the Deviation/Corrective Action records generated from the SSOP program. The Pathogen Reduction/HACCP regulation requires each establishment to have a written SSOP. The records generated from the SSOPs may help identify problem areas and should be useful as you evaluate your overall process. 2. Plant production practices (Standard Operating Procedures). If your establishment has an operational standard operating procedures guide, then it could be used by the team. If not, then it is important to make sure that the team members know and understand the actions required to produce your product(s). 3. Product descriptions and/or recipes. The regulation requires that each plan contains a product description providing information about the product and its end users. Therefore, if you already have written information about the products, it can be incorporated into the HACCP plan as needed. 4. Information on the establishment’s prior recalls and customer complaints that are related to food safety. The HACCP team should be aware of the plant’s history. One way to help is to provide information on recalls and customer complaints that are related to food safety. 5. Establishment data. If you have been collecting micro data, then it will be important for the HACCP team to have this information to help identify trends. Information on such factors as room/cooler/freezer temperatures, oven temperatures, and line speeds also may help the team members. After as much information as possible has been identified and gathered, the HACCP team should review and use the information as it develops the HACCP plan. The team also should keep a copy of all the supporting documents that they used to make their decisions. This will help with future revisions of the HACCP plan. Identifying the lead individual(s) participating in a training program, appointing the HACCP team, and gathering the necessary information are the basic steps to getting started. Then the HACCP team can begin using its knowledge and information to develop a flow chart, provide the product description, and design a HACCP plan that can be implemented successfully to help your establishment continue to produce the safest food supply possible. Developing a HACCP plan is not something that occurs overnight, but it is something that you can accomplish. All you have to do is get started. However, when you are working on a HACCP plan, SAFETY should be the most important concern. C. Potential for Recall of Meat and Poultry Products Theoretically, under a HACCP system, the likelihood of product recalls should be reduced greatly. If the manufacturing process is controlled properly, then the output of that process also should be under control, and the finished product should meet all company and regulatory safety requirements. When a process failure occurs, the HACCP monitoring procedures should alert the operator and the proscribed corrective action procedures should prevent nonconformities in the finished product. However, even under the best of circumstances, unforeseen events may sometimes result in the need to remove adulterated or misbranded products from the marketplace.

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Therefore, all companies involved in food production, processing, distribution, and retailing must establish procedures for conducting a product withdrawal or recall. In addition, the designation of a “crisis management team” and the establishment of policy to address the public relations concerns that may arise in conjunction with a product withdrawal or recall are necessary components of a HACCP system. A recall is a voluntary procedure initiated by a company in an effort to remove an adulterated product from the marketplace. The USDA does not have the authority to order a recall, but in a situation when a company is deemed to be insufficiently cooperative, it can initiate its own seizure actions against the products in question (43). As far as we know, in all instances where product withdrawal or recalls have been necessary, companies regulated by the USDA have been cooperative, and the USDA has not had to resort to product seizures. Indeed, in most instances, the proper and necessary strategy for a company to pursue is to cooperate with the government regulatory agency, and show its customers and consumers that it is able to promptly, decisively, and effectively deal with the problem and move on. Because of the general industry adherence to this pattern and a generally high level of government professionalism in this area, recalls and recall procedures historically have not been sources of controversy. Recently, questions have arisen as to whether the USDA and FDA have sufficient authority with respect to recalls and on record-keeping and trace-back requirements for meat and poultry products. Controversy also has surrounded several recent recalls where recall action has been triggered entirely upon epidemiological supposition unsupported by any hard data, and inaccurate information was provided to consumers in USDA press releases. Each of these areas of controversy is addressed, at least in part, by industry initiative on trace back; by authorizing legislation that has been proposed by USDA; and by the proposed regulation on pathogen reduction and HACCP, which ultimately will result in the mandatory implementation of HACCP in all federally inspected meat and poultry plants. For example, the record-keeping requirements associated with a HACCP plan should enhance a company’s ability to conduct a product withdrawal or recall, and the trace-back component of voluntary producer quality assurance programs may facilitate the identification of the source of contamination in a foodborne illness outbreak. D. USDA Policy on Recalls In 1988, USDA’s FSIS published a revised directive to address the recall of meat and products (34). The directive established a system for classification of recalls based on the public health hazard presented by the product being recalled. 1. Class I Involves a health hazard situation where there is a reasonable probability that the use of the product will cause serious, adverse, health consequences or death. 2. Class II Involves a potential health hazard situation where there is a remote probability of serious, adverse, health consequences from the use of the product. 3. Class III Involves a situation where the use of the product is not likely to cause adverse health consequences.

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4. Nomenclature Used in Recall The FSIS also provided recall oversight; monitoring the effectiveness of the recall; and coordinating activities between federal, state, and local agencies and foreign governments. The directive also provides definitions that are useful in establishing a corporate crisis management strategy. a. Recall. The voluntary removal by a firm from commerce of distributed meat or poultry products when there is reason to believe that such products are adulterated or misbranded under the provisions of the Federal Meat Inspection Act or the Poultry Products Inspection Act. “Recall” does not include a market withdrawal or a stock recovery. b. Correction. The firm’s modification, relabeling, or destruction of a product with the concurrence of FSIS. c. Recalling Firm. The firm that initiates a recall or, in the case of an FSIS-requested recall, the firm that has primary responsibility for the manufacturing and/or marketing of the product to be recalled. d. Firm-Initiated Recall. A recall that is initiated by a firm without a formal request from FSIS. e. FSIS-Requested Recall. A recall initiated by a firm in response to a formal request from FSIS. f. Case Number. The number or code assigned to recall incident for use by FSIS to identify the investigation or product recall. g. Health Hazard Evaluation. An evaluation of the health hazard presented by a product being recalled or considered for recall. The evaluation will be conducted by a team of FSIS experts with access to other individuals or agencies as deemed necessary. h. Emergency Program Team. A team of representatives from various FSIS divisions and staffs assembled to respond to potential or real health hazard incidents reported to EPA. Representatives from the following Agency units may be members of the team: Chemistry Division, Compliance, Emergency Programs Staff, Epidemiology Branch, Export Coordination Staff, Foreign Programs Division, Field Service Laboratories Division, Mathematics and Statistics Division, Import Inspection Division, Information and Legislative Affairs, Microbiology Division, Residue Operations Staff, Processed Products Inspection Division, Residue Evaluation and Planning Division, and Regional Operations, MPIO. The team may be activated by the Director, EPA, whenever deemed necessary, and its members will report to the Director, EPA, for the purpose of conducting assignments related to the health hazard incident. i. Recall Strategy. The action plan recommended by or to the recalling firm and followed by FSIS in monitoring a recall. j. Depth of Recall. The level of product distribution to which the recall is to extend: Consumer or user level, including any intermediate wholesale or retail level. Retail level—the level immediately preceding the consumer or user level. Wholesale level—the distribution level between the manufacturer and the retailer. This level may not be encountered in every recall situation; i.e., the manufacturer may sell directly to the retail outlet. k. Public Notification. A public notification to alert the public and trade that either a product is being recalled because it presents a serious health hazard or that a situation exists for which such notification is deemed to be in the public interest. The necessity

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for public notification will be considered on a case-by-case basis for each recall. However, all Class I recalls will result in the issuance of a press release or another form of public notification, unless so exempted by the Administrator, FSIS. The situations where this exemption will be permitted are (a) when data are sufficient to support the conclusion that the suspect product in commerce is under control by FSIS or another federal or state agency and that the likelihood that any product is in the hands of consumers is extremely remote or (b) when public notification already has been made by another government agency (state or federal) and for which the text, format, and method of notification are acceptable to FSIS. However, information on any recall action of any classification will be made available to the public or press when inquiry is made, provided that such information is not exempt under the Freedom of Information Act. l. Effectiveness Reviews. Reviews for the purpose of verifying that adequate notice about the recall has been provided to all consignees. Adequacy of notice is determined by the degree to which the implicated product in fact is retrieved by, or on behalf of, the official establishment and is disposed of properly. The number of effectiveness reviews to be conducted will be determined on a case-by-case basis by the Assistant Deputy Administrator, Compliance Program. The reviews will be conducted in accordance with Compliance procedures and will focus upon the following elements: Recall level Health hazard Initial effectiveness review findings Recall firm’s actions A sufficient number of effectiveness reviews will be made to provide assurance that recall action is conducted in an effective manner and that appropriate efforts are made to locate and return the product being recalled. In the event that effectiveness reviews disclose recalled product remaining in commerce, the recalling firm must be notified. If the firm does not take prompt action to properly dispose of the product, the Assistant Deputy Administrator, Compliance program, may detain and seize product or initiate other action as appropriate. m. Monitor. To observe and record data concerning a firm’s recall and to conduct effectiveness reviews. n. Recall Evaluation. The evaluation of final reports after recall action is completed to determine the recall’s effectiveness. It will include the percent of product returned, its disposition, and the number and level of consignees reviewed. The recall evaluation will for the basis for terminating the recall. o. Termination of a Recall. Officially, when the Agency determines that all reasonable efforts have been made to remove or correct the violative product and proper disposition has been made according to the degree of hazard. For monitoring purposes, FSIS will classify a recall action “completed” at the time when the firm has actually retrieved and impounded all outstanding product that could reasonably be expected to be recovered, or has completed all product corrections. p. Market Withdrawal. A firm’s removal or correction at its own volition of a distributed product involving a minor infraction that would not warrant legal action by FSIS or that involves no violation of the FMIA or PPIA or health hazard. q. Stock Recovery. A firm’s removal or correction of product that has not been marketed or that has not left the direct control of the firm, i.e., the product is located on premises owned by, or under the control of, the firm, and no portion of the lot has been released for sale or use. Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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E. Processes of Conducting a Recall The process of conducting a recall includes the following six steps: 1. Health Hazard Evaluation An evaluation of the health hazard presented by a product being considered for recall, or being recalled, will be conducted by a team of FSIS experts in cooperation with other individuals or agencies as deemed necessary. The evaluation will include at least the following factors: Nature of the violation or defect Whether any illness or injuries already have occurred from the use of the product Assessment of the likelihood of occurrence of the hazard Assessment of the consequences (immediate or long range) of occurrence of the hazard 2. Recall Classification A recall classification will be assigned to product recalls based on the health hazard evaluation or the assessment of the nature of the deception or other defect. The FSIS will assign the classification, i.e., Class I, Class II, Class III, to indicate the relative degree of health hazard of the product being considered for recall or being recalled. 3. Recall Strategy A recall strategy will be developed to assist in the conduct of a recall and take into account the following factors: Recall classification assigned by FSIS Depth of recall Extent of notification being made to the trade or public Action plan to coordinate the removal and return or correction of the product from the marketplace Effectiveness reviews Elements of a recall strategy will include: Results of the health hazard Ease in identifying the product Degree to which the product’s deficiency is obvious to the consumer Degree to which production remains unused in the marketplace Amount of product involved Area of distribution Action taken or planned by the recalling firm 4. Recall Recommendation The EPA Staff will prepare the recall recommendation for submission to the Deputy Administrator, MPIO. The recall recommendation will include: Health hazard evaluation Recall classification Recall strategy Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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5. Recall Request The recall recommendation will be reviewed by the Deputy Administrator, MPIO. The final decision to request a product recall will be made with the concurrence of the Administrator, FSIS. After the decision to recall, the Deputy Administrator, MPIO, or designee will contact the firm to make the formal request for a recall. The appropriate federal, state, or local agencies will be notified of the product recall. 6. Termination of Recall A recall will be considered officially terminated when FSIS determines that the recall action is completed, that proper disposition has been made of the violative product, and that no further emergency action is pending. The Director, EPA, will assemble the information and reports necessary to make this determination and will make the recommendation for terminating the recall to the Deputy Administrator, MPIO. The recommendation will be reviewed by the Deputy Administrator, MPIO. It is important to note that a memorandum of understanding between FSIS and FDA has been established to set forth the working arrangements between the two regulatory agencies in conducting Class I and Class II recalls of food. HACCP by design is a preventive system. If implemented and operated properly, food produced under a HACCP system should be safe, and the potential for product recalls should be reduced greatly. In the event that a recall becomes necessary, however, a crisis management policy that is in place and periodically tested also will serve as a means of limiting the potential financial loss and loss of consumer confidence that may be associated with a product recall. The combination of a HACCP system and a well-designed crisis management policy will provide the most effective assurance that food products will be safe and insurance against the potential devastating effects of a food safety crisis and public recall. F. Imported Products: State vs. Federal Programs and Agencies Involved Imported food products must meet the same regulatory requirements as products manufactured in the United States. The USDA office of field operations has an import/export division that oversees plants outside the United States that import meat and poultry products. Following are names and Web site addresses for some government agencies that can be contacted for further information and updates on HACCP and guidelines and regulations related to food safety: FSIS CDC CVM CFSAN

www.fsis.usda.gov www.cdc.gov www.fda.gov/cvm www.cfsan.fda.gov

G. Definitions 1.

Control: (a) to manage the conditions of an operation to maintain compliance with established criteria; (b) the state wherein correct procedures are being followed and criteria are being met. 2. Control point: any point, step, or procedure at which biological, physical, or chemical factors can be controlled.

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5. 6. 7. 8.

9.

10. 11. 12. 13. 14.

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Corrective action: procedures to be followed when a deviation occurs. Critical control point (CCP): a point, step, or procedure at which control can be applied and a food safety hazard can be prevented, eliminated, or reduced to acceptable levels. CCP decision tree: a sequence of questions to determine whether a control is CCP. Critical limit: a criterion that must be met for each preventive measure associated with a critical control point. Deviation: failure to meet a critical limit. HACCP plan: the written document that is based upon the principles of HACCP and delineates the procedures to be followed to assure the control of a specific process or procedure. HACCP plan revalidation: one aspect of verification in which a documented periodic review of the HACCP plan is done by the HACCP team with the purpose of modifying the plan as necessary. HACCP plan validation: the initial review by the HACCP team to ensure that all elements of the HACCP plan are accurate. HACCP system: the result of the implementation of the HACCP plan. HACCP team: the group of people that is responsible for developing a HACCP plan. Hazard: a biological, chemical, or physical property that may cause a food to be unsafe for consumption. Monitor: to conduct a planned sequence of observations or measurements to assess whether a CCP is under control and to produce an accurate record for future use in verification. Preventive measure: a physical, chemical, or other factor that can be used to control an identified health hazard. Risk: an estimate of the likely occurrence of a hazard. Severity: the seriousness of a hazard. Verification: the use of methods, procedures, or tests in addition to those used in monitoring to determine if the HACCP system is in compliance with the HACCP plan and/or whether the plan needs modification and revalidation.

MEAT SAFETY IN THE FUTURE

A. Food/Meat Safety and Research Needs During the past several decades, meat scientists and food microbiologists have been performing a variety of valuable and fruitful research activities to promote meat science and meat safety. A number of factors are important to the livestock and meat industry. For example, product quality (i.e., tenderness consistency) and processing efficiencies (i.e., centralized processing and precooking) are important targets for the industry. However, those considerations must be consistent with safety of the product and vice versa. With that focus in mind, it is imperative that all segments from producers to processor to retailer to consumer be involved to help assure meat safety. Practices are needed at the live animal level that can be implemented realistically and may reduce hazards that can be carried to the final product. Identification of those practices

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has been difficult, and demonstrating quantitatively that a preharvest practice reduces the incidence of foodborne disease is unlikely. Therefore, producers may implement practices on the theoretical contingency that they will help reduce the incidence of a hazard. Perhaps, future research will lead to such improvements as vaccines or competitive exclusion agents that will function as classical control technologies at a critical control point in preharvest HACCP. That research demands significant attention by the scientific community. The most progress in meat safety research has been made at the processing level, because this is a logical point to achieve broad-spectrum hazard control or elimination. As compared to the number of live animal producers, there are fewer processing facilities through which the product passes and can be subjected to hazard control. Nonetheless, the ideal system realizes hazard control preharvest and this is coupled with the postharvest gains (49). Postharvest intervention systems that integrate chemical, physical, and thermal strategies require further investigation to determine their synergy. However, those systems must be coupled with appropriate subsequent handling of the product, whether it be by purveyors, food service, retail stores, or the ultimate consumers. There is a need to discern the most correct information transfer system(s) to ensure that postprocessing education completes the concept of safety from live animal to consumer (50). In summary, progress toward safer meat is realized ideally by integration of preharvest and postharvest intervention strategies. No one step in the system is “the place” where safety can be achieved totally. Even though good progress has been made, progress through additional research still holds significant potential. However, this must be integrated with aggressive technology and information transfer. B. Domestic and International Meat Safety in the Future and Meat Safety Standards Just as they do in other areas of life, political and economic interdependencies exist between nations engaged in the trade of meat and meat products. Consequently, a discussion of domestic meat safety issues cannot be separated from international political issues. In keeping with the move from a principally organoleptic (sight-, smell-, touch-, taste-based) inspection system to the more science-based HACCP system, the U.S. regulatory agencies and meat industry have worked together toward implementation. The U.S. implementation of HACCP has not been easy or ideologically complete. Nonetheless, HACCP has been implemented, and the meat inspection system likely will continue to reflect a hybrid of the organoleptic-based inspection and the new science-based HACCP. The U.S. meat industry and regulatory agencies are committed to going beyond an inspection system limited largely to processing operations to include a farm (preharvest) to table (postharvest) approach. It is reasoned that by addressing safety issues at the live animal, processing, marketing, food service, and retail levels using the HACCP approach, safety gains at each level will be at least additive and likely synergistic. No one point in the chain (e.g., live animal) is the ultimate intervention point but rather the combination of interventions at CCPs throughout the chain from producer to consumers will reduce risk to both the domestic and international communities (12). The international community has worked through several avenues to harmonize food/meat safety issues in recognition of ever-expanding international trade. The joint FAO/WHO Food Standards Programme and the Codex Alimentarius Commission (CAC) effort in 1962 is a pivotal example.

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To further advance that effort, food safety guidelines were reinforced at the TwentySecond Session of the CAC held in Geneva, Switzerland in 1997. From that meeting, the Commission adopted the following guidelines: Recommended International Code of Practice—General Principles of Food Hygiene Guidelines for the Application of the Hazard Analysis and Critical Control Point (HACCP) System Principles for the Establishment and Application of Microbiological Criteria for Foods Guidelines for the Exchange of Information between Countries on Rejections of Imported Food Guidelines for the Design, Operation, Assessment, and Accreditation of Food Import and Export Inspection and Certification Systems Guidelines for the Assessment of the Competence of Testing Laboratories Involved in the Import and Export Control of Foods (55). Additionally, Lupien (55) reported that Codex decided on the following statements to balance the role of science and “other factors” in food/meat safety. 1.

The food standards, guidelines, and other recommendations of Codex Alimentarius shall be based on the principle of sound scientific analysis and evidence, involving a thorough review of all relevant information, in order that the standards assure the quality and safety of the food supply. 2. When elaborating and deciding upon food standards, Codex Alimentarius will have regard, where appropriate, for other legitimate factors relevant for the health protection of consumers and for the promotion of fair practices in food trade. 3. In this regard, it is noted that food labeling plays an important role in furthering both of these objectives. 4. When the situation arises that members of Codex agree on the necessary level of protection of public health but hold differing views about other considerations, members may abstain from acceptance of the relevant standard without necessarily preventing the decision by Codex. Considering that the HACCP system was recognized in the aforementioned guidelines, the U.S. approach to meat safety is consistent with international efforts to harmonize meat safety issues. Although progress in harmonization is being made, much work remains. That work entails standardized international training in, for example, HACCP, sanitation, food microbiology, hygiene, toxicology, and related issues. This harmonization also will be impacted by how well the World Trade Organization (WTO) members base their national food safety measures on international standard guidelines and other recommendations adopted by Codex Alimentarius (55). These considerations must be balanced with other factors that impact compliance with WTO “science-based” recommendations. The politics of nontariff trade barriers have resulted in science-based decisions by the WTO being circumvented. Legitimate concerns that go beyond the “science” of safety and impact trade decisions include other actions and consideration by the WTO. Specifically, the Uraguay Round Agreement of the WTO also took into consideration (a) public and moral values and (b) the health and life of not only humans but animals and plants when arbitrating trade issues (78). Though legitimate, those considerations must not be misused to avoid responding to otherwise science-based food safety concerns.

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VIII. SUMMARY Meat safety is a concern of meat producers, processors, retailer, distributors, food handlers, and eventually the consumers. This chapter started with the current status of meat safety and new developments in issues such as meat irradiation, dietary supplements, genetically modified foods, and consumers’ knowledge and practices in meat safety. It continued with a discussion of the history of meat industry safety, microbiological hazards, and rapid methods and automation in microbiology testing related to monitoring microbial meat safety. Chemical and physical hazards related to meat were then presented and discussed. The chapter concluded with a detailed discussion of HACCP and current regulatory policies and inspection. A look into the future of meat safety completed the chapter, which contains a wealth of information along with some speculations into the needs, predictions, and future directions relative to the safety of this important food commodity. REFERENCES 1. 2. 3. 4. 5. 6.

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Cheever, L.O. 1948. The House of Morrell. The Torch Press, Cedar Rapids, IA. CFR. 1999a. Code of Federal Regulations, Title 9-Animal and Animal Products, Parts 200 to End, Part 318.7(b)-Approval of substances for the use in the preparation of products. US Government Printing Office, Washington, DC. CFR. 1999b. Code of Federal Regulations, Title 40-Protection of Environment, Parts 185–189, Tolerances for pesticides in food. US Government Printing Office, Washington, DC. Concon, J.M. 1988. Man-Made Organic Chemical Food Contaminants. Ch. 19 in Food Toxicology: Part B. p. 1133–1229. Marcel Dekker, New York. Davies, A. and R. Board (eds). 1998. The Microbiology of Meat and Poultry. Blackie Academic and Professional, London, UK. Department of Agriculture. 1894. Yearbook of the United States Department of Agriculture. GPO, Washington, DC. Department of Agriculture. 1899. Yearbook of the United States Department of Agriculture. GPO, Washington, DC. Department of Agriculture. 1905. Yearbook of the United States Department of Agriculture. GPO, Washington, DC. Dorsa, W.J., C.N. Cutter, G.R. Siragusa, and M. Koohmaraie. 1996. Microbial decontamination of beef and sheep carcasses by steam, hot water spray washes, and a steam-vacuum sanitizer. J Food Prot 59:127–135. Doyle, M.P., L.R. Beuchat, and T.J. Montville. 1997. Food Microbiology: Fundamentals and Frontiers. American Society of Microbiology Press. Washington, DC. Fiddler, W., J.W. Pensabene, and R.A. Gates. 1997. N-Nitrosodibenzylamine in boneless hams processed in elastic rubber nettings. J AOAC Int 80:353–358. Food and Drug Administration. 1999a. Center for Veterinary Medicine. Antimicrobial Resistance. http://www.fda.gov/cvm/fda/mappgs/antitoc.html Food and Drug Administration. 1999b. Chemical Contaminants, Dioxins and PCBs. http://vm.cfsan.fda.gov/~Ird/pestadd.html Food and Drug Administration. 1999c. Food and Drug Administration Pesticide Program. http://vm.cfsan.fda.gov/~dms/pesrpts.html Food Marketing Institute. 1998. Trends in the United States: Consumer Attitudes and Supermarkets. 1998. FMI, Washington, DC. Food Marketing Institute—Grocery Manufacturers of America. 1998. Consumers’ Views on Food Irradiation. Washington, DC. Foreman, C.T. 1996. Afterward to E. coli O157:The Story of a Mother’s Battle with a Killer Microbe by Mary Heersink. New Horizon Press, Far Hills, N.J. FSIS Directive. 8080-1, January 12, 1988. “Recall of Inspected Meat and Poultry Products.” Fung, D.Y.C. 1986. Microbiology of Meats MF-792. Kansas State University Cooperative Extension Service, Manhattan, KS. April 1986. Fung, D.Y.C. 1992. Historical development of rapid methods and automation in microbiology. J. Rapid Methods and Automation in Microbiology 1(1):1–14. Fung, D.Y.C. 1995. What’s needed in rapid detection of foodborne pathogens. Food Technol 44(6):64–67. Fung, D.Y.C. 1997. Overview of rapid methods of microbiological analysis. In Tortorello, M.L. and S.M. Gendel (eds). Food Microbiological Analysis: New Technologies. Marcel Dekker, New York. Chapter 1. Fung, D.Y.C., A.N. Sharpe, B.C. Hart, and Y. Liu. 1997. The Pulsifier: A new instrument for preparing food suspension for microbiological analysis. Rapid Methods and Automation in Microbiology 6(1):43–50. Gottesman, R. 1986. Introduction to the Jungle. Penguin Books, New York. Gravani, R., D. Williams, and O. Blumenthal. 1992. What do consumers know about food safety? FSIS Food Safety Rev 2(1):12–14. Hatch, F. 1999. Mutagens in cooked foods database, Lawrence Livermore National Laboratory, http://www-bio.llnl.gov/mutagens/html/db.intro.text.html.

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Fung et al. Hilbert, R. 1995 “Recall Confusion with USDA can be Resolved.” Meat Processing. May 1995. Hoban, T. 1999. Consumer acceptance of biotechnology in the United States and Japan. Food Tech. 53(5):50–53. Hurt, D. 1994. American Agriculture: A Brief History. Iowa State University Press, Ames, IA. ICMSF. 1996. Microorganisms in Foods. Blackie Academic and Professional. Jay, J.M. 1992. Modern Food Microbiology. Chapman Hall, New York. Jay, J.M. 1996. Modern Food Microbiology. 5th Ed. Chapman & Hall, New York. Kastner, C.L. 1999a. Food/Meat Safety and Research Needs. Presented at Excellence in Food Science Symposium X. Sept. 17, 1999. Kansas State University, Manhattan, KS. Kastner, C.L. 1999b. Pre- and Post-Harvest Meat Safety. Presented at Annual Meeting of American Association of Bovine Practitioners. Sept. 25, 1999. Nashville, TN. Kastner, J.J. 1997. The history of U.S. food safety policy. Report to History of Am. Agri. 557, Kansas State University, Manhattan, KS. Katsuyama, A.M. 1995. Physical Hazards and Controls in Establishing Hazard Analysis Critical Control Point Programs. Editors: K.E. Stevenson and D.T. Bernard, Pub. National Food Processors Association. Washington, DC. p. 6–1 to 6–6. Knize, M.G., R. Sinha, E.D. Brown, C.P. Salmon., O.A. Levander, J.S. Felton, and N. Rothman. 1998. Heterocyclic amine content in restaurant-cooked hamburgers, steaks, ribs, and chicken. J Agric Food Chem 46:4648–4651. Levy, S.B. 1998. The challenge of antibiotic resistance. Sci. Am. 278(3):32–39. http://www.sciam.com/1998/0398issue/0398levy.html Lupien, J.R. 1998. Food Safety, Efficiency, and Security. 5. Food Quality and Safety: International Dimensions. Special Publication No. 21. Council for Agriculture Science and Technology, Ames, IA p. 26–31. Lupton, J.R., and H.R. Cross. 1994. The contribution of meat, poultry and fish to the health and well being of man. In Pearson, A.M., and T.R. Dutson (eds). Quality Attributes and their Measurement in Meat, Poultry, and Fish Products. Advances in Meat Research Series Vol. 9. Blackie Academic and Professionals. London, UK. Milner, J. 1996. LFRA Microbiology Handbook. Leatherhead Food RA, Surrey. UK. National Academy of Sciences, National Research Council. 1981. The Health Effect of Nitrate, Nitrite, and N-Nitroso Compounds. National Academy Press, Washington, DC. National Academy of Sciences, National Research Council. 1982. Diet, Nutrition, and Cancer. National Academy Press, Washington, DC. Newmann, K. 1990. Synthetic Pyrethroid Insecticides: Structures and Properties, Vol. 4, Springer-Verlag, Berlin, Germany. Nutsch, A.L. 1998. Bacterial Decontamination of Meat Surfaces Through the Application of a Steam Pasteurization Process. Ph.D. Dissertation. Kansas State University Library, Manhattan, KS. Nutsch, A.L., R.K. Phebus, M.J. Riemann, J.S. Kotrola, R.C. Wilson, J.E. Boyer, Jr., and T.L. Brown. 1998. Steam pasteurization of commercially slaughtered beef carcasses: Evaluation of bacterial populations at five anatomical sites. J Food Prot 61:571–577. Nutsch, A.L., R.K. Phebus, M.J. Riemann, D.E. Schafer, J.E. Boyer, Jr., R.C. Wilson, D.J. Leising, and C.L. Kastner. 1997. Evaluation of a steam pasteurization process in a commercial beef processing facility. J Food Prot 60:485–492. Phebus, R.K., A.L. Nutsch, D.E. Schafer, R.C. Wilson, M.J. Riemann, J.D. Leising, C.L. Kastner, J.R. Wolf, and R.K. Prasai. 1997. Comparison of steam pasteurization and other methods for reduction of pathogens on surfaces of freshly slaughtered beef. J Food Prot 60:476–484. Price, J.F., and B.S. Schweigert. 1971. The Science of Meat and Meat Products. W.H. Freeman, San Francisco, CA. Sinclair, U. 1906. The Jungle. Penguin Books, New York. Sofos, J. 1994. Microbial growth and its control in meat, poultry and fish. In Pearson, A.M. and T.R. Dutson (eds). Quality Attributes and their Measurement in Meat, Poultry, and Fish Prod-

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ucts. Advances in Meat Research Series Vol. 9. Blackie Academic and Professionals. London, UK. Sperber, W. 1991. The modern HACCP system. Food Technol 45(6), 116–118, 120. Statutes of the United States of America, 51st Congress, 1st Session (1889–1890), 414–416. Sugionura, T., K. Wakabayashi, M. Nagao, and H. Ohgaki. 1989. Heterocyclic amines in cooked food. Ch 3 in Food Toxicology: A Perspective on the Relative Risk, S.L. Taylor and R.A. Scanlon (eds)., p. 31–55. Marcel Dekker, New York. Todd, E.C.D. 1989. Preliminary estimates of costs of foodborne diseases in the United States. J Food Protect 52:595–601. United Nations Environment Programme (UNEP). 1999. Elimination of 10 intentionally produced persistent organic pollutants favoured by treaty negotiators; health need for DDT exemption seen. http://www.grida.no/inf/news/news99/news130.htm United States Department of Agriculture. Food Safety and Inspection Service. 1999a. http://www.fsis.usda.gov/ United States Department of Agriculture, Food Safety and Inspection Service, 1999b. Office of Public Health and Science (OPHS), http://www.fsis.usda.gov/OPHS/ophshome.htm United States Environmental Protection Agency. 1998. Office of Prevention, Pesticides, and Toxic Substances; Endocrine disruptors. http://www.epa.gov/opptintr/opptendo/index.htm United States Environmental Protection Agency. 1999. The Food Quality Protection Act (FQPA) of 1996. http://www.epa.gov/oppfead1/fqpa/ Weaber, D. 1999. The Statistics. National Cattlemen. June/July Issue. World Trade Organization. 1994. The Results of Multilateral Trade Negotiations of the Uruguay Round: the Legal Texts, Geneva, GATT Secretariat. As reported by E. Burgoyne, 1997. Proceedings of Symposium on Farm Animal Welfare in Canada. International Trade and Animal Welfare. Organized by Dairy and Swine Research and Development Centre Canadian Expert Committee on Farm Animal Welfare and Behavior in collaboration with Canadian AgriFood Research Council and Agriculture and Agri-Food Canada Research Branch. Zhang, P., K.P. Penner, and J. Johnston. 1999. Prevalence of selected unsafe food consumption practices and their associated factors in Kansas. J Food Safety 19(1999):289–297. Zheng, W., D.R. Gustagson, R. Sinha, J.R. Cerhan, D. Moore, C.P. Hong, K.E. Anderson, L.H. Kushi, T.A. Sellers and A.R. Folsom. 1998. Well-done meat intake and the risk of breast cancer. J Natl Cancer Inst 90–1724–1729.

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9 Drug Residues in Meat: Emerging Issues SHERRI B. TURNIPSEED U.S. Food and Drug Administration, Denver, Colorado

I. ANIMAL DRUGS AND THEIR USAGE A. Background B. Emerging Issues II. DESCRIPTION OF CLASSES OF ANIMAL DRUGS A. Antibiotics and Sulfonamides B. Antiparasitics C. Hormones and Growth Agents D. Other Animal Drugs E. Emerging Health Concerns with Specific Classes of Animal Drugs III. REGULATIONS AND RESPONSIBILITIES A. Background B. Recent Changes in Regulation and Monitoring of Drug Residues IV. ANALYTICAL METHODOLOGY A. Background B. Emerging Issues in the Analysis of Animal Drug Residues V. CONCLUSIONS REFERENCES

I. ANIMAL DRUGS AND THEIR USAGE A. Background Modern animal husbandry practices involve the mass production of food animals. Animals are held together in dense populations under controlled conditions where the potential for disease outbreak is such that the use of antibiotics or other medications may be necessary. These drugs can be used therapeutically, to cure existing disease, or prophylactically to minimize the potential for disease threat across a population. Most often, however, they are

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used subtherapeutically as growth promotants to increase feed conversion. As a result, the use of animal drugs in meat production is standard practice, with 60% to 80% of all animals consumed in the United States having been treated with an approved drug (Institute of Medicine, 1989). Obviously the concern with widespread drug therapy in food animals is the possibility of residues remaining in the edible product and the potential human health problems associated with exposure to these residues. Several measures can be taken to minimize exposure to residues in the food supply while still allowing for cost-effective agricultural practices. Characterization of the depuration times for drugs from edible tissue is an important part of the drug approval process. Appropriate withdrawal times (the time producers must wait to harvest animal or by-product after drug therapy has been given) can be set to minimize the incidence of drug residues. The tolerance levels set by the U.S. Food and Drug Administration (FDA) in meat products for approved veterinary drugs are listed in the Code of Federal Regulations (CFR, 1999). The public heath significance of drug residues in food of animal origin is an area of much debate (Wilson, 1994; Sundlof and Cooper, 1996; Long and Roybal, 1994; Natural Research Council [NRC], 1999). While the importance of drug residue exposure can be argued, it is generally accepted that potential problems could arise either because of adverse reactions to the drug itself or because of the development of a resistant bacterial population in the food supply. Most adverse reactions to drug residues in foods are caused by allergic responses. For example, sensitization to aminoglycosides (Tinkleman and Bock, 1984) and

-lactams (Schwartz and Sher, 1984) in beef has been reported. Some drugs such as chloramphenicol (which will be discussed later in this chapter) have potentially more serious acute adverse effects. B. Emerging Issues The issue of antibiotic resistance has come to the forefront in recent years. The concern is increased human exposure to a bacterial population that has grown resistant to antibiotics. Animals fed low (subtherapeutic) levels of antibiotics may develop bacterial infections that evolve to be impervious to these drugs. Humans may be exposed to these bacteria during the preparation or consumption of food. There is general support for the fact that resistant bacteria are present in meat commodities (Quednau et al., 1998; Wegener et al., 1999) due to the use of subtherapeutic antibiotics. There is also growing evidence that this may translate to increased resistance to antibiotics in the human population, although this remains an area of debate. For although there seems to be an increase in resistant bacteria due to the use of animal drugs, many of these organisms are nonpathogenic and will not cause infection when ingested by humans. The appearance of resistance among pathogenic organisms, such as Salmonella DT-104 and Campylobacter, is of more concern (Endtz et al., 1991; Smith et al., 1999; Glynn et al., 1998). For example, there appears to be a correlation between the use of fluoroquinolone antibiotics in chickens and an increase in Campylobacter drug-resistant infections in humans. The link between resistance to glycopeptides and their use in animal feeds has also been investigated (Klein, 2000; Klare et al. 1999; Woodford, 1998). The issue of increased resistance to antibiotics in humans caused by subtherapeutic use of these drugs in animal production is currently the subject of discussion among the United States Food and Drug Administration (FDA) and various animal health and drug industry representatives (FDA, 2000a, b). Recently the National Research Council convened a group to look at the benefits and risks of using drugs in the animal food industry (NRC, 1999). They identified antibiotic re-

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sistance as the major risk with the continued use of drugs in food animals. This group recommended the animal health, medicine, consumer advocate and academic communities work together with the various government agencies to formulate integrated research plans and policy development to address this issue. In addition, the group recommended continued reevaluation of the animal drug approval process to expand the number of drugs that are available and the investigation of possible alternatives to the use of drugs in food animals. II. DESCRIPTION OF CLASSES OF ANIMAL DRUGS The following is a brief description of some of the more common classes of animal drugs used in meat production (CFR, 1999; Booth and McDonald, 1982; Long and Roybal, 1994; Oka et al., 1995; Turnipseed and Long, 1998). A. Antibiotics and Sulfonamides Antibiotics are the most common type of drugs given to food animals. These are given both therapeutically and subtherapeutically to prevent disease and increase feed efficiency. There are several classes of antibiotics. Sulfonamides are a class of drugs that, although not technically classified as antibiotics, are widely used in both humans and food-producing animals to prevent infectious disease. Sulfonamides are derivatives of sulfanilamide, with substitutions at the N1 position. These drugs are bacteriostatic agents that effectively interfere with the synthesis of folic acid in susceptible organisms. They can be administered via feed, water additives, bolus, or injection. Relevant approved uses include the treatment of pneumonia and other respiratory diseases, mastitis, diarrhea, diphtheria, and foot rot in cattle, as well as for diarrhea, enteritis, pneumonia, bronchitis, and septicemia in swine. Diaminopyrimidines, such as trimethoprim and ormetoprim, can be used as potentiators with sulfonamides to give a synergistic antimicrobial effect, and there are several approved veterinary products that use these drugs in combination.

-lactams are a group of antibiotics, both naturally occurring and semisynthetic, that contain a -lactam ring. These drugs are effective against infections such as colibacillosis, bacterial enteritis, salmonellosis, etc. Penicillin is one of the -lactams used for meat-producing animals. Other -lactams approved for use in cattle or pigs in the United States include ampicllin, amoxicillin, cloxacillin, cephapirin and ceftioflur. These drugs can be administered by injection, orally, or as a medicated feed, depending on the drug and the animal species. Aminoglycoside drugs consist of linked amino sugar groups that interfere with bacterial protein synthesis. They are currently used both therapeutically and prophylactically. Examples of aminoglycosides include streptomycin, apramycin, dihydrostreptomycin, gentamicin, and neomycin. These drugs are not absorbed orally, and so (unless infections of the gut are being treated) are usually administered via intramuscular injection. Aminoglycosides are generally excreted through the kidney, and the drugs tend to concentrate in that organ. Chloramphenicol and related analogue drugs are dichloroacetamide derivatives of 1phenyl-2-amino-1-propanol. These drugs effectively inhibit bacterial cell wall formation. Chloramphenicol is used for humans and companion animals; however, it is prescribed cautiously because it may cause aplastic anemia in susceptible individuals. It is not approved in food-producing animals so that unintended exposure, even at residue levels, can

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be avoided (World Health Organization [WHO], 1988). Because chloramphenicol has been found to be effective in treating infections in cattle, monitoring food for residues that may occur as a result of off-label use is important. Florfenicol, which does not cause the same potentially dangerous side effects, has been approved for treatment of bovine respiratory disease. Tetracyclines are antibiotics originally isolated from Streptomyces. They are broadspectrum antibiotics that inhibit protein synthesis in bacterial cell walls. The most common tetracyclines used for animal health are tetracycline, oxytetracycline, and chlortetracycline. These drugs are used in swine and cattle to prevent bacterial infections such as enteritis, pneumonia, and anaplasmosis; chlortetracycline and oxytetracycline are also used as feed additives to promote weight gain and increase feed efficiency. Macrolide antibiotics consist of a multi-membered lactone ring with one or more sugars attached. The macrolides are isolated from Streptomyces species and are most effective against gram-positive organisms and some strains of Listeria and Mycoplasma. Erythromycin and tylosin are the most commonly used in food-producing animals. Other macrolides, including oleandomycin, spiramycin, sedamycin and tilmicosin, are also used in animal husbandry. These drugs have used therapeutically to treat bovine respiratory diseases or mastitis or as a feed additive to promote growth efficiency. Although their chemical structure is quite different, the lincosamide antibiotics (lincomycin, pirlimycin) have similar antibacterial activity, clinical applications, and cellular mechanisms as the macrolides. Coccidiosis is most commonly associated with poultry, but it occurs in other species as well. Antibiotics administered to cattle to treat coccidiosis include amprolium, lasalocid, and monensin. The ionophores (monensin, lasalocid) are also used to promote growth efficiency. There are established tolerances for clopidol, another coccidiostat, in swine and cattle. Other classes of drugs, such as the nitroimidazoles or nitrofuran antibiotics, which have been used as coccidiostats in poultry, also have a history of use in cattle and pigs. Some data suggest that nitroimidazoles and nitrofurans may be carcinogenic, and therefore these drugs are not approved for use in food animals in the United States. Other antibacterial agents that are used in veterinary medicine to treat infection or promote growth efficiency are carbadox, roxarsone, bambermycin, and bacitracin. B. Antiparasitics Antiparasitics are drugs that are used to control parasitic infections. Benzimidazoles are anthelmintics containing a common 1,2 diaminobenzene nucleus; most also have a carbamate functional group. These drugs work by inhibiting the enzyme necessary for the parasite’s mitochondrial production of ATP. Albendazole is a common drug in this class. The benzimidizoles are usually administered in feed or via bolus. The use of these drugs (fenbendazole) can also increase the rate of gain in growing-finishing swine. Avermectins are anthelmintics isolated from Streptomyces avermitilis. They are macrocyclic lactone derivatives and are used to treat roundworms, mites, and grubs. The most commonly used drug in this class is ivermectin; others used in animal production include eprinomectin, doramectin, and the synthetic derivative moxidectin. Levamisole is a broad-spectrum synthetic anthelmintic used for the control of lungworms and gastrointestinal nematodes in cattle, sheep, swine, and other food animals. It is administered orally, topically, via injection, or in a medicated feed. Tetrahydropyrimidines, i.e., pyrantel or morantel (the methyl ester of pyrantel), are used in swine for the treatment

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of Ascaris and Oesophagostomum and are considered broad-spectrum anthelmintics in ruminants. Organophosphates (i.e., Dichlorvos, Coumaphos) are also used as anthelmintics as well as for external pest control. C. Hormones and Growth Agents The use of hormones for increasing the weight gain of food animals is a controversial global issue. Historically, sex hormones or drugs that mimic these compounds have been used for this purpose. Naturally occurring sex hormones such as estradiol, progesterone, and testosterone have been given to increase feed efficiency and produce animals with a more favorable muscle/fat ratio. Synthetic compounds such as melengestrol acetate, trenbolone acetate, and diethylstilbestrol (DES) mimic the natural hormones and have also been used in agricultural production. Zeranol is a compound with similar activity derived from plants. Because of the concern to human health, the use of DES was universally banned in 1978. In 1988 the European Community also banned the use of any anabolic steroids in food-producing animals. In the United States, some of these compounds are approved, providing the manufacturer adheres to proper treatment and withdrawal requirements (Franco and Adams, 1994). Corticosteroids, compounds derived from cortisone (a naturally occurring compound produced in the adrenal cortex), include dexamethasone, prednisolone, betamethazone, flumethasone, prednisone, and triamcinolone. These compounds are effective anti-inflammatory and gluconeogenic agents. These drugs can be used as therapeutic agents for cattle in some instances, but their use as growth promoters or as aids to pass inspection at slaughter is not allowed. Another controversial issue is the use of somatotropines. These compounds are produced naturally by the pituitary glands and regulate various processes such as milk production in cattle and muscle/fat ratio in swine (NRC, 1999; Rochut et al., 2000). These compounds can also be produced using recombinant DNA techniques and administered to the animals to positively effect these processes. Bovine somatotropine products are used in the United States but are not allowed for use in Europe, for fear that the increased milk production may cause an increase in mastitis and therefore the need for therapeutic antibiotics. D. Other Animal Drugs In addition to antibiotics and growth promotants, other drugs that may be given to food animals include tranquilizers and anti-inflammatory drugs. These are of concern because they are often given to animals shortly before they are slaughtered; therefore, high levels of residues may persist in edible tissue. For example, tranquilizers such as acepromazine, azaperone, chlorpormazine, propionlypromazine, and xylazine are sometimes administered to reduce stress or aggressiveness during transport for certain species of pigs (Govaert et al., 1998). The -blocker carazolol may also be given. These drugs have also historically been used as a preanesthetic agent in cattle. In the European Union, maximum residue limits have been set for carazolol and azaperon; the use of promazines in food animals is not allowed. In the United States, azaperone is approved for the control of aggressiveness in small pigs. The nonsteroidal anti-inflammatory drugs used in veterinary medicine include flunixin, phenylbutazone, and naproxen. In general, these drugs are approved for companion and nonfood animals (horses) and are not to be used in cattle or swine. Flunixin is approved in the United States for some therapeutic applications in cattle. There is some evidence,

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however, that these drugs are used to disguise lame or sick food animals to allow them to pass federal meat inspections (Clark et al., 2000). Residues of phenylbutazone, in particular, may be a human health concern because this drug has been shown to induce tumors in some species and exposure to the drug can cause serious adverse reactions such as aplastic anemia and gastrointestinal bleeding in susceptible people (Gowik et al., 1998; De Veau, 1999). E. Emerging Health Concerns with Specific Classes of Animal Drugs Quinolones are pyridone carboxylic acid derivatives that are effective against gram-negative bacteria. Fluoroquinolones (FQs) are a fluorine-containing subclass of the quinolones that have been found to be more effective and also exhibit activity against some gram-positive bacteria. Examples of FQs include sarafloxacin, enrofloxacin, difloxacin, ciprofloxacin, flumequine, and norfloxacin. Sarafloxacin and enrofloxacin were approved for use in poultry in the United States, and these drugs are believed to be used in cattle in other parts of the world. Quinolones, including the FQs, act by interfering with the bacterial DNA gyrase enzyme. Residues of fluoroquinolones in tissue of food animals are of concern because of reports of development of antibacterial resistance to these drugs in humans. For example, the studies mentioned earlier show a strong link between the use of FQ antibiotics in chickens and an increase in Campylobacter-resistant infections in humans (Smith et al., 1999; Endtz et al., 1991). Because of these concerns, the FDA has banned the extra-label use of these drugs in food-producing animals. There is a great deal of concern about the continued use of another group of drugs in animals: those used to treat resistant Enterococci faecium in humans. Avoparcin is a glycopeptide antibiotic closely related to vancomycin, an antibiotic of choice for infections of E. faecium that do not respond to more traditional antibiotics. Although the link between the use of these avoparcin in animal feed and an increase in vancomycin-resistant infections in humans is still an issue of debate (Woodford, 1998; Wegener et al. 1999; Klein 2000; Klare et al., 1999), the use of avoparcin in food animals was discontinued in Europe. Avoparcin is not approved for use in food animals in the United States. Recently the FDA approved a streptogramin for treatment of vancomycin-resistant E. faecium in humans. Virginamycin, also a streptogramin, has been used in food-producing animals for several years. The FDA is currently conducting a risk assessment to determine if the use of this drug in food animals increases the risk that E. faecium infections resistant to this class of drugs will also develop (FDA, 2000b). Beta-agonists are a class of drugs where the health concern in not antibiotic resistance but acute poisoning from the drug residues themselves. Clenbuterol, the b-agonist most commonly given to animals illegally to increase their muscle mass, has been reported to cause human illness (headache, tremors, cardiac palpitations, and nausea) in Europe (Mitchell and Dunnavan, 1998). Although extensive sampling by USDA for this drug in bovine liver tissue did not show a problem with residues, a monitoring program targeting the retinal tissue (the drug exhibits very slow withdrawal in this tissue) of show animals did reveal that the drug was being used in this application. There are many analogues of clenbuterol that may also be used to increase the proportion of lean meat in an animal. Studies indicate that some of these compounds may eventually prove to be safe and effective animal drugs (Smith, 1998; including the recently approved drug ractopamine); however, the potential human health effects dictate that regulatory agencies must be aware of new analogues that are being produced and used illegally (Brambilla et al., 2000).

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III. REGULATIONS AND RESPONSIBILITIES A. Background The responsibility of reducing the incidence of animal drug residues in the nation’s meat supply is shared between the United States Department of Agriculture (USDA) and the FDA. The USDA is responsible for monitoring meat and poultry products for animal drug residues. The Food Safety Inspection Service (FSIS) of the USDA conducts the National Residue Program (NRP) to prevent animals containing violative amounts of drug residues from being marketed (FSIS, 1998). A large part of the NRP involves collecting samples for analysis in the slaughter plants. Extensive on-site sampling is done; samples are also sent to FSIS field laboratories for further testing. The FDA Center for Veterinary Medicine (CVM) is responsible for approving new animal drugs, setting tolerances, and enforcement actions as a result of FSIS findings. FDA CVM and the Office of Regulatory Affairs may also run follow-up analyses and conduct investigations to document use of illegal drugs or prohibited off-label use. B. Recent Changes in Regulation and Monitoring of Drug Residues In the past several years, two laws have passed that affect the way animal drugs are regulated (Baxter, 1998). The first is the Animal Medicinal Drug Use Clarification Act of 1994. This law gives veterinarians greater flexibility to prescribe extra-label uses of approved human drugs for animal use under certain conditions. The Animal Drug Availability Act of 1996 describes measures designed to increase the availability of animal drugs in the marketplace, including streamlining the new drug approval process. The use of certain drugs in food animals may be still be banned (or just their extra-label use may be banned) because of the possible human health risks mentioned already. These include chloramphenicol, diethylstilbestrol, clenbuterol, nitroimidazoles, fluoroquinolones, and glycopeptides. Another change in the way animal drug residues are regulated is the introduction of hazard analysis at critical control points (HACCP) into the FSIS meat inspection program. The use of HAACP as a tool to ensure food safety extends to residue monitoring as well. Under the HAACP system the meat slaughter and processing plants must have systems in place to prevent hazards in their products. These hazards include not only pathogenic organisms and physical hazards, but also chemical residues such as illegal drugs or pesticides. The rule indicates that the industry must evaluate significant residue hazards and develop a HAACP plan for controlling residues (FSIS, 1999). Another change in the way animal drug residues are monitored deals with the issue of international harmonization and the global economy. In the past several years there have been many issues concerning food safety, relating not only to drug residues but to other issues, such as genetically modified foods, that have been viewed very differently in different parts of the world. The organizational structure of the groups responsible for monitoring the food supply for violative drug residues also varies globally (Oka et. al, 1995). There are organizations, such as the Codex Alimentarius Commission (part of the Food and Agriculture Organization of the United Nations) and the World Health Organization, as well as scientific bodies, such as AOAC International, that are trying to reach international consensus on the subjects of drug usage, acceptable residue levels, and appropriate monitoring programs.

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IV. ANALYTICAL METHODOLOGY A. Background Because of the potential health risk from exposure to some animal drugs and the low levels at which they are expected, monitoring the food supply for these residues is an important analytical challenge. Several broad types of analytical methods can be described; these include screening, determinative, and confirmatory procedures. Screening methods are designed to be rapid, easy-to-use tests that will give a positive or negative response for a drug at a given concentration level in a matrix. Traditionally, microbial inhibition tests (MITs) have been used to screen large amounts of samples for antibiotics, and these tests are still widely used today. All MITs are based on the inhibition of bacterial growth by antibacterial residues present in a biological fluid that results in zones of inhibition on bacterial plates. MITs are relatively simple to use and detect many classes of antibacterial compounds. Selective sensitivity for specific classes of antibacterials can be obtained by changes in the culture medium, indicator bacteria, or pH. However, these methods often lack the specificity and sensitivity required for residue detection at maximum residue limits, may be affected by nonspecific inhibitors, do not detect microbiologically inactive metabolites, and often have a 20- to 24-hour incubation time. MITs that have been used by the FSIS for screening red meat and poultry tissues for antibacterial residues include the Swab Test On Premises (STOP), and the Calf Antibiotic and Sulfa Test (CAST) (Sundlof, 1989). The STOP test was one of the original MITs used to detect antibiotic residues in kidney and other tissues of slaughter animals (Johnston et al., 1981). The Fast Antibiotic Screen Test (FAST) is a test used by USDA-FSIS and provides results within 6 hours. It has undergone field trials involving 10,000 samples for comparison with STOP and CAST for sensitivity. The FAST assay is similar to the previous procedures, but the FAST growth medium contains sugar and a purple dye. Bacterial metabolism of the sugar results in acid production and a lowering of the pH that causes a color change from purple to yellow. A sterile cotton swab is saturated with fluid from a tissue sample and placed on a plate of growth medium streaked with bacterial spores, which is then incubated for 6 hours. A purple zone surrounding the sample swab indicates the presence of antimicrobial agent(s). There are several other MITs used globally, including the New Dutch Kidney Test, the German Three Plate Assay, the European Four Plate Test, and the Charm Farm Test. These screening tests, however, are not always accurate at or below the test threshold; are often class, not compound, specific; and may or may not give quantitative information. Therefore, additional analytical tests may be needed to determine if a sample is actually violative for an animal drug residue. Determinative methods are designed to separate, quantitate, and perhaps provide some qualitative information on the analyte of interest. For many of the drugs used in animal agriculture, the method of choice is liquid chromatography (LC) with UV detection, using a variable wavelength or diode array detection system. Liquid chromatographs using fluorescence, chemiluminescence, or post column reaction detectors are also available and have been successfully used to determine drug residues in animal tissue. Gas chromatography (GC) can also be used to analyze drug residues in food matrices. GC is very suitable for volatile analytes such as organophosphates. Many animal drugs are of large molecular weight and are relatively nonvolatile and thermally labile. To overcome these characteristics, chemical derivatization is generally required to obtain sufficient volatility and stability for GC analysis. Several reviews have been written on the determinative methods in use today (Oka et al., 1995, Turnipseed and Long, 1998).

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B. Emerging Issues in the Analysis of Animal Drug Residues 1. Rapid Screening Tests There are some inherent problems with MITs in general, including the FAST test (Korsrud et al., 1998). Imprecision occurs as a result of zone size differences between replicate plates. Zone size may vary as a result of differences in agar layer thickness, agar quality, uneven seeding of bacterial spores on the agar surface, or incubator temperature variation (Brady and Katz, 1987). Additionally, bacteriostatic drugs such as sulfonamides may result in a diffuse zone, whereas bactericidal drugs provide a sharply defined zone of inhibition, and this may complicate the interpretation. In fact, studies indicate that the sensitivity to the FAST test for different classes of drugs can vary widely (Korsrud, 1998; Clark et al., 1999). In addition to MITs, new rapid test kits, generally based on bacterial cell receptor or enzyme immunoassay, are being used to screen samples for specific drugs. The Charm II test is a proprietary competitive microbial receptor binding assay that can detect residues of seven classes of antibiotics. Because the bacterial receptors bind to a functional group on the drug, rather than a side chain as with immunoassay tests, the Charm II test provides detection of a class of antibacterial compounds rather than a single compound. Although this test can detect a number of drugs within a class, the relative sensitivity of the test to individual drugs varies. It is more commonly used for monitoring antibiotics in milk but has been tested for use in bovine muscle and kidney samples. (Korsrud et al., 1994) and was found to be an acceptable, more cost-effective, alternative to thin-layer chromatographybioautography for beta-lactams, (dihydro)streptomycin and erythromycin, and sulfonamides Immunoassays are widely used to monitor therapeutic drugs and drugs of abuse in human medicine, for dog and racehorse testing, and on dairy farms and processing plants to screen milk samples for veterinary drug residues. However, with appropriate extraction methodology, many of these assays may be used for residue analysis of food animal tissues. Recent examples of the use of immunoassays to meat analysis include the analysis of levamisole in meat (Silverlight and Jackman, 1994) aminoglycosides in porcine kidney (Haasnoot et al., 1999), tetracyclines in pork and chicken meat (De Wasch et al., 1998), and nonsteroidal anti-inflammatory drugs in bovine kidney (Clark et al., 2000). Immunoassay may offer a cost-effective and rapid alternative to conventional methodology for drug residue screening. 2. Emerging Technology for Sample Preparation Sample preparation, isolation, and cleanup are major rate-limiting factors in sample analysis, and improvements in this area will advance the development of analytical methods. This fact is especially important in the light of efforts to introduce rapid residue screening tests such as immunoassays. Historically, the classic approach to isolation of drugs from tissues has most often been reported. This approach involves tissue homogenization followed by liquid–liquid partitioning of the homogenate, with or without additional cleanup or concentration steps. These methods may provide adequate separation of the drug from matrix but are often expensive in terms of time, labor, material use, and organic solvent disposal costs. Such approaches also tend to be highly nonspecific in their isolation of the target drug(s). Recent advances in the field of residue analysis offer several promising techniques as possible solutions to the problems caused by outmoded and complex analytical methods. Three techniques, solid phase extraction (SPE), matrix solid phase dispersion (MSPD), and

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immunoaffinity are receiving particular attention because they have the potential to greatly reduce analysis costs and reduce analyst-generated waste and pollution. In the SPE process, a compound is isolated from a liquid sample based on its relative solubility in the liquid mobile phase compared to its solubility in a solid support-bound liquid stationary phase of differing polarity or its affinity to a solid support stationary phase of differing polarity. Isolation is accomplished by passing the analyte dissolved in solvent (organic or aqueous) through a column containing the stationary phase with subsequent elution using an appropriate solvent. Before SPE can be used with solid tissue (e.g., muscle and liver), a separate homogenization step and often multiple filtration, sonication, centrifugation, and liquid–liquid cleanup steps are required. Although SPE may improve cleanup of these solid tissue samples, the additional labor and materials costs make SPE less suitable in some cases. Solid phase extraction methods published for meat and fish tissues are often combinations of SPE with other techniques such as homogenization, liquid–liquid partition, filtration, sonication, and centrifugation. Because the choice of SPE column depends on the matrix and on the particular compound of interest, a wide range of solid-phase extraction columns of differing polarities have been used for drug extraction from tissue and include silica, alumina, C18, NH2, and ion-exchange resins. High throughput processing of samples using arrays of SPE columns developed for combinatorial chemistry applications in drug discovery may have relevance to the analysis of animal drug residues as well (Harrison and Walker, 1998). Matrix solid phase dispersion, a variation of the SPE technique, has some definite advantages. In general terms, MSPD involves blending a tissue sample (0.1 to 1.0 g) with lipophilic polymer-derivatized silica particles (e.g., octadecylsilyl [ODS]-derivatized silica [C18]), which simultaneously disrupts and disperses the sample. This blend of C18 and tissue becomes part of a potentially multiphasic column that possesses unique chromatographic character. Elution of the MSPD column with a solvent or solvent sequence can provide a high-resolution fractionation of target analytes that can be further purified by simultaneous use of co-columns. The final eluate can, in most cases, be directly analyzed, further concentrated, or manipulated to meet the demands of the individual analysis. MSPD has been used in the analysis of furazolidone (Long et al. 1991), penicillins (McGrane et al., 1998), and sulphamethazine (Shearan et al., 1994) in swine tissue as well as clenbuterol in bovine liver (Horne et al., 1998). The advantage of MSPD is that it allows for rapid turnover of samples and hence, access to timely data on residue levels present in samples. Because of its required small sample size, it considerably decreases solvent use compared to the classic methods, which in turn decreases environmental contamination and increases worker safety; it is also suitable for robotics automation. The simplest method of extraction, however, is one that requires minimal or no sample manipulation. These are the methods that extract the drug directly from the sample matrix by means of specific or selective antibodies or receptors. High performance immunoaffinity chromatography (HPIAC) is one such method that combines immunoassay and LC. In HPIAC, a flow-through cartridge containing antibody immobilized on glass or porous beads is used in place of a LC analytical column. Following sample injection, the drug (antigen) is captured on antibody-coated beads, the cartridge is washed to remove unbound material, and the drug is then eluted from the cartridge. Detection may be by UV or other LC detection methods. Immunoaffinity isolation techniques are also used for sample cleanup prior to conventional analysis. Recent examples where immunoaffinity techniques have been used in animal drug residue analysis include the determination of 19-nortestosterone and trenbolone in animal tissue (Stubbings et al., 1998) and avermectins in cattle meat (Li and Qian, 1996). Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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3. Expanded Use of Mass Spectrometry Confirmatory methods are meant to provide absolute identification of the drug residue in question. Because of its sensitivity and specificity, mass spectrometry is the preferred method for confirmation. Guidelines as to what should constitute a positive identification with mass spectral data have been discussed (Sphon 1978; Bethem and Boyd, 1998). As mass spectrometry, particularly modern LC/MS, becomes more practical and affordable, it has been also used to screen and perform quantitative analyses as well as provide qualitative confirmation. For example, corticosteroids could be both screened and confirmed in beef tissue using LC/MS (Stolker et al., 2000). Clenbuterol, along with other -agonists, can be determined and confirmed in bovine retinal tissue using LC/MS techniques (Doerge et. al., 1996). Blanchflower et al. (1996) successfully determined levels of monensin, salinomycin, and narasin in muscle, liver, and eggs from poultry at the low ppb level using liquid chromatography–electrospray mass spectrometry. In another example, Volmer (1996) used electrospray LC/MS/MS for the screening, quantitation, and confirmation of 21 sulfonamides as well as trimethoprim and ormetoprim in milk at the low ppb to ppt level. These combined screening, determinative, and confirmatory methods will most likely be applied more extensively to residue analyses in meat in the near future. In addition, mass spectrometry has also been used to distinguish naturally occurring bovine somatotropin from that produced by recombinant DNA techniques in milk (Rochut et al., 2000), and can be used to identify new analogues of drugs such as -agonists that might otherwise elude the regular screening test (Brambilla et al., 2000). 4. Biosensors and Biochips Technological advances made in other areas of analytical, process, and food chemistry will have an effect on the analysis of drug residues in animal tissues. For example, the Biocore company in Switzerland has developed optical biosensors that have been used for the analysis of animal drug residues, including the detection of sulfonamides in meat (Crooks et al., 1998, Bjurling et al., 1999). Biochip array technology using immunoassay with chemiluminescence detection has been developed for the detection of growth promoters and sulfonamides (McConnell et al., 2000). All of these new technologies will have an impact on how animal drug residues are monitored and regulated in the future. V. CONCLUSIONS The use of drugs is prevalent in the food animal industry to control bacterial and parasitic infections as well as to enhance growth efficiency. Although there are many economic advantages to continuing these practices, the effect on human health, particularly as it relates to antibiotic resistance, must be frequently evaluated. Better analytic monitoring techniques and more global regulatory cooperation will be needed to effectively manage the use of drugs in meat production. REFERENCES Bethem, R.A., and R.K. Boyd. 1998. Limits to confirmation, quantitation and detection by mass spectrometry. Proceedings of the 46th ASMS Conference on Mass Spectrometry and Allied Topics. May 31–June 4, Orlando, FL Baxter, B.K. 1998. Compliance requirements for animal drugs. In: S.B. Turnipseed and A.R. Long (ed.) Analytical Procedures for Drug Residues in Food of Animal Origin. p 9. Science Technology System, West Sacramento, CA.

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Bjurling, P., B. Persson, C. Jonson, M. O’Conner, A. Baxter, and C.T. Elliott. 1999. Detection of sulphamethazine and sulphadiazine in meat using biosensor technology. Paper presented at the 113th AOAC International Annual Meeting and Exposition. September, 29, Houston, TX. Blanchflower, W.J., and D.G. Kennedy. 1996. Determination of monensin, salinomycin and narasin in muscle, liver and eggs from domestic fowl using liquid chromatography-electrospray mass spectrometry. J. Chromatogr B. 675:225–233. Booth, N.H., and L.E. McDonald. 1982. Veterinary Pharmacology and Therapeutics. Iowa State University Press, IA Brady, M.S., and S.E. Katz. 1987. Simplified plate assay diffusion system for microbial assays of antibiotics. J Assoc Official Anal Chem 70:641–646. Brambilla, G., M. Fiori, C. Testa, B. Neri, F. Busico, M. Di Pietrogiacomo, R. Cozzani, and G. Boatto. 2000. Identification of new clenbuterol-like substances in urine and hair by hyphenated techniques. Paper presented at Euroresidue IV Conference on Residues of Veterinary Drugs in Food, May 8–10, Veldhoven, The Netherlands. Clark, S.B., C.R. Kiessling, J.A. Hurlbut, J.N. Sofos, and M.R. Madson. 1999 Sensitivity of detection of various veterinary drugs using the FAST antimicrobial screen test. Paper presented at the 113th AOAC International Annual Meeting and Exposition. September, 29, Houston, TX. Clark, S.B., S.B. Turnipseed, G.J. Nandrea, M.R. Madson, E.R. Singleton, J.A. Hurlbut, J.N. Sofos, and C.E. Shultz. 2000. Identification and confirmation of flunixin meglumine and phenylbutazone residues in animal kidney by ELISA screening and liquid chromatography mass spectrometry. FDA/ORA Laboratory Information Bulletin (in press). Code of Federal Regulations (CFR). 1999. Food and Drugs. Parts 500–599. Revised 4/1. Crooks, S.R., G.A. Baxter, M.C. O’Connor, and C.T. Elliot. 1998. Immunobiosensor—an alternative to enzyme immunoassay screening for residues of two sulfonamides in pigs. Analyst 123:2755–7. De Veau, I.E.J. 1999. Determination of non-protein bound phenylbutazone in bovine plasma using ultrafiltration and liquid chromatography with ultraviolet detection J Chromatogr B 721:141–145. De Wasch, K., L. Okerman, S. Croubels, H. De Brabander, and J.D. Van Hoof. 1998. Detection of residues of tetracycline antibiotics in pork and chicken: correlation between results of screening and confirmatory tests. Analyst 123:2737–2741. Doerge, D.R., M.I. Churchwell, C.L. Holder, L. Rowe, and S. Bajic. 1996. Detection and confirmation of -agonists in bovine retina using LC/APCI-MS. Anal Chem 68:1918–1923. Endtz, H.P, G.J. Ruijs, B. van Klingeren, W.H. Jansen, T. van der Reyden, and R.P. Mouton. 1991. Quinolone resistance in Campylobacter isolated from man and poultry following the introduction of fluoroquinolones in veterinary medicine. J Antimicrob Chemother. 27:199–208. Food and Drug Administration (FDA). 2000a. Draft Risk Assessment on the Human Health Impact of Fluoroquinolone Resistant Campylobacter Associated with the Consumption of Chicken. Available at: http://www.fda.gov/cvm/fda/mappgs/ra/risk.html. Accessed May 23, 2000. Food and Drug Administration (FDA). 2000b. Risk assessment of the public health impact of streptogramin resistance in Enterocuccus faecium attributable to the use of streptogramins in animals; request for comments and for scientific data and information. Available at http://www.fda.gov/OHRMS/DOCKETS/98fr/041900c.txt. Accessed May 23, 2000. Food Safety Inspection Service, United States Department of Agriculture. 1998. National Residue Program Report, Washington, D.C. Available at: http://www.fsis.usda.gov/OPHS/red98/index.htm. Accessed May 23, 2000. Food Safety Inspection Service, United States Department of Agriculture. 1999. The Impact of Pathogen Reduction/HACCP On Food Animal Production Systems. Washington, D.C. Available at: http://www.fsis.usda.gov/oppde/ap/presentations/district%20ma nagers.htm. Accessed May 23, 2000. Franco, D.A. and C.E. Adams. 1994. Hormones. In: L.M. Crawford and D.A. Franco (ed) Animal Drugs and Human Health, p. 103–111, Technomic Publishing, Lancaster, PA

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Glynn, M.K., C. Bopp, W. Dewitt, P. Dabney, M. Mokhtar, and F.J. Angulo. 1998. Emergence of multidrug-reistant Salmonella eterica serotype typhimurium DT104 infections in the United States. N Engl J Med 19:1333–1338. Govaert, Y., P. Batjoens, K. Tsilikas, J.-M. Degroodt, and S. Srebrnik. 1998. Multi-residue analysis of tranquilizers in meat: confirmatory assays using mass spectrometry. Analyst 123:2507–2512. Gowik, P., B. Julicher, and S. Uhlig. 1998. Multi-residue method for non-steroidal anti-inflammatory drugs in plasma using high-performance liquid chromatography—photodiode array detection. J Chromatogr B. 716:221–232. Haasnoot, W., P. Stouten, G. Cazemier, A. Lommen, J.F. Nouws, and H.J. Keukens. 1999. Immunochemical detection of aminoglycosides in milk and kidney. Analyst 124:301–305. Harrison, A.C., and D.K. Walker. 1998. Automated 96-well solid phase extraction for the determination of doramectin in cattle plasma. J Pharm Biomed Anal 16:777–783. Horne, E., M. O’Keefe, C. Desbrow, and A. Howells. 1998. A novel sorbent for the determination of clenbuterol in bovine liver. Analyst 123:2517–2520. Institute of Medicine. 1989. Human health risks with the subtherapeutic use of penicillin or tetracyclines in animal feeds. National Academy Press, Washington, D.C. Johnston, R.W., R.H. Reamer, E.W. Harris, H.G. Fugate, and B. Schwab. 1981. A new screening method for the detection of antibiotic residues in meat and poultry tissues. J Food Prot 44:828–831. Klare, I., D. Badstubner, C. Konstabl, G. Bohme, H. Claus, and W. Witte. 1999. Decreased incidence of VanA-type vancomycin-resistant enterococci isolated from poultry meat and from fecal samples of humans in the community after discontinuation of avoparcin in animal husbandry. Microb Drug Resist 5:45–52. Klein, G. 2000. Food as a potential vector for antibiotic resistances. Berl Munch Tierarztl Wochenschr 113:46–52. Korsrud, G.O., C.D.C. Salisbury, A.C.E. Fesser, and J.D. MacNeil. 1994. Investigation of Charm Test II receptor assays for the detection of antimicrobial residues in suspect meat samples. Analyst 119:2737–2741. Korsrud, G.O., J.O. Boison, J.F.M. Nouws, and J.D. MacNeil. 1998. Bacterial Inhibition Tests Used to Screen for Antimicrobial Veterinary Drug Residues in Slaughtered Animals. J AOAC Int. 81:21–24. Li, J., and C. Qian. 1996. Determination of avermectin B1 in biological samples by immunoaffinity column cleanup and liquid chromatography with UV detection. J AOAC Int 79:1062–1067. Long, A.R., L.C. Hsieh, M.S. Malbrough, C.R. Short, and S.A. Barker. 1991. Matrix solid phase dispersion (MSPD) isolation and liquid chromatographic determination of furazolidone in pork muscle tissue. J Assoc Off Anal Chem 74:292–294. Long, A.R., and J.E. Roybal. 1994. Drug Residues in Foods of Animal Origin. In: Y.H. Hui, J.R. Gorham, K.D. Murrell, and D.O. Cliver (eds) Foodborne Diseases Handbook, Vol 3. pp 529–554. Marcel Dekker, New York, NY. McConnell, R.I., J.V. Lamont, S.P. Fitzgerarld, L.T. Farry, and J.A. Mills. 2000. The development of biochip technology for the detection of drug residues. Paper presented at Euroresidue IV Conference on Residues of Veterinary Drugs in Food, May 8–10, Veldhoven, The Netherlands. McGrane, M., M. O’Keefe, and M.R. Smyth. 1998. Multi-residue analysis of penicillin residues in porcine tissue using matrix sold phase dispersion. Analyst 123:2779–2783. Mitchell, G.A., and G. Dunnavan. 1998. Illegal use of adrenergic -agonists in the United States. J Animal Sci 76:208–211. Natural Research Council (NRC). 1999. The Use of Drugs in Food Animals: Benefits and Risks. National Academy Press, Washington, D.C. Oka, H.H. Nakazawa, K.-I. Harada, and J.D. MacNeil. 1995. Chemical Analysis for Antibiotics Used in Agriculture. AOAC International, Arlington, VA. Quednau, M., S. Ahrne, A.C., Petersson, and G. Molin. 1998. Antibiotic-resistant strains of Enterococcus isolated from Swedish and Danish retailed chicken and pork. J Appl Microbiol 84:1163–1170.

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Rochut, N., B. Le Bizec, F. Monteau, and A. Francois. 2000. Detection and identification of proteic growth hormones in cattle-analysis of bovine and porcine somatotropin by LC-MS” Paper presented at Euroresidue IV Conference on Residues of Veterinary Drugs in Food, May 8–10, Veldhoven, The Netherlands. Schwartz, H.J., and T.H. Sher. 1984. Anaphylaxis to penicillin in a frozen dinner. Ann Allergy 52:342–343. Shearan, P., M. O’Keefe, and M.R. Smyth. 1994. Comparison of matrix solid phase dispersion (MSPD) with a standard solvent extraction method for sulphamethazine in pork muscle using high performance liquid and thin layer chromatography. Food Addit Contam 11:7–15. Silverlight, J., and R. Jackman. 1994. Enzyme immunoassay for the detection of levamisole in meat. Analyst 119:2705–2706. Smith, K.E., Besser, J.M., Hedberg, C.W., Leano, F.T., Bender, J.B., Wicklund, J.H., Johnson, B.P., Moore, K.A., and M.T. Osterholm. 1999. Quinolone-resistant Campylobacter jejuni infections in Minnesota, 1992–1998. Investigation Team. N Engl J Med 340:1525–1532. Smith, D. 1998. The pharmacokinetics, metabolism and tissue residues of -adrenergic agonists in livestock. J Anim Sci 76:173–194. Sphon, J.A. 1978. Use of mass spectrometry for confirmation of animal drug residues. J Assoc Off Anal Chem 61:1247–1252. Stolker, A.A.M., P.L.W.J. Schwillens, C.J.P.F. Kuijpers, C.A. Kan, and L.A. van Ginkel. 2000. The use of LC-MSn for screening and confirmation of corticosteroids in biological matrices. Paper presented at Euroresidue IV Conference on Residues of Veterinary Drugs in Food, May 8–10, Veldhoven, The Netherlands. Stubbings, G.W., A.D. Cooper, M.J. Sheperd, J.M. Croucher, D. Airs, W.H. Farrington, and G. Shearer. 1998. Determination of 19-nortestosterone and trenbolone in animal by high-performance liquid chromatography with immunoaffinity clean-up. Food Addit Contam 15:293–301. Sundlof, S.F. 1989. Veterinary Clinics of North America: Food Animal Practice. 5:411–444. Sundlof, S.F., and J. Cooper. 1996. Human health risks associated with drug residues in animal-derived foods. In: W.A. Moats and M.B. Medina (eds) Veterinary Drug Residues Food Safety. pp. 5–17., American Chemical Society, Washington, D.C. Tinkleman D.G., and S.A. Bock. 1984. Anaphylaxis presumed to be caused by beef containing streptomycin. Ann Allergy 53:243–244. Turnipseed S.B., and A.R. Long. 1998. Analytical Procedures for Drug Residues in Food of Animal Origin. Science Technology System, West Sacramento, CA. Volmer, D.A. 1996. Multiresidue determination of sulfonamide antibiotics in milk by short-column liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 10:1615–1620. Wegener, H.C., F.M. Aarestrup, L.B., Jensn, A.M. Hammerum, and F. Bager. 1999. Use of antimicrobial growth promoters in food animals and Enterococcus faecium resistance to therapeutic antimicrobial drugs in Europe. Emerg Infect Dis 5:329–335. Wilson, R.C. 1994. Antibiotic residues and the public health. In: L.M. Crawford and D.A. Franco (eds) Animal Drugs and Human Health. pp 1–10. Technomic Publishing Company, Lancaster, PA. World Health Organization. 1988. Toxicological Evaluation of Certain Veterinary Residues in Food, Geneva. Woodford, N. 1998. Glycopeptide-resistant enterococci: a decade of experience. J Med Microbiol 47:849–862.

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10 Antemortem Handling and Welfare TEMPLE GRANDIN Colorado State University, Fort Collins, Colorado

I. INTRODUCTION II. CONTINUOUS MEASUREMENT AND MONITORING III. MEAT QUALITY CORRELATIONS IV. HOW STRESSFUL IS SLAUGHTER? V. CAUSES OF POOR WELFARE AUDIT SCORES VI. ANIMAL VISION, HEARING, AND SMELL A. Vision B. Hearing C. Smell VII. BASIC HANDLING PRINCIPLES VIII. HANDLER MOVEMENT PATTERNS IX. DESIGN OF HANDLING FACILITIES A. Nonslip flooring B. Pen Space C. Design of Races and Crowd Pens X. DESIGN AND OPERATION OF RESTRAINT DEVICES XI. STUNNING A. Electrical Stunning B. Captive-Bolt Stunning C. Carbon Dioxide Stunning D. Assessing Insensibility E. Ritual Slaughter XII. STUNNING METHOD AND BLOOD-SPLASH A. Quick Bleeding B. Electrical Stunning of Cattle C. Resting and Handling Animals D. Captive-Bolt Stunning E. Ritual Slaughter

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XIII. BRUISING AND MEAT QUALITY A. Death Losses in Pigs B. PSE in Pigs C. Preventing Dark Cutters XIV. CONCLUSIONS REFERENCES

I. INTRODUCTION The Humane Slaughter Act of 1978 in the United States and laws in many other countries require that animals be rendered insensible to pain prior to any slaughtering procedures (1). The meat-buying public is becoming increasingly concerned about how farm animals are raised, transported, and slaughtered (2). Maintaining high standards during handling and stunning should also be done because it is the right thing to do. Quiet handling of livestock and proper stunning procedures will also provide economic benefits by reducing meat quality defects such as PSE (pale, soft, exudative) pork, dark cutters, and toughness in beef and bruises (3,4). In plants processing large animals such as cattle, careful, quiet handling will also help improve safety and reduce employee injuries. Large animals are dangerous when they become agitated. II. CONTINUOUS MEASUREMENT AND MONITORING People who handle and stun hundreds or even thousands of animals often become numb and desensitized to animal suffering (5). The author has observed that handling and stunning procedures have a tendency to become rough and careless unless they are continuously monitored. The manager who is most effective in maintaining high standards of animal welfare must be involved enough to care, but not so involved in day-to-day operations that he or she becomes desensitized. The author strongly recommends using a HACCP-type approach to measuring the efficacy of stunning and the performance of animal handlers. The objective scoring system is described fully in Grandin (6,7). The five major critical control points of stunning and animal handling are briefly outlined here and more information on proper stunning methods will be in the section on stunning. Each critical control point is measured on a yes/no basis for each animal. Fifty to one hundred animals should be scored each week. 1. Stunning efficacy. Percentage of animals rendered insensible on the first attempt. 2. Bleed rail insensibility. Percentage of animals that remain insensible before and after bleeding. 3. Vocalization. Percentage of cattle or pigs that vocalize (bellow, moo, or squeal). Vocalization in cattle and pigs is highly correlated with physiological stress measurements and adverse events such as missed stuns, excessive electric prod use, excessive pressure from a restraint device, slipping or falling, surgery, and isolation of a single animal (8–11). Each pig or bovine is scored as either a vocalizer or non-vocalizer during handling and stunning. Vocalization scoring is done in the crowd pen, single file race, stunning box, restrainer, shackle area, and bleed rail. Vocalization is not scored while animals are standing in the holding pens because cattle standing undisturbed often vocalize to each other. In large plants where counting of indi-

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vidual pigs squeals is difficult, a sound level meter can be used. If an animal is immobilized with electricity it may still be conscious but unable to vocalize. This is extremely distressful for animals and must not be used as a method to keep conscious animals still (12–14). Electro-immobilization must not be confused with electrical stunning, in which a high amperage current is passed through the brain. Vocalization scoring should not be used on sheep because sheep walking quietly up a race often vocalize to each other. 4. Slipping and falling. Percentage of animals that slip or fall during handling and stunning. This is scored in all areas from the unloading ramp to the stunning box or restrainer. 5. Electric prods. Percentage of animals prodded with an electric prod (goad). Reducing the percentage of animals shocked with an electric goad reduces stress and improves welfare. Audits of these five critical control points must be done on a regular basis, the same way microbiological audits are conducted. Bacteria counts would increase and sanitation procedures would become sloppy unless continuous monitoring was done. Handling and stunning must be audited the same way. One factor that contributes to a deterioration of handling and stunning practices is that the only variable that is measured in the stunning area of many plants is gaps in the production line and speed. When worker evaluations are based on gaps in production, this tends to encourage abuse. III. MEAT QUALITY CORRELATIONS Measurements of meat quality and bruises are important. Progressive plant managers have found that quiet handling in the stunning area will reduce PSE (pale, soft, exudative) in pork. In four different plants, the author found that reducing electric prod use and the quiet handling of pigs resulted in 10% more pork that was suitable for high quality export to discriminating customers in Japan. Improved export pass rates will be correlated with reduced squealing. An overemphasis on preventing gaps in the production line may result in more animal stress and increase meat quality problems. Many plant managers base plant performance on keeping the processing line full because processing line gaps are measured and losses due to poor animal handling are often not measured. Handling must be measured on a regular basis to maintain high standards. Animal welfare is also part of quality. Two of the major hamburger chains are using HACCP type audits of animal handling and stunning. They are done in the same manner as microbiological audits. IV. HOW STRESSFUL IS SLAUGHTER? Animal handling, both on the farm and in the slaughter plant, will cause physiological measures of stress to increase. When animals become agitated during handling, it is most likely because of fear. The fear circuits in animal brains have been completely mapped (15,16,17). Grandin (6) reviewed numerous studies of cortisol (stress hormone) levels during handling both on the farm and at slaughter. The range of values were similar for cattle on farm restraint in a squeeze chute and during slaughter. The range of average values was 24 ng/ml to over 63 ng/ml (8, 18–24). Rough handling, slipping on the floor, and electric prod use resulted in higher cortisol levels of 63 ng/ml. The highest average level recorded in a slaughter plant was 93 ng/ml (8). Cattle were inverted on their backs for 103 seconds prior to ritual slaughter. Properly performed cattle slaughter seems to be no more stressful

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than on farm restraint. One must remember that cortisol is a time-dependent measure. Twenty minutes is required to reach peak value (22,23). V. CAUSES OF POOR WELFARE AUDIT SCORES When an audit reveals poor performance, management must determine the exact cause of the problem. The correct diagnosis of problems can help avoid costly purchases of new equipment. Many managers have a tendency to assume that equipment may have to be replaced when a problem could be easily fixed without a major expense. The major causes of high percentages of animals vocalizing or excessive electric prod use are: 1. People using improper handling procedures. This is usually the number one problem. The author has observed that the two most common animal handling mistakes are overloading crowd (forcing) pens and overuse of electric prods. Pigs and cattle need room to turn. Crowd pens should be filled only half full (Fig. 1). Moving small groups of pigs and cattle will facilitate handling. Sheep can be moved in larger groups because this species has more intense following behavior. 2. Distractions that cause balking. This is the second most common problem. All species of animals may balk and refuse to move when they see things in the race that scare them, such as sparkling reflections, dangling chains, moving people or equipment, shadows, or water dripping (24,25). A calm animal will stop and look right at the distractions that scare it (Fig. 2). You should crouch down and

Figure 1 The crowd pen should be filled only half full, as shown in this photo. These people are using excellent handling methods. The crowd gate should not be pushed up tight against the animals.

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Figure 2 A calm animal will stop and look at distractions that scare it. This pig is looking right at a sparkling reflection. You need to get down on the animal’s level to see it.

look up the race to see what the animals are seeing. It is important to get right down at the animal’s eye level. Shields can be installed to prevent animals from seeing moving people or objects up ahead. One of the worst causes of balking is air blowing down the race into the faces of approaching animals. Animals also balk and may refuse to enter a dark place. They have a tendency to move from a darker place to a brighter place (26,27). Adding a light to illuminate a race entrance (Fig. 3) or moving a lamp to eliminate a sparkling reflection will often improve animal movement. If air is blowing toward the animals, the plant ventilation should be changed. Simple inexpensive changes can often greatly improve animal movement. People who are working to remove the distractions that impede animal movement must be very observant of small details that may be insignificant to them. A person may not notice a sparkling reflection, but the animal does. Animals should move through the system easily. If they balk, you should find the distraction that is causing balking instead of increasing electric prod usage. In the very best systems, 95% of the cattle could be moved through a slaughter line that processed over 200 cattle per hour without an electric prod.

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Figure 3 This spotlight illuminates the restrainer entrance and will facilitate cattle entry. Animals tend to approach lighted areas.

There will be more information on facilitating animal movement in the behavior and equipment design sections. 3. Equipment maintenance. Poor maintenance of captive bolt guns is a major cause of poor stunning (7). Employees will become frustrated and will be more likely to handle animals in a rough manner if they are frustrated because gates are broken or other equipment is malfunctioning. 4. Equipment design. This is discussed in the equipment section. 5. Genetic predisposition to excitability. Some animals have a very excitable temperament and are difficult to drive. Some lean pigs and cattle are very excitable (28–30). These animals will often have high vocalization scores. Pigs from certain farms were more difficult to drive than pigs from other farms (31). Plant management should work with producers to solve this problem. Pigs with “excitable genetics” will be easier to handle at the slaughter plant if producers have walked through the pens every day during the finishing period. Only 10 to 15 seconds per pen is needed. Such interaction trains excitable pigs to be more comfortable with human handling. Pigs that had been walked in the alley during finishing were less excitable and easier to handle (25,28,32). Producers should be encouraged to produce animals that will be reasonably easy to handle. Another problem the author has observed are extremely wild cattle that become highly agitated and difficult to handle at a slaughter plant (33). This problem is caused by both genetics and previous experience with handling. Cattle with an excitable temperament that are raised on large pastures where they seldom see people should be exposed to people on foot, months before they arrive at a slaughter plant. The author has observed that cattle that have never seen a person on foot can be difficult and dangerous to handle. These very wild cattle are also more likely to get bruises or have meat with quality problems such as dark cutters. Ranchers should be encouraged to get their animals accustomed to people on foot.

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VI. ANIMAL VISION, HEARING, AND SMELL A. Vision Ruminant animals can discriminate among different colors (34). The latest research shows that sheep, goats, and cattle are dicromats, which means that they may be partially color blind. The cones in the ruminant eye are most sensitive to yellowish green light (552 to 555 nm wave length) and blue (444 to 455 nm) (35). Ruminants lack cones that are maximally sensitive to the color red. Practical experience has shown that cattle and pigs are very sensitive to anything that has high contrast. This causes them to balk at drain grates or a change from a concrete to metal floor (27,29,36). Lighting should be even and diffuse, and harsh contrasts of light and dark should be avoided. Cattle and other grazing animals have wideangle vision and they can see in excess of 300° (37). To prevent the animals from becoming scared of distractions outside the race, stunning boxes, races, and crowd pens should have solid fences. The crowd gate should also be solid. B. Hearing Cattle and sheep have very sensitive hearing. They are more sensitive to high-frequency noise than people and they are especially sensitive to high-frequency sound, around 7000 to 8000 Hz (38). Humans are more sensitive to 1000–3000 Hz (38). Cattle can easily hear up to 21,000 Hz (39) and there is also evidence that cattle have a lower hearing threshold than people (40). This could mean that sounds that may not bother people may hurt the animals’ ears. Intermittent noise is very aversive to pigs (41). Reducing noise will improve animal movement. High-pitched noise is worse than low-pitched noise. Employees should not yell, whistle, or make loud noises; clanging and banging equipment should be silenced by installing rubber stops, and noisy air exhausts should be piped outside or silenced with inexpensive mufflers (muffling devices wear out and should be replaced every 6 months to keep noise levels low). Hissing air is one of the worst noises, but it is also the easiest to eliminate. A high-pitched whine from a hydraulic pump or undersized plumbing is disturbing to animals and can make them balk. At one plant, installing larger-diameter plumbing to eliminate a high-pitched whine from a hydraulic system resulted in calmer, easier to move animals. In another plant, excessive noise from ventilation fans made pigs balk. Noise from the fans increased as the pigs approached the restrainer. Noise increases physiological stress levels. Slaughter in a quiet research abattoir resulted in lower cortisol levels compared with slaughter in a large noisy commercial plant (42). When new systems are built, there needs to be more emphasis on noise reduction. Recently, the author visited an up-to-date pork slaughter plant. Over 800 pigs per hour were quietly moved through the plant with very little balking. The race system, overhead conveyors and restrainer system were engineered to greatly reduce noise. Gates had rubber pads to prevent clanging and banging; motors and conveyors were designed to reduce highpitched noise. Well-trained handlers quietly moved the pigs up the race with very little squealing. The type of building used in the animal handling area will also influence sound levels. Buildings constructed from pre-cast concrete with a high ceiling will have higher sound levels due to echoes than a building constructed from cooler board panels that have foam insulation sandwiched between two pieces of metal. Lowering the ceiling can sometimes help reduce sound levels. Hanging baffles from the ceiling may also help.

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C. Smell Many people interested in the welfare of livestock are concerned about animals seeing or smelling blood. Cattle will balk and sniff spots of blood on the floor (36); washing blood off facilitates movement. Balking may be a reaction to novelty. A piece of paper thrown in the race or stunning box elicits a similar response. Cattle will balk and sometimes refuse to enter a stunning box or restrainer if the ventilation system blows blood smells into their faces at the stunning box entrance. They will enter more easily if an exhaust fan is used to create a localized zone of negative air pressure. This will suck smells away from cattle as they approach the stunning box entrance. Observations in kosher slaughter plants indicate that cattle will readily walk into a restraining box that is covered with blood. In Jewish ritual (Kosher) slaughter, the throat of a fully conscious animal is cut with a razor sharp knife. The cattle will calmly place their heads into the head restraint device and some animals will lick blood or drink it. Kosher slaughter can proceed very calmly with few signs of behavioral agitation if the restraining box is operated gently (43). However, if an animal becomes very agitated and frenzied during restraint, subsequent animals often become agitated. An entire slaughter day can turn into a continuous chain reaction of excited animals. The next day after the equipment has been washed the animals will be calm. The excited animals may be smelling an alarm pheromone from the blood of severely stressed cattle. Blood from relatively low-stressed cattle may have little effect. However, blood from severely stressed animals, which have shown signs of behavioral agitation for several minutes, may elicit a fear response. Eibl-Eibesfeldt (44) observed that if a rat is killed instantly in a trap, the trap can be used again. The trap will be ineffective if it injures and fails to kill instantly. Research with rats supports this idea. Rats showed a fear response to the blood of rats and mice that had been killed with carbon dioxide (45,46). Recent research with pigs and cattle indicates that stress pheromones are secreted in the saliva and urine. Vieville-Thomas and Signoret (47) and Boissey (48) both report that pigs and cattle tend to avoid objects or places that have urine on them from a stressed animal. This stress response is not instantaneous. The stressor was applied for 15 to 20 minutes to induce the effect. In the cattle experiment, cattle were given repeated shocks during a 15-minute period. VII.

BASIC HANDLING PRINCIPLES

The first principle of animal handling is to avoid getting the animal excited. It takes up to 30 minutes for an animal to calm down and have its heart rate return to normal after it has been handled roughly (49). Calm animals move more easily and they are less likely to bunch together and be difficult to remove from a pen. Handlers should move with slow deliberate movements and refrain from yelling. Recent research by Joe Stookey and DR Waynert at the University of Saskatchewan (50) has shown that whistling and yelling is more stressful than hearing a gate slam. All species become agitated when they are isolated from other animals. In sheep and cattle, isolation can cause cortisol levels to rise (52,53). Cattle, elk, bison, and other large animals can become agitated and very dangerous when isolated. If an isolated animal becomes agitated, other animals should be put in with it. Electric prods should be replaced as much as possible with other driving aids such as sticks with flags on them or

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Figure 4 Cattle can be easily turned and moved by shaking a stick with plastic streamers on it by their head.

panels for pigs (Fig. 4). A piece of plastic fabric that is stiffened on the top with a rod makes a good tool for moving pigs down an alley. Handlers also need to learn how to use following behavior. The crowd pen should not be filled until there is room in the single file race for the animals to enter. If the single file race is full, animals in the crowd pen will turn around. Good handling requires paying attention to many small details of exactly how to do a procedure. The crowd pen and the alley that leads to it from the yard should be filled only half full. Handlers must also be careful not to force animals with crowd gates. This is especially a problem with power crowd gates. If a system is designed and operated correctly, animals should walk up the race without being forcibly pushed. When animals are pushed up too tightly with a power crowd gate, handling becomes more difficult. Tightly packed animals are unable to turn around to enter the race. VIII. HANDLER MOVEMENT PATTERNS People who handle animals need to understand the principles of the flight zone and point of balance (Fig. 5) (25,53,54). Handlers should work on the edge of the animal’s flight zone. Flight zone size depends on the wildness or tameness of the animal. A completely tame animal has no flight zone and may be difficult to drive. To make an animal move forward, the handler must be behind the point of balance at the shoulder. To back it up, he or she stands in front of the point of balance. Figures 6 illustrates handler movement patterns that make it possible to greatly reduce the usage of electric prods. Cattle, pigs, or sheep will move forward in a race when a handler walks quickly past the animal in the opposite direction of desired movement. The handler must move quickly past the point of balance at the shoulder to induce the animal to move forward. The animal will not move forward until the handler passes the shoulder and reaches the hips.

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Figure 5 Diagram of the flight zone and point of balance. Handlers should work on the edge of the flight zone. Entering the flight zone in the area marked by the letters will make the animals move.

IX. DESIGN OF HANDLING FACILITIES A. Nonslip Flooring A minimum essential for all species is nonslip flooring. Careful, quiet handling is impossible if animals slip or fall. Slipping in a cattle stunning box will cause animals to become agitated and difficult to stun. A grating constructed from 2 cm diameter steel bars welded in a 30 cm by 30 cm grid will prevent slipping in high traffic areas where floors have become worn.

Figure 6 Handler movement pattern to induce an animal to move. When the handler walks back past the point of balance in the opposite direction of desired movement, the animal will move forward. The animal moves in the opposite direction when the handler passes the balance line.

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B. Pen Space Stockyard or antemortem pens must provide enough space. The American Meat Institute (55) has guidelines for minimum pen space requirements. Many countries have Codes of Practice that stipulate the amount of pen space required. A good rule of thumb is that there should be sufficient space for all the animals to lie down at once. In the United States, the Humane Slaughter Act of 1978 requires that all holding pens be equipped with water troughs or some other watering device. In hot weather, pigs require additional space to prevent death losses due to heat stress. C. Design of Races and Crowd Pens Detailed information of race and crowd pen designs can be obtained in Grandin (28,29,56,57). There are three major design mistakes that can make quiet, calm handling extremely difficult: a single-file race that is too wide; a race that appears as a dead end; and a crowding pen on a ramp. Single-file races and stunning boxes must be narrow enough to prevent animals from turning round or becoming wedged beside each other. A cattle race should be 76 cm wide, and races for pigs should have only 3 cm of clearance on each side of the largest pigs. For cattle, a curved race is more efficient (29). Curved races work well because animals entering the race cannot see people or other activity up ahead (Fig. 7). However, a curved race must be laid out correctly. If it is bent too sharply at the junction between the single-file race and the crowding pen, the animals may refuse to enter because the race entrance appears to be a dead end. Curved races must be laid out so that animals standing in the crowding-pen can see a minimum of three body lengths up the race before

Figure 7 A curved race is more efficient than a straight one because the animals cannot see people up ahead. Solid sides that prevent the animal from seeing outside the race will keep animals calmer.

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it turns. Correct curved race layouts are shown in references (28,56,57). Weeding (58) illustrated a pig race that is laid out wrong: the race looks like a dead end to the pigs and this system increased stress. Another serious design mistake is to build a crowding pen on a ramp. In facilities where a ramp is required to reach the stunning box or restrainer, it should be located in the single-file race. Groups of animals in a crowding pen will tend to pile up on the back gate if the crowding pen is located on a ramp. Cattle and sheep will readily move up a ramp but pigs will move easier in a level system with no ramps. New pig handling facilities should be level. X. DESIGN AND OPERATION OF RESTRAINT DEVICES In small plants with line speeds of under 240 pigs per hour, it was less stressful to electrically stun pigs with hand-held tongs while they were standing in groups on the floor (10) compared to moving them through a single-file or double-file race. At higher speeds, floor stunning with tongs tends to become rough and sloppy. Whereas cattle and sheep move easily through a single-file race, pigs tend to be more difficult to drive. This is a species difference: cattle and sheep naturally move in single file whereas pigs do not. Cattle and sheep will move very easily and quietly through a well-designed single-file race. Design of animal restraint devices for both conventional slaughter where the animal is stunned and ritual slaughter are covered in detail in Grandin (5,28,29,43,59,60). The behavioral principles of low stress restraint are as follow: 1. 2.

3.

4.

5.

Animals should never be left in a stunning box or restraint device. Stun or ritually slaughter immediately after the animal enters. Animals should enter the device easily. If they balk, check for distractions discussed previously. A lamp can be used to illuminate the entrance. It must provide indirect lighting. On devices that are above the floor, install a false floor to prevent the entering animal from perceiving the visual cliff effect (59,60). Ruminants can perceive depth (61). Block the animal’s vision so that it does not see people or suddenly moving objects. Install metal shields around the animal’s head on box-type restrainers. This is not necessary on conveyor restrainers because the next animal sees the animal in front of it. Block the animal’s vision of an escape route until it is fully held in a restraint device (60). This is especially important on restrainer conveyors. Cattle often become agitated in conveyor restrainers if they can see out from under the hold-down cover before their back feet are off the entrance ramp. Extending the solid hold down cover on a conveyor restrainer will usually have a calming effect and most cattle will ride quietly. Solid hold downs can also be beneficial for pigs on conveyor restrainers. Experiment with pieces of cardboard to figure out the best locations for shields to block the animal’s vision. Provide nonslip flooring in box-type restrainers and a nonslip cleated entrance ramp on conveyor restrainers. Animals tend to panic when they lose their footing. Parts of a restraint device that press against the animal’s body should move with slow steady motion. Sudden jerking motion excites animals. On existing equipment, install inexpensive flow controls to provide smooth steady movement of moving parts that press against the animal.

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7.

8.

9.

10.

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Use the concept of optimum pressure. The restraint device must apply sufficient pressure to provide the feeling of being held, but excessive pressure that causes pain should be avoided. Install a pressure regulator to reduce the maximum pressure that can be applied. Very little pressure is required to hold an animal if it is fully supported by the device. If an animal bellows or squeals in direct response to the application of pressure, the pressure should be reduced. A restraint device must either fully support an animal or have nonslip footing. Animals panic if they feel as if they may fall. Restraint devices should hold fully sensible animals in a comfortable, upright position. Equip restraint devices with controls that enable the operator to control the amount of pressure that is applied. Different-sized animals may require differing amounts of pressure. Hydraulic or pneumatic systems should have controls that enable a cylinder on the device to be stopped in mid-stroke. Restraint devices should not have sharp edges that dig into an animal. Parts that contact the animal should have smooth rounded surfaces and be designed so that uncomfortable pressure points are avoided. The operator must be adequately trained and supervised. One big problem in many slaughter plants is that the people who handle and stun animals are the lowest paid in the plant. In England, their pay has been raised and people who stun animals must be licensed.

XI. STUNNING A good stunning method must render an animal completely insensible, similar to a state of surgical anesthesia. Some stunning methods are reversible, such as head-only electrical stunning, and unless the animal is bled quickly it will return to consciousness. Other methods such as penetrating captive bolt or cardiac arrest electrical stunning irreversibly start the process of death. A. Electrical Stunning To induce instantaneous insensibility electrical stunning must induce an epileptic state in the brain (62–64). The electrodes must be positioned so that the electric current passes through the brain (Fig. 8). Electrical parameters such as amperage, voltage, and frequency must be verified with the use of either electrical or chemical measurements from the brain to induce instant insensibility (6). Insufficient amperage can cause an animal to be paralyzed without losing sensibility. This would cause suffering. Berghaus and Troeger (65) evaluated animal welfare implications of higher-frequency (500 or 800 Hz) head-only electrical stunning in comparison to “normal” (50 Hz) stunning. (In head-only stunning an electric current is passed through the brain by two electrodes positioned on the head.) They concluded that: (a) All stunning frequencies tested (50, 500, 800 Hz) caused an effective stun (epileptic fit) within a minimum current flow time (1.3 ampere constant) of 0.3 seconds. (b) The minimum electrical charge (ampere  seconds) to induce epilepsy under laboratory conditions can be calculated within 0.4 Coulomb; this is less than 1/10 of the amount resulting after usual stunning operations (current flow time of 4 seconds). Higher frequencies are being used to help reduce petechial hemorrhages (blood splash) in the meat. The use of higher stunning frequencies did not result in a reduction of time of unconsciousness under laboratory conditions, as was de-

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Figure 8 Head-to-back cardiac arrest electric stunning of sheep. The head electrode must be positioned so that the current passes through the brain.

scribed by Anil and McKinstry (66). On the contrary, the duration of the tonic phase was longer with 800 Hz stunning frequency than with 50 Hz stunning and the recovery of breathing was delayed after 500 Hz stunning as compared with 50 Hz stunning. (d) All stunning frequencies tested were in conformance with animal welfare demands. The difference may be explained by the fact that Anil and McKinstry used very high frequencies of over 1,500 Hz. More information on the use of high frequencies to stun pigs can be found in Simmons (67). For market-weight pigs, a minimum of 1.25 amps is required (63). For sheep, a minimum of 1.00 amp is required (68,69). These amperages must be maintained for one second, during stunning, to induce instant insensibility. The Council of Europe (1991) recommends use of the aforementioned minimum amperages during electrical stunning for pigs and sheep. There must be sufficient voltage, during electrical stunning, to deliver the recommended minimum amperage; 250 volts is the recommended minimum voltage for pigs to ensure insensibility (70). Research has also shown that too high an electrical frequency will result in failure to induce insensibility. Warrington (64) found that insensibility was most effectively induced at frequencies of 50 cycles. Frequencies at 2000 to 3000 Hz failed to induce instant insensibility and may cause pain (71,72). However, in pigs weighing under 200 lbs (80 kg), Anil and McKinstry (66) found that high-frequency, 1592 Hz sinewave or 1642 Hz square-wave, head-only for stunning at 800 ma (0.80 amp) would induce seizure activity and insensibility in small pigs. One disadvantage of stunning under the aforementioned conditions is that the pigs regain sensibility more quickly than do pigs stunned using frequencies of 50 to 60 cycles. The pigs in the latter experiment (66) weighed one-third less than comparable U.S. market pigs and this probably explains why the lower amperages were effective.

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Some plants stun animals using amperages below those recommended as the minimum by the Council of Europe (1991) in an attempt to reduce blood spots in the meat. Stunning market-weight pigs with less than 1.25 amps should not be permitted (63). Grandin (6) believes that because only a 1-second application at 1.25 amps is required to induce instant insensibility in market-weight pigs, plants should be permitted to use circuits that lower the amperage setting after an initial 1-second stun, at 1.25 amps for pigs or at 1 amp for sheep. Plants should also be encouraged to use electronic constant amperage circuits that prevent amperage spiking (6,73). Both practical experience and research have shown that the aforementioned types of circuits greatly reduce petechial hemorrhages (blood spots) in carcass muscles (73,74). High-frequency stunning has never been verified to induce instant insensibility when applied with a head-to-body, cardiac arrest, stunning electrode (the type of electrode used in almost all large U.S. pork slaughter plants). In this type of stunning, the electric current is passed from an electrode on the head to an electrode on the body. In the Velarde (75) study, the pigs were stunned with a high-frequency 800 Hz current through the brain and then a second 50 Hz current was passed through the heart to induce cardiac arrest. The highfrequency 800 Hz current was effective with this “split stun procedure.” In most U.S. plants, a single current is passed from head to body, and frequencies of over 50 to 60 Hz are still not verified when used with an electrode where a single current is passed from the head to the body. Grandin (6) recommends that when a single current is passed from head to body, the first 1 second should be a minimum of 1.25 amps at 50 to 60 Hz. The author recommends that higher frequencies should only be used when they are passed through two electrodes on the head. Research is still needed to verify insensibility when frequencies over 60 Hz are passed from head to body. Electrical stunning of cattle requires a two-phase stun, whereas pigs and sheep are electrically stunned by use of a single-phase application of current. Due to the large size of cattle, a current must first be applied across the head to render the animal insensible before a second current is applied from the head to body to induce cardiac arrest (76). A single 400 volt, 1.45 amp current passed from the neck to the brisket failed to induce epileptic form changes in the brain (77). To ensure that the electrodes remain in firm contact with the bovine animal’s head for the duration of the stun, the animal’s head must be restrained in a mechanical apparatus. The Council of Europe (1991) requires a minimum of 2.5 amps applied across the head to induce immediate epileptic form activity in the EEG of large cattle. A frequency of 50 to 60 cycles should be used unless higher frequencies are verified by either electrical or neurotransmitter measurements taken form the brain. For all species, electrodes must be cleaned frequently to ensure that a good electrical connection occurs between stunner and animal. The minimum cleaning schedule is once a day; and, for safety, the electrode wand must be disconnected from the power supply before cleaning. Adequate electrical parameters for cardiac arrest stunning cannot be verified by clinical signs, because cardiac arrest masks the clinical signs of a seizure. Measurement of brain function is required to verify any new electrical parameters that may be used in the future (6). If head-only stunning is used, the tongs must be placed so that the current passes through the brain (71,64). Tongs may be placed on both sides of the head or one tong can be placed on the top and the other tong placed on the bottom of the head. Another scientifically verified location for head-only stunning is with one electrode placed under the jaw, and the other electrode placed on the side of the neck right behind the ears. For cardiac-arrest stunning of pigs and sheep, one electrode must be placed on the head and the other elec-

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trode may be placed at any location on the body to induce cardiac arrest. The head electrode may be placed on the forehead, side of the head, top of the head, or in the hollow behind the ear, but must never be placed on the neck because this would cause the current to bypass the brain. Electrodes must not be applied to sensitive areas such as inside the ear, in the eye, or in the rectum. When head-only reversible stunning is used, the animal must be bled promptly to prevent return to sensibility (78). Dutch scientist Hoenderken states that pigs must be bled within 30 seconds (63), whereas Blackmore and Newhook (79) recommend that they be bled within 15 seconds, to ensure that they remain insensible throughout bleed-out. The author has observed that in some small locker plants that used a slow hoist for elevating electrically stunned pigs, proper bleed-out was not accomplished within 30 seconds. B. Captive-Bolt Stunning A captive-bolt stunner must be positioned on the correct position on the animal’s head (Fig. 9). The most common cause of low efficacy scores for use of captive-bolt stunning in the USDA Survey was poor maintenance of the captive-bolt stunner (7). Captive-bolt stunners must be cleaned and serviced, following the manufacturer’s recommendations, to maintain maximum hitting power and to prevent misfiring or partial-firing. High-bolt velocity causes a concussion that induces instantaneous insensibility (80,81). Each plant should develop a system of verified maintenance for captive-bolt stunners. Pneumatic-powered captive-bolt stunners must be operated at the air pressure recommended by the manufacturer. A major cause of failure to render animals insensible with one captive-bolt shot is poor ergonomic design (some pneumatic stunners are so bulky it is very difficult to achieve correct stunner placement on the animal’s forehead). Ergonomics can sometimes be improved by use of a handle extension and improved balancers.

Figure 9 Correct positions for captive bolt stunning.

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Aversive methods of restraint that cause 3% or more of the cattle or pigs to vocalize must not be used as a substitute for improvements in ergonomics of captive-bolt stunners. Electrical immobilization must never be used as a method for restraining sensible animals prior to or during stunning. Several scientific studies have shown that electrical immobilization is highly aversive (12–14). Assessment of animal discomfort by counting vocalizations is impossible to achieve in electrically immobilized animals because paralysis prevents vocalization. Electrical immobilization must not be confused with electrical stunning. Properly done, electrical stunning passes a high amperage current through the brain and induces instantaneous insensibility. Electrical immobilization with a low amperage current holds a sensible animal still, by paralyzing its muscles, and does not induce epileptiform changes in the EEG (82). A third cause of missed captive-bolt stunner shots is an overloaded or fatigued operator. Assessment of stunning efficacy at the end of the shift will pinpoint this problem. In some large plants, prevention of the overloading/fatigue problem may require employment of two captive-bolt stunner operators or frequent rotation of cross-trained operators. For cattle, the most effective position for captive-bolt placement, to induce instantaneous insensibility, is in the middle of the forehead. The hollow behind the poll should be avoided as a site for captive-bolt stunning except in large Bos indicus cattle, which have a bony ridge in the forehead that makes captive-bolt stunning more difficult. Observations of cattle stunning indicate that under field conditions, penetrating captive-bolt stunners are more effective than nonpenetrating captive-bolt stunners that have a mushroom-type head; observations in many plants indicate that there is less margin for error with nonpenetrating captive-bolt stunners and the shot must be exactly on target to render the animal instantly insensible. The use of mechanical head restraint will improve the accuracy of captive-bolt stunning, but it can increase stress if it is improperly used (83). To minimize stress, the animal should be stunned within 5 seconds after its head is restrained. If more than 3% of the cattle vocalize (moo or bellow), the head restraint device will have to be modified to reduce stress. Animals should enter the head restraint easily, with a minimum of prodding. C. Carbon Dioxide Stunning There has been controversy about the humaneness of carbon dioxide (CO2) stunning. Velarde (75) reported that, as in other countries, the use of CO2 stunning has recently increased in popularity in the European Union but its acceptability on welfare grounds has been questioned by several researchers. Gregory (84) examined the effectiveness of a compact stunner and suggested that insensibility is not instantaneous and narcosis began 30 to 39 seconds after the start of immersion procedure. Additionally, the exposure to the gas stimulates breathing frequency and may lead to respiratory distress (85). On the other hand, from the study of the changes occurring in the EEG patterns of pigs, Forslid (86) observed that purebred Yorkshire pigs reach insensibility before the onset of the violent motor activity. Some people who are interested in animal welfare claim that CO2 stunning is extremely aversive to pigs while other people claim it is humane. Both practical experience and scientific studies indicate that genetic factors play a large role in determining the aversiveness of CO2 gas to pigs. For pigs of some genetic types, use of CO2 stunning is probably very humane but for other pigs it may be very stressful. Ring (87) concluded that because pigs stunned with N2 in his study, despite lower PaO2 than the CO2-stunned pigs, did not show any signs of restlessness, choking attacks,

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collapsing, or flight reflexes for 2 minutes, stunning with CO2 cannot be considered to be caused by hypoxia. Raj (88) found stunning pigs in argon resulted in a faster loss of consciousness than CO2. They measured both electrocardiogram and somatosensory evoked potentials from the brain. German researchers observed that during the time before the stage of analgesia was experienced, the pigs were fully conscious; so, during this stage, unpleasant feelings cannot be excluded, but obvious signs of unpleasant feelings were not noticed. After the stage of excitation, the animals passed the stage of asphyxia when they were exsanguinated. Barfod and Madsen (89) concluded that the loss of consciousness during CO2 anesthetization is rapid and is similar to other forms of narcosis and, therefore, it appears to be an acceptable method for pre-slaughter stunning. Forslid (86) in his work on purebred Yorkshire pigs reported that determinations of plasma cortisol, adrenaline, and noradrenalin did not provide any direct evidence that the inhalation of CO2 imposed any emotional strain in addition to that induced by the mere transport of the swine to the intermediate preexposure situation. Dutch research indicated that the excitation phase that occurs during CO2 stunning starts prior to the onset of unconsciousness (63); this study raised the question of potential distress in pigs during the induction of CO2 anesthesia. More recently, research by Forslid (90) indicated that unconsciousness occurred prior to the onset of the excitation phase; therefore, CO2 stunning is definitely humane. All of the research conducted by Anders Forslid at the Swedish Meat Research Institute has been on Yorkshire pigs (Anders Forslid, Swedish Meat Research Institute, personal communication). In Yorkshire X Landrace crossbred pigs, exposure to CO2 was less aversive than were electrical shocks (90). Aversion was measured by determining the time required to enter and reenter a CO2. Dodman (91) and Grandin (92) observed, in a commercial slaughter plant in the United States, that white crossbred pigs (with Yorkshire breed-type characteristics) had a much milder reaction to CO2 than black, white-striped crossbred pigs (with Hampshire breed–type characteristics). Grandin (5,93) concluded that the effect of genetic factors on the reaction to CO2 may make it acceptable from an animal welfare standpoint for some breeds or genetic lines within a breed and not acceptable for other breeds or genetic lines within a breed. Excitement and rough handling prior to entry into the compact plant may also affect the animal’s reaction; so, there is a possibility that rough handling may have a large effect on pig reaction in one breed and little effect on pig reaction in another breed or genetic line within a breed (5). Many of the Hampshire-type pigs, when stunned in a Wernberg Compact plant, started to react in the first few seconds after they contacted the gas. Hampshire-type pigs rode quietly in the gondola until they contacted the gas; they then attempted to rear up to avoid the gas while they were fully conscious (93). Grandin (5) observed that Danish pigs (which have a very low incidence of the Halothane gene) remained calm when they breathed CO2, but that Irish pigs (which have a high incidence of the Halothane gene) became highly agitated within seconds after sniffing the gas. Experiments with Pietrain X German Landrace pigs indicated that Halothane-positive pigs had a more vigorous reaction to CO2 than Halothane-negative pigs (94). These pigs had little or no reaction during initial contact with the gas; the reaction started about 20 seconds after the animals contacted the gas. Seventy percent of the Halothane-positive pigs had strong motoric reactions while only 29% of the Halothane-negative pigs reacted in this manner. Troeger and Woltersdorf (94) expressed concern that reactions in Halothane-positive animals may possibly be of animal welfare concern but concluded that

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the use of high CO2 concentrations (80% or greater) reduced the incidence of vigorous reaction. An earlier German study with pigs of unspecified genetics indicated that the animals were anesthetized before the excitation phase (87). It is likely that some Halothane-positive pigs were tested in the Ring (87) investigation but further studies with both Halothane-positive and Halothane-negative Hampshire pigs are still needed. The effect of the Napole gene in the Hampshire breed also needs to be researched. Human beings also vary in their reaction to CO2. People who have panic attacks, which have a strong genetic basis, will react very badly to CO2; the gas may induce panic attacks in these people (95,96). Neville Gregory from the Meat Research Institute in England reviewed a number of studies that indicated that most people find the smell of CO2 gas to be pungent when it is breathed at a concentration of 50%. In conclusion, CO2 stunning is probably very humane for use with certain genetic types of pigs and stressful for pigs of other genetic types. The use of a mixture of CO2 and argon gas may create an improved gas stunning system for poultry (97). It is possible that a combination of CO2 and argon might make CO2 stunning less stressful for genetic types of pigs that react badly to CO2. Any research studies conducted to determine animal welfare aspects of gas stunning should use populations of pigs that include those that are both positive and negative for the Halothane gene and for the Napole gene. One welfare advantage of CO2 stunning is that CO2 systems can be designed so that lining pigs up in single file races can be eliminated. In Denmark, pigs are moved into the CO2 chamber in groups of five. Handling pigs in groups makes quiet handling easier. Whereas cattle and sheep are animals that will naturally walk in single file, pigs resist lining up in a single file race. Systems in which cattle and sheep are moved through singlefile races can be made to work extremely well. However, pigs move more easily in small groups. D. Assessing Insensibility Only measurements taken directly from the brain in a laboratory are appropriate for assessing any change in stunning parameters. However, if an animal shows any signs of return to sensibility on a slaughter line, immediate corrective action must be taken. To assess insensibility in a meat plant one should look at the stunned animal’s head and ignore the reflexes occurring in the body (5). Reflexive movements and kicking will occur in insensible animals that have been properly stunned with electricity or a captivebolt stunner. The mistake many people make is to look at leg kicking. Random limb movement can create a safety hazard when large cattle kick, and this activity can occur in an unconscious animal. When cattle or sheep are shot with a captive bolt, the animal should instantly drop to the floor if it is stunned in a box. In a conveyor restrainer, the head should drop down. It is normal for the head to go into a spasm for a few seconds before it drops. Insensibility in cattle can be evaluated immediately after the head spasm. In electrically stunned cattle, sheep, or pigs, stunning induces a grand mal seizure that causes instant unconsciousness. This seizure causes rigid spasms that last for at least 30 seconds; these spasms can mask signs of sensibility such as blinking. After the stunner amperage is set correctly the animal should not be evaluated for insensibility until 30 seconds after electric stunning. At no time, either during or after stunning, should the animal vocalize (squeal, moo or bellow). Vocalization is a sign that a sensible animal may be feeling pain. It is easy to

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evaluate insensibility after an animal is hanging vertically on the bleed rail; it should hang straight down and have a straight back, and the head should be limp and floppy (5,6,98). If the stunned animal has kicking reflexes, the head should flop like a limp rag. If the animal makes any attempt to raise its head, it may still be sensible. An animal showing a righting reflex must be immediately re-stunned. There should also be no rhythmic breathing and no eye reflexes in response to touch. Blinking is another sign of an animal that has not been properly stunned and thus may still be sensible. Gasping is permissible; it is a sign of a dying brain (99). If the tongue is hanging straight down and is limp and floppy, the animal is definitely stunned; if the tongue is stiff and curled, this is a sign of possible sensibility. The heads of chickens or turkeys that have been stunned with electricity or gas should hang straight down after stunning. Birds that have not been properly stunned will show a strong righting reflex and raise their heads. Both mammals and birds that have been stunned with CO2 should be limp and floppy. Gas-stunned mammals and birds should not have reflexive movements and should not display kicking actions. The entire body and head should be flaccid and floppy. For all stunning methods, blinking where an open eye closes and then fully re-opens is a sign of returning to sensibility. E. Ritual Slaughter Ritual slaughter is performed according to the dietary codes of Jews or Muslims. Cattle, sheep, or goats are exsanguinated by a throat cut without first being rendered unconscious

Figure 10 Diagram of an upright restraint device for ritual slaughter. Animals should be held in a comfortable upright position during ritual slaughter.

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Figure 11 Head-holding device for ritual slaughter. This head holder could also be easily modified to apply electrical stunning of cattle. The head holder is mounted on a center track conveyor restrainer.

by pre-slaughter stunning. Ritual slaughter is exempt form the Humane Methods of Slaughter Act of 1978 in order to protect religious freedom in the United States; in Europe and Canada, however, ritual slaughter is covered by humane slaughter regulations. Because ritual slaughter is exempt in the United States, some plants use cruel methods of restraint, such as suspending a conscious animal by a chain wrapped around one hind-limb. In more progressive plants, the animal is placed in a restrainer that holds it in a comfortable, upright position (100). The latest guidelines for ritual slaughter, published by the American Meat Institute (6,55), strongly recommend the use of upright restraint devices (Figs. 10–12). Most large cattle slaughter plants are using more comfortable methods of restraint, but there are still some plant managers who have no regard for animal welfare. They persist in hanging large cattle and veal calves upside down by one hind-leg. There is no religious justification for use of this cruel method of restraint. The plants that suspend cattle/calves by one hind-leg do so in order to avoid paying the cost of installing a humane restraint device. Humane restraint devices can often pay for themselves by improving employee safety. When ritual slaughter is being evaluated from an animal welfare standpoint, the variable of restraint method must be separated from the act of throat cutting without prior stunning. Distressful restraint methods mask the animals’ reactions to its throat being cut. Four state-of-the-art restraint devices have been designed, built, and operated that hold cattle and calves in a comfortable upright position during kosher (Jewish) slaughter (5,100). To determine whether cattle feel the act of having their throat cut, Grandin (5), at one plant, de-

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Figure 12 Diagram of center track conveyor restrainer equipped with a head-holding device for ritual slaughter. The chin lift yoke is attached to two biparting sliding doors.

liberately applied the head restrainer so lightly that the animals could pull their heads out; none of the 10 cattle moved or attempted to pull their heads out. Observations of hundreds of cattle and calves during kosher slaughter indicated that there was a slight quiver when the knife first contacted the throat (5). Invasion of the cattle’s flight zone by touching its head caused a bigger reaction (5,100) than did the act of having its throat cut. The animal’s head must be restrained in such a manner that the incision does not close back over the knife. Cattle and sheep will struggle violently if the edges of the incision touch during the cut (5). The design of the knife and the cutting technique are critical for preventing the animal from reacting to an incision of its throat. In kosher slaughter, a straight, razor-sharp knife that is twice the width of the throat is required, and the cut must be made in a single continuous motion; for halal (Muslim) slaughter, there is no knife-design requirement. Halal slaughter performed with short knives and multiple hacking cuts results in vigorous reactions of cattle being treated in this manner. Fortunately, many Muslim religious authorities accept pre-slaughter stunning. Muslims should be encouraged to stun the cattle or to use long, straight, razor-sharp knives that are similar to the knives used for kosher slaughter. Investigators agree that throat-cutting without stunning does not induce instantaneous unconsciousness. In some cattle, consciousness is prolonged for over 60 seconds (101,102). Grandin (5) observed that near-immediate collapse can be induced in over 95% of cattle if the ritual slaughterer makes a rapid, deep cut close to the jawbone. Further observations indicated that calm cows and bulls lose sensibility and collapse more quickly than do cattle with visible signs of agitation. Cattle that fight restraint are more likely to have prolonged sensibility; gentle operation of restraint devices facilitates rapid loss of sensibility (5). To provide the best possible animal welfare, restraint devices must be operated correctly. The most common problems in restraining animals involve applying excessive pressure to the body. If more than 5% of the cattle vocalize or struggle in the restraint device, it is either poorly designed or it is operated too roughly. A survey done in plants perform-

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ing kosher slaughter in an upright restraint system indicated that under 5% of the cattle vocalized when the system was operated correctly (103). Dunn (8) found that significantly more cattle vocalized when they were inverted onto their backs for ritual slaughter as compared to the number of cattle that vocalized when they were held in a restrainer that kept them in an upright position. Higher cortisol levels were also correlated with higher rates of vocalization (8). Plants that shackle and hoist large cattle often have loud bellowing by more than 50% of the animals treated in that manner. In some cases, those vocalizations can be heard outside the building. Instructions for proper operation and design of comfortable upright restraint devices can be found on the Internet at www.grandin.com and in papers by Grandin (5,28,29,59,60,100,104). The use of comfortable restraining equipment complies with the religious principles of both halal and kosher slaughter. Kosher and halal slaughter were originally developed to spare the animal pain (100). XII.

STUNNING METHOD AND BLOOD-SPLASH

The major negative effect on meat quality of poor stunning methods is that they affect the incidence of petechial hemorrhages or blood-splash in the meat. Blood-splash is a cosmetic defect and it occurs when small capillaries in the muscle rupture while the circulatory system is still intact. Blood-splash can appear as small red spots or it can cover a larger area and look like a bruise. Lambooy and Sybesma (105) stunned pigs with 70 volt or 475 volt electricity in groups, in the shackling pen, or in a conveyor restrainer or with CO2 stunning. They reported that (a) high voltage and stunning in a pen resulted in lower incidences of bloodsplash and (b) CO2-stunned pigs showed no blood-splash. Grandin (106) compared pighandling treatments that consisted of an experimental treatment (shortened stunning time; no electric prods and overnight rest prior to stunning) to a control treatment in which electric prods were used. Special treatment handling provided the greatest reduction in petechial hemorrhages when low winter temperatures had greater day-to-day variability. The incidence of blood-splash and hemorrhages is increasing in both beef and pork. The most likely cause of this increased incidence is greater emphasis in animal selection on leanness in beef and pork. Leanness is probably associated with increased fragility of the animal’s capillaries and, thus, with an increase in the incidence of blood-splash. Recent audits of pork and beef slaughter plants by the National Pork Producers Council and the National Cattlemen’s Beef Association indicate that blood-splash is costing the pork industry almost 50 cents for every hog marketed (106,107) and is costing the beef industry about 12 cents for every slaughter steer or heifer marketed (107). In the pork industry, damage to loins and hams is costing approximately $43 million annually (106). Sensitivity of individual animals to blood-splash differs greatly (108). Unstable weather, especially circumstances in which ambient temperatures quickly fluctuate, can make animals more sensitive to such damage. Grandin (109) found that fluctuating weather affected the efficacy of improved electrical stunning in terms of reductions in blood-splash in pigs. Therefore, when a new stunning or handling procedure is studied for its effectiveness in reducing blood-splash, it must be tested on alternate days, against a control method, for several weeks to remove confounding due to temperature and weather fluctuations. Seasonal differences in blood-splash have also been reported in pigs (109). Research has demonstrated that an automatic stunning system mounted on a center track (double rail) restrainer reduced blood splash by 20% compared with a standard Vconveyor restrainer. On V-conveyor restrainers, blood-splash will increase if one side of

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the conveyor runs faster than the other (76). Carbon dioxide stunning will produce less blood-splash than does the average electrical stunner (110). However, tests run in a single plant with CO2 versus a well-maintained and operated electrical stunner showed no differences in incidence of blood-splash. Electrical stunning is comparable to CO2 stunning from a blood-splash standpoint, if it is done perfectly. The most serious problem with bloodsplash incidence is in terms of its association with ultra-lean pigs with very heavy muscling. CO2-stunning probably has little advantage over electrical stunning for use with normal pigs but it is most likely to provide a reduction in blood-splash in ultra-lean pigs. Some pigs with extremely lean muscling have very weak skeletons, which increases the incidence of broken backs and damage to the loins during stunning. The first step in reducing blood-splash is to stop double-stunning; double-stunning occurs when an animal is given two separate jolts of electricity. Practical experience has shown that blood-splash can be greatly reduced by following the stunning practices that are outlined below. Double-stunning damages the capillaries because it makes the muscles contract twice. To prevent this, the operator must press the wand firmly against the pig before the stunning current is turned on. The wand must remain pressed against the pig until the timer stops the flow of current. If the wand slides during the stun, blood-splash incidence may increase. A stunning switch with dirty contacts or a cord with frayed wires inside may also cause blood-splash, because they can allow fluctuations in the electrical current due to the momentary making and breaking of the circuit. Wiring and switches need to be changed frequently to prevent fluctuations in the electrical current. It is also essential to clean electrode contacts every day and make sure the pigs are wet. Quiet handling is essential. Pigs that rear or struggle during stunning are more likely to be double stunned, which may increase blood-splashing. Blood-splash will also increase in pigs left in the restrainer during breaks. Practical experience has also shown that bloodsplash will increase in a V-restrainer conveyor if one side runs faster than the other. This causes stretching of the animals’ skin. Some plants have attempted to reduce blood-splash by reducing the stunning amperage to 0.5 amps. This is a practice that should be banned. Scientific research has shown that a minimum of 1.25 amps at 250 to 300 volts must pass through the pig’s brain to induce instantaneous unconsciousness (63). Use of lower amperages will kill the pig, but the animal may feel the symptoms of a heart attack. The plants with the lowest incidence of blood-splash in North America use 1.25 amps with an electronic amperage-controlled circuit. Unlike old-fashioned voltage-regulated stunners, the amperage in electronic amperage-controlled circuits is kept constant at 1.25 with the electronics and the voltage automatically changing with resistance encountered in individual hogs. A survey of seven large pork plants conducted by a major processed-products firm indicated that the one plant that used an electronic amperage-controlled stunner had a 100% reduction in blood-splash compared to use of the old-fashioned, voltage-regulated stunner. The concept is very simple; the amperage should be held absolutely constant at 1.25 amps while the voltage is allowed to vary within certain limits. There is no reason to stun hogs with 0.5 amp. An electronic system with constant amperage will generate less blood splash than will use of a voltage-regulated system at 0.5 amp. Electronic amperage-controlled electrical stunners are now available with computer outputs that count double-stuns and misapplied stuns. The printouts show that an operator’s performance declines after about 2 hours; the operators should be rotated every few hours to prevent fatigue from increasing blood-splash.

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There has never been a test comparing CO2 stunning to electronically controlled constant amperage electrical stunning in the same plant. The test described previously, in which CO2 stunning and electrical stunning were compared in the same plant, involved use of an old-fashioned voltage-regulated stunner. It is likely that electronically amperage-controlled electrical stunning, if it is perfectly maintained and operated, will generate the same incidence of blood-splash as will CO2 stunning. The principles described above apply to both manual and automatic stunners. A. Quick Bleeding It is essential that animals are bled quickly after electric stunning. The animal should be stuck within 15 seconds after stunning. This will reduce blood-splash in all species (72,111). Bleeding very quickly reduces blood-splash because quickly lowering the blood pressure helps prevent small capillaries from bursting. B. Electrical Stunning of Cattle Blood-splash problems are the main reason why electrical stunning has not become more popular for use with cattle. Plant managers in New Zealand have found that blood-splash incidence is low in grass-fed cattle, but when the Australians tried electrical stunning in fed cattle, blood-splash levels were too high to make it commercially viable. Practical experience in New Zealand has shown that very quick bleeding (within 10 seconds) is required to keep blood-splash incidence low. C. Resting and Handling Animals Practical experience in many pork slaughter plants reveals that pigs should be rested for 2 to 4 hours to reduce incidences of both blood-splash and pale, soft, exudative (PSE) lean. In cattle, long resting periods are not recommended, but it is advisable to allow the animals about 30 minutes to settle down after unloading. Such rest will help prevent excitement during subsequent handling. Reducing or eliminating electric prod usage also helps to reduce occurrence of blood-splash. Calkins (112) found that electric prods increased incidence of blood-splash in pigs. Practical experience in many slaughter plants has shown that using very low voltage prods, with 24 volts or less, reduced blood-splash. If pigs squeal when electrically prodded, they are receiving a shock that is too strong. D. Captive-Bolt Stunning Some beef plants have found that a cartridge-fired stunner causes less blood-splash than does use of a pneumatic stunner. There are two possible explanations for these differences. First, some air-operated stunners inject air into the brain. The second explanation is that poor maintenance of captive-bolt stunners or differences in shooting position can cause increases in blood-splash. Some operators using air-operated stunners will shoot cattle behind the poll because the air-powered stunner may be harder to position on the forehead than is the cartridge-fired stunner. A test conducted in one plant with a highly skilled operator using perfectly maintained equipment showed no difference in incidence of bloodsplash. Pneumatic guns require careful maintenance, and some plants skimp on maintenance, resulting in excessive recoil and poor stunning. There is also concern that pneumatic stunners that inject air could contaminate beef by forcing brain tissue throughout the body. Recent research (113,114) showed that air in-

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jection sometimes forced pieces of neurological tissue as large as a pencil into the heart and other organs. This problem was not observed when a cartridge-fired stunner was used. For food-safety concern reasons, the use of stunners that inject air into the head is not recommended. One major beef packing company has found that the extra cost of blank cartridges for a cartridge-fired stunner was economically justified because eliminating air injection reduced occurrence of blood-splash. E. Ritual Slaughter Cattle and calves slaughtered without stunning (Jewish ritual slaughter—kosher, Moslem—halal) are more prone to blood-splash than are cattle stunned by use of either type of captive-bolt stunner. Cutting the throat of cattle and calves without first stunning them almost always increases incidence of blood-splash. Blood-splash incidence in cattle stunned with a captive-bolt stunner is almost always under 0.5% percent; but in ritually slaughtered cattle, blood-splash often occurs in 3% to 10% of cattle. Keeping cattle calm during ritual slaughter will reduce occurrence of blood-splash because excited cattle often have a bigger spasm when they lose consciousness (5). Restraint devices should be designed to apply as little pressure as possible to the animal’s body. After the throat is cut, restraining devices should be loosened immediately. Cattle should be ritually slaughtered within seconds after their heads are restrained in the neck yoke; allowing an animal to fight restraint will increase the incidence of blood-splash. The technique used in throat-cutting can also affect the incidence of blood-splash and the speed of bleed-out. Cutting the throat too far back on the neck slows bleed-out and increases blood-splash. The incidence of blood-splash can differ greatly between two different ritual slaughterers. Stunning cattle with a captive-bolt device will greatly reduce incidence of blood-splash caused by ritual slaughter. Some religious authorities will permit either pre or post-slaughter stunning of animals. Stunning with a captive-bolt device, immediately after ritual slaughter, will lower blood-splash incidence from 3%, in the best ritual slaughter plant, to about 1%; such incidence will still be slightly elevated as compared to stunning the bovine prior to bleeding. Gregory et al. (115), studying bobby (newborn) calves in Australia, determined that a faster bleed-out was obtained when the thoracic, compared with the neck, sticking protocol was used, and when electrical stunning rather than captive-bolt stunning was used. New Zealand researchers reported that the amounts of jugular blood that were collected from calves that were exsanguinated by throat-cutting indicated that cerebral blood perfusion is very severely impaired after gash-cutting while cortical dysfunction occurred at a time when mean arterial blood pressure was sufficiently high to maintain cerebral circulation. They regarded those findings as indirect evidence of a retrograde flow of blood from the vertebral arteries, through the occipto-vertebral anastomis, to the open cephalic ends of the severed carotid arteries. Due to differences in the anatomy of the blood vessels in cattle and sheep, ritual slaughter will not increase blood-splash in sheep. Almost the entire blood supply to the brain of a sheep is in the front of the neck whereas cattle have some small vessels in the back of the neck. This difference in anatomy causes sheep to bleed-out more quickly. XIII. BRUISING AND MEAT QUALITY Rough handling is a major cause of bruising in all species. Grandin found that cattle handled quietly had half as many bruises (3). Bruises cost the U.S. beef industry $4.03 for ev-

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ery fed steer or heifer marketed (116). Forty-nine percent of the fed cattle marketed in the United States have bruises (117). Bruises on cull cows that have passed through many auctions is even higher (108). Cattle that have been handled at auctions had more bruises than animals marketed directly (118,119). Contrary to popular belief, animals can be bruised right up until the moment of bleeding (120). Cattle with horns or tipped horns had more bruises than cattle that were completely dehorned (121–124). Tipping the horns did not reduce bruising. Overloading of trucks will increase bruises but increase the incidence of downed animals (125). There is an optimal truck loading density (126). If animals are either too loose or too tight, bruising will increase. Recommended stocking truck densities for different types of animals can be found in National Institute of Animal Agriculture, Bowling Green, Kentucky, Grandin (29,104), and in Codes of Practice published by producer organizations. A. Death Losses in Pigs High heat combined with high humidity can be deadly to pigs. The Livestock Conservation Institute Livestock Safety Index provides an easy-to-use guideline for safe transport of pigs. When the temperature is 30°C with a relative humidity of 50%, pigs should be transported in the early morning or at night. Lean hybrid pigs with heavy muscling are more prone to death losses than old-fashioned fatter pigs. The author has observed that death losses in pigs doubled and tripled when lean hybrids were first introduced. Growing pigs to very heavy weights (over 114 kg) increases the problem. B. PSE in Pigs Both scientific research and practical experience has shown that genetics is a major factor in the cause of PSE. Several studies have shown that pigs that are homozygous positive for the stress (Halothane) gene will have a greater incidence of PSE (127). Pigs that carry one gene will be intermediate in pork quality. Experience in many plants indicates that genetics contributes to 50% of the PSE, poor handling 10% to 12%, and chilling about 30%; the last 10% is caused by fluctuating weather and other factors. Resting pigs for a minimum of 1 hour prior to stunning will reduce PSE (128,129). Quiet handling in the stunning race will reduce PSE 10% to 12% according to tests conducted in several large plants. Meat quality in pigs can be degraded during the last 5 minutes in the stunning race. This is why quiet handling is so important. Pigs that get hot and overheated are more likely to have poor pork quality. Infrared measurements of high-skin temperature are correlated with quality problems (130). C. Preventing Dark Cutters Dark cutters cost the beef industry millions of dollars annually. The latest National Beef Quality Audit figures estimate that dark cutters cost the U.S. industry $1.36 for every fed steer marketed. In 1975, the incidence of dark cutting in fed steers was estimated at 0.5% (131). During the early to mid-nineties, dark cutting doubled to 1% according to the National Beef Quality Audit. Dark cutting meat is darker than normal and has a shorter shelflife. Many factors contribute to dark cutting, such as fluctuating temperatures, mixing strange cattle, and hormone implant programs. Grandin (132) and Price and Tennessen (133) both report that fighting between strange cattle will increase dark cutting. When cattle are mixed, they fight to determine a new dominance hierarchy. Mixing young fed bulls can result in up to 73% dark cutters (133). Cattle breed will also affect the incidence of dark

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cutting. Zebu crossbreds had fewer dark cutters compared with shorthorns (134). Observations by the author at slaughter plants have indicated that the incidence of dark cutting can be very high in cattle from certain feedlots. Dark cutters from high-incidence feedlots can run up to 30% during periods of fluctuating temperatures. The introduction of trembolone acetate implants correlates with the increase in dark cutters. Scanga (135) reported that the overuse of trembolone acetate (synthetic male hormone) implants significantly increased the incidence of dark cutters in fed steers. This study utilized the computer records of two large meat plants, and over two million cattle were studied. The Scanga (135) study verified many anecdotal observations on dark cutting. Fluctuating temperatures and extremely high temperatures also contributed to dark cutters. Ambient temperatures of over 35°C for 24 to 48 hours prior to slaughter increased dark cutters in both fed steers and heifers. Other factors that increase dark cutters include spending the night at the slaughter plant and cattle that are extremely wild and agitated. Voisinet found that cattle that become agitated during handling had lower weight gains, more borderline dark cutters, and tougher meats (4, 136). Several studies have shown that bulls are much more susceptible to dark cutters than steers or heifers (133). XIV. CONCLUSIONS Improving animal welfare is both the right thing to do and it is economically advantageous. During a 25-year career, the author has observed that the single most important factor that determines how animals are treated is the attitudes of management. Good equipment makes good welfare easier, but it is useless unless it has good management to go with it. REFERENCES 1. 2. 3. 4.

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Humane Slaughter Act, 1978. MC Appleby, BO Hughes. Introduction. In Animal Welfare. M.C. Appleby and B.O. Hughes (Eds.) Wallingford: CAB International, UK, 1997. T Grandin. Bruises on southwestern feedlot cattle. J. Anim. Sci. 53(Suppl. 1):213, 1981. BD Voisinet, T Grandin, SF O’Connor, JD Tatum, MJ Deering. Bos indicus cattle with excitable temperaments have tougher meat and a higher incidence of borderline dark cutters. Meat Science 46:367–377, 1997. T Grandin. Euthanasia and slaughter of livestock. J Am Vet Med Assoc 204:1354–1360, 1994. T Grandin. Good Management Practices for Animal Handling and Stunning. American Meat Institute, Washington, DC. 1997. T Grandin. Objective scoring of animal handling and stunning practices in slaughter plants. J Am Vet Med Assoc 212:36–39, 1998. CS Dunn. Stress reaction of cattle undergoing ritual slaughter using two methods of restraint. Veterinary Record 126:522–525, 1990. T Grandin. The feasibility of using vocalization scoring as an indicator of poor welfare during slaughter. Appl Anim Behavior Sci 56:121–128, 1998. PD Warris, SN Brown, SJM Adams. Relationships between subjective and objective assessments of stress at slaughter and meat quality in pigs. Meat Science 38:329–340, 1994. RG White, JA DeShazer, CJ Tressler. Vocalizations and physiological response of pigs during castration with and without anesthetic. J Anim Sci 73:381–386, 1995. PJ Pascoe. Humaneness of electro-immobilization unit for cattle. Am J Vet Res 10: 2252–2256, 1986. T Grandin, SE Curtis, TM Widowski, and JC Thurman. Electro-immobilization versus mechanical restraint in an avoid choice test. J Anim Sci 62:1469–1480. 1986.

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R Hoenderken. Electrical and carbon dioxide stunning of pigs for slaughter. In: Stunning of Animals for Slaughter. G. Eikelenboom (Ed.), pp. 59–63. Martinus Nijhoff Publishers, Boston, 1983. PD Warrington. Electrical stunning: A review of literature. Veterinary Bulletin 44:617–633, 1974. A Berghaus, K Troeger. Electrical stunning of pigs: Minimum current flow time required to induce epilepsy at various frequencies. Proc. International Congress of Meat Science and Technology (Barcelona, Spain) 44:1070–1071, 1998. MH Anil, JL McKinstry. The effectiveness of high frequency electrical stunning in pigs. Meat Science 31:481–491, 1992. NJ Simmons. The use of high frequency currents for the electrical stunning of pigs. M.S. thesis, University of Bristol, Langoford, UK, 1995. NG Gregory, SB Wotton. Sheep slaughtering procedures. III. Head to back electrical stunning. British Veterinary Journal 140:570–575, 1984. KV Gilbert, CJ Cook, CE Devine. Electrical stunning in cattle and sheep: Electrode placement and effectiveness. Proc International Congress of Meat Science Technology (Kulmback, Germany) 37:245–248, 1991. K Troeger, W Woltersdorf. Measuring stress in pigs during slaughter. Fleischwirtsch 69(3):373–376, 1989. PS Croft. Problems with electrical stunning. Veterinary Record 64:255–258, 1952. PG Van der Wall. Chemical and physiological aspects of pigs stunning in relation to meat quality: a review. Meat Science 2:19–30, 1978. T Grandin. Cardiac arrest stunning of livestock and poultry. In: Advances in Animal Welfare Science. M.W. Fox and L.D. Mikley (Eds.) Humane Society of the U.S. Washington, D.C., 1985. DK Blackmore, GV Peterson. Stunning and slaughter of sheep and calves in New Zealand. New Zealand Veterinary Journal 29:99–102, 1981a. A Velarde, L Faucitano, M Gispert, MA Oliver, A Diestre. A survey of the efficacy of electrical and carbon dioxide stunning on insensitivity in slaughter pigs. Proc. International Congress of Meat Science and Technology (Barcelona, Spain) 44:1076–1077 (Abstract C124), 1998. NG Gregory. Slaughter technology. Electrical stunning of large cattle. Meat Focus International. pp. 32036. CAB International, Wallingford, Oxon, UK, 1993. CJ Cook, CE Devine, KV Gilbert. Electroencephalograms and electrocardiograms in young bulls following upper cervical vertebrae to brisket stunning. New Zealand Veterinary Journal 39:121–125, 1991. SB Wotton, NG Gregory. Pig slaughtering procedures: Time to lose brain responsiveness after exsanguination or cardiac arrest. Research in Veterinary Science 40:148–151, 1986. DK Blackmore, JC Newhook. Insensibility during slaughter of pigs in comparison to other domestic stock. New Zealand Veterinary Journal 29:219–222, 1981b. CC Daly, PF Whittington. Investigation into the principle determinants of effective captive bolt stunning of sheep. Res Vet Sci 46:406–408, 1989. DK Blackmore. Energy requirements for penetration of heads of domestic stock and developments of a multiple projectile. Vet Rec 116:36–40, 1985. E Lambooy. Electro-anesthesia or electro-immobilization of calves, sheep and pigs by Feenix Stockstill. Vet Quart 7:120–126, 1985. R Ewbank, MJ Parker and CW Mason. Reactions of cattle to head restraint at stunning: A practical dilemma. Animal Welfare 1:55–63, 1992. N Gregory, B Moss, R Leeson. An assessment of carbon dioxide stunning in pigs. Veterinary Record 121:517–518, 1987. AM Raj, NG Gregory. Welfare implications of gas stunning of pigs. 2. Stress of induction of anesthesia. Animal Welfare 5:71–78, 1995.

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A Forslid. Transient neocortical, hipocampal and amygdaloid EEG silence induced by one minute inhalation of high concentration CO2 in the swine. Acta Physiologica Scandinavica 130:1–10, 1987. C Ring. Two aspects of CO2 stunning methods for pigs. Animal protection and meat quality. Proc. International Congress of Meat Science and Technology (Brisbane, Australia). 34:98–100, 1988. AB Raj, SP Johnson, SB Wotton, JL McInstry. Welfare implications of gas stunning of pigs. Vet J 153:329–339, 1997. K Barford, KB Madsen. Carbon dioxide anesthetization of pigs. Proc International Congress of Meat Science and Technology (Brisbane, Australia) 34:91–92, 1988. A Forslid. Pre-slaughter CO2-anesthesia for swine. Proc International Congress of Meat Science and Technology (Brisbane, Australia) 34:93–95, 1988. EC Jongman, JL Barnett, PH Hemsworth. The aversiveness of carbon dioxide stunning in pigs. Manipulating Pig Production VI, p. 128. Pig Research and Development Corporation, Australia, 1998. NH Dodman. Observations on the use of the Wernberg dip-lift carbon dioxide apparatus for pre-slaughter anesthesia pigs. Br Vet J 133:71–80, 1977. T Grandin. Possible genetic effect on pigs reaction to CO2 stunning. Proc. International Congress of Meat Science and Technology. Brisbane Australia 34:96–97, 1988a. K Troeger, W Woltersdorf. Gas anesthesia of slaughter pigs. Fleischwirtsch International 4:43–49, 1991. E Griez, J Zandbergen, J Pols. Response to 35% CO2 as a marker of panic and severe anxiety. Am J Psychiatry. 147:796–797, 1990. L Bellodi, P Giampaolo, D Caldirola, C Arancro, A Bertani, D DiBelle. CO2 induced panic attacks: A twin study. Am J Psychiatry 155:1184–1188, 1998. AM Raj, NG Gregory. An evaluation of humane gas stunning methods for turkeys. Veterinary Record 135:222–224, 1994. NG Gregory. Animal Welfare and Meat Science. CAB International, Wallingford, Oxon, UK, 1998. NG Gregory. Preslaughter handling, stunning and slaughter. Meat Science 36:45–56, 1994. T Grandin, JM Regenstein. Religious Slaughter and Animal Welfare: A Discussion for Meat Scientists. Meat Focus International. pp. 115–123. CAB International, Wallingford, Oxon, UK, 1994. DK Blackmore. Differences between sheep and cattle during slaughter. Vet Sci 37-223–226, 1984. CC Daly, E Kallweit, F Ellendorf. Conventional captive bolt stunning followed by exsanguination compared to shechitah slaughter. Vet Rec 122:325–329, 1988. T Grandin. Survey of Handling and Stunning in Federally Inspected Beef, Pork, Veal and Sheep Slaughter Plants. ARS Research Project No. 3602-32000-002-08G, United States Department of Agriculture, Washington, DC, 1997c. T Grandin. Animal Handling Guidelines for Meat Packers, American Meat Institute, Washington, DC. 1991b. E Lambooy, W Sybesma. The effect of environmental factors such as preslaughter treatment and electrical stunning on the occurrence of haemorrhages in the shoulder of slaughter pigs. Proc. International Congress of Meat Science and Technology (Brisbane, Australia) 34:101–103, 1988. JG Morgan, JG Cannon, FK McKeith, D Meeker, GC Smith. National Pork Chain QUality Audit (Packer-Processor-Distributor). Final Report to the National Pork Producers Council. Colorado State University, Fort Collins, and University of Illinois, Champaign-Urbana, 1993. GC Smith, JB Morgan, JD Tatum, CC Kukay, MT Smith, TD Schnell, GG Hilton, C Lambert, G Cowman, B Lloyd. Improving The Consistency and Competitiveness of Non-Fed Beef; and, Improving the Salvage Value of Cull Cows and Bulls. The Final Report of the National Non-

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Fed Beef Quality Audit—1994. Final Report to the National Cattlemen’s Beef Association. Colorado State University, Fort Collins, 1994. GV Peterson, DR Wright. Observations of subcutaneous speckling in lambs. New Zealand Veterinary Journal 27:166–168, 1982. T Grandin. Effect of temperature fluctuation on petechial hemorrhages in pigs. Proc. International Congress of Meat Science and Technology. Brisbane, Australia 34:104–105, 1988b. HA Channon, GR Trout, AM Pengelly, RD Warner. Stunning of pigs: Effect of method, current level and duration on carcass quality of pigs. Manipulating Pig Production VI, p. 125, Pig Research and Development Corporation, Australia, 1998. DE Burson, MC Hunt, DE Schafer, D Beckwith, JR Garrison. Effects of stunning method and time interval from stunning to exsanguination on blood splashing in pork. J Anim Sci 57:918–21, 1983. CR Calkins, GW Davis, AB Cole, DA Hutsell. Incidence of blood splashed hams from hogs subjected to certain antemortem handling methods (abstr). J Anim Sci 50 (Suppl. A):15, 1980. T Garland, N Baver, M Bailey. Brain emboli in the lungs of cattle after stunning. Lancet 348:610, 1996. GR Schmidt, KL Hossner, RS Yemm, DH Gould. Potential for disruption of central nervous system tissue in beef cattle by different types of captive bolt stunners. J Food Prot 62:390–393, 1999. NG Gregory, FD Shaw, RW Rowe. Effect of stunning and slaughter method on brain function and bleeding efficiency in calves. Proc. International Congress of Meat Science and Technology (Brisbane, Australia) 34:112–113, 1988. SL Armstrong, JAB Robinson, WR Hayes. Tracking and reducing cattle and carcass bruising through the use of management improvement tools. Final Report to the Canadian Cattlemen’s Association. Beef Improvement Ontario; Centre for Genetic Improvement of Livestock, University of Guelph; and Better Beef Limited, Guelph, Ontario, Canada. pp 1–14. SL Boleman, SJ Boleman, WW Morgan, DS Hale, DB Griffin, JW Savell, RP Ames, MT Smith, JD Tatum, TG Field, GC Smith, BA Gardner, JB Morgan, SL Northcutt, HG Dolezal, DR Gill, FK Ray. National Beef Quality Audit-1995: survey of producer-related defects and carcass quality and quantity attributes. J Anim Sci 76:96–103, 1998. DE Hoffman, MF Spire, JR Schwenke, JA Unruh. Effect of source of cattle and distance transported to a commercial slaughter facility on carcass bruises in mature beef cows. J Am Vet Med Assoc 12(5):668–672, 1998. PW McNally, PD Warriss. Recent bruising in cattle abattoirs. Vet Rec 138:126–128, 1996. HRC Meischke, JC Horden. A knocking box effect on bruising in cattle. Food Tech. in Australia. 28:369–371, 1976. JR Wythes, JC Horder, RC Cheffins. Effect of tipped horns on bruising. Vet Rec 104:390–392, 1979. FD Shaw, RI Baxter, WR Ramsey. The contribution of horned cattle to carcass bruising. Vet Rec 98:255–257, 1976. WR Ramsay, HRC Meischke, B Anderson. The effect of tipping horns and interruption of the journey on bruising in cattle. Aust Vet J 52:285–286, 1976. HRC Meischke, HR Ramsay, FD Shaw. The effect of horns on bruising in cattle. Aust Vet J 50:432–434. PV Tarrant, FJ Kenny, D Harrington. The effect of stocking density during 4 hour transport to slaughter on behavior, blood constituents and carcass bruising in Friesian steers. Meat Sci 24:209–222, 1988. GA Eldridge, and CG Winfield. The behavior and bruising of cattle during transport at different space allowances. Aust J Exp Agric 28:695–698, 1988. JL Aalhus, SDM Jones, AKW Robertson, Tong, AP Sather. Growth characteristics and carcass composition of pigs with known genotypes for stress susceptibility over a weight range of 70 to 120 kg. Animal Production 52:347–353, 1991.

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FJM Smulders, AMTS Romme, CHG Woolthuis, et al. Pre-stunning treatment during lairage and pork quality. In: Eikelenboom, G. (Editor) Stunning of Animals for Slaughter Martinus Nijhoff, Boston. pp 90–95, 1983. SD Milligan, MF Miller, CB Ramsey, LD Thompson, MP Springer. Improving pork quality by resting pigs, hot fat trimming and freeze chilling. J Anim Sci 74 (Supl 1):167 (Abstract), 1996. C Gariepy, J Amiot, S Nadai. Ante-mortem detection of PSE and DFD by infrared thermography of pigs before stunning. Meat Science 25:37, 1994. RL Epley. Dark Cutting Beef. Fact Sheet No. 17, Agric. Extension Service, University of Minnesota, St. Paul, MN, 1975. T Grandin. The effect of pre-slaughter handling and penning procedures on meat quality. J Anim Sci 49(Suppl. 1):147 (Abstract), 1979. MA Price, T Tennessen. Preslaughter management and dark cutting carcasses of young bulls. Canadian J Anim Sci 61:205–208, 1981. R Tyler, DJ Taylor, RC Cheffins, MW Rickard. Bruising and muscle pH in Zebu crossbred and British breed cattle. Vet Rec 110:444–445, 1982. JA Scanga, KE Belk, JD Tatum, T Grandin, GC Smith. Factors contributing to the incidence of dark cutting beef. J Anim Sci 76:2040–2047, 1998. BD Voisinet, T, Grandin, JD Tatum, SF O’Conner, and JJ Struthers. Feedlot cattle with calm temperaments have higher average daily gains tham cattle with excitable temperaments. J Anim Sci 75:892–96, 1997.

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11 Slaughtering and Processing Equipment MARÍA DE LOURDES PÉREZ-CHABELA and ISABEL GUERRERO LEGARRETA Universidad Autónoma Metropolitana–Iztapalapa, Mexico City, Mexico

I. INTRODUCTION II. PLANT LOCATION AND FACILITIES III. ANTEMORTEM HANDLING IV. STUNNING A. Electric Shock B. Captive-Bolt or Pneumatic Pistol C. Carbon Dioxide V. LANDING VI. STICKING VII. BLEEDING VIII. DRESSING A. Beef Dressing B. Sheep Dressing C. Pig Dressing IX. OFFAL HANDLING AND INSPECTION A. Beef Offal Handling B. Sheep Offal Handling C. Pig Offal Handling D. Inedible By-products X. COOLERS XI. CONCLUSIONS ACKNOWLEDGMENTS REFERENCES

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I. INTRODUCTION Slaughtering—the first step in the transformation of muscle into edible meat—affects the quality and quantity of meat. Meat quality depends on postmorten biochemical changes, which relate to sanitation, as well as physicochemical and physical attributes (aroma, color, and texture among others). From the quantity point of view, carcass yield is related to preand postmortem handling. II. PLANT LOCATION AND FACILITIES Because of noise and odor generation, slaughtering plants must not be located near urban areas. The U.S. Department of Agriculture (USDA, 1981) recommends that plants be located far from areas where objectionable odors or particles are generated, such as dumps or chemical plants. Slaughtering plants should also have accessibility; therefore, they should be connected to streets or highways but separated from other plants or buildings. Water supply must be in good quantity because washing is a continuous operation throughout the plant (Fig. 1). Non-potable water is a hazard and must be avoided. Carcasses are washed after dressing, so any bacterial contamination in the water supply will be passed to the meat substrate (Murray and Madden, 1996). Water disposal is equally important, because the wastewater contains grease, blood, hair, and tissue and bone particles. Slope in floors is required (no less than 10 cm for each 6 m in working areas, and 15 to 20 cm for each 6 m in the coolers) to avoid accumulation of effluents (USDA, 1981). Federal or local legislation related to waste disposal varies among countries or regions of the same country, but almost every country where a slaughtering plant is built has its own legislation.

Figure 1 Water pipelines and a water-cooled saw.

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Figure 2 Hand-washing facilities and a pedal-operated platform. Ceilings should be no less than 3 m high or more in certain working areas, such as those for evisceration and cleaning. They should be flat and smooth and free of unnecessary structures (Libby, 1986). Building materials for walls, floors, drains, ceilings, and equipment are also subject to regulations. All these materials should provide easy cleaning. In general, these materials comprise concrete, ceramic floor tile, floor-glazed brick, glazed tiles, smooth surface Portland cement plaster, plastic, or Portland cement plaster for ceilings. Certain materials in particular are not acceptable, such as lead, porcelain, wood, leather, fabrics, or any material that undergoes chemical reactions (Ockerman, 1980b). Square angles or joints where material can accumulate must be avoided. Floors must be of any nonslippery material. The size of the killing room may vary according to plant capacity, but in any case it must have enough space for animal handling and equipment operation, with walking areas around operative sections. Adequate lighting is also necessary, either natural or artificial. In any case, 220 lux in the working areas is necessary, 540 lux in the inspection areas, and 110 lux in the coolers. The U.S. Department of Agriculture describes lighting necessary for each operation and lamp placement. All lamps must have a protective shield. Ceilings must be painted in white or a light color. Ventilation and refrigeration also must be controlled for comfort as well as for reducing microbial growth. A maximum temperature of 10°C is necessary in all working areas. Hand-washing facilities and drinking fountains should be located in the working rooms (Fig. 2). These must be pedal operated (Figs. 3 and 4). Facilities for boot washing before walking into the working area must be supplied (Gracey, 1989). Product transferred in the plant should not come in contact with the doorways; 1.50 m doorways are necessary. There must be double-acting doors, constructed of rust-resistant

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Figure 3 Cleaning a beef carcass from a pedal-operated platform. materials, with a transparent panel at eye level. All windows, doors, and other openings must have insect and rodent barriers such as screens or seals (USDA, 1981). III. ANTEMORTEM HANDLING Antemortem facilities include livestock pens, inspection facilities, and holding pens. Transportation conditions from the production area to the slaughtering plant deeply affect meat quality, besides being part of humane animal handling. Handling, time, climatic conditions, and general health of the animals determine transportation losses. For example, weight losses in cattle can reach the proportions shown in Table 1. Once animals are in the livestock pens, water must be provided. All parts of the pen, as well as runways and ramps, must be paved with concrete or brick and well drained. The number of animals in each pen depends on animal size. For instance, cattle require 7.5  6

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Figure 4 A pedal-operated platform and a pig scalding tank.

m for 20 to 25 animals. Sheds must be provided to prevent excessive sun exposure. Good lightning (54 lux) is necessary. Holding pens allow the animals to walk into the stunning area without unnecessary stress. Grandin (1991) describes specific handling of animals in the holding pens in depth. IV. STUNNING Once the animals are inspected premortem, they are taken through the holding pens to the stunning area. Except for specific ritual slaughtering (kosher and halal), animals must be stunned before bleeding. Use of a specific stunning method depends on factors such as animal species, breed, age, and production costs. There are three main stunning methods: captive bolt or pneumatic pistols, electric shock, and carbon dioxide tunnels.

Table 1 Weight Losses During Transportation in Cattle Transportation time (hours) Up to 24 24 to 36 36 to 72 72 or more

Weight loss (%) 1.05 to 3.91 3.45 to 5.40 3.88 to 6.37 3.96 to 7.00

Source: Aldana, 1984.

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A. Electric Shock Electric shock stunning it is usually applied to pigs and sheep. It consists in applying electricity through the animal’s brain, using two electrodes to induce an epileptic shock, or through the heart. If the electric shock goes through the brain, the animal is only stunned, and bleeding must be carried out within 30 seconds because the animal can recover consciousness. In such a case, the electrodes must be placed on each side of the head (Fig. 5). Intensity must be no less than 250 mA and 75 V during 10 seconds (Grandin, 1980). The electrodes are kept moist in a 20% saline solution to assure electricity conduction. If a pig is left unbled for 2 to 3 min it will recover, and in 5 min will be completely normal. If electricity goes through the heart, the shock is irreversible and kills the animal by electrocution. If electric shock is adequately applied, the animal will not feel any pain. Conversely, if amperage is not high enough, the animal may feel a painful shock. Most large processing plants use electric stunning through the heart, applying the electric current from the

Figure 5 Electric stunning.

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Figure 6 An electric stunning restraint pen. head to the back of the animal. In this method the animal must be restrained in a confined area (Fig. 6). As electricity is used, the stunning area must be isolated and kept dry. For safety reasons the operator must wear rubber boots and stand on insulated ground. B. Captive-Bolt or Pneumatic Pistol Stunning with a captive bolt or pneumatic pistol can be applied in several species, although the specific way of application varies slightly. In calves, swine, horses, and cattle (Bos taurus), the muzzle is applied to the forehead. In Cebu cattle (Bos indicus), in sheep and in plants industrializing the brains, it is applied in the back of the head. This is because of the thickness of the forehead in Bos indicus and sheep (De la Puente, 1996). Captive-bolt pistols eject a metallic cylinder through the animal’s skull and return to their original position for the next shot. Alternatively, pneumatic pistols impact the animal’s head without pene-

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tration. In both cases, the brain cortex is permanently damaged. Captive-bolt pistols are used when the slaughtering rate is less than 240 or more animals per hour, because recharge is time consuming. Air pressure in pneumatic pistols may vary if there is a malfunction in the air compressor. If air pressure is to high, harm to the operator’s hands, back, or arms is possible. When this stunning method is used in pigs, the muzzle is applied just above the eye level, in the center of the forehead (Aldana, 1984). Animals are confined to a restraint area where movements are limited for the operator’s safety, as well as to prevent animal self-injuries. C. Carbon Dioxide The stunning action of carbon dioxide is accomplished by blockade of the animal’s neural terminals, therefore reducing the nervous impulses. Carbon dioxide concentrations of 65% to 70% during 45 seconds work most efficiently. Bleeding must be carried out within the next 30 seconds (Velazco, 2000). If the gas concentration is lower, the animals are not adequately stunned. If it is too high, there is a tendency for stiffness, reflex muscular activity, and inadequate bleeding. If time of exposure is too long, skin congestion can occur and the carcass can take a bluish hue after scalding. CO2 is heavier than air, so all devices operate on the basis of keeping the gas at low levels, making the animals descend to an area where suitable gas concentrations are fed, such as the oval tunnel (for slaughtering 120 to 240 animals per hour) or the Ferris wheel (Libby, 1986). The advantages of using CO2 are that nontoxic residues are found in meat or of by-products, and the animal’s body is relaxed, facilitating skinning and evisceration; it produces less noise than other stunning equipment; it requires fewer operators; and it does not promote muscle hemorrhages as is the case with other devices. The disadvantages of CO2 are that it is slower than other stunning methods, although at more constant rate, and initial investment is high (Gracey, 1989). V. LANDING Once the animal is stunned, it is landed in an area separated from the bleeding area (Fig. 7). This landing area must be at least 2 m wide (USDA, 1981) and is designed to prevent improperly stunned animals from running away. VI. STICKING It is desirable to keep the animal alive, but stunned, in order to eliminate the blood. Thorough bleeding can be achieved when the heart and respiratory functions are still working. After stunning, death occurs due to massive bleeding by sectioning the carotid arteries and the jugular vein with a sticking knife. Because blood is a vehicle for microbial distribution throughout the animal body, organisms introduced during sticking can be found after a few hours in other parts of the carcass. Carcass temperature also affects microbial proliferation (Roberts et al., 1980). For this reason, it is important to remove any dirt in the area where the sticking knife was introduced. Oversticking (i.e., puncturing the pleura) causes the blood to flow into the chest cavity, increasing the risk of microbial contamination. Sticking knives are 15 to 25 cm. The point of the knife is inserted about 2 cm in front of the breastbone, and an incision is made toward the jaw, penetrating 12 to 15 cm and sectioning the carotid artery and jugular veins. In sheep, the sticking knife is inserted immedi-

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Figure 7 Beef restraint pen and landing area. ately below and behind the ear, severing the jugular vein. In beef, an incision is made just in front of the sternum, also cutting into the main blood vessels (De la Puente, 1996). VII.

BLEEDING

Bleeding, to a large extent, reduces microbial contamination. It also prevent formation of “blood-splash” due to pressure built within the muscles, which diminishes the meat’s acceptability and represents a hygienic risk (Palumbo et al., 1996). Blood is also of economic importance as it can be transformed into meal for animal consumption or used in pharmaceutical applications. Usually, sticking is done while the animal is hung from its hind leg; in this position, better bleeding is achieved although pressure can built up in some organs. Rails 4.8 m high above the floor should be used for beef bleeding; 3.3 m for pig, sheep, and calf; 2.74 m for goat (USDA, 1981). Bleeding is carried out in curbed-in areas with enough space to prevent blood from splashing on stunned animals lying in dry areas or on carcasses being skinned. In beef, once the animal is hung on the bleeding rail, the carcass is not lowered until the completion of the process. In pigs, the carcass is later lowered for scalding and de-hairing. VIII. DRESSING Once the animal has been bled, the hide, or skin, and viscera are detached from the carcass and it is then trimmed. The head, feet, and tail are left attached to the pig carcass, whereas head and hide are removed from beef carcasses. Finally, the carcasses are washed with high-pressure spraying.

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A. Beef Dressing Dressing starts with dehiding: an incision is made in front of the brisket on the centerline of the neck towards the head. The animal is then scalped, the horns chopped, and the head is skinned out. A cut is made across the larynx and the head is detached by cutting through the occipital joint. The front legs are cut, leaving the knucklebones on the carcass (Fig. 8); the hind legs are cut through the tendons. An incision is made down the hide, removing it from the carcass with the use of a small saw (Fig. 9). The abdominal cavity is opened by cutting behind the brisket; the liver is removed by cutting it from the diaphragm, which in turn is cut. The content of the chest cavity is removed and the aorta is trimmed. The body is then sawn, dividing the carcass into two sides (Fig. 10).

Figure 8 Beef de-hiding.

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Figure 9 De-hiding saw.

To prevent microbial contamination it is important to trim excessive tissue, the area where the sticking knife was introduced, and the spinal cord. The carcass is then washed by high-pressure spraying (19 kg/cm2) (Velazco, 2000) (Fig. 11). Head and hide are not included in the carcass weight. Dressing of beef is carried out in rails at least 3.3 m (USDA, 1981). B. Sheep Dressing The sheep’s legs are cut from the knuckle down the front legs; the skin is removed, starting from the neck up to the root of the tail; and the hind legs are cut. Evisceration and washing are similar to that described above. Head, skin, forefeet and hindfeet, and viscera should be removed before weighing (Libby, 1986).

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Figure 10 Splitting a beef carcass.

C. Pig Dressing After bleeding, pig carcasses are scalded in tanks with water at 60° to 65°C heated by steam (Fig. 12a). After 5 minutes the hair can be easily removed. The scalding vat should be constructed of metal, and the floor must be well drained. The vats are equipped with a cradle to remove the pig after scalding and before scraping (Fig. 12b). In de-hairing machines, the pig revolves and beaters remove the hair (Fig. 13a, b). De-hairing can also be carried out by hand (Aldana, 1984). Carcasses are then hung from the hind legs in a shaving rail equipped with a drained cabinet washer. The remaining hair is then burned off by using a blowtorch (Fig. 14) or by passing the carcass through a furnace, allowing the skin to become dry and hard. Evisceration is carried out in a similar way as with beef (Fig. 15).

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Figure 11 Beef carcass high-pressure spraying.

A

Figure 12 Scalding a pig carcass.

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B

Figure 12 Continued.

A

Figure 13 Revolving pig de-hairing equipment.

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B

Figure 13 Continued.

Figure 14 Burning off the remaining hair. 269 Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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Figure 15 Splitting a pig carcass. The carcass is then washed by high-pressure spraying (Fig. 16). Pig, sheep, goat, and calf dressing rails should allow the leg hooks from which the carcass is suspended to be 2.25 m above the floor or inspector’s platform (USDA, 1981). IX. OFFAL HANDLING AND INSPECTION Viscera inspection may vary from country to country. However, all legislations require examination of indicative organs providing information about the health of the animal. Therefore, identification of carcasses and their respective viscera is necessary. The movement of the viscera on the inspection table should be synchronized with the movement of the carcass conveyor. If any indication of an unhealthy animal is detected in the viscera, the carcass can be easily removed from the line. Depending largely on tradition, certain viscera are used for human consumption or are considered inedible. This is the case with pigskin, brains, and genitalia. Commercial aspects are also considered in directing viscera for uses other than human consumption, as their use in the pharmaceutical and other industries generally gives added value to various internal organs. A. Beef Offal Handling Brains and tongue are removed from the head. The tongue is removed first, inspected, thoroughly washed, scraped, and hung from the root end to preserve the shape. The head is split

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Figure 16 Pig carcass high-pressure spraying. and the brain removed and inspected. Other parts of the head, such as cheek meat and lips, are used in some countries for edible product manufacturing. Trachea, lungs, and heart are removed and examined. Lungs and heart are used for edible purposes and must be thoroughly examined, as sometimes food material from the stomach can be found in the bronchial tubes (Ockerman and Hansen, 1988). The liver must be carefully detached from the gall bladder, examined and cut across the thin end to allow blood to drain. Spleen, stomachs, and sweetbread are removed, trimmed of adhering fat or excessive tissue from other internal structures, and placed in a cooling room. Intestines are used in the preparation of tripe; they are thoroughly cleaned and trimmed of adhering fat. The esophagus is carefully stripped from the outer muscles and dried before use (Gracey, 1989). Blood is used either for edible purposes in several countries (black pudding in Scotland, moronga in Mexico, butifarra negra in Spain)—sheep or pig blood generally being preferred—or directed to animal feed production. When paunches are used for edible purposes, they should be emptied on tables (USDA, 1981). B. Sheep Offal Handling In a similar way as beef, some sheep viscera are used mainly for edible purposes: brain, tongue, head meat, liver, heat, lungs, spleen, sweetbread, heart, and liver. They are handled as described for beef offal. Tripe from sheep stomach is used in several traditional European sausages. Lamb kidneys are usually left attached to the carcass, but in mutton carcass they are separated and sold apart (Ockeman and Hansen, 1988).

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C. Pig Offal Handling The offal to live animal ratio is less in pig than in beef or sheep because the skin, head, feet, and tail are taken into account for total carcass weight. Its simple stomach weighs less than the various stomachs of ruminants. As with beef, the trachea, lung, heart, liver, and esophagus are used for edible purposes; the stomach is used as a container of some products; feet, tail, and kidneys are generally detached from the carcass and sold separately (Ockeman and Hansen, 1988). The head is separated from the carcass; the tongue, brains, and cheek meat are removed in the same way as in beef carcasses. The skin is used for gelatin production, but is also eaten, after frying, as a snack. D. Inedible By-products By-products such as viscera are aimed for industries other than the human food industry. Their use varies according to animal species and is influenced by cultural aspects. In general, inedible offals are used for pharmaceutical purposes; for animal feed formulation as meat, bone, and blood meal; or for other industries, as in the case of hide and wool. These by-products are gallbladder, bones, and hooves from beef, sheep, and pigs; horns and feet from sheep and beef; wool and skin from sheep. Beef hide and hair from pigs are inedible by-products. Scraps and condemned parts can also be used for meat meals. A number of pharmaceutical products are obtained from slaughter by-products, mainly from the thymus and thyroid of beef, sheep, and pig; pig stomach lining; sheep prostate and intestine; beef and sheep pancreas and suprarenal glands; pig and beef ovaries and spleen; and beef pituitary and pineal glands, corpus luteum, parathyroid, and tested (Libby, 1986). Inedible products and catch basins for grease recovery must be located to avoid contact with handling of edible products. Flow of inedible products must assure that they will not come in contact with edible products (USDA, 1981). X. COOLERS The average temperature of a cooler must be between 2° and 6°C. A certain degree of “sweating” occurs when relative humidity is high (70% or more) (Ockerman, 1980a). Required building material in coolers must be easy to clean; floors must have a slope to allow proper drain. Rails should be at least 3.3 m above the floor for halves of beef, 9 m for calves and hogs, and 2.25 m for quarters of beef; goat and sheep carcasses should be suspended 1.95 m to the hook. Carcasses must hung be 1 m from the walls and 0.60 m from the refrigeration equipment, with a separation among carcasses of 0.30 m (USDA, 1981). Edible organs and offal should be placed in a separate cooler of the carcasses. Retained carcasses or parts should be located in a refrigerated separate compartment (Aldana, 1984). XI. CONCLUSIONS Slaughtering methods are based on obtaining the optimum quality and quantity of meat. Humane killing, in addition to its ethical implications, results in better-quality meat. Regulations may vary from one country or region to another, but in all cases the objective is to assure sanitation for the food market and safety for the workers. Use of edible and inedible by-products, although largely based on economic and cultural factors, is an important part of the investment return.

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ACKNOWLEDGMENTS The authors thank Dr. Marcelo Signorini, Universidad Nacional del Litoral, Argentina, for his comments. All photographs are courtesy of the University of Guelph. REFERENCES Aldana, L.L. Tecnología de la carne y sus productos. Editorial Pueblo y Educación. Havana, Cuba, 1984. De la Puente, J. Personal communication, 1996. Gracey, J.E. Higiene de la carne. 8th edition. pp. 72–94. Interamericana-McGraw Hill. Madrid, Spain, 1989. Grandin, T. Is your hog stunner insulated? Meat Process. February 1980. p. 69. Grandin, T. Guias recomendadas para el manejo de animales para empacadores de carne. American Meat Institute. Washington, DC, 1991. Libby, J.A. Matanza Humanitaria. In: J.A. Libby (Ed.). Higiene de la carne. pp. 47–53. Editorial Continental, Mexico City, 1986. Murray, K.A., and R.H. Madden. Assessment of materials or microbiological sampling of carcasses. 42th Internat. Conf Meat Sci Tech pp. 197–198, 1996. Ockerman, H.W. Quality control of postmortem muscle tissue. Volume 2: Environmantel control. The Ohio State University. Washington D.C., 1980a. Ockerman, H.W. Quality control of postmortem muscle tissue. Volume 4: Microbiology. The Ohio State University. Washington D.C. 1980b. Ockerman, H.W., and C.L. Hansen. Animal by-product processing. Ellis Horwood Ltd., Chichester, UK, 1988. Palumbo, S.A., B.S. Eblen, A.J. Miller, and J.G. Phillips. Comparison of techniques to evaluate the bacteriological quality of pig carcass surfaces. 42th Internat Conf Meat Sci Tech pp. 195–196, 1996. Roberts, T.A., H.J.H. MacFie, and W.R. Hudson. The effect of incubation temperature and site of sampling on assessment of the number of bacteria on red meat carcasses at commercial abattoirs. J Hyg Camb 85:371–380, 1980. U.S. Department of Agriculture. 1981. U.S. Inspected meat and poultry packing plants. Agriculture Handbook 570. Washington, D.C. Velazco, J. Problemas de calidad en el sacrificio de bovinos. Carne Tec. Mexico City. January/February, pp. 18–21, 2000.

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12 Carcass Processing: Factors Affecting Quality OWEN A. YOUNG MIRINZ Centre AgResearch, Hamilton, New Zealand NEVILLE G. GREGORY South Australian Research and Development Institute (SARDI), Flaxley, South Australia, Australia

I. INTRODUCTION II. THE BIOCHEMISTRY OF THREE QUALITY PROBLEMS IN MEAT A. Dark-cutting Meat B. Pale, Soft, Exudative (PSE) Meat C. Enduring Toughness in Meat III. SOME PRACTICES LEADING TO QUALITY PROBLEMS IN MEAT A. Production Practices B. Breed and Cloning Effects C. Processing Practices IV. CONCLUDING REMARKS REFERENCES

I. INTRODUCTION This chapter is the first of three (12 through 14) that examine processing the live animal from the moment of slaughter to the preparation of meat cuts for a variety of food uses. Basically, processing is an uncomplicated activity accomplished with cutting tools and differing little from its practice in the days of hunter-gatherers, who would disassemble slaughtered animals in a set sequence that suited the simple needs of the tribe. In modern societies, however, the needs are very complex, and carcass processing has become a highly organized activity in response to these needs. They include hygienic, pathogen-free meat; defined eating qualities; minimal impact on the environment; and worker well-being, to name just a few.

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One clear need that is often unstated in meat processing is the need for a consistency in the attributes of the outputs—meat, hides, effluent, and so on. Consistency is a fundamental goal of all industrial activities, but meat processing has lagged behind other industries. For example, a given model of automobile tire is, from the perspective of the consumer, identical from one tire to the next, and any irregularity is obvious and decrees replacement or refund. Not so with meat. Meat is inherently variable, arising not only from

Table 1 Origins of Some Quality Problems in Meat Cause Genetic factors Stress-sensitive pig breeds Callipyge lamb Belgium Blue cattle Production factors Emaciation Disease Staggers conditions Wildness, handling difficulty Lameness Poor sanitation on pig farms Trauma Bruising in sheep, cattle Overcrowding in farmed fish Fighting Injection-site infections Old animals Growth promotants Preslaughter factors Excessive exercise

Heat stress Cold stress Mixing of unfamiliar groups Long-distance transport Swim washing Overcrowding in pens Diarrhea Dehydration Slaughter factors Electrical stunning Carbon dioxide stunning Delayed bleeding Postslaughter factors Overstimulation Very fast cooling

Quality problem PSE in pork Enduring toughness Increased risk of enduring toughness Increased risk of dark-cutting condition Off-flavours with facial eczema Off-flavours in lamb Increased risk of dark-cutting condition Hockburn, breast blisters in poultry Boar taint Meat rejection Fin erosion, scarring Dark-cutting condition Meat rejection Enduring toughness Enduring toughness Dark-cutting condition Heat shortening in poultry meat Burnt tuna condition in fish Risk of PSE in pork PSE in pork, turkey Dark-cutting condition Dark-cutting condition Dark-cutting condition in bull beef and pork Increased risk of dark-cutting condition Dark-cutting condition in lamb, enduring toughness Dark-cutting condition in pork Risk of carcass contamination Dark, dry, sticky meat Risk of blood-splash in many species Risk of bone breaks in poultry and pigs Skin damage in salmon, PSE in pork Risk of blood-splash in many species Excessive drip, enduring toughness Enduring toughness in ruminant meat

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variations between muscles but also from the between-animal variability that arrives at the slaughterhouse gate each day. The processor is faced with making the most of an often highly variable raw material. In an ideal world the slaughterhouse management would adjust the process to suit each animal, aiming to reduce the variability of the outputs. Currently little is done in the way of tailoring processing. In contrast, much slaughterhouse practice adds to variability through poor control. Before discussing processing and its control in more detail (Chapter 13), it is useful to gain an overview of how certain variations in the the raw material—the live animal arriving from feedlot or ranch—affects the quality of meat as it emerges from processing, and how the processor can affect the outcomes for better or worse. The overview is the subject of this chapter. Many meat quality problems can be traced to causes acting at various points in the chain from conception to consumption. Some of these are listed in Table 1, grouped according to where in the chain they occur. This list is not exhaustive and concentrates on causes that arise during animal production and through to the hours shortly after slaughter. A host of quality problems can and do arise later in the chain. However, if meat enters the latter part of the conception to consumption chain in poor condition, good practices often cannot reverse the existing poor quality. Even if that is possible, it is better to eliminate quality problems upstream than to repair damage downstream. To illustrate factors affecting outcomes of carcass processing, this chapter describes the biochemistry of three common quality problems along with some predisposing practices in production and processing. The conditions are the dark-cutting condition, the pale, soft, exudative (PSE) condition, and the phenomenon of enduring toughness in meat. It is clear from Table 1 that they can arise from more than one practice and so they are good examples of how outcomes can be affected. II. THE BIOCHEMISTRY OF THREE QUALITY PROBLEMS IN MEAT A. Dark-cutting Meat The term dark-cutting refers to the darker color of meat cuts with this condition. Another designation, DFD, refers to dark-cutting meat’s physical properties: dark in color, and firm and dry to the touch. This problem occurs most commonly in beef, vension, and pork. The key difference between dark-cutting meat and normal meat is its pH after rigor processes are complete. Meat pH falls during rigor development and the value finally reached is frequently called the ultimate pH (Chapter 2). The pH of dark-cutting meat is higher than the normal 5.4 to 5.6, occasionally being as high as 7.0. The usual pH criterion defining dark-cutting meat is an ultimate pH above 6.0. However, this value is arbitrary, and the properties of dark-cutting meat evolve as a continuum from pH 5.6 upwards. 1. Biochemical Basis of the Dark-cutting Condition In postmortem muscle adenosine triphosphate (ATP) is continually lost through hydrolysis: ATP → ADP  Phosphate  H

(Reaction 1)

where the value of  depends on the ionic conditions in the cell. To maintain the normal cellular concentration of ATP, it is continually regenerated in reactions linked to the

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metabolism of energy stores, principally glycogen, muscle’s starch energy reserve composed of polymerized glucose molecules: 3H  3ADP  3Phosphate  nGlucose → 3ATP  (n  1)Glucose  2Lactate  2H

(Reaction 2)

The net sum of these two reactions is: nGlucose → (n  1)Glucose  2Lactate  2H

(Reaction 3)

For every molecule of glucose metabolized, two hydrogen ions are produced along with two lactates. The key to whether muscle will become dark-cutting meat is the concentration of glycogen in the live muscle. In a well-fed and rested animal, the concentration of glycogen in muscle is about 1% by weight. When expressed as lactate—because that is what glycogen is metabolized to—this is equivalent to about 100 mole of lactate per gram of muscle. Clearly, if all the glycogen were metabolized to lactate, 100 mole of hydrogen ions would be produced, as demanded by Reaction 3. In pure water, the pH would be very acidic at pH 1.0. The pH of meat is much higher than 1.0, typically 5.5 and seldom falling below 5.4. This difference is explained by the high buffering capacity of muscle tissue, and the fact that one or more glycolytic enzymes becomes inactive as the pH falls. Glycolysis slows and finally stops at around pH 5.4, leaving some residual glycogen in the muscle tissue. Figure 1 illustrates a hypothetical time course of pH fall for muscles containing between 40 and 100 mole of lactate equivalents (as glycogen) per gram of muscle. Irrespective of the glycogen concentration, the rate of pH fall in the first few hours is independent of the glycogen concentration. However, as glycogen becomes depleted in the muscle containing the least glycogen (40 mole equivalents), the pH can fall no lower than 6.5, and that would become the ultimate pH for that muscle. There would be no residual glyco-

Figure 1 Hypothetical time course of pH fall for muscles containing different concentrations of glycogen (expressed as mole lactate equivalents per gram). Low concentrations result in a high ultimate pH and no residual glycogen.

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gen. For the muscle with 60 mole equivalents, the ultimate pH would be lower, 6.0, but still with no residual glycogen. Both these muscles would develop the dark-cutting condition. The muscle containing 75 mole equivalents has sufficient glycogen to achieve a low and normal ultimate pH, 5.5, and so would not develop the condition. Neither would the muscle containing 100 mole equivalents. However, only that muscle would have residual glycogen at its ultimate pH. 2. Properties of Dark-cutting Meat With some exceptions, dark-cutting meat adversely affects quality. The exceptions are those comminuted products where the pH is required to be high, and in the hamburger trade, where a higher meat pH confers better cohesiveness and fluid holding capacity. But as a primal cut, dark-cutting meat poses significant quality problems (Hood and Tarrant, 1981), not the least being the dark-red appearance. If all meat were dark-cutting, the consumer would see little variation in color. However, when one steak is dark among bright red companions, it is often rejected. Defects other than raw meat color include toughness (sometimes), flavor effects, reduced microbiological stability, and a more rare cooked meat color, each dependent on how elevated the pH is. Bouton et al. (1957), Purchas and Aungsupakorn (1993), and Watanabe et al. (1996) found that the toughness of beef or lamb was maximal at pH 6 (Fig. 2). Whatever the cause of this effect, the toughness does not necessarily “age out” on storage prior to retail sale. Dark-cutting meat is less flavorful (Dransfield, 1981; Purchas et al., 1986), has more off-flavors, (Fjelkner-Modig and Ruderus, 1983), and evokes more negative comments than normal-pH beef (Dransfield, 1981; Dutson et al., 1981). Braggins (1996) explored the

Figure 2 Changes in shear force with ultimate pH in longissimus muscle from bulls and steers. Dotted lines are 95% confidence limits. (Adapted from Purchas and Aungsupakorn, 1993.)

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Table 2 Mean Sensory Panel Intensity Scores for Cooked Semimembranosus Muscle from Sheep in Three Ultimate pH Groups Ultimate pH range Attribute Overall odor Overall flavor

5.44–5.60

5.75–6.13

6.30–6.45

Statistical effect of pHa

51b 50

45 42

40 27

*** ***

Source: Data are from Braggins (1996). a ***, P  0.001. b The scale was from 0 (none) to 100 (extreme) for 10 sheep in each group.

chemical origins of these flavor changes by examining the headspace volatiles of fat from cooked sheepmeat. Total volatiles decreased with increasing meat pH, as foreshadowed by Madruga and Mottram (1995). The reduction in aldehydes and alcohols was particularly marked (Braggins, 1996) and probably contributed to odor and flavor loss in the meat (Table 2, Fig. 3). The concentration of free glucose is lower in dark-cutting meat than in normal meat (Table 3). As a reducing sugar, glucose can be flavor active as a reactant in the Maillard reaction. Glucose is also important for the microbiological stability of meat. This is particularly important for countries exporting chilled product to remote markets. Ideally, the meat should be transported at the lowest possible temperature above its freezing point, 1.5°C. Provided the bacterial contamination is low at the time of vacuum packing, meat of normal pH will remain wholesome for 12 or more weeks. This storage life is compromised with high pH meat for two reasons. First, putrefactive bacteria such as Shewanella putrefaciens and Yersinia enterocolitica are able to grow anaerobically on meat at pH values above about 5.9 (Barnes and Impey, 1968; Grau, 1981), and second, the glucose concentration is lower as pH increases (Table 3). When glucose is absent, or becomes absent due to micro-

Figure 3 Frequency of flavor descriptors for low and high ultimate pH sheepmeat. Full scale was 50 for the ‘Bland/Flat/Low’ descriptor. (Adapted from Braggins, 1996.)

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Table 3 Ultimate pH and Glucose Concentration in Longissimus Muscle of Beef in Rigor Ultimate pH range

Mean glucose concentration (g/g)

5.40–5.49 5.60–5.69 5.80–5.89 6.00–6.09 6.20–6.29 6.40–6.70

118 70 59 13 15 0

Source: Data are from Newton and Gill (1978).

biological activity, the microflora begin to use amino acids as an energy source, generating offensive catabolic products. Cooked meat color is affected by pH. During cooking, myoglobin is denatured to brown pigments. However, at high pH values, myoglobin is more heat stable and less prone to denaturation, so redness is more likely to persist in dark-cutting meat, creating the impression that it is undercooked. This variation in cooked-meat appearance due to pH is important in commercial food catering where a standard cooking regimem may yield meat of various apparent levels of doneness if the pH of the raw product varies. B. Pale, Soft, Exudative (PSE) Meat PSE meat is caused by the denaturation of muscle proteins that takes place when muscles simultaneously experience a low pH, from postmortem metabolism, and high temperature (Bendall and Wismer-Pedersen, 1962). This can happen when the cooling regimem is normal but glycolysis and the related pH fall is excessively rapid (Fig. 4), so that the meat

Figure 4 Hypothetical time course of pH fall for pig muscles that will yield normal and PSE pork. In PSE pork, pH falls more rapidly and usually attains a slightly lower pH than normal.

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achieves ultimate pH when still warm. Among meat animals, the phenomenon occurs most commonly in pigs. This is primarily because Reactions 1 and 2 are intrinsically fast in pigs and can be further enhanced if the pig is genetically prone to stress, as is common in certain breeds, e.g., Landrace. The PSE condition can also develop in turkeys, athletic fish species such as tuna, and in unusual circumstances in broiler chickens. Accelerated muscle acidification can also occur in beef, especially in double-muscled breeds (Boccard, 1981), in very large animals, and when post-slaughter cooling is slow. 1. Biochemical Basis of the PSE Condition Whatever the fundamental cause of PSE meat, it is useful to understand the biochemical basis common to PSE-like conditions irrespective of species. Between pH 7 (the pH of live muscle) and pH 6, hydrogen ions are released when ATP is hydrolysed to ADP. The faster this happens, the faster the pH will fall. To generate large quantities of hydrogen ions requires Reaction 2 to regenerate fresh ATP for Reaction 1, but it is important to realize that ATP hydrolysis is the regulating reaction. PSE-like conditions develop only when ATP hydrolysis is rapid and the muscle temperature is high. The combination of low pH and high temperature leads to a denaturation of the soluble sarcoplasmic proteins and of myofibrillar proteins, notably myosin, the protein principally responsible for gel formation in cooked meat. According to Offer (1991), myosin denaturation is the decisive event in determining the soft and exudative state of PSE meat. The denaturation of the myosin molecule occurs at the molecule’s head in such a way as to draw the thick and thin filaments—the principal myofibrillar components—more closely together at rigor than is normal, so more water is expelled. Stated another way, less water is retained (Fig. 5), an undesirable feature in meat or products made from it.

Figure 5 Water retention of myofibrils from normal and PSE pork. At any pH in the range experienced in pork or its processed products, myofibrils from normal pork retain more water. (Adapted from Bendall and Wismer-Pedersen, 1962.)

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Figure 6 Relationship between the pH of pork muscles 45 min after slaughter, and myosin denaturation and percent drip. (Redrawn from Offer, 1991.)

The time course of myosin denaturation can be monitored by the loss in the ATPase activity of the myosin heads, which is reduced by low pH at high temperature (Penny, 1967a). However, ATP protects the heads from denaturation to some extent, and its presence in prerigor muscle prevents catastrophic damage that would otherwise result. Actin is even more protective than ATP (Penny, 1967b). Although protection by ATP is lost when a carcass enters rigor, this loss is more than compensated for by the cross-linking of myosin heads with actin. Thus, it is the conditions that the musculature experiences in the prerigor period that determine the extent to which muscles become PSE. Figure 6 summarizes the effect of muscle pH at 45 minutes post slaughter on myosin denaturation and drip. 2. Properties of PSE Meat When the myofibrillar and sarcoplasmic proteins become denatured they become more reflective, so the meat looks paler. PSE meat tends to release drip because the denatured proteins hold less water than normal. The myoglobin in the fluid that is remains is more prone to convert to the brown pigment, metmyoglobin, compounding the unpleasant appearance. In some cases only certain muscles present in a meat cut are PSE, so the cut becomes multihued and unattractive to consumers expecting a consistent colour. Denaturation also makes the myofibrillar proteins less soluble in the salt used in many comminuted meat products, and so gel formation is retarded in products made from PSE meat (Wismer-Pedersen, 1960). C.

Enduring Toughness in Meat

Consumer surveys have established that meat tenderness (lack of toughness) is the most important attribute in the eating quality of red meats. The range of prices for cuts of different tenderness (e.g., grilling vs stewing cuts) and the retail marketing claims for tenderness confirm that tenderness is an important consumer issue. Figure 7 shows how tenderness can

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Figure 7 Frequency of tenderness values in one cut (longissimus lumborum) from a nationwide beef tenderness survey taken at retail (New Zealand, mid-1990s). Values above 10 kgF are considered tough, so 10% of consumers would have had a bad eating experience.

vary within one muscle (longissimus lumborum) in beef offered for retail sale throughout one country, New Zealand, in the mid 1990s. Samples with shear force values 6 kgF or below have a highly acceptable tenderness to most consumers, whereas above 10 kgF, consumers describe the meat as tough. Meat tends to become more tender during chilled storage, so had these meat samples been stored chilled for several weeks before cooking and testing, the profile would have moved slightly to the left. In some cases, however, meat remains irrevocably tough. Some samples with high shear force values can remain so to the point that the meat spoils. 1. Biochemical Basis of Enduring Toughness in Meat Compared to bony fishes, land mammals are subject to greater gravitational stresses. Mammals have a greater proportion of musculoskeletal connective tissues (bone, tendon, cartilage), and these tissues are also more robust, as reflected in the higher melting point of mammalian collagens (Piez, 1967). It is not surprising, therefore, that toughness arising from connective tissue is a problem in meat animals, particularly large animals, but not fish. Moreover, meat from muscles with high concentrations of collagen (many forequarter muscles, shank muscles) is tougher—and cheaper—than from muscles with low concentrations (psoas, longissimus). The proteolysis of intramuscular collagen in stored meat is slow and difficult to detect (Stanton and Light, 1988). For this and other reasons, collagen is largely responsible for the aptly named “background toughness” in meat (Bailey, 1988) that is often responsible for enduring toughness. This toughness is exacerbated by the stable cross-linking that occurs in collagen molecules as animals age. These cross-links endure no matter how long meat is hygienically stored. Furthermore, a fraction of these cross-links is heat stable, and these links persist in cooked meat. The expression “tough as old boots” has a basis in collagen chemistry.

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Although some production practices impinge on toughness from connective tissue (see later), toughness arising from certain myofibrillar proteins can be affected by both meat production practice and postmortem processing. Among the muscle proteins that undergo significant postmortem proteolysis, and are thus strongly implicated in models of tenderization, are connectin (Locker and Leet, 1976), now more commonly called titin, desmin (Young et al., 1980), vinculin, and other related proteins of muscle (Taylor et al., 1995). These proteins have structural roles in the muscle cell, so their fragmentation by proteases fits comfortably with tenderization models (Koohmaraie, 1992). Of the protease classes active in postmortem muscle, the calpain family is generally held to be primarily responsible for tenderization of meat. In the live animal, calpains are one of a number of proteolytic systems involved in protein turnover in cells. From the perspective of meat tenderization, three calpain system components are important. The - and m-calpains are proteolytic whereas calpastatin, as the name implies, is an inhibitor of the calpains. In the live animal calpastatin modulates calpain activities. Calpains and other candidates for a role in tenderizing are discussed in Chapter 13 and elsewhere. For the moment, if capastatin activity is more dominant than - and m-calpain activities by way of production or processing effects, enduring toughness can often result. 2. The Consequences of Enduring Toughness in Meat At first sight, the principal characteristic of enduring toughness is just that, toughness that lasts. However, toughness arising from connective tissue can often be overcome by prolonged heating as in casserole-style dishes. By contrast, myofibrillar toughness is truly enduring. Blade tenderization or grinding are the common methods of overcoming toughness from either cause. In the case of grinding, the end result is a lower value commodity product suitable for the sausage and hamburger trades. III. SOME PRACTICES LEADING TO QUALITY PROBLEMS IN MEAT This section describes a number of practices, both historical and contemporary, that lead to dark-cutting meat, PSE-like conditions or enduring toughness. The practices discussed are not exhaustive and in many cases, animal, production, and processing effects interact to exacerbate poor quality or, in rarer instances, rectify the quality problem. The practices can usefully be grouped under production, breeding, and processing. A. Production Practices Before domestication of farm animals, meat was obtained by hunting and trapping of wild animals. The cave paintings of Lascaux, France, and elsewhere depict the pursuit that must have been common in hunting. In many instances, animals would have been pursued to exhaustion. Frequently these would be weaker animals, perhaps in poorer physical condition than their escaping peers. In the fight-or-flight response, adrenaline is released from the adrenal medulla into the bloodstream, activating glycolysis in skeletal muscle. Along with noradrenaline, adrenaline also helps to mobilize liver glycogen as glucose, and body fat as free fatty acids into the bloodstream, also for use in muscle. In this way, adrenaline, and in an indirect manner noradrenaline, help to refuel muscle. They do not drive muscle contraction and metabolism, but rather facilitate these activities. Exercise can reduce muscle glycogen before slaughter (Chrystall et al., 1982). When exercise is excessive, and when glycogen supplies in the liver run out and fat mobilization

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does not keep up with the demands of the muscle, the glycogen concentration in muscle becomes depleted. Depletion of muscle glycogen before slaughter results in the dark-cutting condition with all its adverse consequences. When human societies began to farm animals, selection for slaughter was done at leisure and the “fatted calf” was chosen rather that an animal in poor condition. The slaughter would seldom involve a chase. Modern pastoral farming continues these traditions. In many countries throughout the world—but particularly in Australasia and South America—finishing ruminants for slaughter on pasture is big business. However, even without the chase, pastoral farming often leads to high pH meat (Tarrant, 1981). Stresses often arise when free-range cattle—unused to human contact or close contact with their peers—are corralled, transported and slaughtered at a central facility. Once muscle glycogen has been depleted by stress arising from these activities, it takes many hours, or even days, to restore it to normal levels, and there is usually insufficient time to allow recovery before slaughter (Lacourt and Tarrant, 1981). Therefore, resting the stressed animal for a few hours before slaughter is not necessarily effective in preventing the dark-cutting condition. Graafhuis and Devine (1994) surveyed the ultimate pH of beef and sheepmeat in New Zealand slaughterhouses, and found that for thousands of cattle and sheep, 30% of each species had a meat pH (longissimus lumborum) above 5.8, summarized for bulls and steers in Fig. 8. There was also clear sex effect, assuming other factors were equal. Bulls are particularly prone to psychological and physical stressors, frequently leading to loss of glycogen and the dark-cutting condition. Because high ultimate pH is largely peculiar to pastoral farming systems, diet might also be involved. The energy intake of animals fed a pastoral diet is lower than that from a grain-based diet, and several researchers have reported that the higher the energy intake, the greater the accumulation of muscle glycogen (McVeigh and Tarrant, 1982; Melton et al., 1982; Pethick and Rowe, 1996). However, the effect of diet is not mediated by energy

Figure 8 Frequency of ultimate pH values in the longissimus lumborum muscle from pasture-finished steers (540) and bulls (770). (Adapted from Graafhuis and Devine, 1994.)

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alone. Grain diets promote the accumulation of muscle glycogen by altering the way dietary energy is used (Gross et al., 1988; Huntington et al., 1996; Daly et al., 1999). With grain diets, propionate absorption from the rumen is enhanced at the expense of acetate absorption. The former fatty acid but not the latter is the substrate for gluconeogenesis resulting in glycogen accumulation. The implication for pasture finishing is that whereas the initial level of glycogen is nominally sufficient to produce a normal ultimate pH, the concentration of glycogen to buffer against stress-induced losses is marginal. Add to this the greater potential for stress in extensive pastoral grazing systems in the few days before slaughter, and it is clear why the high ultimate pH issue is so important in pastoral finishing systems. Because grain-based diets are much more expensive than pasture diets, feedlot-finishers of cattle maintain animals for only as long as it takes to produce the desired muscle growth and marbling. Producers who finish animals on pasture have much lower on-ranch holding costs. As a result, cattle can be and often are held on-ranch for extended periods to capture anticipated higher prices. The downside of this is that as animals become older, the connective tissue that permeates all muscles becomes tougher, adding to meat toughness due to other structural components of muscle. Connective tissue in muscle largely comprises strands of collagen. When immature collagen, such as is found in young animals, is heated above about 60°C, its rigid, organized helical structure ‘melts’ to produce gelatine. Gelatine does not cause toughness, but with increasing maturity, collagen becomes increasingly cross-linked with a variety of covalent intermolecular bonds, some of which are heat stable. On melting, these persist to produce a shrunken, cross-linked mass of melted collagen. Shrinkage generates tension, which relaxes only slowly as bonds break on extended casserole-style cooking. However, this event is particularly slow in older, strongly cross-linked muscle. Figure 9, from Kopp and Bonnet (1987), compares tension development and relaxation in bovine collagen from animals of different ages.

Figure 9 Effect of bovine age on the isometric tension of muscle sheath collagen during heating at 80°C for 30 minutes. (Adapted from Kopp and Bonnet, 1987.)

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Unlike toughness due to other structural components, collagen-derived toughness does not markedly decrease during chilled storage (Etherington, 1987). On extended hightemperature cooking, even the most resilient collagen will eventually dissolve, but beef cuts conventionally defined as grilling or frying steak by anatomical location will be disappointingly tough if derived from an old animal. In this sense the collagen-derived toughness is enduring. The phenomenon of increased collagen cross-linking with age has an added layer of complexity. During periods of slow animal growth, the proportions of crosslinking in collagen increase, resulting in tougher meat (Rompala and Jones, 1984; Bailey and Light, 1989). In pasture-based production systems, seasonal differences in nutrition often result in periods of slow animal growth, perhaps leading to increased toughness and variability in toughness.

-Adrenergic agonists are drugs that mimic the effects of adrenaline. It is well known that supplementation of animal diets with these drugs can, without increasing food intake, alter carcass composition by increasing lean deposition while minimizing fat deposition (Moloney et al., 1990). The economic benefit of this effect has been exploited, licitly or illicitly, by many meat producers throughout the world. However, meat from animals supplemented with these drugs is tougher and does not age to the same extent (Fiems et al., 1990; Kretchmar et al., 1990; Simmons et al., 1997), as exemplified in Fig. 10. The increased meat toughness is linked to alterations in protein synthesis and degradation ratios. Kretchmar et al. (1990) suggested that protein accretion is due to enhanced levels of cal-

Figure 10 Effect of postmortem storage at 15°C on shear force values of cooked meat from lambs supplemented for 8 days (triangles) or 55 days (circles) with clenbuterol (filled symbols) or equivalent controls (open symbols). Data points are means of 14 lambs. (Adapted from Simmons et al., 1997.)

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pastatin. In a postmortem environment, these enhanced levels will inhibit calpain-mediated proteolysis, thus giving rise to tougher meat. B. Breed and Cloning Effects In pigs, the most important factor leading to PSE meat formation is the genetics of the pig. Proneness to producing PSE meat is one expression of a condition in pigs known as stress susceptibility. Stress susceptibility is part of the same syndrome that causes live pigs to go into rigor when they are administered with the pharmaceuticals, halothane or suxamethonium. The formation of PSE meat after slaughter and the induction of rigor with halothane in the live animal share common features: they both occur during a rapid fall in muscle pH in response to either preslaughter handling and the slaughter process (in the case of PSE meat), or to the anaesthetic agent (in the case of halothane). The rigor response to halothane is an inherited condition, and even when inherited from just one parent, excessive preslaughter exercise and heat stress can trigger the PSE condition in these animals. Stress-sensitive pigs are also prone to dying if they are excessively stressed during preslaughter handling. Here again the animal’s muscle pH falls markedly. Some doublemuscled cattle are also prone to preslaughter muscle pH fall if they are severely stressed. They develop an awkward gait because of antemortem contracture in leg muscles, but it is unusual for them to die from the stress response. The meat from double-muscled breeds of cattle can be paler than that from other beef (Boccard, 1981), but this does not usually cause any negative customer or consumer reactions. There is some evidence that breed can affect ultimate pH. Young et al. (1993) compared the ultimate pH of Coopworth and Merino lambs, raised on the same pasture and slaughtered under identical conditions. The mean ultimate pH values were 5.77 and 6.16, respectively. A similar result was recorded by Hopkins et al. (1996). There are no equivalent data for cattle, but it seems possible that selective pressures over many years of pastoral farming to maximize an important production variable, say pounds of lean meat/acre, could have inadvertently selected an undesirable but less easily measured trait, such as propensity to the dark-cutting condition. In 1993 Jackson and Green reported a genetic trait in sheep that resulted in better lean meat yields at a given liveweight (Fig. 11). (The gene name, callipyge, is from the Greek, meaning ‘beautiful rump.’) The downside of the better yield is enduring toughness, reportedly arising from a reduction in calpain-mediated proteolysis caused by an increase in calpastatin activity that was presumably active in muscle growth in the live animal (Koohmaraie et al., 1995). Developments in reproductive technologies now permit cloning of embryos or, more recently, of mature adult tissue, producing Dolly the sheep and other examples (Willadsen, 1986; Wilmut et al., 1997; Wells et al., 1999). Although these technologies might not be used in the routine production of meat animals, they allow rapid penetration of favorable traits into the production gene pool, thereby reducing variation between animals and resulting in more consistent meat products. Equally, unfavorable traits can also spread rapidly. If producers are paid only by the weight of meat produced, fast muscle growth would be a desirable trait to spread through cloning. However, if the resulting meat were tough, the extra profit from weight might be offset by the loss in sales due to enduring toughness. Similar arguments apply to transgenic animals whose favorable and unfavorable traits can be rapidly spread by cloning.

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Figure 11 Lean meat yield from callipyge and normal lamb carcasses over a range of carcass weights. (Adapted from Freking et al., 1998.)

C. Processing Practices The meat quality from the most carefully raised animals can be markedly reduced by poor treatment before slaughter (see earlier). The 24 hours following slaughter are also critically important in determining quality, largely through the interaction of temperature, time, and the rate of rigor onset. The commercial development of refrigeration in the nineteenth century spawned an international trade in frozen meat, and increasing reliance on low temperatures as a means of controlling spoilage, so extending the storage and display life of meat. As time was money, and rapid chilling and freezing minimized spoilage, rapid cooling technologies were applied to meat processing. These technologies undoubtedly resulted in hygienic meat and in this respect were an outstanding success. The downside was often enduring toughness in the meat. In the case of the New Zealand export lamb industry to Europe in the 1960s, lamb carcasses were often blast frozen at the end of the slaughter line, well before rigor was attained. When muscle tissue is chilled below a certain temperature, typically 10°C, before the onset of rigor, it contracts and is said to be “cold shortened.” If frozen before rigor, it contracts on thawing. Either way, the cooked meat is tough no matter how long it is aged prior to cooking. The fact that enormous quantities of tough lamb were foisted on the British market in the 1960s was lost on the New Zealand meat industry, which at the time had a production rather than a consumer focus. The tenderness problem was vividly exposed when a research technician mistakenly held meat, excised prerigor, in a chiller. The resulting landmark research by Locker and Hagyard (1963) set the contemporary scene for processing that can guarantee tenderness as described in the next chapter. A key part of the technology is electrical stimulation, which accelerates glycolysis and thus the onset of rigor. Quite simply, with electrical stimulation the meat can be chilled soon after slaughter and still avoid cold shortening and, therefore, enduring toughness from that source.

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Tenderness can be promoted by the judicious use of temperature and stimulation, and much research has been directed at explaining tenderness development in terms of the behavior of proteases after slaughter (see Chapter 13 and elsewhere). However, none of this is of interest to the processor who must juggle temperature, stimulation, and time to the best effect. The processor has one other weapon to promote tenderness—namely muscle posture. During processing, quadrupeds are normally suspended by the hind-leg hocks. As the carcass muscles enter rigor, some are stretched, some show no change in length, and some are free to contract because for these muscles the points of skeletal attachments are brought closer together than would be the case in a standing animal. For reasons described in Chapter 13, stretched muscles are frequently more tender (as meat) than normal-length muscles. Shortened muscles are usually tougher. If a carcass is suspended by the pelvic bone (aitch bone hanging), hind muscles adopt a more natural posture, resulting in more consistent tenderness of these muscles (Hostetler et al., 1972; Quarrier et al., 1972). In another technology (Tendercut™, Wang et al., 1994), selected cuts are made through bone and connective tissue on suspended carcasses such that commercially valuable muscles are stretched as they enter rigor. IV. CONCLUDING REMARKS This chapter has given a overview of three meat quality outcomes that are affected by current practices in the production, breeding, and processing of several species. Against this background, the next chapter describes the processing detail of the first two days from slaughter and how it can be controlled. During this time the carcass is disassembled and the meat dissected into categories for further treatment aimed at satisfying market needs. As will be seen, the quality problems examined in the present chapter—particularly the PSE condition and enduring toughness—can be minimized by process control. REFERENCES Bailey, A. 1988. Connective tissue and meat quality. In: Proc 34th Int Cong Meat Sci Technol, Brisbane, Australia. pp 152–160. Bailey, A.J., and N.D Light. 1989. Connective Tissue in Meat and Meat Products, p 204. Elsevier, London. Barnes, E.M., and C.S. Impey. 1968. Psychrophilic spoilage bacteria of poultry. J Appl Bacteriol 31:97–107. Bendall, J.R., and J. Wismer-Pedersen. 1962. Some properties of the fibrillar proteins of normal and watery pork muscle. J Food Sci 27:144–159. Boccard, R. 1981. Facts and reflections on muscular hypertrophy in cattle: double muscling or culard. In: R. Lawrie (Ed.). Developments in Meat Science—2, pp 1–28. Applied Science Publishers, London. Bouton, P.V., A. Howard, and R.A. Lawrie. 1957. Studies on beef quality. CSIRO Div. Food Preservation Melbourne, Australia., Tech. paper no. 5, p 1. Braggins, T.J. 1996. Effect of stress-related changes in sheepmeat ultimate pH on cooked odor and flavor. J Agric Food Chem 44:2352–2360. Chrystall, B.B., C.E. Devine, M. Snodgrass, and S. Ellery. 1982. Tenderness of exercise-stressed lambs. N Z J Agric Res 25:331–336. Daly, C.C., O.A. Young, A.E. Graafhuis, and S.M. Moorhead. 1999. Beef quality effects from pasture and grain finishing diets. N Z J Agric Res 42:279–287. Dransfield, E. 1981. Eating quality of DFD beef. In: D.E. Hood and P.V. Tarrant (Eds.). The Problem of Dark-Cutting Beef, pp 344–358. Martinus Nijhoff, The Hague.

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Dutson, T.R., J.W. Savell, and C.G. Smith. 1981. Electrical stimulation of ante mortem stressed beef. In: D.E. Hood and P.V. Tarrant (Eds.). The Problem of Dark-Cutting Beef. pp 253–268, Martinus Nijhoff, The Hague. Etherington, D.J. 1987. Collagen and meat quality: effects of conditioning and growth rate. In: A.M. Pearson and T.R. Dutson (Eds.). Advances in Meat Research, Vol. 4, Collagen as a Food. pp. 351–360, van Nostrand Reinhold, New York. Fiems, L.O., B. Buts, Ch. V, Boucqué, D.I. Demeyer, and B.G. Cottyn. 1990. Effect of a -agonist on meat quality and myofibrillar fragmentation in bulls. Meat Sci 27:29–39. Fjelkner-Modig, S., and H. Ruderus. 1983. The influence of exhaustion and electrical stimulation on the meat quality of young bulls: Part 2—physical and sensory properties. Meat Sci 8:203–220. Freking, B.A., J.W. Keele, M.K. Nielsen, K.A. Leymaster. 1998. Evaluation of the ovine callipyge locus: II. Genotypic effects on growth, slaughter, and carcass traits. J Anim Sci 46:2549–2559. Graafhuis, A.E., and C.E. Devine. 1994. Incidence of high pH beef and lamb. II: Results of an ultimate pH survey of beef and sheep plants in New Zealand. In: Proc Twenty-Eighth Meat Ind Res Conf, pp 133–141, MIRINZ, Auckland. Grau, F.H. 1981. Role of pH, lactate and anaerobiosis in controlling the growth of some fermentative Gram-negative bacteria on beef. Appl Environ Microbiol 42:1043–1049. Gross, K.L., D.L. Harmon, and T.B. Avery. 1988. Net portal nutrient flux in steers fed diets containing wheat and sorghum grain alone or in combination. J Anim Sci 66: 543–551. Hood, D.E., and P.V. Tarrant. 1981. The problem of dark-cutting in beef. Martinus Nijhoff, The Hague. Hopkins, D.L., N.M. Fogarty, and D.J. Menzies. 1996. Muscle pH of lamb genotypes. Proc Aust Soc Anim Prod 21:347. Hostetler, R.L., B.A. Link, W.A. Landmann, and H.A. Fitzhugh. 1972. Effect of carcass suspension on sarcomere length and shear force of some major bovine muscles. J Food Sci 37:132–135. Huntington, G.B., E.J. Zetina, J.M. Whitt, and W. Potts. 1996. Effects of dietary concentrate level on nutrient absorption, liver metabolism, and urea kinetics of beef steers fed isonitrogenous and isoenergetic diets. J Anim Sci 74:908–916. Jackson, S.P., and R.D. Green. 1993. Muscle trait inheritance, growth performance and feed efficiency of sheep exhibiting a muscle hypertrophy genotype. J Anim Sci 71 (Suppl 1):241. Koohmaraie, M. 1992. Ovine skeletal muscle multicatalytic proteinase complex (proteasome): purification, characterization, and comparison of its effects on myofibrils with -calpains. J Anim Sci 70:3697–3708. Koohmaraie, M., S.D. Shackelford, T.L. Wheeler, S.M. Lonergan, and M.E. Doumit 1995. A muscle hypertrophy condition in lamb (callipyge): Characterization of effects on muscle growth and meat quality traits. J Anim Sci 73:3596–3607. Kopp, J., and M. Bonnet. 1987. Stress-strain and isometric tension measurements in collagen. In: A.M. Pearson and T.R. Dutson (Eds.). Advances in Meat Research, Vol. 4, Collagen as a Food. pp. 163–185, van Nostrand Reinhold, New York. Kretchmar, D.H., M.R. Hathaway, R.J. Epley, and W.R. Dayton. 1990. Alterations in postmortem degradation of myofibrillar proteins in muscle of lambs fed a -adrenergic agonist. J Anim Sci 68:1760–1772. Lacourt, A., and P.V. Tarrant. 1981. Selective glycogen depletion and recovery in skeletal muscles fibre types of young bulls subjected to a behavioural stress. In: D.E. Hood and P.V. Tarrant (Eds.). The Problem of Dark-Cutting in Beef. pp 417–429, Martinus Nijhoff, The Hague. Locker, R.H., and C.J. Hagyard. 1963. A cold shortening effect in beef muscles. J Sci Food Agric 14:787–793. Locker, R.H., and N.G. Leet. 1976. Histology of highly stretched beef muscle. II Further evidence on the location and nature of gap filaments. J Ultrastr Res 55:157–172. Madruga, S., and D.S. Mottram. 1995. The effect of pH on the formation of Maillard-derived aroma volatiles using a cooked meat system. J Sci Food Agric 68:305–310. McVeigh, J.M., and P.V. Tarrant. 1982. Glycogen content and repletion rates in beef muscle, effect of feeding and fasting. J Nutr 112:1306–1314.

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Melton, S.L., J.M. Black, G.W. Davis, and W.R. Backus. 1982. Flavor and selected chemical components of ground beef from steers backgrounded on pasture and fed corn up to 140 days. J Food Sci 47:699–704. Moloney, A.P., P. Allen, D.B. Ross, G. Olson, and E.M. Convey. 1990. Growth, feed efficiency and carcass composition of finishing Friesian steers fed the -adrenergic agonist L-644,969. J Anim Sci 68: 1269–1277. Newton, K.G., and C.O. Gill. 1978. Storage quality of dark, firm, dry meat. Appl Environ Microbiol. 36:375–376. Offer, G. 1991. Modelling of the formation of pale, soft and exudative meat: effects of chilling regime and rate and extent of glycolysis. Meat Sci 30:157–184. Penny, I.F. 1967a. The influence of pH and temperature on the properties of myosin. Biochem J 104:609–615. Penny, I.F. 1967b. The effect of post-mortem conditions on the extractability and adenosine triphosphatase activity of myofibrillar proteins of rabbit muscle. J Food Technol 2:325–338. Pethick, D.W., and J.B. Rowe. 1996. The effect of nutrition and exercise on carcass parameters and the level of glycogen in skeletal muscle of Merino sheep. Aust J Agric Res 47:525–537. Piez, K. 1967. Soluble collagen and the components resulting from its denaturation. In: G.N. Ramachandran (Ed.). Treatise on collagen, Vol. 1, Chemistry of collagen, pp 207–252, Academic Press, London. Purchas, R.W. and R. Aungsupakorn. 1993. Further investigations into the relationship between ultimate pH and tenderness of beef samples from bulls and steers. Meat Sci 34:163–178. Purchas, R.W., C.B. Johnson, E.J. Birch, R.J. Winger, C.J. Hagyard, and R.G. Keogh. 1986. Flavour studies with beef and lamb. Massey University, New Zealand. Quarrier, E., Z.L. Carpenter, and G.C. Smith. 1972. A physical method to increase tenderness in lamb carcasses. J Food Sci 37:130–131. Rompala, R.E., and S.D.M. Jones. 1984. Changes in the solubility of bovine intramuscular collagen due to nutritional regime. Growth 48:466–472. Simmons, N.J., O.A. Young, P.M. Dobbie, K. Singh, B.C. Thompson, and P.A. Speck. 1997. Postmortem calpain-system kinetics in lamb: effects of clenbuterol and preslaughter exercise. Meat Sci 47:135–146. Stanton, C., and N.D. Light. 1988. The effects of conditioning on meat collagen: Part 2. Direct biochemical evidence for proteolytic damage in insoluble perimysial collagen after conditioning. Meat Sci 23:179–199. Tarrant, P.V. 1981. Selective glycogen depletion and recovery in skeletal muscles fibre types of young bulls subjected to a behavioural stress. In: D.E. Hood and P.V. Tarrant (Eds.) The Problem of Dark-Cutting in Beef. pp 3–36, Martinus Nijhoff, The Hague. Taylor, R.G., G.H. Geesink, V.F. Thompson, M. Koohmaraie, and D.E. Goll (1995). Is Z-disk degradation responsible for postmortem tenderization? J Animal Sci 73:1351–1367. Wang, H., J.R. Claus, and N.G. Marriott. 1994. Selected skeletal alterations to improve tenderness of beef round muscles. J Muscle Foods 5:137–147. Watanabe, A., C.C. Daly, and C.E. Devine. 1996. The effects of ultimate pH of meat on tenderness changes during ageing. Meat Sci 42:67–78. Wells, D.N., P.M. Misica, and H.R. Tervit. 1999. Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells. Biol Reprod 60:996–1005. Willadsen, S.M. 1986. Nuclear transplantation in sheep embryos. Nature 320:63–65. Wilmut, I., A.E. Schnieke, J. McWhir, A.J. Kind, and K.H.S. Campbell. 1997. Viable offspring derived from fetal and adult mammalian cells. Nature 385:810–813. Wismer-Pedersen, J. 1960. Effect of cure on pork with watery structure. I. Binding of salt and water to the meat. Food Res 25:789–798. Young, O.A., A.E. Graafhuis, and C.L. Davey. 1980. Post-mortem changes in cytoskeletal proteins of muscle. Meat Sci 5:41–55. Young, O.A., D.H. Reid, and G.H. Scales. 1993. Effect of breed and ultimate pH on the odour and flavour of sheepmeat. NZ J Agric Res 36:363–370.

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13 Carcass Processing: Quality Controls OWEN A. YOUNG, SIMON J. LOVATT, and NICOLA J. SIMMONS MIRINZ Centre AgResearch, Hamilton, New Zealand CARRICK E. DEVINE HortResearch, Hamilton, New Zealand

I. INTRODUCTION II. THE MECHANICS OF CARCASS PROCESSING A. From Slaughter to Scales B. From Scales to Primal Cut C. Distribution and Consumption III. THE CONTROL OF CARCASS PROCESSING A. Quality Outcomes from Variations in Carcass Processing B. Effects of Processing on the Kinetics of Meat Tenderization C. Modeling Meat Aging and Carcass Processing IV. CONCLUSION REFERENCES

I. INTRODUCTION The past century witnessed the evolution of slaughtering methods from solo butchering, where each man or small team performed all unit operations, to powered rail and chain systems manned by many slaughtermen, each specializing in one or few unit operations. Whatever the technology employed, we can be sure that a high throughput of animals per day has always been an industry focus, because it minimizes costs per unit value of meat. From an accountant’s perspective that outcome is ideal, but it overlooks quality problems that can and frequently do arise when the focus is on throughput alone. Meat quality can be defined in a variety of ways depending on the properties required by the end user. For example, a sausage manufacturer wants good binding and low drip but is unconcerned about tenderness and color, whereas a retailer of table cuts wants bright sta-

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ble color and predictable tenderness. These qualities are affected by the comparative properties of the muscles chosen, the age of the animal, and several production and preslaughter handling factors. They are also affected by carcass treatment in the hours following slaughter. Some of these factors were broadly examined in Chapter 12 and include the physiological status of the animal, the biochemistry of rigor development, and the tenderization process during meat storage. This chapter describes the nuts and bolts of carcass processing as it is commonly practiced, but devotes more attention to the control of carcass processing. This is because in the 30 or so hours that processing can take, many of the quality attributes of meat are acquired or preordained, such as tenderness, drip loss, color, and time to spoilage. By manipulation of the process, desirable qualities can be maximized and undesirable qualities minimized. Therefore, this chapter begins with a section on processing from the time of slaughter to the activities in boning rooms, where the various primal cuts are prepared and distributed for many end uses. Then, muscle to meat conversion is described in the context of how meat qualities, particularly tenderness, can be controlled during carcass processing. II. THE MECHANICS OF CARCASS PROCESSING A. From Slaughter to Scales 1. Cattle Figure 1 summarizes the main unit operations in a typical beef slaughterhouse. The scheme is indicative only, because there is variation in detail among states, countries, and slaughterhouses. In all cases, however, the hide is removed before evisceration and carcass splitting. The carcass is then weighed, graded, and dissected into the cuts required. In Chapter 10, antemortem handling and humane slaughter were examined, and it remains to summarize the principal events in slaughter as they affect later processing. Cattle are stunned mechanically or electrically as a means of rendering the animal unconscious and thus insensitive to pain. Mechanical stunning is typically achieved with a captive bolt that not only concusses but also penetrates the skull and damages a portion of the brain. Electrical stunning is applied in a number of configurations, head-only across the brain, head-to-back, and head-to-thorax (brisket) as further described in Chapter 14. The last two configurations stop the heart whereas a head-only stun does not. Stuns that stop the heart have the advantage of minimizing postslaughter convulsive activity that increases the risk of worker injury. Internationally, mechanical stunning by captive bolt or like devices is much more common than by electrical methods. After stunning, the animal is ejected from the stunning chamber, usually onto some form of stainless steel table. A cut is made in the throat to locate the esophagus (weasand), which is sealed to prevent draining of rumen contents. Next the slaughterman performs a thoracic stick. This entails puncturing the large vein that returns circulating blood to the heart. Large volumes of blood issue immediately. For subsequent carcass suspension, one hind leg is shackled to a link chain that in turn is connected to the mobile chain and rail transport system, which moves the carcasses from one operation to another. Religious strictures demand that animals be slaughtered in particular ways. Under Islamic law the animal must die by bleeding—Halal slaughtering—so a stun that stops the heart is unsuitable. The key feature of Halal-approved stunning is that the animal must be able to recover if slaughter does not proceed. One humane Halal protocol involves a head-

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Figure 1 An outline of the main unit operations of carcass processing for cattle. only stun that leaves the animal completely insensible but the heart continues to beat. Once stunned, the throat is cut transversely resulting in massive blood loss and, within a short time, brain death. Judaic-prescribed slaughtering has many features in common with Halal slaughtering, but preslaughter stunning of any kind is forbidden. Stunned cattle can kick involuntarily while sticking and shackling are performed. Kicking presents a significant hazard to workers. Therefore, an immobilizing current is often applied to the carcass (see Chapter 14). The animal’s muscles contract, minimizing reflex kicking. If not applied on the sticking table, the current can be applied when the animal is subsequently shackled to the link chain (see Fig. 13 in Ch. 14). An immobilizing current uses the same waveform as low voltage electrical stimulation. The voltage to achieve the current is low enough to permit worker contact during current flow, although in some jurisdictions worker contact is forbidden. After immobilization, or specified low voltage stimulation, work begins on the free leg. The aim is to clear the hide from the leg while maintaining the hide as a single entity. Other activities at this time include hoof removal (above the shackle), udder/testicle/pizzle removal, and clearing of the anus that is then clamped to prevent release of feces. Subsequently, carcass suspension is transferred to the other leg for its hide clearance and hoof removal.

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About this time the carcass is resuspended by both hind legs, using conventional beef hooks passing under the Achilles tendons. Work continues to clear the hide from the belly, flank, front legs and head, so that the hide can be removed in a single sheet with the familiar shape of a game animal hide. Horns, if any are removed. The cleared flap of skin from each hind leg is clamped to two chains that are pulled down toward the floor stripping the hide as they progress. This operation is assisted by slaughtermen using air-powered rotating knives to prevent the removal of lumps of subcutaneous fat along with the hide. In many slaughterhouses a form of electrical stimulation is applied at this time. The muscles along the spine (longissimus dorsi and others) contract, preventing spinal fractures occurring as the hide is stripped. (Fractures damage valuable meat cuts.) Once removed from the carcass, the hide usually falls through a chute and is of no further interest to the carcass processing line. The head is usually removed at this point but not discarded. Moreover, its identity link to the carcass is maintained, because the condition of lymph glands in the head—as revealed by regulatory inspection—is a good indicator of animal health. At some later point, tongues and cheek muscle tissue are recovered. Brains may or may not be recovered in other departments of the slaughterhouse. (In some slaughterhouses the head is removed before the hide is pulled, requiring that the head be skinned manually.) Meanwhile, the carcass is prepared for evisceration. A brisket saw is used to split the rib cage along its ventral line. A knife cut continues the ventral split along the abdomen. The diaphragm muscle is cut from its carcass insertions, and the entire viscera including heart and lungs is spilled onto an endless belt of trays so that the various tissues can be conveniently inspected and ultimately separated before progressing to other slaughterhouse departments. The carcass is then split vertically along the spine with a saw, and is ready for regulatory inspection. Any diseased or otherwise contaminated carcass side(s) are transferred to another rail (the detain rail) for trimming or removal of defective parts before return to the mainstream of carcass sides. Some defects result in complete condemnation to rendering or pet food. Excess fat is often trimmed at this point, immediately prior to treatments designed to remove surface contamination of fecal matter, dirt, hair, and blood. Generally, fecal matter is trimmed with a knife. Other matter is washed off by methods that achieve varying degrees of decontamination. Methods include cold water or chemical sprays, warm water sprays (82°C), steam-vacuum sanitization, steam pasteurization tunnels, and combinations of these. The carcass sides are weighed. In some countries, carcasses are graded at this point according to relevant criteria. These include conformation, subcutaneous fat color, gender, age (rib ossification, dentition, animal origin), or by combinations of these factors. In other countries carcasses are assessed in chillers according to these and other criteria, notably intramuscular marbling. In some slaughterhouses, carcass sides are subjected to high-voltage electrical stimulation after weighing and grading. Because of the capital cost of high-voltage equipment, this form of stimulation is most common in high-capacity slaughterhouses with multiple shifts. In a typical configuration, the sides enter a stimulation enclosure where the anterior lateral surface of the forequarter contacts an electrode system, whose length (and chain speed) determines the total stimulation time. The electric circuit is completed as charge flows through carcass to the transport chain that is held at earth potential.

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In a high-capacity beef slaughterhouse, more than 2,000 cattle can be processed per 8-hour shift per process line. The residence time for a carcass between stunning and scales is normally not less than 20 minutes. At the other extreme, some slaughterhouses may process only a handful of animals per day, the difference being reflected in the number of workers and the degree of automation. Processing speeds are a compromise between capital costs of slaughterhouse buildings, automation, labor costs, worker number and safety, hygiene, and chiller capacity, to name a few factors. Hygiene is a particularly important constraint where the meat is destined for long chilled storage before consumption, because microbe densities established on meat surfaces during processing strongly affect time to spoilage. Several unit operations of hide/pelt removal must be carefully controlled to minimize the transfer of microbes to the surface of the carcass. Control of these and other critical operation (evisceration, washing) can significantly restrain throughput. 2. Some Differences for Other Species Pigs are usually stunned electrically but the stimulation of the central nervous system can cause broken bones and muscular damage arising from the simultaneous contraction of antagonistic muscles. The degree of damage depends on the characteristics of the stunning current and electrode placement. (Other problems that can arise from electrical treatments, and which can occur in all livestock species, are discussed in Chapter 14.) In some slaughterhouses, pigs are rendered insensible by gaseous stunning, accomplished with a carbon dioxide–rich atmosphere. A subsequent conventional thoracic stick results in death if not already achieved by asphyxiation. For other species except pigs, the same pattern of hide/pelt removal, evisceration, carcass splitting, and weighing is followed. For pigs the skin is seldom removed. Instead, the carcass is steam-or hot water–scalded then flamed to remove hairs. In many sheep slaughterhouses, a technique called inverted dressing is applied. As the name suggests, most of the operations are carried out on carcasses suspended from their front legs. The advantages of this technique are speed, reduction of labor through increased automation, and the resultant reduced potential for carcass contamination from reduced human contact. Sheep are particularly prone to reflexive kicking after slaughter and an additional current–spinal discharge–is sometimes applied before pelt removal. A pH measurement in the longissimus muscle by probe electrode at 45 minutes postmortem is a useful way of detecting the PSE condition in pork. Excessively rapid glycolysis that leads to this condition is revealed by a pH of 6.0 or less at that time. B. From Scales to Primal Cut 1. Carcass Chilling Hygiene requirements demand that carcass sides be cooled as soon as possible after weighing. The higher the temperature, the more contaminating microbes will proliferate, so setting the pattern of future meat spoilage. The criteria of adequate chilling differ around the world depending on legislated standards. For example, meat-exporting countries adopt the criteria of the importing country. In many countries a specified deep leg temperature, typically 7°C, must be attained in a specified time, say 12 hours. At that time all other tissues of the carcass will be cooler than the specified site, with some approaching the temperature of the surrounding cold air. A typical beef chiller has a bank of fans directing cold air over the carcass sides. Chillers are operated in either batch or continuous modes, the former mode being much

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more common. Batch chilling introduces variability, because product that is first loaded into the chiller is typically subjected to a different time-temperature regime than that loaded last. Carcass cooling also results in surface drying. On one hand, drying reduces carcass weight, but on the other, the drier surface inhibits microbial growth. In some slaughterhouses, cold water is intermittently sprayed over carcass sides from nozzles above the suspension rails. While this has some cooling effect, the main advantage is maintenance of carcass weight. Carcass sides require a minimum floor area for effective chilling, requiring that the slaughterhouse chillers be sufficiently large to accommodate the output of the slaughter lines. Chiller space represents a major capital cost that can be reduced by the adoption of boning before chilling. 2. Boning Before Chilling This technique, known as hot boning, involves the removal of muscles from carcasses immediately after the scales. Hot boning probably has its origins in antiquity. After the hunt, the immediately edible parts of the kill, like muscle and viscera, would be recovered as soon as possible. In its modern application, meat is vacuum packed and boxed before the chilling process begins. Hot boning offers a number of commercial advantages: the use of chiller space is more efficient since the meat is now chilled in boxed form, and bone is not chilled; there is a small but significant increase in the amount of meat removed from the carcass, and less physical effort is required to recover the meat, which is still soft because rigor has not been attained. Since hot boning is usually performed within an hour of slaughter, the carcass muscles have not attained their ultimate pH and there is a risk that high pH meat—which is of lesser quality (Chapter 12)—will be included in the boxed beef leaving the boning room. With conventional boning (see next), pH measurements made 24 hours or so after slaughter can identify carcasses with high ultimate pH, a luxury not currently available to hot boning slaughterhouses. However, a new technology, RapidpH™ (Young et al., 1999) allows on-line detection of high pH meat within 10 minutes of slaughter. This technology may facilitate the uptake of hot boning, which to date has been largely limited to manufacturing meat rather than prime table cuts. 3. Conventional Boning Carcass sides are held in chillers for a minimum of 12 hours before boning operations begin. In many slaughterhouses carcass sides are graded in chillers just prior to boning, as this is the first opportunity to evaluate marbling and lean meat color. To do this a cut is usually made between the 12th and 13th vertebrae to expose the longissimus (striploin) muscle in cross section. This cut marks the conventional division between forequarter and hindquarter. After grading decisions have been made, sometimes on the basis of ultimate pH, work begins on what is essentially a disassembly operation of meat from bone and excess fat, according to customer specification. At its simplest, the entire lean meat yield is frozen as manufacturing beef. (This is the most common fate of hot-boned meat.) At its most detailed, individual muscles are dissected from the carcass. This process is called seam boning and is most commonly practiced in Europe. Between these extremes is an enormous range and mix of boning specifications, demanding good organizational skills in highthroughput boning rooms. The intensity of activity in a large boning room must be seen to

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be appreciated. It looks like an organized confusion of workers, conveyers and meat. However, inspection reveals an orderly input of carcass sides being split into fore and hindquarters, each branching to work stations where disassembly continues. Generally, forequarter meat is more suited to manufacturing end uses, whereas hindquarter muscles are more suited to table cuts. However, within a quarter there is a range of meat qualities from different muscles. Sophisticated processors recognize this and disassemble the quarters to optimize output. The output of a boning room is—along with bones and fat destined for rendering—a few or a multiplicity of raw meat types, packed in a variety of ways to maintain product integrity during distribution to the customer be it meat grinder, frankfurter manufacturer, consumer at retail, or smokehouse. Their needs differ, demanding a range of packaging: plastic film wraps, vacuum wraps, carbon dioxide atmospheres in aluminized plastic films, film-lined cartons, to name a few. Therefore, packaging equipment is common in boning rooms. In some cases the cuts of meat are further processed on-site, thus requiring no intermediate packaging. In common with the process line from slaughter to scales, meat boning is a continuous linear process. A breakdown of equipment can be extremely costly because workers continue to be paid while idle. Thus, equipment reliability is very important. Boning rooms represent a significant capital expenditure for a slaughterhouse and are understandably as small as possible. However, as room size decreases, worker density increases, resulting in potential health and safety problems. Because workers are usually paid by throughput, consider the dangers of workers wielding razor sharp knives at high speed in close proximity to each other. Noise and repetitive strain injury are also major hazards in this environment. At all points of carcass processing, meat becomes contaminated with microbes from the equipment, particularly knives, and from human contact when meat is handled. The more cuts made and the greater the human contact, the greater the contamination, making boning operations a prime source of contamination that leads to spoilage after chill storage. Moreover, the more workers involved, the greater the chance of contamination with pathogens from an infected worker. For example, a single worker with a Staphylococcus infection on a cut finger can contaminate every piece of meat touched on a boning line. For this reason, boning operations are a critical control point for hygiene. C. Distribution and Consumption In a description of carcass processing, product distribution and consumption may at first sight seem out of place. However, the quality attributes acquired during carcass processing set the scene for quality changes during distribution and storage. These qualities are ultimately experienced by consumers, and many times the experience is unsatisfactory. Even where the time between processing and retail sale is short, less than a week for example, complaints about poor quality seldom get relayed to the processor, who is frequently focused only on throughput. The problem is worse in international meat trade where many months can pass between slaughter and consumption, and where the meat can have several owners before final sale. After experiencing poor quality the consumer is often reluctant to make a repeat purchase, and, over decades, these quality problems have contributed to declining sales of red meat. The solution to quality problems is process control.

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III. THE CONTROL OF CARCASS PROCESSING This section examines the control of processing operations that affect meat quality attributes, principally tenderness, which is arguably the most important attribute in the consumer’s eating experience. The processing operations are examined from a biophysical and biochemical perspective, because an understanding of the mechanisms involved is useful for making rational processing decisions. Within the processor’s control there are three main variables that impact on meat tenderness and other qualities. These are the fall in meat temperature in chillers, the fall in meat pH as manipulated primarily by electrical inputs to the carcass, and extension or restraint of individual muscles. For a given combination of these factors—which to some degree are interrelated—a tenderness outcome is generated. A. Quality Outcomes from Variations in Carcass Processing 1. Meat Tenderness Defined The two major components in muscle that define tenderness are the structural and contractile proteins that together make up the contractile apparatus (or myofibrillar structure), plus the connective tissue proteins that—from the muscle cell’s outer membrane to the tendon— transmit the contractile forces to the skeleton. Both contribute to tenderness but in different ways. As a generalization, connective tissue, which is dominated by collagen, defines the background toughness in meat (Bailey, 1988). This background toughness is not easy to change, being “hard-wired” by a muscle’s role in the live animal and by age. In contrast, tenderness due to the contractile apparatus can readily be modified by processing conditions. 2. Effect of Prerigor Conditions on Sarcomere Length and Tenderness The most obvious change in a muscle after slaughter is its transformation from being soft and pliable just after death to being rigid and inextensible sometime later. This loss of extensibility is directly related to the development of irreversible cross-bridges between the two principal proteins of the contractile apparatus, myosin and actin. The attachment of the myosin heads to actin precludes the free-sliding of thick and thin filaments, so the muscle becomes stiff, the state of rigor mortis. Although not often obvious, muscles shorten during rigor attainment, as individual sarcomeres that compose the myofibrils contract. Therefore, a muscle entering rigor will shorten if unrestrained, or will develop tension if restrained by skeletal attachment. The classic studies of Locker and Hagyard (1963) demonstrated that shortening of unrestrained ruminant muscle is greatest at low temperatures (10°C), somewhat less at warmer temperatures (30°C), and minimal in the temperature range 12° to 20°C (Fig. 2). This U-shaped relationship between shortening and temperature is derived from two distinct shortening mechanisms. One involves greater shortening as temperatures increase and the other involves greater shortening as temperatures decrease. The mechanism acting at higher temperatures is as follows. At any temperature, muscles contract as they enter rigor, and the extent (or force) of this contraction increases with temperature. This mechanism accounts for the right arm of the temperature-shortening curve (Fig. 2). This effect is sometimes referred to as heat shortening but is more accurately termed rigor shortening because it occurs at all temperatures but more so when the muscle entering rigor is warm. The contraction comes about when ATP concentrations fall to very

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Figure 2 Mean shortening of excised beef sternomandibularis held at a range of temperatures during rigor development. Vertical lines are error bars. (Adapted from Locker and Hagyard, 1963.) low levels, so that the myosin heads bind irreversibly to actin and generate a contraction. This binding and resulting contraction is a defining characteristic of rigor shortening. When measured in the whole muscle, the rigor shortening process can take hours. It typically begins around pH 6.0 and becomes maximal when the final (ultimate) pH is attained. In contrast, an individual muscle fiber enters rigor in one to two minutes (Jeacocke, 1984). The individual fibers enter rigor over a range of times due to differences in muscle fiber type, glycogen content and other factors. The net result is an extended time course of rigor development for the entire muscle. The temperature dependence of the degree of rigor shortening can be explained in that the force of the contraction is greatest at a temperature that is close to physiologically normal (38°C) and declines as temperature is reduced. In contrast, the second mechanism, cold shortening, involves increasingly severe contractions as the temperature declines below 10°C while the muscle pH is still above 6.0 (Locker and Hagyard, 1963). Cold shortening is responsible for the left arm in Fig. 2. Cold shortening is very different from rigor shortening. In rigor shortening, force develops when ATP is at very low concentrations. In cold shortening, significant concentrations of ATP are still present and the meat pH is well above ultimate. The shortening is induced by an increased intracellular calcium ion concentration, which results in myosin/actin cross-bridge cycling generating a contractile force. This is the normal, physiological process of force generation by live muscle. It therefore depends on the presence of ATP, and is inhibited by the presence of the by-products of metabolism, in particular the accumulation of inorganic phosphate and the increase in acidity (decrease in pH). Therefore, for any given temperature, the extent of cold shortening decreases as the pH declines, and disappears when the pH falls below about 6.0 (Locker and Hagyard, 1963).

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Figure 3 Effect of stretching or shortening beef sternomandibularis excised prerigor on cooked meat force score values before and after storage. Storage was for 3 days at 15°C. Higher shear force values indicate tougher meat. (From Davey et al., 1967.)

The cause of the elevated intracellular calcium ion concentration when muscle temperature is low may be explained as follows. In living muscle, calcium leaks passively from the sarcoplasmic reticulum, but reuptake by the calcium pump in the reticulum counteracts the leakage and so maintains low intracellular calcium concentrations when the muscle is not being stimulated by motor neurons. The calcium pump is an ATP-dependent enzyme system and, in common with most enzyme systems, works faster at higher temperatures (to a limit). In contrast, passive leakage is relatively temperature insensitive. When muscle is cooled prerigor, a temperature will be reached where the reuptake becomes slower than the leakage. At this point, the intracellular calcium concentration will increase and contraction is triggered. The two relationships between temperature and contraction (Fig. 2) are important because the temperatures involved are in the range often experienced by muscles during carcass chilling. Thus, shortening occurs when carcasses are cooled too quickly while still in the prerigor state (cold shortening), or when carcasses are cooled insufficiently after slaughter when approaching rigor (rigor shortening). Davey et al. (1967) examined the tenderness consequences of a range of cold-induced muscle shortenings, as well as the effects of stretching the muscle during the prerigor period (Fig. 3). All meat was toughened by cold shortening, but the toughening persisted in stored meat* only when the muscle was shortened above 20%. At 40% shortening,† storage of meat did not decrease toughness at all. In contrast, shortening a muscle to more than * For reasons discussed elsewhere, meat tends to become more tender during storage, so-called meat aging. † Extreme shortening does not occur when muscles are restrained by the skeleton, but can occur when meat is hotboned and thus freed from restraint during rigor onset.

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40% reversed some of the toughening effect. Marsh et al. (1974) showed that when muscles shorten by more than 40%, the fibers supercontract to produce localized nodes of massively contracted sarcomeres, interspersed with areas of stretched sarcomeres. The net effect at these excessive shortenings is a limited tenderization rather than toughening (Fig. 3). The tenderness consequences of rigor shortening are more complicated than those of cold shortening. While samples maintained during the prerigor period at around 37°C have highly shortened sarcomeres, the effects of rigor shortening on shear force values (measured objectively) are minor compared with an equivalent shortening produced by low temperatures. In contrast, sensory panel assessments of rigor-shortened meat have shown significant increases in toughness, although the toughening does not match that of cold shortening (Hertzman et al., 1993). The difference between objective measurements and those of sensory panels may relate to textural differences perceived by humans but not objectively by machines. 3. Rate of Glycolysis and Temperature Control of Muscle Shortening The postmortem fall in muscle pH and the attainment of rigor are intimately linked to glycolysis. Glycolysis is the sum of a series of enzyme catalyzed reactions. Since reaction rates usually increase with temperature, one might expect that glycolysis would do likewise. However, as monitored by pH fall, the rate of glycolysis in beef muscle is minimal around 12°C (Fig. 4), and begins to increase at temperatures below and above. This phenomenon of increased metabolic activity at low temperatures is linked to the cold shortening shown in Fig. 2. Increased ATPase activity at low temperatures—due to higher concentrations of calcium ions in the muscles cell—requires that the glycolysis rate be increased to regenerate ATP to meet the increased cellular demand.

Figure 4 Rate of glycolysis as monitored by rate of pH decline in bovine sternomandibularis muscle entering rigor at different temperatures. Note the negative sign for dpH/dt, indicating that pH falls more rapidly with higher numerical values. A parabola has been fitted to the data and is discussed in the section on modeling. (Adapted from Jeacocke, 1977.)

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The rate of glycolysis, and thus pH fall, depends mainly on the activity of myosin ATPase. The activity varies among species, among breeds and animals within species, and among muscles within animal. Rates in modern pig breeds are roughly double those in ruminants. In PSE-prone pigs, the condition is linked to runaway glycolysis. Whiter muscles have higher glycolytic rates than red muscles. The variability in glycolytic rates, particularly between animals, complicates attempts to tailor processing specifications to produce the specific combination of pH and temperature decline needed to prevent toughness from shortening. Generally, the chilling regime should ensure that muscles on a carcass are not exposed to temperature extremes as pH falls. Carcass chilling has to be synchronized with the rate of pH decline, so that muscle is not excessively warm when the pH is low, nor excessively cool when the pH is high. This is not easy to achieve in practice. A significant temperature gradient can exist between the core of a large muscle mass on a carcass and its surface, an effect that becomes increasingly marked as the carcass size increases, or as the air temperature in the chiller is lowered or fan speeds increase. In the case of heavy beef carcasses, which are difficult to cool, deep muscle temperatures are well above 12°C between slaughter and rigor attainment, so the rate of glycolysis will be high and pH will fall rapidly (Fig. 4). Thus there is a risk of rigor shortening (as well as PSE-like conditions), while at the same time superficial muscles may cold shorten when exposed to an aggressive cooling regime designed to remove heat quickly. Small carcasses, such as those of sheep, can easily cold shorten because cooling becomes more effective as the surface to volume ratio of carcasses increases. Cold shortening is particularly common if zealous attention is paid to rapid cooling for hygiene reasons. In pigs, with their inherently rapid glycolysis, cold shortening is unlikely unless carcasses are cooled extremely quickly. In chicken, cold shortening is also not a problem because significant muscle shortening does not occur in this species until temperatures fall to around 0°C. However, rigor shortening can occur and this is particularly prevalent in stressed birds, and is a significant cause of toughness (Khan and Nakamura, 1970). Beyond temperature control in chillers, processors have two other ways of controlling shortening, muscle restraint and electrical stimulation. 4. Effects of Muscle Restraint Between Slaughter and Rigor In carcasses suspended from the Achilles tendons, as is normal practice in chillers, some muscles are stretched while other are free to contract. Some of those free to contract are high value muscles, such as those of the loin. To minimize contraction, or even induce stretching in some higher value muscles, several researchers have developed alternative methods of carcass suspension. According to Hostetler et al. (1972), the most successful of these for beef suspends the carcass from the pelvic bone (aitch bone hanging), allowing the hind limbs to adopt a position roughly perpendicular to the axis of the body, reminiscent of the live animal’s posture. Sarcomere lengths are longer and tenderness is significantly improved by this suspension method, particularly for valuable muscles of the hind leg and the loin. The procedure is also effective for lamb (Quarrier et al., 1972). Unfortunately, this suspension technique has not been widely adopted, due to an increased chiller area requirement, and for some muscles a slightly different shape as a meat cut. In another approach to muscle stretching, Wang et al. (1994) showed that if selected bones and ligaments were severed, stretching could be accomplished in the normal carcass suspension. This Tendercut™ technique results in more tender meat for several muscles (Fig. 5).

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Figure 5 Shear force for muscles from carcasses conventionally suspended (control) or subjected to selected cuts in bone and connective tissue (Tendercut™). (Adapted from Wang et al., 1994.)

Hot boning has been most successful in the production of manufacturing meat. However, hot-boned meat cuts such as the striploin are often destined for the quality table cut market. The muscles in these cuts are freed from all carcass restraint on hot boning,* so chilling must be carefully controlled to avoid excessive contraction and thus toughening. In most hot boning plants, the cooling regime is geared to chilling meat as quickly as possible for hygiene and capital cost reasons. Tenderness can easily suffer from this practice. Viewed another way, however, because large carcasses are reduced to cuts or individual muscles, the substantial temperature gradients that exist in intact carcasses can be avoided. Temperature decline can be controlled more precisely if the effort is made. Recent commercial trials in New Zealand have shown that well-controlled hot boning can generate meat quality at least as good as from well-controlled conventional boning. 5. Electrical Stimulation, Cold Shortening, and Acceleration of Tenderization Electrical stimulation is the application of an electric current to the muscles of a freshly slaughtered carcass† and is useful for minimizing cold shortening and accelerating tenderization, among other effects. The electric current causes muscle contraction and accelerates the associated biochemical events of glycolysis. During stimulation, which usually lasts tens of seconds, the muscle pH falls (pH) in response to the vigorous muscle activity, typically by 0.5 pH units (Fig. 6) but sometimes more (Chrystall and Devine, 1978). In addition, the rate of subse* The freedom from skeletal constraint in hot-boned meat has another consequence in table cuts. Butchers and consumers expect a given cut to be certain shape from their long experience with conventionally hung meat. Hotboned cuts adopt rather different forms. † Electrical stimulation strictly includes all electrical inputs applied to a carcass during processing (stunning, immobilization, spinal discharge, high-voltage stimulation), but is considered a single event for this discussion.

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Figure 6 Schematic of pH fall in unstimulated and stimulated muscle. Unstimulated muscle takes more time than stimulated muscle to pass the period of cold shortening risk. The units of the abscissa axis depend on temperature, up to 30 hours to attain ultimate pH in rapidly cooled, unstimulated carcasses.

quent pH fall after the muscle has returned to a relaxed state can be greater than that of unstimulated muscle. At 35°C, the rate of this pH decline can be 50% greater. The full effect of electrical stimulation is therefore the combination of these two effects. The extent of pH fall during stimulation is affected by a number of stimulation parameters. These include the magnitude of the stimulation current, its waveform, the duration of stimulation and the interval between slaughter and stimulation, as detailed in Chapter 14. If high-voltage electrical stimulation is applied correctly, muscle will reach its ultimate pH in less than half the time required for unstimulated muscle (Fig. 6). Importantly, since cold shortening is essentially prevented once the muscle pH reaches 6, the time in which cold shortening can occur is greatly reduced. This effect has been particularly useful in the lamb industry where cold shortening has been a major problem. An early demonstration of the technology’s effectiveness is described by Chrystall (1989). Loins and legs from unstimulated lamb carcasses were placed in a blast freezer (25°C) within 2 hours of slaughter and later cooked from the frozen state. The meat was exceedingly tough, with only 1% meat samples acceptably tender as judged by shear force values. By contrast, meat from similarly processed but stimulated lamb carcasses had about 75% of shear values in the acceptable range. If stimulation is applied, carcasses can be cooled quickly to maintain hygiene standards with little risk of cold shortening. For this reason, the technology was rapidly adopted in commercial lamb processing in New Zealand during the decade from 1978, and has since spread internationally in a number of forms. Electrical stimulation is also used to accelerate tenderization, resulting is a reduced holding period before retail sale. Beef destined for quick sale in domestic markets is stimulated so that tenderizing begins when muscle temperature is higher. This application of electrical stimulation is discussed in more detail later.

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6. Electrical Stimulation, Drip, and Color Electrical stimulation has a number of other effects, some desirable and others potentially undesirable. Processors often enthusiastically stimulate carcasses in the belief that if a little stimulation is good then more must be better. This is not necessarily the case. When pH falls very rapidly in a warm carcass—as is the case for strongly stimulated carcasses—proteins in the meat denature and cellular membrane integrity is reduced. The result is increased drip, reminiscent of the pale, soft and exudative (PSE) condition that bedevils the pork industry (discussed in Chapter 12 and elsewhere). A combination of high temperature and rapid decline in pH is a recipe for drip. Because the rate of glycolysis is already rapid in postmortem pig muscles, and excessively rapid glycolysis often generates the PSE condition, there is no obvious reason for using electrical stimulation in this species. However, if stimulation is used, it needs to be associated with aggressive chilling to counter the effects of high temperatures on the denaturation of meat proteins (Taylor and Tantikov, 1992). The protein denaturation caused by the combination of high temperature and low pH extends to the proteins of the mitochondrion, the organelle in cells responsible for oxygen consumption in cellular respiration. The meat will therefore consume less oxygen when exposed to air, so that atmospheric oxygen diffuses deeper into meat than would otherwise be the case. As a result, the oxymyoglobin layer thickens and produces a more desirable bright red color early in the postrigor period. Where meat grading is based on lean meat color, electrical stimulation has been introduced specifically to exploit this effect. However, the application of electrical stimulation for this purpose requires care, as the price to pay for excessive stimulation can be reduced color stability and thus reduced retail display life (Ledward, 1985). B. Effects of Processing on the Kinetics of Meat Tenderization To this point, tenderness has been discussed as if it were a quality end-point achieved when the meat emerges from a boning room ready for consumption. However, such is not the case. If meat is chill-stored after slaughter, it usually becomes more tender with time. This phenomenon is often termed meat ageing. The kinetics of tenderization—how it develops with time—is the subject of this section. The kinetics have practical importance. A processor preparing meat for consumption within a week of slaughter requires tender meat in that week. In contrast, an exporter might require tenderness many weeks after processing but not necessarily before. These different needs translate into different processing options that have capital equipment and therefore financial implications. Muscle temperature and pH during processing affect the kinetics of tenderness development through their effects on enzymes that occur in live muscle and retain activity to varying degrees in muscle as meat. This section begins with a brief discussion on enzymes important in tenderizing, then interprets tenderness development during and after processing in the context of changing enzyme activities. 1. Tenderization of Meat Due to Enzymic Proteolysis of Muscle Proteins Meat ageing has little effect on toughness (lack of tenderness) imparted by connective tissue, because enzymes able to degrade collagen are either absent or inactive in meat. Instead, it is the weakening of the myofibrillar structure that is largely responsible for the

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increase in tenderness due to ageing. Proteins making up this structure that are causally involved in tenderness development include nebulin (Fritz and Greaser, 1991), titin (Locker and Leet, 1976), and desmin (Young et al., 1980) and similar proteins (Taylor et al., 1995). Although one model of tenderization invokes only calcium ions causing disassembly of structural filaments (Takahashi et al., 1987), it is more generally believed that two groups of endogenous proteolytic systems—calpains and cathepsins—are responsible. The calpain group has three components: two proteases and one inhibitor. Of the two proteases, -calpain and m-calpain, the former is implicated in tenderization because the calcium ion concentration in postmortem muscle is high enough for its activation. Although calpains are optimally active around neutral pH, they retain some activity at lower pH levels. In experimental models, calpains rapidly degrade the structural myofibrillar proteins cited above. This and other features point to the calpain system playing a leading role in postmortem tenderization. Cathepsins are ubiquitous in muscle tissue but are enclosed within lysosomes and are activated only when released from this organelle. Three muscle cathepsins have been implicated in tenderization (Dutson, 1983). Once released from the lysosome, these proteases are optimally active at acidic pH values. They are therefore likely to be more active during longer term postrigor proteolysis (Wu et al., 1981) when pH is low and membrane integrity is lost, so releasing the enzymes from the lysosomes. 2. Effect of Processing on Meat Aging During and after rigor development, pH and temperature have significant effects on the development of tenderness through their effects on enzyme activities. When the intracellular calcium concentration increases postmortem to around 100 M (Jeacocke, 1993), -calpain is transformed from an inactive to an active form that is capable of proteolytic activity. Under this condition, the ability of the calpastatin to bind calpain, and thus inhibit its activity, is reduced and proteolysis can begin. The higher the temperature, the more rapid the proteolytic activity. Thus, anything that hastens pH fall and the release of calcium (such as electrical stimulation) will in theory increase tenderization due to earlier activation of calpain at a higher temperature. However, there is a complicating factor at play, namely calpain autolysis that eventually renders the calpain inactive. A very rapid rate of pH fall when the carcass temperature is high can result in tough meat (Pike et al., 1993), or at least meat that does not age to its full potential. This paradox may be explained in terms of the balance between proteolytic activity and loss of activity through calpain autolysis. In the case of very rapid pH fall and high temperatures, calpains are activated early, and proteolysis will be rapid but short acting as inactivation through autolysis quickly develops. With a slower pH fall and lower temperatures, the balance between proteolysis and inactivation will shift toward slower proteolysis but over a longer period. This model is consistent with results from Simmons et al. (1996) (Fig. 7), from studies examining the effects of temperature on calpain activity independent of pH. In muscle maintained at 35°C, the activity of -calpain was substantially reduced when the ultimate pH of 5.5 was reached. Shear force values were lower (meat was more tender) when rigor was attained at 35°C than at 25 or 15°C (Fig. 8). However, subsequent ageing under chilled conditions to 7 days was minimal for the 35°C treatment, presumably limited by the low residual calpain activity. In contrast, muscle held at 15°C showed little change in calpain

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Figure 7 Activity of -calpain at several pH values postmortem when muscle was held at three temperatures. Activity measurements were made at a constant pH and thus were independent of the pH values on the abscissa axis. (From Simmons et al., 1996.)

activity in the prerigor period and had higher shear force values in rigor on day 1. Proteolysis occurred mainly in the postrigor period, so that the final tenderness was higher. Thus the relative acceleration of calpain autolysis over proteolytic activity could create the lack of ageing found in some cases of PSE meats or when electrical stimulation is applied followed by slow cooling.

Figure 8 Kinetics of tenderization after muscle entered rigor at three different temperatures. (From Simmons et al., 1996.)

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Under cold-shortening conditions, calpastatin activity appears to decrease more slowly than under normal rigor attainment conditions (Zamora et al., 1998) and this effect may be involved in the increased toughness associated with cold-shortening. The kinetics of calpain activity are thus dependent on pH and temperature, which can be manipulated during processing to ensure that tenderness is attained within a time frame appropriate for the target market. Different processing regimes should be applied to meat that is to be consumed within a few days and meat that is allowed to tenderize during weeks of storage, roughly slow and fast cooling respectively. Judiciously applied electrical stimulation can be useful for accelerating tenderization, as the start of tenderization will coincide with higher carcass temperatures, and therefore the rate will—until the carcass is fully chilled—be higher. This is presumably the basis of the stimulation-accelerated tenderization described by Savell et al. (1978) and George et al. (1980). Increased tenderness associated with direct physical disruption of the muscle contracting in response to the electrical current has also been proposed (Ho et al., 1997). It is plausible that structural weakening may render structural proteins more susceptible to proteolytic attack by calpain and cathepsins. While -calpain is responsible for the tenderizing that occurs during and immediately after rigor, after 3 to 4 days of storage (depending on the temperature), calpain activity is negligible. It is then that cathepsins may assume an important role in meat tenderization. Given that meat retains proteolytic activity from calpain and/or cathepsins, there is a further opportunity for tenderization during storage. Higher storage temperatures accelerate tenderization (examined in the next section) but also promote the growth of spoilage organisms. The ageing regime chosen to optimize tenderness at the point of retail sale must also produce a hygienic product, as is discussed in other chapters. C. Modeling Meat Aging and Carcass Processing The interactions between the many variables affecting meat quality are complex. Indeed, processing regimem to bring about some of the desired outcomes are in conflict, so it is impossible to achieve all desired quality attributes simultaneously. A modeling approach can help control this complexity. Models are a useful and inexpensive way of exploring phenomena. If a model is described by a computer program, as is often the case, it is a simple matter to change the inputs (e.g., temperature, time, weight) to generate new sets of behaviors. This is far cheaper than industrial trials. In seeking to model carcass processing and the resultant meat quality at the point of sale, it is important to recognize the diverse activities of the meat industry. A U.S. meat packer supplying the domestic market with grain-finished beef might need to produce meat that is tender within a week of slaughter, blooms strongly on retail display, and is hygienically acceptable to the USDA. An Australasian processor sending chilled lamb by sea to a distant market would need to produce meat that is tender at retail, but that tenderness would not have to be achieved until many weeks after slaughter. At the same time, the meat must have good color stability and be hygienic. Another processor supplying frozen lamb might require that the meat be aged before freezing because it is common practice to cook frozen meat within a few hours of thawing. Models broad enough to span the practical range of each meat processing variable affecting quality could be applied to each of these diverse meat processing activities. Such models* are being developed by the authors of this chapter and colleagues. * Fig. 6 in Chapter 12 is another example of a model, for the PSE condition in pork.

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1. Examples of Meat Quality Modeling A model to predict tenderness at any time after rigor has its origins in the results of Jeacocke (1977) who measured the temperature dependence of the glycolysis rate for beef (in negative pH units per hour) over the temperature range 0° to 35°C (Fig. 4). The significance of the trough at 12°C has been discussed earlier in this chapter. An equation can be fitted to Jeacocke’s data. The choice of equation depends on personal experience of model forms, but generally simple equations are favored because many natural phenomena are described by them. In this case, the data appear to take the shape of a parabola, although the fit is imperfect, particularly near the lowest point of the curve. From this equation, the pH at any time postmortem can be predicted, provided the temperature is known, either by direct measurement or from a thermal model based on Newton’s law of cooling, thermal conduction through the meat, and other relationships. Some meat science facts and assumptions are then applied. When the muscle pH—computed by integrating the equation in Fig. 4—reaches a certain value, say 5.9, the muscle is fully in rigor and ageing begins. It is assumed that all meat tenderness starts at some initial level of myofibrillar-derived toughness, depending on the extent to which the muscle has shortened (Fig. 3). Call this initial toughness F0, which is the force (kgF) required to shear a standard sample of cooked meat. Given this initial toughness, the tenderness of the meat at any time during storage could be estimated if rate at which toughness decreased at different temperatures were known. Dransfield et al. (1992) and Graafhuis et al. (1992) measured the ageing rates of beef and lamb, respectively, at various temperatures. The measured toughness over time for lamb held at various temperatures is shown in Fig. 9. Most curves in the temperature range studied start at about the same initial toughness (F0), because for this work the meat had been neither rigor shortened nor cold shortened. The curves flatten out to about the same toughness, but do so at different rates. One might therefore suppose that at any given temperature, the toughness at any time, t, could be described by an exponential decay equation: Ft
F  (F0  F) ekt where k is the rate of decay, e is the natural logarithmic base (2.718), F0 is the initial toughness, and F is the shear force value at a theoretical infinite time of ageing. If the value of k were known for each temperature in the range 2° to 35°C, the tenderness at any time after rigor could be calculated for any storage temperature or combination of temperatures. If it is supposed that in the complex chain of enzyme-catalyzed reactions that leads to tenderizing there is a single reaction that is rate-limiting, the rate of this reaction would be expected to depend on temperature according the Arrhenius equation. Thus, a plot of the natural logarithm of k against the inverse absolute temperature (Kelvin scale) might yield a straight line. When this is done using k values derived from Fig. 9, the data form a reasonably straight line (Fig. 10). Values of k derived from the equation in Fig. 9 can then be used in the exponential equation above to predict the tenderness at any time post rigor. The constants in Fig. 9’s equation will vary slightly between species. This simple example of tenderness modeling based on temperature has been extended to include the effects of muscle shortening and electrical stimulation as they respectively affect initial toughness and a second quality attribute of meat, myosin denaturation. Shortening induced by cold shortening or rigor shortening will affect F0 (see Fig. 3 for example); when poorly applied, electrical stimulation increases myosin denaturation Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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Figure 9 Decrease in lamb toughness during aging at different temperatures. Only some curves are labeled. At higher temperatures, lamb tenderizes to an end point more rapidly. The initial toughness (F0) is around 10kgF, decaying to final toughness (F) of around 4 kgF. (Data were extracted from Graafhuis et al., 1992.)

Figure 10 Arrhenius plot showing the effect of temperature on the rate of meat aging, expressed as k values, which were derived from data in Fig. 9.

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through temperature and pH interactions. Denatured myosin increases drip and reduces juiciness, and is clearly a quality defect. If it is supposed that in some particular market, a quality level of meat, Q, depends on initial toughness (F0) and on the degree of myosin denaturation, then Q could be defined in terms of an equation such as: Q
Fraction of myosin denatured  F0/Scale factor The smaller the value of Q the better, because the fraction of myosin denatured should be low and F0, as an initial shear force score value, should also be low. The scale factor is selected to give F0 a suitable level of importance relative to myosin denaturation and could vary, as is discussed shortly. Equations describing shortening and myosin denaturation effects were incorporated in a computer program that was executed for a range of prerigor cooling rates and degrees of electrical stimulation. The degree of stimulation was described as a stimulation fraction from 0, (none) to 1.0 (maximum stimulation). The quality level Q was then plotted for each combination of cooling and stimulation to illustrate the combined effects (Fig. 11). The model predicts that meat quality Q will be poor both at low cooling rates (due to high myosin denaturation) and at combinations of high cooling rates and low stimulation frac-

Figure 11 Contour plot from a meat quality model showing the effects of different cooling rates and the degree of electrical stimulation on one possible measure of overall quality, defined here as follows: fraction of myosin denatured  initial toughness/30. Low values on contour lines mean higher quality; thus the “valley” in the center is a region of higher quality. The choice of 30 as a scale factor is discussed in the text.

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tions (due to cold shortening–induced toughness). On the other hand, the model suggests that there is an optimum degree of electrical stimulation for any given cooling rate. It must be emphasized though that Fig. 11 is simply a useful illustration of how modeling results can be used in a practical way. As it stands, this illustration should not be used to design a meat processing regimem. When modeling a process for optimal quality, the relative importance—the scale factor—given to initial toughness (F0) and protein denaturation, or to other modeled quality parameters, will depend on a customer’s relative preference for various quality attributes. This preference will in turn depend on the type of market the slaughterhouse supplies. The factor will also depend on the dimensions of the quality attributes (e.g., which tenderometer was used). Other modeled quality parameters might include juiciness, the potential for microbial growth, or color stability on display. The resulting quality equation is simply another model, one of customer preference for each of the components of meat quality. IV. CONCLUSION Meat packing is an industrial process. In any industrial process the key to consistent quality is control. Highly acceptable meat could be produced by all meat packers if the process were ideally controlled in each case. Several factors mitigate against this. In many situations the raw material (live animal) entering the process is highly inconsistent (highly variable) with respect to breed, age, and physiological state. This is particularly true when the animal supply is from many growers each making production decisions independently. Moreover, if processing itself is variable, then this compounds the problem. Examples of variable processing include variable electrical stimulation, overcrowding in chillers, different cooling applied to different carcasses and parts of carcasses. Each factor can translate to quality problems. For the meat industry, the difficulty is that meat eating quality cannot be assessed until the cooked product is consumed days or weeks later, with no formal traceability back through the supply chain. In this environment, meat is relatively undifferentiated and therefore assumes a commodity status. Many in the industry are comfortable with this status because it is an environment where quality is averaged and blame is spread. Process control leading to differentiation of quality attributes is essential for any meat packer wishing to break from the commodity mold. Control of the live animal supply may not always be possible, but from the hours before slaughter to the attainment of ultimate pH, the processor can exert control. Control depends on measurement. In this chapter, the importance of temperature measurement with respect to time has been emphasized. Alternatively, temperature can be predicted from models. Superimposed on temperature measurement is measurement of the electrical inputs, such as stunning and stimulation, that all hasten pH fall. Armed with this information and a broad understanding of meat science, meat packers can improve average quality and reduce variability, often with no capital expenditure. This is the path to branding so that premiums can be extracted from the market. For cost reasons, the future of processing may lie in hot boning. Controlled cooling is easier to achieve with meat cuts than with carcasses. In its logical extension, cooling may be accomplished with water, a medium that is several times more efficient at cooling meat cuts than air. In turn, water cooling requires that meat must be packed in impermeable wrapping, suggesting that the preparation of standardized and branded retail cuts may be accomplished by meat packers who currently produce carcasses or at best primal cuts.

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REFERENCES Bailey, A. 1988. Connective tissue and meat quality. In: Proc. 34th Int. Cong. Meat Sci. and Technol., Brisbane, Australia. pp 152–160. Chrystall, B.B. 1989. Trends and developments in meat processing. In: R. Purchas and B. Hogg (Eds.) The Production and Processing of Meat. Chapter 2., Proc N Z Soc Anim Production. Chrystall, B.B., and C.E. Devine. 1978. Electrical stimulation, muscle tension and glycolysis in bovine sternomandibularis. Meat Sci 2:49–58. Davey, C.L., H. Kuttel, and K.V. Gilbert. 1967. Shortening as a factor in meat aging. J Food Technol 2:53–56. Dransfield, E., D.K. Wakefield, and I.D. Parkman (1992). Modelling post-mortem tenderisation. I. Texture of electrically stimulated and non-stimulated beef. Meat Sci 31:57–73. Dutson, T.R. 1983. The measurement of pH in muscle and its importance to meat quality. Proc. 36th Recip. Meat Conf., Fargo, North Dakota, pp. 92–97. Fritz, J.D., and M.L. Greaser. 1991. Changes in titin and nebulin in postmortem bovine muscle revealed by gel electrophoresis, western blotting and immunofluorescence microscopy. J Food Sci 56:607–615. George, A.R., J.R. Bendall, and R.C.D. Jones. 1980. The tenderizing effect of electrical stimulation of beef carcasses. Meat Sci 4:51–68. Graafhuis, A.E., S.J. Lovatt, and C.E. Devine, 1992. A predictive model for lamb tenderness, Proc 27th Meat Ind. Res. Conf., Hamilton, New Zealand, pp. 143–147. Hertzman, C., U. Olsson, and E. Tornberg. 1993. The influence of high temperature, type of muscle and electrical stimulation on the course of rigor, ageing and tenderness of beef muscles. Meat Sci 35:119–141. Ho, C.Y., M.H. Stromer, and R.M. Robson. 1996. Effect of electrical stimulation on postmortem titin, nebulin, desmin, and troponin-T degradation and ultrastructural changes in bovine longissimus muscle. J Anim Sci 74:1563–1575. Ho, C.Y., M.H. Stromer, G. Rouse, and R.M. Robson. 1997. Effects of electrical stimulation and postmortem storage on changes in titin, nebulin, desmin, troponin-T, and muscle ultrastructure in Bos indicus crossbred cattle. J Anim Sci 75:366–376. Hostetler, R.L., B.A. Link, W.A. Landmann, and H.A. Fitzhugh. 1972. Effect of carcass suspension on sarcomere length and shear force of some major bovine muscles. J Food Sci 37:132–135. Jeacocke, R.E. 1977. The temperature dependence of anaerobic glycolysis in beef muscle held in a linear temperature gradient. J Sci Food Agric 28:551–556. Jeacocke, R.E. 1984. The kinetics of rigor onset in beef muscle fibres. Meat Sci 11:237–251. Jeacocke, R.E. 1993. The concentrations of free magnesium and free calcium ions both increase in skeletal muscle fibres entering rigor mortis. Meat Sci 35:27–48. Khan, J.W., and R. Nakamura. 1970. Effects of pre- and postmortem glycolysis on poultry tenderness. J. Food Sci 35:266–267. Ledward, D.A. 1985. Post-slaughter influences on the formation of metmyoglobin in beef muscles. Meat Sci 15:149–171. Locker, R.H., and C.J. Hagyard. 1963. A cold shortening effect in beef muscles. J Sci Food Agric 14:787–793. Locker, R.H., and N.G. Leet. 1976. Histology of highly-stretched beef muscle. IV. Evidence for movement of gap filaments through the Z-line, using the N-line and M-line as markers. J Ultrastr Res 56:31–38. Marsh, B.B., N.G. Leet, and M.R. Dickson. 1974. The ultrastructure and tenderness of highly cold shortened muscle. J Food Technol 9:141–147. Pike, M.M., T.P. Ringkob, D.D. Beekman, Y.O. Koh, and W.T. Gerthoffer. 1993. Quadratic relationship between early-post-mortem glycolytic rate and beef tenderness. Meat Sci 34:13–26. Quarrier, E., Z.L. Carpenter, and G.C. Smith. 1972. A physical method to increase tenderness in lamb carcasses. J Food Sci 37:130–131.

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Savell, J.W., T.R. Dutson, G.C. Smith, and Z.L. Carpenter. 1978. A research note: structural changes in electrically stimulated beef muscle. J Food Sci 43:1606–1607. Simmons, N.J., K. Singh, P. Dobbie, and C.E. Devine. 1996. The effect of prerigor holding temperatures on calpain and calpastatin activity and meat tenderness. Proc. 42nd Int. Congr. Meat Sci. Technol., Lillehammer, Norway, 414–415. Takahashi, K., O.-H. Kim, and K. Yano. 1987. Calcium-induced weakening of Z-disks in postmortem skeletal muscle. J Biochem 101:767–773. Taylor, A.A., and M.Z. Tantikov 1992. Effect of different electrical stimulation and chilling treatments on pork quality. Meat Sci 31:381–395. Taylor, R.G., G.H. Geesink, V.F. Thompson, M. Koohmaraie, and D.E. Goll (1995). Is Z-disk degradation responsible for postmortem tenderization? J Animal Sci 73:1351–1367. Wang, H., J.R. Claus, and N.G. Marriott. 1994. Selected skeletal alterations to improve tenderness of beef round muscles. J Muscle Foods 5:137–147. Wu, J.J., T.R. Dutson, and Z.L. Carpenter. 1981. Effect of postmortem time and temperature on the release of lysosmal enzymes and their possible effects on bovine connective tissue components of muscle. J Food Sci 46:1132–1135. Young, O.A., A.E. Graafhuis, and C.L. Davey. 1980. Post-mortem changes in cytoskeletal proteins of muscle. Meat Sci 5:41–55. Young, O.A., J. West, and A.L. Hart. 1999. Rapid method for measuring complex carbohydrates in mammalian tissue. New Zealand Patent Application No. 337276. Zamora, F., F. Chaib, and E. Dransfield. 1998. Calpains and calpastatin from cold-shortened bovine m. longissimus lumborum. Meat Sci 49:127–133.

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14 Electrical Inputs and Meat Processing PHILIP E. PETCH MIRINZ Centre AgResearch, Hamilton, New Zealand

I. INTRODUCTION II. ELECTRICAL STUNNING III. ASPECTS OF ELECTRICAL STUNNING A. Head-Only Electrical Stun B. Deep Stun C. Electrical Stunning Parameters and Equipment D. Efficacy of Electrical Stunning E. Placement of Electrodes F. Blood-Splash and Broken Bones G. High-Frequency Stunning IV. ELECTRICAL TREATMENTS DURING DRESSING A. Low-Voltage Immobilization B. Spinal Discharge C. Bleeding Treatments D. Hide Pull V. ELECTRICAL STIMULATION A. Overview B. Low-Voltage Electrical Stimulation C. High-Voltage Electrical Stimulation VI. SOME PROBLEMS WITH ELECTRICAL TREATMENTS A. Overview B. Contact and Carcass Resistance C. Special Problems with High-Voltage Stimulation VII. CONTROLLED CURRENT TECHNOLOGY VIII. WORKER HEALTH AND SAFETY IX. MONITORING SYSTEMS AND PROCEDURES A. Process Monitoring Equipment B. The Need for Processing Auditing

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Petch C. Process-Auditing Equipment D. Equipment Calibration

X. AREAS OF RESEARCH XI.

SUMMARY APPENDIX: BASIC ELECTRICAL THEORY REFERENCES

I. INTRODUCTION Pioneering experiments with electricity and muscle began in the 1600s when Swammerdam stimulated an innervated frog muscle with a low voltage, causing the muscle to contract (Bendal, 1980). In 1780 Luigi Galvani carried out a range of electrical experiments (Fig. 1), including hanging freshly killed frogs’ legs on an iron fence during a thunderstorm. When the legs touched the iron railing they twitched violently, even when there was no lightning (Wilson, 1965). In the 1950s, several workers explored the use of electrical stimulation as a means of improving tenderness of meat (Harsham and Deatherage, 1951; Rentschler, 1951). The adoption of high-speed blast freezers by the New Zealand frozen meat trade in the late 1960s led to serious problems with cold shortening, resulting in unacceptably tough meat. Electrical stimulation of carcasses was found to accelerate the onset of rigor so the carcasses could be frozen quickly while avoiding cold shortening. The success of electrical stimulation in New Zealand sparked major scientific interest and led to the commercial application of this technology by meat-exporting nations.

Figure 1 Early experiments with electrical stimulation, by Galvani.

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During the 1970s and 1980s, electricity gained further uses in meat processing as a means of ensuring humane slaughter via electrical stunning, and of enhancing worker safety by preventing reflex movements of the carcass during dressing. This chapter discusses the application of electrical inputs during slaughter and dressing from an engineering perspective rather than a meat quality perspective. It discusses the application of electrical stunning, methods for immobilizing the carcass to prevent carcass movement during dressing, and the application of electrical stimulation to promote tenderness. Some gaps in the knowledge, and topics of current research are also briefly discussed. The chapter concludes with a brief outline of elements of electrical theory that may be helpful when reading the chapter. II. ELECTRICAL STUNNING Seeking to kill a domestic animal in the most painless way possible is the final act of welfare that humankind can offer it. When animals are slaughtered in meat packing plants, they are generally stunned before slaughter. If stunning is properly carried out, the animal’s death will be much less distressing to it than could ever be expected in the wild. This is as it should be. A properly stunned animal will be insensible, so it feels no pain or discomfort during the slaughter procedure. To satisfy this requirement, stunning must itself be acceptably free of pain or stress, and insensibility must be maintained during slaughter until unconsciousness due to hypoxia in the brain occurs. Throughout the world, the meat industry uses various methods of stunning animals, including free bullets, captive-bolt pistols, percussion bolts, carbon dioxide gas, and electrical stunning. Internationally, the captive-bolt pistol is most commonly used to stun cattle; electrical stunning is most widely used for sheep and pigs (Gregory, 1998). The use of electricity to stun animals prior to slaughter has an extended history. Benjamin Franklin is reputed to have carried out experiments with electrical stunning in the eighteenth century (Hill, 1935). The Slaughter of Animals Act, passed in the United Kingdom in 1933, authorized the use of electrical stunning for pigs as one way to ensure that “every such animal shall be instantaneously slaughtered or shall be rendered insensible to pain until death supervenes.” This lead to the widespread use of electrical stunning in British pig plants during the 1930s, and subsequently in many other countries (Warrington, 1974). The objective of electrical stunning is to pass a current through the brain to temporarily disrupt normal brain function. If done correctly, the animal is rendered unconscious and completely unresponsive when the slaughter cut is made. By the time that the animal would have recovered from the stun if not slaughtered, its brain has ceased to function due to oxygen deprivation caused by loss of blood flow to the brain. Electrical stunning technologies can be divided into two main forms: head-only stunning and deep (cardiac arrest) stunning. These technologies are now discussed. III. ASPECTS OF ELECTRICAL STUNNING A. Head-Only Electrical Stun The head-only electrical stun, in which the electric current is passed through the brain but no other vital organs, induces a seizure similar to a grand mal epileptic seizure. Electroen-

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Figure 2 Typical EEG waveforms (a) prior to stunning and (b) immediately after stunning (same scale).

cephalograms (EEGs) recorded immediately after stunning are very similar in form to those recorded during human grand mal epileptic seizures (Devine et al., 1986; Jones et al, 1988). Typical EEG waveforms recorded before and after stunning are shown in Fig. 2. Based on the human experience, it is assumed that the animal exhibiting this brain activity is unconscious in essentially the same manner as a human is during the seizure (Gregory, 1998). Because no electric current passes through the heart, the heart continues to beat during the stun-induced seizure and if slaughter does not proceed, the animal will eventually recover with no ill effect (Leach et al., 1980). Therefore the head-only stun is considered acceptable for Muslim (Halal) slaughter (Gilbert et al., 1986). When a successful head-only stun is applied, the animal displays a sequence of external responses (Anil, 1991; Gregory, 1998). The animal first becomes rigid (the tonic phase), with the head raised and the hind legs tucked up into the body. Breathing stops. The tonic phase typically lasts 10 to 25 seconds. Thereafter, if the animal is not slaughtered, there is a transition to the clonic phase, in which kicking movements take place for 15 to 45 seconds. A quiet phase then sets in, breathing restarts, and the animal shows signs of regaining awareness. Consciousness can be assessed from corneal reflexes (Gregory, 1998). If the animal responds to corneal contact, it is probably conscious. Associated with the physical responses, the brain undergoes a series of chemical changes (Cook et al., 1995). The degree and duration of these changes are linked to the duration of the stunning current, and to the duration of the seizure and subsequent analgesia. Because animals subjected to a head-only stun can begin to recover consciousness within 25 to 30 seconds, it is important that the slaughter cut be made as soon as practicable after the stun has been applied. The cut should sever the major blood vessels (arteries and veins) to ensure that blood flow to the brain is stopped. The time to permanent insensibility after the cut varies with species: it can be as short as 7 seconds with sheep but up to 60 seconds with calves (Hoenderken et al., 1980; Newhook and Blackmore 1982a,b; Blackmore et al., 1983; Grandin, 1999). Cattle have a significant blood supply to the brain through the vertebral arteries, which are not affected by a throat cut. There was therefore concern that in cattle post-stun consciousness could begin to return before permanent unconsciousness resulted from the throat cut. This caused the humaneness of head-only stunning to be questioned for cattle, preventing its early introduction. This issue was resolved by Cook and coworkers in 1993 (Cook et al., 2000), who showed that the effects of stunning and reduction in blood flow due to the slaughter cut combine synergistically to hasten brain death. Therefore head-only stunning gives an adequate length of unconsciousness during slaughter for all species including cattle. However, a chest stick is preferred for optimum welfare assurance when slaughtering cattle. Provided the slaughter cut is made during the tonic phase, an additional benefit of electrical stunning is that the animal is rigid. This makes the cut easier to carry out and helps protect the slaughter personnel from kick or knife injury.

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B. Deep Stun The so-called deep stun (also known as the cardiac arrest stun) involves passing the electric current through both the brain and the heart. The amount of current is chosen so that the heart fibrillates, causing blood circulation to stop. With this type of stun, the animal will not recover. Thus the stun is not suitable for Halal slaughter. Two main current pathways are used for deep stunning: head to back and head to lower chest or leg. In terms of animal welfare, deep stunning is acceptable for the slaughter of calves (Grandin, 1999) and is favored over head-only stunning for other animals (Wotton and Gregory, 1986; Grandin and Smith, 1998), because the animal dies from the stun. The slaughter cut is important only as a means of allowing the blood to drain from the carcass. When stunning cattle, a head-only stun must be applied first to ensure insensibility before the cardiac arrest stun is applied (Grandin, 1999). Another advantage of using a deep stun compared with a head-only stun is that the animal tends to be more still during the slaughter cut and dressing (Gregory, 1998). This is because the current flow through the animal’s body disables the nervous system. Hence the spinal reflexes such as kicking do not occur (Wotton et al., 1992), as discussed later, and the animal quickly becomes flacid. C. Electrical Stunning Parameters and Equipment Electrical stunning can be applied in several ways. In many cases, the animal is restrained in a crush and the electrical current is passed through its head (head-only stun). The current may pass across the head, from top to bottom or from the nose region to the neck. Refer to Fig. 3. Alternatively, the current may pass from the head to the lower chest (thoracic stun)

Figure 3 Head-only stunning of sheep.

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or to the back (head to back stun), as shown in Figs. 4 and 5. For cattle, a head-only stun is applied immediately before the thoracic or head-to-back stun to achieve a deep stun. In smaller European slaughterhouses, pigs are often stunned with hand-held tongs while the animal is free-standing. Stunning is most often carried out using waveforms derived from the normal electrical supply (50 or 60 Hz) via a transformer, although commercial systems are available that use higher frequencies. Automated systems generally use between 400 and 1000 volts a.c. (Troeger, 1991), and restrict the current flow using some form of current control (Gregory, 1998). Where stunning tongs are applied manually, worker safety usually dictates that the voltage be less than 250 volts a.c. (Troeger, 1991). Under these conditions, current control is not practical. This can lead to insufficient current flow and there is no way to ensure that every stun is successful, as discussed later. The duration of the stun can be controlled manually or automatically. The absolute minimum acceptable stun duration is 0.2 second (Cook et al., 1995), but this is too short to apply reliably in practice. Typical parameters for stunning current and duration as used for various types of animals in New Zealand are given in Table 1 (Gilbert, 1993). These are broadly in line with international practice. Electrical stunning of pigs is difficult because the modern pig grown for meat is susceptible to stress, and to muscle hemorrhage and broken bones during handling, stunning, and slaughter (Troeger, 1991). This subject is discussed under blood-splash and broken bones later in the chapter, and has been reviewed by Anil et al. (1997). However, a minimum stunning current of 1.25 to 1.3 amps applied for more than one second has gained acceptance from a number of researchers as being suitable for pigs.

Figure 4 Head-to-chest (or foreleg) stunning of sheep.

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Figure 5 Head-to-back stunning of sheep.

Table 1 Summary of Time and Current Parameters of Electrical Stunning Head-only electrical stunning

Min. currenta (A) Range (A) Min. time (s) Range (s)

Sheep

Lambs

Calves

Cattle

Deerb

1.0 1.0–1.5 1.0 1.0–4.0

0.7 0.7–0.9 0.8 0.8–1.5

0.9 0.9–1.5 1.0 1.0–4.0

1.1 1.1–2.5 1.0 1.1–4.0

1.0 1.0–2.0 1.0 1.0–3.0

Head-to-body electrical stunning

Min. currenta (A) Range (A) Min. time (s) Range (s)

Sheep

Lambs

Calves

Cattle

Deerb,c

1.0 1.0–3.5 1.0 1.0–4.0

0.7 0.7–1.3 0.8 0.8–4.0

0.9 0.9–1.5 0.9 0.9–4.0

1.1 1.1–6.0c 1.0 1.1–18c

1.0 1.0–2.0 1.0 1.0–3.0

a

Minimum current required for humaneness but not necessarily movement control. Hinds only at this stage. c These values have been used in practical situations without definitive studies. Heavier cattle should be given 2.5 amps or more (Council of Europe ruling). b

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The stunning tongs used in Europe for stunning pigs consist of scissorlike devices about 750 mm long, with electrodes attached to the ends (Sparrey and Wotton, 1997). The operator is required to place the electrodes on each side of the head and apply the stunning current. Figures 3, 4 and 5 show some manual stunning equipment. Manual stunning is generally used for sheep when throughput is low to moderate. However, automated stunning systems may be used with sheep at high rates of slaughter (9 to 10 animals per minute). Compared with sheep and pigs, more complex systems of mechanical restraint are required when stunning cattle. While manual electrical stunning is widely used in some countries, automated stunning systems are usually safer and more cost-effective. One system used in New Zealand is the automated electrical stunning system illustrated in Fig. 6 (prototype version). The animal is restrained using a neck bail, which closes from either side onto the narrow part of the neck behind the head. The neck bail forms one electrode, and is connected to the stunner power source. The head is then lifted by a metal plate or bar while a second electrode (the nose electrode) is moved down to contact the head at the base of the nose. The current passes from the nose electrode through the head and brain to the neck electrode. If a thoracic stun is required, current can then be applied from the nose to the

Figure 6 Automatic beef stunning. U.S. patent 4748719 is held by Jarvis Products Corporation, Connecticut.

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chest via a third electrode that makes contact with the lower part of the chest between the forelegs. This current is applied after the head-only stun so that the animal is insensible before the start of the thoracic stun. The stunning sequence is controlled automatically and provides a very reliable stun (Gilbert, 1993; Petch and Gilbert, 1997). D. Efficacy of Electrical Stunning When applied properly, electrical stunning is a highly reliable way of protecting an animal from suffering during slaughter. However, animals come in a wide range of shapes, sizes, and temperaments. It is not as easy as might be imagined to design automated or manual systems capable of delivering a consistent, effective stun to every one of the hundreds or thousands of animals that can pass through a meat packing plant in a single day. Factors that are critical to ensuring a successful and humane stun while preserving the economic value of the carcass include the following: The animal must be properly presented to the stunner. The equipment must be designed so that sufficient current is available to ensure instantaneous insensibility at the start of the stun. The electrodes must make a good electrical contact with the animal (contact resistance needs to be low). Enough of the available current must pass through the brain to ensure instantaneous insensibility. Each animal should be stunned once only, i.e., no interrupted or “double stuns.” The current must not damage valuable parts of the hide. Blood-splash and speckle bruising must be minimized. Bone breaks must be minimized. The issues of sufficient current and acceptably low contact resistance are common to all the electrical treatments applied during slaughter and dressing, and are discussed under contact and carcass resistance later in the chapter. The remainder of this section will discuss the design and positioning of the electrodes, and efforts to minimise blood-splash and other carcass damage that can arise from electrical stunning. E. Placement of Electrodes The head of any animal is a complex, anisotropic structure, containing a brain that is surrounded by membranes and regions of bone, fat, muscle, skin (plus hair or wool), and fluid. The whole structure is permeated by a highly complex network of blood vessels and nerves. This complex structure makes it difficult to predict precisely how the electric current will spread out from one electrode, pass through an animal’s head, and then come together again at the second electrode. With present technology it is not practical to directly measure the amount of current flowing in specific regions within a living animal’s head. However, the current pathways have been modeled for pigs’ heads (Koch et al., 1996), and Lambooij (1994) has explored placement of electrodes directly against the brain. This allowed satisfactory stunning with only 25 volts (approximately 0.13 amps) at 150 Hz, a finding that suggests that with conventional stunning techniques, a significant proportion of the current flows through the structures surrounding the brain rather than directly through it. Because direct measurements of current flows are lacking, present recommendations on electrode positioning are therefore based on common sense reasoning combined with

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observation of the animal and analysis of the chemical or electrical activity in the brain. While this situation may not appear ideal, literally billions of animals have been stunned successfully using this empirical approach. The electrodes should be placed so that the brain lies in the most direct path from one electrode to the other. Figure 6 shows a good example, in that the current passes from the nose through the whole head and out through the neck electrodes. Alternatively, the electrodes can be placed on either side of the head between the eyes and the ears, or one on the top of the head and the other underneath (Grandin and Smith, 1998). With the head-only stunner shown in Fig. 3, both electrodes are placed on the top of the head. In this case the current field can be expected to fan out in a roughly hemispherical shape that includes the brain. In all cases, positioning of the electrodes is less critical and a successful stun is easier to achieve if the stunning voltage is high, so that the current meets or exceeds the recommendations in Table 1. Both electrodes must not be placed low down on the neck. If the electrodes are placed across the neck and further back than approximately 50 mm behind the ears, the spinal cord may be affected, resulting in the expected tonic and clonic responses, but the current will bypass the brain, leaving the animal paralyzed but still conscious. This is unacceptable in terms of humane slaughter. If kicking starts immediately after the current stops, the stun was unlikely to have been successful. F. Blood-Splash and Broken Bones Conditions known as blood-splash and speckle bruising can occur as a result of electrical stunning. The contraction of the muscles during the tonic phase is generally powerful, leading to rupture of fine capillaries. Blood can be forced from these capillaries into the muscle tissue creating large blotches (blood-splash), or into the muscle cover and fat in a rashlike pattern (speckle bruising). The seepage is aggravated by the surge in blood pressure associated with electrical stunning. The muscular contraction during stunning can also cause bones to break, particularly in heavily muscled, weak-boned animals such as pigs. These defects can be adequately controlled for sheep and cattle but remain a significant problem with pigs. Because of their economic consequences (Morgan et al., 1993; Smith et al., 1994), blood-splash and bone breakage have been widely studied (Gilbert, 1993; Grandin, 1994; Grandin and Smith, 1998; Gregory, 1998). Techniques to avoid these problems include: Elimination of interrupted and double stuns Minimization of the interval between stunning and sticking Where possible, use of deep stunning so circulation is stopped immediately Use of high voltage, constant current stunning systems and avoidance of unnecessarily long stuns Proper maintenance of all equipment to ensure the contact surfaces are clean and all electrical wiring is in good order Minimization of stress prior to slaughter, particularly with pigs and deer Ensuring that the restraining system allows the animal’s body to move during the muscle spasm caused by electrical stunning Use of high-frequency stunning, particularly with pigs If carcass damage remains a problem, an alternative method of stunning should be considered, such as CO2 gas (pigs) or captive bolt (beef).

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Another similar problem associated with electrical stunning is shoulder bruising (Gilbert, 1993), caused when a sheep is still in the tonic phase of a head-only stun. As the sheep is put onto the spreaders used to convey the carcass along the slaughter chain, the required spreading of the forelegs causes tearing of muscle tissue and blood vessels in the shoulder region. This problem can be virtually eliminated by improving the shackling and hanging techniques. G. High-Frequency Stunning High-frequency stunning (60 Hz) can alleviate problems with blood-splash and broken bones, particularly with pigs (Simmons, 1995). The muscle spasm induced by high frequency currents is less powerful than with 50 or 60 Hz currents, and the incidence of blood-splash is often reduced. Warrington (1974) reviewed work done by Koledin in 1963, in which he investigated the use of frequencies in the range 2400 to 3000 Hz. However, van der Wal (1977) found when stunning pigs that square wave currents in the 2000 to 3000 Hz range did not cause immediate unconsciousness and appeared to be very painful. Anil and McKinstry (1992) found that sine waves at 1592 Hz and squarewaves at 1642 Hz caused immediate unconsciousness at 150 volts, but recovery times were somewhat shortened. Lambooij et al. (1996) found that a head-only stun (800 Hz, 240 volts, 3 seconds) followed by a cardiac arrest stun (50 Hz, 125 volts, 3 seconds) achieved an efficient and humane stun for pigs. High-frequency stunning is accepted in Europe as a reliable method for stunning pigs. However, more research is needed with the larger U.S. pigs to confirm its acceptability unless the animal is first stunned with a 1 second 50 or 60 Hz stun (Grandin, 1999).

IV. ELECTRICAL TREATMENTS DURING DRESSING The movements of a live animal are under the control of the brain. However, there are a number of reflex pathways mediated by the spinal cord, and once the animal has been head-only stunned, these pathways are less likely to be inhibited by the brain. Violent kicking and walking movements may occur during the first few minutes after stunning, particularly if the animal is being handled. These movements can pose a significant hazard to workers responsible for dressing the animal, both from kicks and from induced knife injuries. Two electrical techniques are available to prevent these reflex movements. The first, called low-voltage immobilization, uses a low-voltage electrical current that causes the muscles to contract moderately and maintain a rigid carcass. The second, called spinal discharge, uses a much larger current to discharge the neurons of the spinal cord so the reflex pathways are disabled. These techniques are widely used in New Zealand in plants that specialize in head-only electrical stunning (Halal slaughter) and have been described by Gilbert and coworkers (Gilbert et al., 1983; Gilbert, 1993). Apart from this work, these techniques have not been systematically studied or widely reported. Importantly, these treatments are known to induce significant reductions in muscle pH and hence they interact with the effects of electrical stimulation (Petch and Gilbert, 1997). This section (A–D) is written largely based on personal observation, unpublished research (Petch and Zhang, MIRINZ Centre), and discussions with Gilbert and associates.

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A. Low-Voltage Immobilization A low-voltage immobilization current stimulates the (still functioning) nervous system, causing the muscles to contract and the carcass to become rigid. The electric waveform used is usually similar to the current used during low-voltage stimulation, described later in the chapter. If applied for 45 to 60 seconds (depending on the current), the resulting muscle contraction depletes the energy reserves in the muscles, which become exhausted, and no further movement is possible. This system is widely used in venison and some beef processing plants in New Zealand. It simultaneously stimulates and immobilizes the carcass. Beef carcasses can be immobilized on the slaughter table using a short metal bar or strip (known as a rubbing bar electrode) that contacts the hindquarters, and a second rubbing bar that contacts the neck region. Alternatively a battery clamp can be attached to the lip, with the return current carried by a clip or probe attached at the anus, or via the chain that is used to hoist the animal via the hock. In a high-throughput operation typical of sheep processing plants there is unlikely to be enough space on the chain at the point of slaughter to allow a full immobilization/stimulation to be carried out. Instead, the immobilization current is usually applied for only 5 to 15 seconds during bleeding so that the carcass is still during the initial stages of dressing. The current can be applied by using a rubbing bar that contacts the carcass at the inside of the hind legs. The current passes through the body and forelegs of the carcass to the transport chain and back to the stimulator. The contact is likely to be poor and variable due to the presence of the pelt. This means that the current flow is generally lower than recommended for low-voltage stimulation and the muscle contractions are correspondingly weaker. B. Spinal Discharge The brief period of low-voltage immobilization applied in a high throughput operation may not be sufficient to ensure that all carcasses will be completely still. To further immobilize the carcasses, a second stage of treatment, known as spinal discharge, is often used. Spinal discharge typically involves a 0.6 to 1.5 amp, 50 to 60 Hz current, applied via a rubbing bar electrode that contacts the shoulder region of the carcass for 2 to 4 seconds. The current passes into the shoulder, along the length of the spinal cord and out via the hind legs and gambrel. This depolarizes the motor neurons in the spinal cord that control the reflexive activity and effectively prevents further carcass movement. As for stunning, the carcass will enter a tonic phase for up to 20 seconds after spinal discharge is applied. During this phase the carcass will be quite rigid, but will subsequently become limp and unresponsive. (The deep stunning techniques discussed earlier also cause a significant current to flow along much if not all the spinal cord, depolarizing the motor neurons in the same way. This is why carcasses stunned in this way are much more still than head-only stunned carcasses.) The electrical circuitry used for producing spinal discharge currents is very similar to that used for stunning. A transformer delivers 400 to 550 volts a.c., and the current is controlled by an inductor connected in series with the carcass. The amount of current flowing through the carcass and its duration can be adjusted to produce the required degree of carcass stillness. However, spinal discharge cannot be applied indefinitely, because spinal discharge will result in a significant fall in muscle pH and therefore complicates the application of electrical stimulation. There is also a risk of contamination if spinal discharge is applied for more than a few seconds, due to contraction of the rumen forcing its contents out through the esophagus.

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C. Bleeding Treatments Some beef plants, particularly in the United States, use a high voltage pulse unit to improve bleeding after slaughter (D. Grose, personal communication). The unit produces a short electrical pulse about once a second, causing the carcass muscles to alternately contract and relax. This acts to squeeze the blood vessels and helps to expel blood. The pulses are similar to those produced by an electric fence; i.e., 200 s to 300 s long, with a peak voltage in the range 2000 to 8000 volts. D. Hide Pull The process of removing the hide from a beef carcass can generate large stretching forces, particularly in the lumbar region of the spine as the hide is drawn over the shoulders and head. This can result in tearing of the ligaments holding the vertebrae together, damage to the vertebrae themselves, and tearing of the valuable longissimus dorsi (LD) muscle. Some beef plants use an electrical device similar to a low voltage stimulator to pass a small electric current through the LD muscles during hide pulling, causing them to contract (Fig. 7). The combined strength of the contracted muscles and spinal column is often sufficient to prevent damage during hide pulling.

Figure 7 Electrically induced stiffening of the back during hide pulling. Current flows along the LD muscle between the anterior and posterior contacts.

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V. ELECTRICAL STIMULATION A. Overview The earliest reported use of electricity to improve meat quality is its purported use by Benjamin Franklin in 1749 to electrocute turkeys with the result that they were “uncommonly tender” (Lopez and Herbert, 1975). Although studies in the 1950s investigated the use of electrical stimulation as a means of improving tenderness of meat (Harsham and Deverage, 1951; Rentschler, 1951), it was not commercially applied at that time. However, the technique was widely adopted when its benefits in avoiding cold shortening (Chrystall and Devine, 2000) and improving tenderness (Bendall, 1980; Taylor and Marshall, 1980) became known. Two forms of electrical stimulation are widely used: low voltage and high voltage. While their purpose is essentially the same, they are quite different processes. Their names provide one distinction: commercially available low-voltage stimulation systems generally deliver voltages below 150 volts, whereas commercial high-voltage stimulation operates at up to 1130 volts peak and beyond. However, this distinction is often not clear in the research literature, where the voltages used in experiments range from 2.5 volts (Taylor and Marshall, 1980) to 9000 volts (Smulders and Eikelenboom, 1985). A more useful distinction between low and high voltage stimulation lies in their mode of operation: stimulation via the nervous system (low voltage) versus direct stimulation of the muscles (high voltage). The types of waveform and the frequencies useful for electrical stimulation vary even more widely than the voltages. The most important variables that have been studied and their ranges can be summarized as follows: Frequency: 0.5 Hz to 60 Hz Duration of stimulation: 0 to 4 minutes Waveform: sine waves, pulse trains derived from sine waves, rectangular pulses, short duration (300 s) impulses Burst length: 0.5 to 8 seconds or continuous current European and New Zealand researchers have tended to use continuous stimulation for the duration of the treatment, whereas researchers in the United States have focused more on bursts of stimulation current with a period of relaxation between. Australian researchers have made wide use of both techniques. In a seminal study, Chrystall and Devine (1978) showed the relationship between pulse frequency and pH fall in the muscles. These workers used unidirectional pulses derived from a 50 Hz a.c. supply (Fig. 8). Pulse frequencies between 5 Hz and 17 Hz result in the greatest rate of pH fall, and frequencies below 5 Hz are much less effective. Above 17 Hz, the effectiveness of stimulation also falls, but even at 100 Hz the pH fall is more than 70% of that at 15 Hz. Several groups have noted that different muscles respond differently to electrical stimulation (e.g., Houlier et al., 1980). Bouton et al. (1980) found that the longissimus dorsi responds better to 14 Hz and the semimembranosus responds better to 40 Hz. Differences in pH fall have been linked to the ratio of slow-twitch fibers and fasttwitch fibers present in each muscle (Swatland, 1980; Devine et al., 1983). Tornberg (1996) discusses this issue further in her review of aspects of meat tenderness. Several groups have obtained satisfactory stimulation using bursts of 60 Hz a.c. waveforms, with a range of burst lengths from 0.5 to 8 seconds (Smith et al., 1977; Riley et al., 1980). Such waveform bursts are widely used in industry.

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Figure 8 Effect of stimulation frequency on the fall in pH. Stimulation consisted of 200 volts (peak) unidirectional pulses derived from the 50 Hz a.c. mains supply for 120 s.

A variety of pulse widths have been tested as well. Bendall (1980) notes that pulse widths of 2 ms or shorter are unlikely to trigger all the muscle fibers when stimulating beef, but pulse widths of 5 ms or more are known to be satisfactory. In summary, stimulation waveform parameters can vary quite widely without seriously reducing the efficacy of electrical stimulation in causing a fall in muscle pH. The choice as to which type of stimulation, where to site it in the meat plant, and which waveform parameters to use will be determined as much by practical considerations such as physical space and the overall parameters of the processing plant as by the direct effect on pH fall. As a generalization, high voltage stimulation is most suitable for high carcass throughput rates, where several carcasses will be stimulated simultaneously. Because of the high voltage and current used, elaborate safety systems are required. These requirements, combined with high-power electronics, mean that a typical high-voltage installation is about 20 times the price of a low-voltage installation. As a result, many high-throughput sheep and beef processing plants use high voltage stimulation to stimulate between 7 and 120 carcasses (or sides) at once, whereas smaller plants tend to use low voltage stimulation, stimulating only 1 or 2 whole carcasses at once. B. Low-Voltage Electrical Stimulation 1. Description Low-voltage stimulation operates by stimulating the nervous system, which then causes the muscles to contract (Chrystall et al., 1980; Chrystall and Devine, 1983). It therefore relies on a functioning nervous system and loses much of its effectiveness once the nervous system ceases to function (approximately 5 minutes postslaughter). However, low-voltage

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Figure 9 Low-voltage stimulation waveforms use in (a) New Zealand and the United States and (b) Australia. stimulation can still produce a useable pH fall up to 20 minutes post slaughter. When applied this long after slaughter, it appears to stimulate the muscles directly as does high voltage stimulation, although the muscle contractions are weaker and the pH fall is less than for high-voltage stimulation because the current density is lower. This form of stimulation is used in some smaller processing plants, as a “poor-man’s” high-voltage stimulation. As outlined earlier, there are a great many possible low-voltage stimulation waveforms. Figure 9 shows two alternative waveforms, widely used in New Zealand, and in parts of the United States and Europe (Fig. 9a), and in Australia (Fig. 9b). Table 2 summarizes parameters for low-voltage stimulation. A wide range of specifications exists for low-voltage electrical stimulators in the United States, but most operate with an output voltage of 20 to 90 volts, with the current applied either in bursts or continuously, for 15 to 20 seconds (Savell, 1985). 2. Application Low-voltage stimulation can be applied in a number of ways. Beef carcasses to be stimulated before dressing (within 5 minutes of slaughter) are generally suspended by one hock Table 2 Low-Voltage Stimulation Parameters Used in New Zealand Peak current (A)

Peak voltage (V)

Lamb

20–90

0.2

Sheep

Beef (side)

Beef (whole)

Pulse width (ms)

Period (ms)

Duration of stimulations (s)

0.2

No spec.

0.3

5–10

58–80

30–90

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Table 3 High-Voltage Stimulation Parameters Peak current (A)

Peak voltage (V)

Lamb

990–1150

1.6

Sheep

Beef (side)

Beef (whole)

Pulse width (ms)

Period (ms)

Duration of stimulation (s)

Mode of operation

1.8

2.5

7.5

7.5–10

60–80

60–120

Continuous

using a chain and shackle. A large spring-loaded clip or a spear is used to connect the live lead from the stimulator to the neck wound or lip. The current returns to the stimulator via the chain and shackle, or preferably via a hook or clip attached to the tail. The clip or hook may include a length of metal rod that is curved so that it is easily inserted into the anus. The anus provides a particularly good contact because the surface membrane in this region is thin and moist. This improves the reliability of stimulation. Carcasses that are to be stimulated using low voltages after dressing (approximately 20 minutes post slaughter) are usually suspended from a gambrel. The live lead is attached to the neck of the carcass using a clip or a spear, while the current returns to the stimulator via the gambrel. However, recent research in the author’s laboratory has shown that the lower hind leg and the junction between the leg and the gambrel offer a high electrical resistance. This may cause the stimulation current to be too low, resulting in ineffective stimulation. See Sec. VI.B for more details. C. High-Voltage Electrical Stimulation 1. Description High-voltage stimulation acts directly on the muscles to induce contraction. High-voltage stimulation involves much higher voltages and currents than low-voltage stimulation and is generally applied 30 to 60 minutes post slaughter. As for low-voltage stimulation, there are a great many possible waveforms and parameters (Table 3). Figure 10 shows one waveform, widely used in New Zealand, Australia, and parts of Europe. The waveform is bipolar: the pulses are alternately positive and negative and are derived from the a.c. power supply using a switching system. The U.S. industry has followed their American researchers in favoring bursts of 60 Hz current, with a typical specification being 550 to 600 volts, 2 second long bursts with one second between, for 15 to 20 bursts (Savell, 1985).

Figure 10 High-voltage stimulation waveform used in New Zealand, Australia, and parts of Europe.

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2. Application High-voltage stimulation is often applied using a rubbing bar electrode. In a typical highthroughput packing house, the carcasses (or sides) are suspended from a gambrel and skid while the rubbing bar contacts them in the shoulder region (Fig. 11). The skid is pulled along by the chain so that the carcass is drawn along the length of the live electrode. The duration of the stimulation is controlled by the length of the electrode and the speed of the chain. In a variation on this, the rubbing bar can be maintained at ground potential while a second live rubbing bar contacts the hock region of the carcass (Smulders and Eikelenboom, 1985). This is possible only if the chain supporting the carcass has an insulating section to isolate the live part of the suspending chain from the transport chain.

Figure 11 High-voltage stimulation of beef sides using a rubbing bar (Photograph courtesy of Auckland Meat Processors and Jarvis Equipment (NZ) Ltd., New Zealand).

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Figure 12 High-voltage stimulation using the Lecto-Tender apparatus.

Another technology widely used in the United States employs a continuous belt of metal plates, linked by insulating material. The plates move with the carcasses, and are livened only as they make contact with the carcasses to provide the current pathway (Fig. 12). In lower-throughput operations, the carcass may be pushed into a small booth with entry and exit doors. The live lead is attached to the carcass by a clip or a spear inserted into the neck, or by a metal plate that is raised until it makes contact with the carcass (Cuthbertson, 1980). Because of the danger posed to personnel by high voltages, the whole installation must be isolated by systems designed to prevent access to the electrically live carcasses or electrodes. Where a rubbing bar or continuous belt system is used, the whole assembly should be enclosed within a tunnel or room, with physical barriers and warnings at the points where carcasses enter or leave the tunnel. Pressure pads or photoelectric sensors are used to disconnect the live electrode if personnel are detected inside the tunnel (Anon.,

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1995, Anon., 1996). Interlocks are also required on all doors or access hatches, so that the electrode is disconnected if these doors are opened. Most importantly, strict safety procedures are required for shutting the system down when personnel need to visit the protected zone, and restarting it after personnel have left the protected zone. VI. SOME PROBLEMS WITH ELECTRICAL TREATMENTS A. Overview With all the electrical treatments discussed so far, the most critical problems lie in ensuring suitable currents flow through the animal or carcass. Several factors can cause variations in the current flow, as follows: 1.

2. 3. 4.

5. 6.

Contact resistance can be large and/or variable. This is a particular problem with low-voltage treatments and where the hide or pelt has not been removed. Sheep with long wool are especially problematic. Animals and carcasses vary in size, shape and conformation. This can complicate the positioning of electrodes, particularly with automated systems. Animals and carcasses vary in their response to electrical treatments. Animal or carcass movement can cause serious problems, particularly with stunning. If an animal moves immediately before the stunning equipment makes contact with it, a poor or unexpected contact position may result, causing an unsatisfactory stun. Manually controlled procedures are subject to human failings such as boredom, error, and fatigue. Equipment failure can cause serious disruption to plant operations.

Many meat processing plants are critically dependent on electrical stimulation. While properly designed and maintained stimulation equipment is very reliable, should the equipment fail, the result is often an immediate and complete stoppage of work, with all employees idle on full pay. Experience shows that such an event attracts the focused attention of plant managers more quickly and completely than virtually any other problem that can occur. Proper maintenance and having some spare parts on hand represent cheap insurance. Of the problems listed above, variations in contact and carcass resistance are probably the least understood in industry, perhaps because they are not readily observable. B. Contact and Carcass Resistance Electrical resistance is a problem only if it is high enough to significantly limit the flow of electric current or if physical damage such as burning results. This is a matter of context. If the required current flow is large or the available voltage is small, the effect of high resistance may be very significant. Similarly, the contact position can be important if unintended conductive pathways are introduced, which allow the current to bypass the expected current path. The key issues are: The hide or pelt of an animal is comparatively highly resistive, particularly if covered with long, dry hair or wool. The resistance of the hide or pelt is highest with low voltages; it reduces as the voltage is increased.

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Some parts of the carcass, such as bone, connective tissue, and fat, are also relatively poor electrical conductors. Perhaps surprisingly, both the tongue and the nose are poor connection points, probably due to their high connective tissue content. When a current flows through a resistance, heat is released in the resistance. This can cause localized burning. Different connection points cause the current to flow through different pathways, each with a possibly significant difference in resistance. This may mean, for instance, that the brain is effectively by-passed during stunning. Contact and carcass resistances are particularly important with electrical stunning and low-voltage stimulation, because of the risk to animal welfare (stunning) and because resistance problems are often exacerbated if the available voltage is low. Consider a practical example. The current specified to stun a large beef animal is at least 2.5 amps. In a trial with 811 beef animals (Petch and Gilbert, unpublished), an automatic stunner made contact between the muzzle and the neck immediately behind the head. Of the head resistances measured during stunning, 95% lay between 44 and 109 ohms. If a hypothetical 180 volt stunner was used and the current not controlled, the current flow would lie between 4.1 amps and 1.7 amps, in which case many of the animals would not receive a satisfactory stun. In this example, the contact resistance is likely to be a major contributor to the variation in the measured resistance, and would pose a serious welfare problem. In another plant tested during the same trial, a slightly different stunner made contact between the top of the head at the midpoint between the muzzle and the eyes, and the neck behind the head. For 95% of the 367 head resistances measured with this stunner, the resistance lay between 67 and 185 ohms. While these resistance populations overlap, they are significantly different. Without current control, the current flow expected with a hypothetical 180 volt stunner at that plant would be between 2.7 amps and 0.97 amps, and almost all of these animals would not receive a satisfactory stun. Fortunately, both groups of animals were actually stunned using well-designed controlled-current stunners, so all received a satisfactory stun. These examples illustrate how inadequate stunner voltage can combine with variable contact resistance to create significant welfare problems. With head-to-back stunning of sheep, the contact resistance between the electrode and an animal’s back can be large if the pelt is unwetted, and particularly if the wool is long. The heat dissipated can lead to burning and costly damage to the pelt. Similar problems can apply to cattle. Low voltage stimulation is also very vulnerable to high contact resistance. One common method of applying low voltage stimulation is to use the hock, shackle, and chain as the return path for the electric current (Fig. 13). In a trial involving low-voltage stimulation of 345 bull carcasses (Petch and Gilbert, unpublished), the current flow in more than 97% of the carcasses fell below the New Zealand specification of 300 mA, and the median was 219 mA. The current had to flow through the hock to reach the hoof region, and then pass through the hide. These regions combined to produce excessive resistance. The live connection to the head was also poor, with the clamp being attached to the hide at the cheek. The overall result was completely unsatisfactory. The following methods can be used to minimize the impact of contact resistance: Use a large contact area Use contact sites that have a moist, thin surface: for example, anal probes

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Figure 13 Low-voltage stimulation of cattle suspended by one hock.

Wet the contact area with water or saline solution Always make the connection in the same way Use controlled-current equipment if at all possible Use automation where practical to minimize variation One further approach to reducing contact resistance during low voltage stimulation has been developed by Smulders and Eikelenboom (1985). These researchers interspersed a low voltage waveform (35 volts, 14 Hz) with short 3000 volt impulses (1.5 ms long, 1 pulse per second). Smulders and Eikelenboom hypothesized that the high-voltage pulses lowered the hide resistance so that the low-voltage current could flow freely. Commercial equipment was developed based on this principle in the early 1980s. C. Special Problems with High-Voltage Stimulation High-voltage stimulation with a rubbing bar electrode involves several issues that must be overcome:

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1. Electrode Positioning The electrode that rubs against the shoulder must be positioned so that the carcass is displaced from the vertical and presses gently but firmly against the electrode. If the carcass presses too lightly against the electrode, it may repeatedly flick away from the electrode and fall back like a pendulum as the current flows in surges. This is known as carcass bounce, and in severe cases can continue for most of the length of the electrode. Alternatively, if the carcass presses too hard against the rubbing bar, the gambrel may turn in the skid, allowing the carcass to roll along the electrode. This can disrupt the stimulation current and may cause the carcass to fall off the gambrel. In addition to correct positioning of the electrode, carcass bounce can be controlled by using an insulated bar fitted on the opposite side of the carcass to limit the distance that it can swing (Fig. 11). Similarly, carcass rolling can be prevented by fitting a stabilizing bar that prevents the gambrel from turning. 2. Carcass Staining In some cases, the carcass can develop a brown stain where it contacts the stimulation electrode. This discoloration is primarily due to rust forming on the electrode, then rubbing off onto the carcass. Staining can be severe when the electrode is new, but it usually reduces after a few hours as a glaze forms over the electrode surface. Staining can be minimized by using a corrosion-resistant grade of stainless steel (U.S. Grade 316 or similar), by mounting the electrode with a fall or rise of approximately 100 mm along its length so that the contact point with the carcass changes along the length of the electrode, and by allowing the surface glaze to accumulate over time. For example, the glaze should not be scoured away during cleaning. Finally, the electrode can be passivated with concentrated nitric acid. 3. Equipment Failure Failure of the switch units that control the stimulation waveform can cause unexpected effects. In particular, should both switches fail permanently on, the current flow in the carcass will be the continuous a.c. frequency rather than the pulsed waveform. Localized contact resistance between the gambrel and the hock can then cause the Achilles tendon to melt and the carcasses to fall off the gambrel in the middle of the stimulation tunnel. It is most disconcerting to find that carcasses are entering the system at one end, but nothing is emerging at the other. VII.

CONTROLLED CURRENT TECHNOLOGY

With any of the electrical treatments discussed, the desired effects are caused by the flow of electrical current rather than the voltage that drives it. This means that it is important to ensure that the current flows is appropriate for the treatment concerned. Current flow can be controlled in a number of ways. These can be separated into two classes: Passive current control, in which the maximum current that can flow is limited by a fixed electrical impedance such as an inductor, or some other limit built into the system. Active current control, in which the current flow is controlled by some variable mechanism that continually adjusts the output to achieve the desired current.

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Provided that worker safety can be ensured, high-voltage current-controlled equipment can offer very significant advantages (Gregory, 1998; Grandin, 1999), and often with only slight increases in cost and complexity. For the first beef stunning example outlined in the previous section, the voltage could be set at 550 volts, with a single inductor included to limit the current to no more than 3 amps. This simple passive control system eliminates the risk of large current spikes that have been associated with blood-splash and broken bones. The inductor also dominates the total impedance of the circuit and reduces the effects of contact and carcass variability on the stun current. The stun current for the resistance range quoted (44 to 109 ohms) would therefore range from 2.9 amps to 2.5 amps, rather than between 4.1 amps and 1.7 amps as before. This represents a huge improvement in stunning repeatibility, with advantages in terms of both animal welfare and reduced blood-splash. Higher voltages also help break down total carcass resistance including the resistance of the hide (Anon., 1991), and allow a more appropriate current to flow. This is useful with head-only stunning, where localized burning of the hide or pelt on the head is of little consequence. Active control systems can act in a variety of ways to limit the current flow. Provided they are properly designed, active systems can achieve very precise current control. Petch and coworkers (Petch and van Royen, unpublished; Petch, 1999) have developed systems that can deliver a wide range of controlled-current waveforms. These are being used for ongoing research into controlled-current stunning, immobilization, and stimulation. A number of organizations manufacture controlled-current stunners. VIII. WORKER HEALTH AND SAFETY There are several potential areas of risk for workers involved with electrical treatments applied during slaughter and dressing. The first is the obvious risk of injury or death due to acute electric shock. This risk is clearly greatest with high voltage treatments such as stunning, spinal discharge, and high-voltage stimulation. Considerable effort has been directed at developing systems to prevent mishaps with high voltage stimulation (Anon. 1995; Anon. 1996). Rather less attention has been paid to ensuring safety with stunning or spinal discharge, although European legislation requires that the voltage used with manual stunning of pigs be maintained below 230 volts to protect worker safety (Troeger, 1991). The second area of risk is more subtle: the potential risk of injury or disability resulting from accumulated damage caused by long-term exposure to low levels of electric current, as may be experienced when working on carcasses as they receive a low-voltage treatment. This is a difficult issue to manage because of the difficulties in obtaining enough information to accurately quantify the risks. A realistic assessment will require extensive records covering many workers for many years. To further complicate the issue, many operations during slaughter and dressing can be carried out more efficiently and safely on carcasses while they are being immobilized using electric currents. There are many incidences of workers being severely injured from their own knives when a carcass moved unexpectedly. Without a solid base of knowledge about the long-term risks from low level electrical currents, it is difficult to make judgments about best practice for such electrical treatments. Current policy in many areas is to minimize exposure to electrical currents above very low levels. Recent legislation in Europe (Anon., 1999) seeks to prevent large-area

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contact in potentially damp areas with voltages exceeding 6 volts a.c. or 15 volts d.c. This effectively prevents workers from touching carcasses undergoing any of the electrical treatments outlined in this chapter. If risk due to carcass movement is to be minimized at the same time, a significant redesign of the slaughter area of many meat processing plants would be required, particularly those carrying out Halal slaughter procedures where the head-only stun does not extinguish spinal reflexes. IX. MONITORING SYSTEMS AND PROCEDURES Any industrial production system involves a process that converts raw materials into one or more products that meet specific market requirements. In the case of meat products, these market requirements will include a variety of measures, including tenderness, texture, taste, color, wholesomeness, and storage life. Animal welfare is also a key issue. The electrical treatments applied during slaughter and dressing can affect all of these parameters. It is therefore important to monitor and audit the electrical treatments as they are applied. Furthermore, systems and procedures must be in place to ensure that any deficiencies are immediately detected and corrected. A variety of equipment is available to help meat processors and regulatory authorities carry out monitoring and auditing procedures. A. Process Monitoring Equipment Electrical devices are commercially available that analyze the electrical voltages and currents applied during stunning and stimulation (Petch and Peach, 1994; Coulton, personal communication; Ross, 1999). These continually provide feedback to operators that the electrical process is being carried out properly, and warn of any problems. Some stunning monitors (Coulton, personal communication) also check that the electrical contact with the animal’s head is good before the stunning current is applied. This ensures that the stun will be effective and humane. B. The Need for Process Auditing Regular checking is essential to ensure that the electrical treatments continue to be applied correctly. Many factors can alter the effectiveness of the treatments, including: 1. 2. 3. 4.

Changes in employees, leading to changes in the way that treatments are applied Partial or complete failure of the electrical equipment Variations in the animals, due to seasonal variations or different suppliers Fluctuations in the quality of the electrical supply caused by routine or extraordinary operation of plant facilities such as chillers, water heaters, etc. 5. Changes in the processing plant, such as the installation of new equipment, which may affect the quality of the electrical power supply 6. Changes in the local electricity supply demand (for example, construction of a major new industrial or residential complex), which may alter the quality of the electrical power supply to the packing plant as a whole Extended records should be kept and reviewed to detect seasonal or longer-term variations, so corrective action can be taken.

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C. Process-Auditing Equipment Quite sophisticated equipment has been developed that allows a detailed analysis of both high and low voltage stimulation waveforms. The waveform analyser (Petch and Peach, 1994) analyzes the current waveform passing through individual carcasses undergoing electrical stimulation, and records the number of pulses that meet the New Zealand specification for high and low voltage stimulation. This device is used to audit the operation of electrical stimulation equipment. A waveform recorder (Petch and Peach, 1994) is used to record the current flowing through a carcass. The current waveform is then downloaded onto a computer and analyzed to determine the nature of any problems with the stimulator. The waveform recorded is used primarily as a diagnostic tool for meat processors that are having problems with stimulation. A sophisticated datalogger system has been developed (Petch and Lynch, 1997) that can record and analyze all of the electrical currents applied during slaughter and dressing for large numbers of animals. This system was used to record the pulse width, pulse frequency, peak current and peak voltage, and the duration where relevant, of the stunning, immobilization, and stimulation treatments applied to nearly 20,000 sheep and 3000 cattle in four plants. The information gained is continuing to provide insights into the performance of the electrical equipment used to apply these treatments. Analysis revealed, among other things, the erratic performance of a cattle stunning box operator who was chronically drunk. D. Equipment Calibration All equipment used for monitoring or auditing electrical treatments must be regularly tested to ensure that it still meets its specified accuracy and precision. There is little point is monitoring a process with faulty equipment. Where regulatory bodies are involved, regular calibration using test equipment that is itself traceably calibrated against national standards is essential. X. AREAS OF RESEARCH Most of the standards set down for electrical stimulation during the 1970s were based on the assumption that stimulation was the only electrical treatment applied during slaughter and dressing. While this assumption was largely true at that time, it is known that the electrical treatments introduced subsequently can cause a significant pH fall. Furthermore, these treatments suffer from significant variability. Present research is focused on minimizing the variability of the electrical treatments, understanding the interactions between the various treatments, quantifying and understanding the variability in the carcass response to the treatments and developing ways to control the treatments to achieve the desired meat quality outcomes. This research is particularly important for the chilled meat trade. The determination of the current pathways during electrical treatments is difficult. Some work has inferred the pathways by measuring the voltage at different points (Houlier and Sale, 1984), while both magnetic resonance imaging and electromagnetic tomography hold some promise for the direct measurement of the current density and pathways.

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XI. SUMMARY This chapter describes electrical treatments applied during slaughter and dressing, including stunning, immobilization, and stimulation. Electrical stunning is used to ensure that animals are insensible during slaughter. It is an important method of preserving the welfare of animals and has been successfully applied throughout the world. However, it needs to be applied with care to ensure that every animal is properly stunned and either remains insensible until death through loss of blood occurs (head-only stunning), or is killed by the stun (deep stunning). Electrical stunning can result in meat quality problems, particularly with pigs. Immobilization is used to prevent carcass movement during dressing. Its primary function is to protect workers from injury. Electrical stimulation is used to induce the early onset of rigor. It is a vital tool in bringing about the rapid ageing and conditioning of carcasses. Two main forms are widely used in industry: low-voltage stimulation and high-voltage stimulation. A key issue with any of these treatments lies in ensuring that the current that flows is correct and follows the desired pathway. Contact resistance can cause significant problems and must be managed effectively. All the electrical treatments discussed can have important effects on meat quality attributes and must be properly monitored. APPENDIX: BASIC ELECTRICAL THEORY To appreciate what happens when an electrical treatment is applied to a carcass, it is helpful to understand some basic electrical concepts. This section will briefly discuss those concepts needed to understand the essence of these electrical treatments. Electric current: Electric current is the nett movement of electrons through a material. An electric current can be viewed as the electrical equivalent of water moving from point to point through a pipe. The ampere (amp) is the unit of electric current. Conductor: Any material that allows electrons to flow through it freely is called a conductor. Copper wire is a good example of a conductor. Insulator: Any material that does not allow electrons to flow through it freely is called an insulator. Plastics are usually good insulators. Resistance: Any material has a tendency to oppose, or resist the movement of electrons. This effect is known as resistance. The resistance of a material can be very small, such as in a superconductor, or very large, as a piece of plastic. The unit for resistance is the ohm. Copper is widely used as a conductor because it has a low resistance to the flow of electrons. Plastics used to insulate wires have a high resistance to the flow of electrons. Voltage: Voltage describes how much electrical “pressure” is being applied to electrons between two given points. The unit for voltage is the volt. Ohm’s law: Ohms’ law defines the relationship between voltage, current and resistance. If a piece of material has a resistance of one ohm between two points, then a voltage of one volt will be needed to drive one amp of current between those points. Ohms law is usually written: Voltage
amperes  resistance Electric circuit: An electric circuit is any path that an electric current can flow through. An electric circuit is always a complete loop, with current always moving at every point within that loop. See Fig. 14 for an example.

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Figure 14 A simple electrical circuit. Load: Most electrical circuits contain some form of intentional electrical load, in which some useful function is carried out. In this chapter, the animal or carcass is usually the load for the circuit concerned. Direct current: In many circuits, the supply voltage or current is essentially constant with time. This is referred to as a direct current (abbreviated d.c.). A good example is the voltage (or current) from a dry cell as used in a flashlight Alternating current: In many circuits, the voltage or current continually alternates between a positive peak value and a negative peak value, typically following a sine wave pattern as shown in Fig. 15. This is referred to as an alternating current (abbreviated to a.c.). The number of times the pattern is repeated per second is called its frequency, measured in hertz (Hz). Almost all domestic and industrial power supplies are a.c. supplies, and Fig. 15 shows the voltage waveform expected from a 110 volt 60 Hz a.c. supply (continuous curved line) as typically used in the United States. Peak voltage: The peak voltage of an a.c. supply refers to the maximum positive or negative voltage reached by that supply. In Fig. 15, the peak voltage is 156 volts.

Figure 15 Waveform of the U.S. 60 Hz domestic electric power supply.

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Power in a resistor: When an electrical current flows in a resistor, heat is dissipated in the resistor as energy is expended to drive the current through. This is the principle used in an electric heater. The power dissipated in a load is often written: Power
voltage  current

REFERENCES Anil, M.H. Studies on the return of physical reflexes in pigs following electrical stunning. Meat Sci 30:13–21, 1991. Anil, M.H., and J.L. McKinstry. The effectiveness of high frequency electrical stunning in pigs. Meat Sci. 31:481–491, 1992. Anil, M.H., J.L. McKinstry, and S.B. Wotton. Electrical stunning and slaughter of pigs: Guidelines for good welfare assurance. Fleischwirtschaft 77 (7):632–635, 1997. Anon. Guide to the effects of current passing through the human body. Australian Standards Association, NSW, Australia, 1991. Anon. New Zealand electrical code of practise for installation, operation, maintenance and testing of animal stunning and meat conditioning electrical appliances (Draft) NZECP 38, Office of the Chief Electrical Inspector, Ministry of Commerce, New Zealand, 1995. Anon. High voltage electrical stimulation systems (HVES). Guidance Note 56, Meat and Livestock Commission, Milton Keynes, England, 1996. Anon. Electrical installations of buildings—Part 4: Protection for safety. IEC 60364-4-41. Commission Electronique Internationale, Geneva, Switzerland, 1999. Bendall, J.R. The electrical stimulation of carcasses of meat animals. In: R. Lawrie (Ed.) Developments in Meat Science: 37–59. Applied Science Publishers, England. ISBN 0-85334-866-9, 1980. Blackmore, D.K., J.C. Newhook, and T. Grandin. Time for onset of insensibility in four- to six-weekold calves. Meat Sci 9:145–146, 1983. Bouton, P.E., R.R. Weste, and F.D. Shaw. Electrical stimulation of calf carcasses: response of various muscles to different waveforms. J Food Sci 45:148–149, 1980. Chrystall, B.B., and C.E. Devine. Electrical stimulation, muscle tension and glycolysis in bovine sternomandibularis. Meat Sci 2:49–59, 1978. Chrystall, B.B., and C.E. Devine. Electrical stimulation of rats: a model for evaluating low voltage stimulation parameters. Meat Sci. 8:33–41, 1983. Chrystall, B.B., and C.E. Devine. Meat and electrical stimulation. In: G. Collins (Ed.) Encyclopedia of Food Science and Technology, 2nd ed. Wiley-Interscience, New York 2000. Chrystall, B.B., C.E. Devine, and C.L. Davey. Studies in electrical stimulation: post-mortem decline in nervous response in lambs. Meat Sci 4:69–78, 1980. Cook, C.J., C.E. Devine, and D.A. Blackmore. Preslaughter humaneness: assuring humaneness of slaughter. In F. Smulders (Ed.) Assuring animal welfare during meat processing. European Consortium for Continuing Education in Advanced Meat Science and Technology, Utrecht, Netherlands 2000. (In press) Cook, C.J., C.E. Devine, K.V. Gilbert, D.D. Smith, and S.A. Maasland. The effect of electrical headonly stun duration on electroencephalographic-measured seizure and brain amino acid neurotransmitter release. Meat Sci 40:137–147, 1995. Coulton, D. Private communication. Meat and Livestock Commission, Milton Keynes, England, 1998. Cuthbertson, A. The added values becoming available through meat handling developments. Catering and Hotel Management, Oct. 1980, 41–43, 1980.

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Devine, C.D., S. Ellery, and S. Averill. Responses of different types of ox muscle to electrical stimulation. Meat Sci 10:35–51, 1983. Devine, C.E., K.V. Gilbert, A.E. Graafhuis, A. Tavener, H. Reed, and P. Leigh. The effect of electrical stunning and slaughter on the electroencephalogram of sheep and calves. Meat Sci 17:267–281, 1986. Gilbert, K.V. Electrical stunning and slaughter in New Zealand. Technical Report 908. Meat Industry Research Institute of New Zealand, Hamilton, 1993. Gilbert, K.V., C.E. Devine, R. Hand, and S. Ellery. Electrical stunning and stillness of lambs. Meat Sci. 11:45–58, 1983. Gilbert, K.V., R. Hand, and C.E. Devine Electrical stunning and beef cattle slaughter in New Zealand. In: Proc Eur Meeting Meat Res Workers 32, I: 117–119, 1986. Grandin, T. Methods to reduce PSE and bloodsplash. Allen D. Leman Swine Conference 21:206–209, 1994. Grandin, T. Good manufacturing practices for animal handling and stunning. Dept. of Animal Science, Colorado State University, Fort Collins, 1999. Grandin, T. and G.C. Smith. Animal welfare and humane slaughter. Dept. of Anim. Sci., Colorado State University, Fort Collins, 1998. Gregory, N.G. Animal welfare and meat science, CABI Publishing, Oxford, U.K. ISBN 0 85199 296X, 1998. Harsham, A., and F.E. Deatherage. Tenderization of meat. U. S. Patent 2,544,681, 1951. Hill, L. Electric methods of producing humane slaughter. Veterinary J 91:51–57, 1935. Hoenderken, R., E. Lambooy, J.G. van Logtestijn, and W. Sybesma. Dutch research on stunning of slaughter animals. In: Proc Eur Meeting Meat Res Workers 26, I:100–101, 1980. Houlier, B., and P. Sale. Electrical stimulation efficiency and distribution of electric potential and electric field along lamb carcasses In: Proc Eur Meeting Meat Res Workers, 30, 2:9, 1984. Houlier, B., C. Valin, G. Monin, and P. Sale. Is electrical stimulation efficiency muscle dependent? In: Proc Eur Meeting Meat Res Workers 26 vol. II J5:81–83, 1980. Jones, P.N., F.D. Shaw, and N.L. King. The comparison of electroencephalograms recorded before and after electrical stunning of cattle. Meat Sci 22:255–265, 1988. Koch, R., F. Feldhusen, J. Hartung, and W.W. Giese. Modell zur messung der stroverteilung im schweinkopt bei der elektrobetäubung. Fleischwirtschaft 76:692–696, 1996. Lambooij, B, G.S.M. Merkus, N. van Voorst and C. Pieterse. Effect of low voltage with a high frequency electrical stunning on unconsciousness in slaughter pigs. Fleischwirtschaft 76 (12):1327–1328, 1996. Lambooij, E. Electrical stunning by direct brain stimulation in pigs. Meat Sci. 38, 433–441, 1994. Leach, T.M., R. Warrington, and S.B. Wotton. Use of a conditioned stimulus to study whether the initiation of electrical pre-slaughter stunning is painful. Meat Sci 4:203–208, 1980. Lopez, C.A., and E.W. Herbert. The private Franklin: the man and his family. W.W. Norton, New York, 1975. p. 44. Morgan, J.B., J.B. Cannon, F.K. McKeith, D. Meeker, and G.C. Smith. National Pork Chain Quality Audit (Packer-Processor-Distributor). National Pork Producers Council, Colorado State University, Fort Collins, and University of Illinois, Champaign-Urbana, 1993. Newhook, J.C., and D.K. Blackmore. Electroencephalographic studies of stunning and slaughter of sheep and calves: part 1. Meat Sci 6:221–223, 1982a. Newhook, J.C., and D.K. Blackmore. Electroencephalographic studies of stunning and slaughter of sheep and calves: part 2. Meat Sci 6:295–300, 1982b. Petch, P.E. A 1350 volt 5 amp linear amplifier. Proceedings, IPENZ Technical Conference, Auckland, New Zealand, 1999. Petch, P.E., and K.V. Gilbert. Interaction of electrical processes applied during slaughter and dressing with stimulation requirements. In: Proc 43rd Int Congr Meat Sci Technol Auckland, 1997. Petch, P.E., and B.J. Lynch. A novel datalogger using an FPGA for data reduction. Proceedings of the Fourth Electronics New Zealand Conference. Auckland, New Zealand, 1997.

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Petch, P.E., and B.M. Peach. Developments in electrical stimulation and monitoring. Proceedings Twenty-eighth Meat Industry Research Conference, Auckland, New Zealand, 1994. Rentschler, H.C. Apparatus and method for the tenderization of meat. U.S. Patent 2,544,724, 1951. Riley, R.R., J.W. Savell, G.C. Smith, and M. Shelton. Quality, appearance and tenderness of electrically stimulated lamb. J Food Sci 45:119–121, 1980. Ross, M. Stand alone stunner monitor. Applied Control Electronics, Greenville, USA, 1999. Savell, J.W. Industrial applications of electrical stimulation. In: A.M Pearson and T.R. Dutson (Eds.) Advances in Meat Research. AVI Publishing, Westport, Connecticut, 1985. Simmons, N.J. The use of high frequency currents for the electrical stunning of pigs. Ph.D. thesis, University of Bristol, 1995. Smith, G.C., T.R. Dutson, Z.L. Carpenter, and R.L. Hostetler. Using electrical stimulation to tenderize meat. In: Proc Meat Ind Res Conf, Virginia, pp. 147–155, 1977. Smith, G.C., J.B. Morgan, J.D. Tatum, C.C. Kukay, M.T. Smith, T.D. Schnell, G.G. Hilton, C. Lambert, G. Cowman, and B. Boyd. Improving the consistency and competitiveness of non-fed beef; and improving the salvage value of cull cows and bulls. National Cattleman’s Association. Colorado State University, Fort Collins, 1994. Smulders, F.J.M., and G. Eikelenboom. Electrical stimulation of carcasses. Fleischwirtschaft 65:1356–1358, 1985. Sparrey, J.M. and S.B. Wotton. The design of pig stunning tong electrodes—a review. Meat Sci. 47:125–133, 1997. Swatland, H.J. “‘lular heterogeneity in the response of beef to electrical stimulation. Meat Sci 5:451–455, 1980. Taylor, D.G., and A.R. Marshall. Low voltage electrical stimulation of beef carcasses. J Food Sci 45:144–145, 1980. Tornberg, E. Biophysical aspects of meat tenderness. Meat Sci 43:S175–S191, 1996. Troeger, K. Slaughtering: animal protection and meat quality. Fleischwirtschaft 71:298–302, 1991. van der Wal, P.G. Chemical and physiological aspects of pig stunning. Meat Sci 2:19–30, 1977. Warrington, R. Electrical stunning: a review of the literature. Vet Bull 44:617–635. Wilson, M. Electricity: The willing genie in a wire. In Energy, Life Science Library, Time International, The Netherlands. 118–120, 1965. Wotton, S.B., M.H. Anil, P.E. Whittington, and J.L. McKinstry. Pig slaughtering procedures: headto-back stunning. Meat Sci 32:245–255, 1992. Wotton, S.B., and N.G. Gregory. Pig slaughtering procedures: time to loss of brain responsiveness after exsanguination or cardiac arrest. Res Vet Sci 40:148–151, 1986.

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15 Meat and Meat Products YOULING L. XIONG and WILLIAM BENJY MIKEL University of Kentucky, Lexington, Kentucky

I. INTRODUCTION II. EXPORT AND IMPORT III. CANNED MEATS IV. FROZEN MEATS V. COOKED REFRIGERATED MEATS VI. DRY-PRESERVED MEATS VII. CURED MEATS A. Ingredients and Functions B. Hams C. Bacon VIII. SAUSAGES A. Ingredients B. Fresh Sausages C. Cured Sausages D. Fermented Sausages IX. LUNCHEON MEATS X. PREPARED DINNER MEATS REFERENCES

I. INTRODUCTION Meat and meat products, referred to here as “red meats” or postmortem muscles from mammalian species (beef, veal, pork, and lamb/mutton), are an important component in the American diet. Despite the surge in poultry product consumption in the past two decades, red meats, with a current annual production totaling about 20 million metric tons (or 53 kg consumed per capita), remain to have a dominant market share in all muscle foods produced in the United States (American Meat Institute [AMI], 1999). Today, meat and meat

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Figure 1 Examples of processed and prepared meat and meat-based products. (This is an illustrative example only. The use or illustration of a trade name does not mean that the authors and their affiliation endorse the product.)

Table 1 Meat and Meat Products by Variety Meats Canned meats

Frozen meats

Dry-preserved meats Cured meats

Sausages Dinner meats

Luncheon meats

Processing/characteristic

Example

Retort to sterilize; fully cooked; cured or noncured; metal or plastic containers Cooked or raw; most microwavable; include home-meal-replacement items; breakfast items Low water activity; cured; refrigeration not required Cured with nitrite/nitrate, salt and adjuncts by injection or dry rub Fresh, cured, or fermented; comminuted or emulsified; spiced Prepared meals (HMR); pumped products; battered/breaded meats; precooked or raw; frozen or refrigerated Deli meats; lunchables; fully cooked and ready to consume; restructured meats

Ham; pork luncheon meat; corned beef; beef stew; beef in chili sauce Breaded boneless pork cutlet; pork sausage; meat loaf; beef stew and steaks; deli pouch; meatball; Beef jerky; pastrami Hams; bacon; jowl; most deli meats Bratwurst; frankfurters; salami; pepperoni Steak with vegetables; barbecue smoked pork; seasoned pork roast Sliced ham; bologna, and salami; ham and cheese; head cheese

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products available in the marketplace are no longer limited to the traditional items; in the past few years, numerous new items of processed meats, particularly those in the form of “convenience foods,” have emerged. Many of the products are fully cooked and ready to consume, whereas others are portion-controlled and “case-ready,” to be cooked in a microwave or conveniently heated in an oven at home. These products are widely distributed at retail and deli markets, and in a typical large food store, over 100 meat items can be readily found. Figure 1 shows examples of meats that are sold in grocery stores. Despite the great variety, meat products can be divided into seven groups based on their product characteristics and the general processing procedures required (Table 1). Postmortem muscle that has gone through major physical or chemical alterations is generally considered processed meat. Thus, in a broad sense, meat processing may include protein extraction, chemical and enzymatic treatments, massaging or tumbling, curing, stuffing, canning, smoking, and other related preliminary preparations, such as meat particle size reduction and mixing of meat with various additives. It is noteworthy that simple handling of fresh meat in retail stores and in homes (e.g., cutting, grinding, and packaging) is generally excluded from the definition of meat processing. II. EXPORT AND IMPORT Today, food production and merchandise have become a globalized enterprise. This has allowed the United States to export or import greater amounts of fresh and processed meats. Despite the large U.S. trade deficit in textiles, electronics, and a number of other economic sectors, U.S. agriculture maintains its trade surplus with foreign countries. For examples, through July of fiscal 1999, U.S. agriculture exported $41.0 billion worth of commodities, in contrast to $31.6 billion imported goods during the same period (United States Department of Agriculture [USDA], 1999). Among the leading commodities that contribute to the trade surplus are meats and meat products, whose 1.7 million-ton exports amount to a $3.7 billion value, versus $2.5 billion for imports. The surplus, however, is expected to decrease as a result of the U.S. dollar strength and relatively low prices of domestic meat and meat products. Countries that lead in exporting fresh chilled and frozen meat and meat products to the United States include Canada (451  106 tons), Australia (209  106 tons), and New Zealand (183  106 tons), whereas those that contribute to most of the U.S. import tonnage of further processed meats are Denmark (50  106 tons, about 40% are canned pork), Argentina (37  106 tons, all are processed beef products), and Uruguay (23  106 tons, with 10% canned beef products) (USDA, 1998). Table 2 compares U.S. exports and imports of meats by product type, and the changes that occurred from 1990 to 1996. III. CANNED MEATS Canning is a thermal process that employs heat (steam) to sterilize the food material placed in a sealed container. Thus, canning produces shelf-stable meat products that can be conveniently consumed in outdoor activities or situations where refrigeration is not readily available. The annual production of canned meats (including poultry) has stayed at about 1 billion pounds in recent years (Pearson and Gillett, 1996). Pasta with meat, chili, and canned hams represent about 70% of total canned meat products produced in the United States. Vienna sausage, canned luncheon meat, and meat spread are some other major forms of canned meats. The internal temperature of canned meat products must reach

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354

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Table 2 United States Meat Exports and Imports by Type of Products (in thousand metric tons), 1990 and 1996 Beef and veal

Year

Fresh chilled, and frozen

Canned

Pork

Prepared/preserved

Lamb, mutton, and goat

Fresh, chilled, and frozen

Hams and bacon

Other, prepared

Sausages, all types

Mixed sausages

31.5 55.2

13.4 15.3

3.5 2.4

1.9 1.6

1.0 5.3

4.5 15.8

— —

14.2 92.5

Canned

Variety meats fresh and chilled

Other livestock meats

Total

11.4 32.5

18.6 21.9

1100.7 1053.3

226.6 469.3

70.6 26.2

743.8 1507.8

Imports 1990 1996

699.3 640.7

57.6 53.4

10.9 13.6

19.1 33.1

233.5 183.6

— — Exports

1990 1996

339.9 596.9

— —

7.8 14.6

2.5 2.5

66.8 267.4

10.1 25.6

Source: USDA Agricultural statistics, 1998.

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Figure 2 Pork ham processed (pasteurized) in a plastic “can” (left) and beef stew processed in semirigid retortable plastic bowls.

121°C to achieve commercial sterility. However, this severe heat treatment may adversely affect the product flavor, texture, and color. Therefore, an increased number of canned meats are now only pasteurized to an internal temperature between 65° and 75°C. In a typical pasteurization process, canned meat is cooked in water at about 80°C for several hours, or steam pasteurized. It is desirable to keep the temperature difference between the heating medium and meat to a minimum to minimize cooking losses and jelly formation. All canned pasteurized meats must be cured to comply with federal regulations. They must be labeled “Perishable—Keep Under Refrigeration.” Thus, canned pasteurized meat products require refrigeration during distribution and storage. Meat canning is similar to fruit and vegetable canning in processing operations. Pretreated and prepared meat, which may contain various ingredients, is packed into cans varying in size and shape, e.g., pullman, round sanitary, pear-shaped, and oblong. Most cans used for meat are made of thin sheets of steel coated with a thin film of tin to prevent rusting. An enamel consisting of sulfur-resistant resins is formed on the surface of tin to prevent corrosion of metal due to interaction with sulfur compounds produced from meat. Plastic “cans,” such as semi-rigid retortable bowls and trays, have also been developed in recent years. Although the products are not exactly “canned,” they are processed in the same manner as metal-canned meats. Plastic can-like containers (e.g., D-shaped) made of nylon, surlyn, and other ethylene/vinylacetate copolymers are used for producing “cookin” hams that receive pasteurizing treatments (Fig. 2). Vacuum-sealed cans are either sterilized or pasteurized in retorts, and are subsequently cooled, labeled, and marketed. IV. FROZEN MEATS Frozen meat and meat products make up a major sector of the meat industry; with the increased consumer demand for convenient foods, production of this group of meats has con-

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tinued to grow in recent years. Freezing as a means of preserving meat became known following the development of efficient mechanical refrigeration during the 19th century; however, the freezing technology was not fully commercialized in the meat industry until the 1960s, during which decade there was a rapid growth in home freezers and refrigerators. The availability of efficient freezing systems has greatly facilitated meat exportation as well as importation (Table 2). Domestically, it has enabled meat producers and processors to market various convenient, packaged, and prepared foods (e.g., the so-called home meal replacements that have entered the market in the past few years) at the retail level. The freezing process involves placing meats in a cold room, typically 18 to 20°C. Water in the product exposed to the freezing temperatures will transform into ice. Due to the presence of various water-soluble and hydrophilic compounds, ice crystals actually do not form until the temperature of meat reaches a few degrees below 0°C. Quality of frozen meat and meat products is influenced by many factors. The rate of freezing has a profound effect on meat tenderness and drip loss. In general, fast freezing is conducive to the formation of small ice crystals that are located intracellularly and cause little physical damage to meat components. On the other hand, a slow freezing process favors large ice crystals to form, extracelluarly, which results in disruption of muscle cells and causes exudation (Fennema, 1975). In almost all commercial meat plants, forced air circulation (blast freezing) is used to achieve rapid freezing. Blast freezing may operate on either a batch or a continuous basis and normally employs 20 to 40°C cold air. Air with higher velocity allows a greater heat transfer coefficient, and hence, a more rapid temperature drop in meat. To avoid thaw rigor, beef and lamb must have gone through rigor mortis prior to freezing. To initiate early onset of rigor, thereby preventing thaw rigor, electrical stimulation can be employed. To prevent quality loss due to protein denaturation, cryoprotectants are often included in the formulation of meat products. Among them, Polydextrose®, polyphosphate, and to a lesser extent, sorbitol, are used. Packaging is another important factor affecting the shelf-life of frozen meats. Large, wholesale meat cuts are often vacuum sealed to prevent lipid oxidation and formation of metmyoglobin. For retail meats, cooked or raw, films with low water permeability and adequate mechanical strength, e.g., Sarlyn®, are required. To prevent the products from being exposed to light, thereby eliminating oxidation catalyzed by light-sensitive compounds, nontransparent outside cardboard-type packages are commonly used. There are various kinds of frozen, cooked meat products in today’s food market. A quick survey has shown many specific items manufactured by different companies, e.g., entree items: breaded boneless pork cutlet; country fried beef steaks; meat loaf; beef pot roast; and corn dog (batter-dipped wieners on a stick); dinner items: gravy and sliced beef; tomato sauce and meat loaf; steak and gravy; noodles with beef stew; steak with mushroom gravy; meat loaf in gravy; deli pouches (cheese burgers inside); croissant pockets; stuffed sandwiches (beef, ham, pepperoni); and meatballs in sauce with pasta (Fig. 3). Most of these meats are packaged with side dish items, such as green beans, tomatoes, carrots, mashed potatoes, onions, macaroni and cheese, and others, and are microwavable. V. COOKED REFRIGERATED MEATS This group includes various kinds of ready-to-consume products, many of which are sandwich-type prepared meats and belong to the “HMR” category (discussed later). Side dish items are normally included in the same package and the product needs only to be warmed

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Figure 3 Cooked frozen meat products: croissant pockets with ham, cheddar, and selected vegetables (left), and beef steak with peas and mashed potato (right). either with a microwave or conventional oven before serving. In some cases, flavor and freshness appearance may be a problem. For example, a precooked porkchop may develop warmed-over-flavor upon reheating, rendering the product unacceptable. To enhance product shelf-stability, antioxidants and specific flavor ingredients are often included. VI. DRY-PRESERVED MEATS Drying of meat is a very old process that was originally used to “preserve” meat, namely, to keep the meat at ambient temperature for an extended period of time. Historically, sundrying was the only drying method, but today hot air-drying is prevalent commercially. Alternatively, freeze-drying is done, but because of its prohibitively high costs, it is restricted to some specialty products that require rapid rehydration. The most well-known dried meats are beef jerkies and dried meat bits as a soup base. Dried meats prepared in the United States are cooked before drying. The usual process is to cut meat into slices and then cook, mince, or cut it into small cubes to provide a large surface-to-volume ratio, and dry it. Precooking is important not just because of food safety, but also because it facilitates the removal of a large amount of water from meat following protein aggregation. For beef jerkies, marination is a required extra pretreatment: meat slices are marinated (e.g., in teriyaki soy sauce, brown sugar, liquid smoke, black and red pepper, and other seasonings) before cooking and drying. During the drying process, meat undergoes extensive physical and chemical changes. Conventional air-drying involves the extraction of almost all of indigenous water in muscle. The process is fundamentally a diffusion of water to the meat surface, which is driven by the difference in water vapor partial pressure between the air and the meat surface. Both continuous belt oven and fluidized bed driers are used to dry meat. The removal of water from the muscle cells as well as between muscle fibers results in stronger hydrophobic interactions of the myofibrils, thereby hardening the texture of meat for desirable chewiness. VII.

CURED MEATS

Curing of meat refers to treatment of fresh meat with salt, nitrite or nitrate compounds, and adjuncts for the purpose of preservation and obtaining desirable color and flavor. It is not

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known where and when the use of curing first began, but some evidence suggests that around 3000 B.C. the Chinese began using salt that was contaminated with nitrate to preserve meat. Many cured meat products are available in the current food market. They include traditional pork products such as ham and bacon, and to a lesser extent, beef products such as corned beef. More recently, a number of new cured meats have been developed, with most belonging to the restructured and sliced deli meat category. This section will concentrate on ham and bacon because both not only are popular products in the cured meat category, but are also of a long tradition, have unique characteristics, and involve some special processing technologies. A. Ingredients and Functions 1. Curing Agents Salt in the form of NaCl is commonly used in cured meats. Occasionally, KCl is used to replace or partially substitute for NaCl to alleviate its potential adverse effect on health of the humans. The main functions of salt, other than providing flavor, are solubilizing proteins, dehydration, and altering osmotic pressure so as to inhibit bacterial growth and subsequent spoilage. Salt can be applied to meat directly in its dry crystal form, or dissolved in solution (pickle) before incorporation into meat. Although salt is an indispensable ingredient in cured products, the true “curing” agent is nitrite (NO2) or nitrate (NO3). Nitrate was originally approved for color fixation in cured meats, but now it has largely been replaced by nitrite. This is because nitrate is reduced to nitrite either by organisms or by reducing compounds before the actual curing process begins, and because direct application of nitrite can be more easily controlled. In the United States, the use of nitrate is now restricted to drycured products, such as country cured hams and dry sausages. Nitrite (or nitrate) is a multifunctional compound. It induces and stabilizes the pinkish color of lean meat, contributes to the characteristic flavor of cured meat, inhibits the growth of spoilage and pathogenic microorganisms (especially Clostridium botulinum), and retards development of oxidative rancidity. The level of nitrite or nitrate allowed in cured meats, both ingoing and residual, is strictly regulated by the USDA. For instance, the maximum level of ingoing NaNO2 in pumped bacon is 120 ppm, and the residual level (in the finished product) shall not exceed 40 ppm. Establishment of nitrite regulation is based on the concern that nitrite can form nitrosamines by reacting with secondary amines in cooked cured meat as well as in the intestines of the human body. In response to consumer concern, an increased number of meat processors are now reducing the nitrite level in cured products. A recent survey showed that residual nitrite levels in cured meats (ham, bacon, wiener, and bologna) produced in the United States average about 10 ppm, which represents only 10% of the recorded levels in cured meats two decades ago (Cassens, 1997). The pinkish red color characteristic of cooked cured meats results largely from the reaction of the heme group in myoglobin with nitric oxide forming the nitrosylmyoglobin pigment. Nitric oxide is derived from nitrite in the presence of reducing compounds such as erythorbic acid. Part of nitrite dissolved in water can form nitrous acid (HNO2). Under a reducing condition, nitrous acid decomposes to nitric oxide. When nitric oxide binds to the heme iron, it changes the electron distribution or resonance of the heme structure, thereby producing a pinkish color. Under heating conditions, nitrosylmyoglobin is converted to the more stable forms, i.e., nitrosylhemochromogen and dinitrosylhemochrome. The concerns with possible carcinogenic effect of residual nitrite present in cured meats have prompted researchers to search for alternative curing methods. One of the pos-

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sible methods was the so-called nitrite-free curing (O’Boyle et al., 1990). In this curing system, the three major functions of nitrite—color fixation, antimicrobial activity (antibotulism in particular), and inhibition of oxidative rancidity—are conferred through the use of combined materials. Dinitrosylhemochrome is presynthesized by reacting nitric oxide gas with hemin extracted from beef red blood cells, an abundant animal by-product. Dinitrosylhemochrome is not very stable, so the pigment is microencapsulated with carbohydrate-based encapsulating agents to prevent it from direct contact with air. To reproduce the antioxidative activity of nitrite, antioxidants such as ascorbate, tripolyphosphate, and various phenolics (TBHQ, BHA, etc.) are added to the nitrite-free mixture. In addition, antimicrobial agents, such as sorbate, sodium hypophosphite, methyl fumarate, and sodium lactate, are also used. The mixture is blended with meat in sausage production. Frankfurters and wieners manufactured using the nitrite-free curing system cannot be distinguished from products made by the traditional nitrite-cured method. 2. Curing Adjuncts Reducing compounds are added in meat-curing mixtures to hasten color development via converting nitrite to nitric oxide, and ferric iron of the heme to ferrous iron. The most commonly used reducing agent is sodium ascorbate (vitamin C), or its isomer, erythorbate, which is less expensive and more stable. Muscle itself also contains endogenous reductants and enzymatic reducing activity, but the reducing power of these factors is relatively small. In addition to reducing metmyoglobin (Fe3) to myoglobin (Fe2), and nitrite to nitric oxide, ascorbate (or erythorbate) also serves as an antioxidant to stabilize both color and flavor, and to decrease the formation of nitrosamines. Another curing adjunct is phosphate, usually sodium pyrophosphate, tripolyphosphate, and hexametaphosphate, that can be used individually or in various combinations. In either case, the total addition cannot exceed 0.5% of the meat product weight as regulated by the USDA. Phosphates do not directly enter the curing reactions; they are added mainly to increase water-holding capacity of muscle, thereby reducing shrinkage of finished products. Phosphates are also effective antioxidants and can retard rancidity development. However, because nitrite and ascorbate are stronger antioxidants, the antioxidative effect by phosphates may not be significant in cured meats. Because phosphates are corrosive (they bind with metal ions), the equipment used must be made of stainless steel or plastic, or metal with a plastic coating. B. Hams Hams can be separated into two groups: bone-in hams (processed using the whole pork legs) and boneless hams (made from deboned pork meat). The latter type also belongs to the deli meat group and therefore will be further discussed later. A typical ham is cured with a mixture consisting of salt, sugar, sodium nitrite, sodium erythorbate, and sodium tripolyphosphate. Occasionally, corn syrup in lieu of sugar is utilized. Hams can be cured by two fundamental methods: dry curing and pickle (wet) curing. In dry curing, the curing ingredients are applied to fresh pork leg by rubbing without the addition of water. Thus, the curing ingredients, particularly salt, draw enough water from the meat due to the osmotic pressure gradient to form a brine, which serves to transport the curing ingredients into the meat through diffusion. This curing method is obviously labor-intensive and distribution of the curing ingredients is not efficient. Furthermore, dry-cured meats are often too salty and may be unacceptable to many consumers. Therefore, except for country ham, commercial

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processing of hams is now accomplished mostly by pickle injection. Dry curing is also still used for fatty cuts, such as fat backs, clear plates, and jowls. 1. Dry Curing Country hams are prepared using dry curing methods. Country hams are not cooked but must be free of Trichinae and have a salt content in the finished product of at least 4%, and shrink a minimum of 18% during processing. The flavor and appearance of country hams distinctly differ from common pickle-cured hams. Country hams are salty, somewhat dry, and rather hard products with a rich flavor. They are primarily produced in the southern regions of the United States, including the states of Kentucky, Virginia, North Carolina, Tennessee, Georgia, and Missouri. Most commercial country ham processors rely on added nitrite or nitrate for curing. However, a small percentage of processors do not add nitrate or nitrite and still make highly flavored and well-colored country hams. Salt and seasoning may be contaminated with nitrate or nitrite and produce the color and flavor characteristic of country hams. The curing mixture is divided into three equal portions, which are applied by rubbing around the entire cut immediately, and 7 and 14 days later, respectively. It is important to make sure that the flesh surface is adequately rubbed with the cure mixture because the cure penetrates into the ham through the flesh surface, not the skin surface. For best product quality, country hams should be cured under refrigeration conditions with a temperature of 2.2° to 4.4°C (36–40°F) and a relative humidity of 70% to 90%. Hams are stacked with overhauling two to three times during a 30- to 50-day curing period. The exact curing time depends on the size of ham (~2 days/pound). Following curing, country hams are held for approximately 20 more days for salt equalization. The equalization room should be maintained at 7.2° to 12.8°C (45° to 55°F) at a relative humidity of 75% to 90% and can be done in the curing room. The hams should then be hung by the shank using a string, or placed in stockinette and suspended from a rack, and allowed to age for 6 to 9 months but no longer than 12 months. The temperature of the aging room is relatively high (21° to 35°C) and the humidity relative low (50% to 60%) to facilitate dehydration and development of firm texture and distinct flavors. It is generally believed that both cathepsins (which degrade proteins to small flavor-active peptides) and lipases (which generates volatile compounds by catalyzing lipid oxidation) are involved in the flavor development. Frequently, molds will grow on the surface of country hams during salt equalization and subsequent aging. These molds are generally unharmful and can be removed by wiping with a cloth dampened with edible oil. Finished country hams should have a moisture content of 50% to 60% and an average salt content of 4.5% to 5.5%. There are other types of dry-rubbed hams made in other countries that are more or less similar to country hams made in the United States. Examples are Chinese Jinhua ham, Italian prosciutto ham, German Black Forest ham, and Spanish serrano ham. 2. Pickle Curing This is the most widely used curing method for hams. The method differs from dry curing in that the curing mixtures are dissolved in water to form a brine or pickle. Furthermore, nitrite instead of nitrate is used in pickle curing. Among the different ways to incorporate curing solution into meat, stitch pumping is by far the most widely used. In a typical commercial operation, multiple needles are used to inject the curing solution into the ham flesh. The

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pickle, in the amount of ~10% of the raw pork leg weight, is injected from different sides of the leg, allowing a uniform incorporation of the curing ingredients. This amount of added solution is usually lost by the time the ham is fully cooked. The amount of residual brine after cooking is regulated. If the finished ham does not weigh more than it did before cure injection, no label restrictions are imposed. However, if the finished ham is heavier than its fresh cut, i.e., the protein fat-free (PFF) value falls below 20.5, then it must be labeled “cooked ham with natural juice” (PFF 18.5) or “cooked ham with water added” (PFF 17.0) (USDA, 1984). C. Bacon Similar to ham processing, bacon can be cured by both dry rub and pickle curing. Since pork bellies are relatively thin, they do not require a long time to cure before cooking and smoking. A typical pork belly requires only 10 to 14 days to cure by dry rub, compared with hams, which require several months. Most commercially processed bacon is now cured through multi-needle stitch pumping. Finished bacon is usually partially cooked; the final internal temperature of the belly reaches only 52° to 56°C. Before consumption, bacon slices must be fully cooked to ensure a complete destruction of possible pathogenic microorganisms and the parasite Trichinae. Recently, microwavable bacon has been developed. The process involves frying of cured belly for sufficient length to develop the characteristic flavor and texture of cooked bacon. The fully cooked bacon is packaged in a special pack designed to enhance microwave heating (5 to 10 seconds). Additional procedures may be necessary, including the use of binders to minimize moisture loss during heating and texturizing through rollers to prevent excessive distortion of the rashers. The product is rather stable, requiring no refrigeration until opened. There are several other kinds of bacon, e.g., jowl bacon, beef bacon, and Canadian bacon. Canadian bacon, made from pork loin muscle, differs sharply from conventional bacon, which is made from the pork bellies.

VIII. SAUSAGES Sausages are a unique type of comminuted meat products that are usually spiced or seasoned to obtain various flavor intensities and profiles. The development of sausages was initially driven primarily by economic factors, i.e., it utilizes low-quality meats such as trimmings, head and shoulder meat, and edible by-products. Convenience and variety are other important reasons why sausages are widely consumed in modern society. In the United States, about 4 billion kg of sausage products are produced annually, and the per capita consumption is estimated to be 15 kg per year. Based on the product characteristics and the specific processing method used, sausages can be classified into three major groups: fresh sausages, cured sausages, and fermented sausages. A comprehensive list of sausages produced in the world is provided by Roman et al. (1994). Technologically, sausage making consists of several common steps—comminution to reduce meat and fat particle size (grinding, mincing, chopping, or flaking), mixing with ingredients, stuffing into a specific casing, linking to obtain specific lengths, and finally, packaging.

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A. Ingredients 1. Raw Meat A proper selection of meat ingredients is essential for the production of sausages of uniform quality. Raw meats used for sausages are generally low-valued materials, but they must be fresh, i.e., with very low microbial counts. These include cuts high in connective tissue or fat, tough meat from mature animals, carcass trimmings, mechanically separated meat, and edible animal by-products. The function of each selected raw meat ingredient may be unique. Meats used for binding should have a sufficiently high protein content and the proteins should be readily extracted and form gels during cooking. Skeletal muscle from cows, bulls, and sows is an excellent meat binder, whereas high-fat trimmings are generally poor binders. There is another group of meats that are included in sausage formulations to “fill” the void space in sausages. Filler meats have little or no binding ability, and they include offal meats (e.g., tripe, snouts), skin, and partially defatted beef and pork tissue. The rapidly increased use of poultry meat in the sausage industry is worth particular mentioning. Poultry meat has been blended into pork and beef sausages. The increased use of poultry meat in sausage production has resulted mainly from the relatively low cost for poultry meat (particularly turkey), and increased consumer demands for “light meat,” which is perceived as more healthy than red meat. 2. Salt and Nitrite Salt is the single most critical nonmeat ingredient. The main form of salt utilized in sausage production is sodium chloride. Its principal function is to solubilize and extract the myofibrillar proteins needed to form a bind during cooking. Of course, it also imparts flavor and has antimicrobial effects. Thus, salt is responsible for the textural characteristics and integrity of finished sausage products. Most commercial sausages contain 1.5% to 2.5% added salt. Phosphates at a level up to 0.5% in finished products are used to improve water-binding capacity of meat by increasing fiber swelling and solubilizing proteins. Phosphates may also help to stabilize flavor and color in finished product, presumably by sequestering transitional metal ions (Fe and Cu), thus reducing oxidation. Many sausage products are cured with nitrite. Sodium nitrite is commonly used, although in certain cases it may be substituted for by potassium nitrite. The maximum level of nitrite allowed in sausage is 156 ppm. The use of nitrate is more restricted; it can be used only in dry and semi-dry, fermented sausages. Nitrite is used in conjunction with the reducing agent ascorbate or erythorbate, and phosphates. 3. Water and Extenders Water, sometimes together with ice, is added in sausage making to help distribute nonmeat ingredients and increase the product yield. In 1988, the USDA implemented a new regulation to permit water addition to partially substitute for fat in cooked sausages, so long as the sum of fat (30% maximum) and added water in the final product does not exceed 40% of the product weight. As a result of this regulatory change, numerous new, low-fat sausage products—for example, “reduced-fat” sausages ( 25% fat reduction over traditional products), “low-fat” or “light” sausages (contain 10% fat), “extra lean” sausages (contain

5% fat), and “fat-free” sausages (contain 1% fat)—have been produced. Along with the addition of water, nonfat dry milk, whey protein concentrate, sodium caseinate, wheat gluten, cereal flours, tapioca dextrin, soy flour, soy protein concentrate, and more recently,

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polysaccharide gums, at limited levels, are used as extenders or fillers in sausages. The terms extender and filler are often used interchangeably. Their main functions are to improve functional properties related to product texture and flavor, and to aid in meat particle binding and water retention. 4. Seasonings Unlike most other processed meats, sausages are seasoned products. Different spices and flavorings are added in sausages, and their use levels are primarily dictated by product identity standards and not by regulations. Spices are aromatic vegetable substances in whole, broken, or ground form. Spices may be added as natural spices or spice extracts. In the latter case, they must be labeled as “flavoring.” Flavorings refer to extractives that contain flavor constituents from fruits, vegetables, herbs, roots, meat, seafood, poultry, eggs, dairy products, and other food sources. Flavoring compounds can also be synthesized. A good example is monosodium glutamate, which is a potent flavor enhancer. Most flavorings are oil-based extracts. Because of their high flavor intensity, they can be more accurately applied in sausage to obtain desired flavor intensity than their natural counterparts (spices). Sugars in a variety of forms—sucrose, dextrose, corn syrup, and so on—are most commonly used in sausages. Almost all sausage products contain sugar in one form or another. B. Fresh Sausages As the word “fresh” indicates, fresh sausages include a variety of uncooked sausages, such as breakfast sausage and sausage patties, whole hog sausage, bratwurst, Italian-style sausage, and Polish-style sausage, all of which can have a fat content up to 50% of the raw product weight. They are salted but not cured with nitrite, and are generally coarsely ground and not emulsified. Fresh smoked sausages such as Kielbasa also belong to this product group. Fresh sausages are manufactured by grinding meat through plates with holes ranging from 0.32 cm (1/8) to 0.95 cm (3/8) in diameter. Particle size reduction is achieved through extrusion and cutting in a screw auger operating in a horizontal chamber. The ground meat is mixed with salt, seasonings, and other ingredients by blending in a mixer or similar equipment. Stuffing is done by extrusion of the batter into casings through a smallopening tube called the horn. There are two major types of sausage casings—natural or synthetic. Natural casings are small intestines from hog and sheep. The intestine is inverted and thoroughly washed in a dilute chlorine solution (0.5%) followed by water rinsing. Excess fat and connective tissue are removed by brushing with a soft brush. Natural casings are normally packed in saturated salt solution and stored in a cold room or a freezer. Natural casings are denatured upon cooking. Shrinkage of the casing allows it to firm up the sausage links. Synthetic casings are made of edible collagen materials or inedible cellulose. For fresh sausages, edible casings are always used. After stuffing, sausage is linked to make individual links of equal length. Because casings contribute a significant cost to sausage products, technology has been developed to manufacture sausages without the use of casings (Frye, 1996). In one method (“co-extrusion”), collagen solution is extruded around an endless rope of sausage batter, forming a thin collagen layer on the surface of the sausage. Subsequent treatment with a saturated salt solution causes dehydration of collagen and transforms it into a fibrous structure or coating. After heating and cooling, a thin invisible film is formed as the result of collagen cross-linking, thereby encasing the sausage. Another method (using “sintered

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mold”) is based on the principle that myofibrillar proteins extracted to the surface of sausage can coagulate upon acidification. The meat batter is extruded into a porous mold. When a small amount of acetic acid is pumped through the mold, it lowers the pH and causes the proteins on the surface to coagulate, forming a natural “skin.” Fresh sausages are sold uncooked, and require either refrigeration or freezing. C. Cured Sausages Nitrite-cured sausages are mostly finely chopped and emulsified. The most popular products in this group are frankfurters and bologna, which account for about 25% and 20% of all sausages (including both red meat and poultry) consumed in the United States. Frankfurters are prepared in various forms, and those with a relatively large diameter are often referred to as wieners. Most frankfurters in the United States are a blend of beef and pork, with or without poultry, mildly seasoned with paprika and other spices, and smoked. The processing technology for bologna is similar to that for frankfurters; however, bologna is much larger in diameter (e.g., ~10 cm compared to less than 2.5 cm for frankfurters) for the purpose of sandwich preparation, and the spices used may be different. Many luncheon meats with distinctly different standards of identity actually also belong to the “cured sausage” category. In a typical cured sausage processing, the mixture of meat and ingredients is finely chopped and emulsified. Frequently, mixing and chopping are done simultaneously in a bowl chopper where a series of vertically positioned rotating knives cut through meat that is forced to move with the horizontally positioned rotating bowl. The chopping process creates sufficient shear to comminute meat and fat into fine particulates. Because myofibrillar proteins are extracted in the presence of added salt and phosphate during chopping, they will form coatings on the surface of the fat droplets and produce protein matrices surrounding the emulsified fat particles, thereby reducing the surface tension. Some sausage manufacturers use a separate emulsifier to produce fine emulsions after the meat is already chopped. However, with a good chopping machine, a separate emulsifier is often found unnecessary. The finely chopped meat system is completely stabilized during cooking, where three-dimensional gel structures are formed and fat particles are imbedded in the gel matrices. Most cured sausages are also smoked. One of the most critical factors in the production of emulsified meats is the temperature of meat batters during chopping. This temperature should be maintained low enough to prevent emulsion collapse, but not too low to keep fat soft. It varies depending on type of fat added or meat used. For most frankfurter emulsions, a chopping temperature of about 10° to 12°C is desirable. Due to friction between the high-velocity rotating blades and meat particles, a 10°C temperature rise in meat batters is not uncommon. To prevent heat rise during chopping, ice is used to replace water in the product formulation. Finely chopped sausages are stuffed mostly in synthetic casings. Therefore, after cooking, casings are removed by peeling prior to vacuum packaging. Natural casings described above are also used for certain finely chopped products, and they are not removed after cooking. D. Fermented Sausages The first use of fermentation in meat is lost in the mists of antiquity but may date back to the Babylonian culture around 1500 B.C. Fermented sausages can be divided into two main groups based on the processing procedure and product characteristics: dry and semi-dry. For both groups, lactic acid is produced; thus, meat is “fermented.” Dry and semi-dry

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sausages, as their names imply, differ in moisture content, which averages between 30% to 40% and 40% to 50%, respectively. Of the different fermented meat products produced domestically or imported, pepperoni and salami are two of the most popular items, with pepperoni alone amounting to 180 million kg consumed annually (Hinkens et al., 1996). The most crucial processing step in sausage fermentation is to timely lower the pH of fresh meat (which averages about 5.6 to 5.8 post rigor) so as to curtail the growth of spoilage microorganisms The final pH of fermented sausages typically ranges from 4.8 to 5.2, depending on tanginess, firmness, and other product characteristics desired. Lactic acid bacteria, which produce lactic acid through glycolysis, can be introduced into meat either by “chance inoculation” (natural fermentation) or by inoculating a starter culture. In natural fermentation, lactic bacteria are inoculated by chance from the processing environment (e.g., processing equipment). Sometimes, a portion of already fermented meat from a previous batch is added to a new batch to start fermentation. This procedure, called “backslopping,” reduces the incubation time for the bacteria to reach a productive level. Natural fermentation has been used for centuries but it has many obvious disadvantages. The fermentation usually takes a long time (e.g., more than one week). The population and type of lactic acid bacteria in fresh meat are difficult to control. If the initial population of lactic bacteria is small and the meat pH cannot be rapidly lowered, spoilage microorganisms will predominate and the product will fail. Moreover, pathogens can grow well in meat when the pH is not sufficiently low, especially when they do not have to compete with lactic bacteria. Many lactic bacteria from chance inoculation are heterofermentative, i.e., in addition to producing lactic acid, they also produce acetic acid, alcohol, gas, etc. Because different bacteria species may be introduced each time, batch-to-batch variability in product flavor, acidity, and textural characteristics can be very high. Today, almost all commercial production of fermented sausages is done by using selected starter cultures. Starter cultures are available in two forms: frozen concentrate and lyophilized dry powder. Starter cultures available commercially are typically blends of two or more different microorganisms and sometimes different strains of the same microorganism. The most commonly used microorganisms are Lactobacillus, Pediococcus, Lactococcus (all three are homofermentative), and Micrococcus (used to reduce nitrate to nitrite). Specific examples are L. plantarum, P. acidilactici, and L. lactis sub sp. lactis. Fermented sausages are salted and cured, and both nitrate and nitrite can be used. Salt is needed to facilitate dehydration and impart flavor. Fermented sausages are usually heavily spiced, making the product particularly palatable. Organic acidulants, such as encapsulated glucono-delta-lactone and lactic acid, are sometimes mixed with fresh meat at the beginning of fermentation. They are used to quickly establish an acidic environment that will stimulate the growth of lactic acid bacteria and inhibit spoilage microorganisms. In fact, dry or semi-dry sausages can be produced by direct acidification with proper acidulants such as lactic acid and glucono-delta-lactone, a slow acid-releasing compound. Sausages prepared by direct acidification have a characteristic tangy flavor closely resembling that of fermented products, and they are most widely used in pepperoni production for pizza toppings. In order to produce lactic acid during fermentation, sugar must be present, which serves as substrate for glycolytic enzymes inside the bacterial cells. Simple sugars, such as sucrose and dextrose, are preferred because they can be readily transported through the bacterial cell wall. The amount of sugar added to dry or semi-dry sausages are typically in the range of 0.5% to 2.0%. The lower the desired pH, the more sugar will be needed. After ground meat is blended with all ingredients, including the starter culture, the mixture is stuffed and subsequently incubated in a closed chamber (sometimes a smoke-

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house) to allow fermentation to take place. Temperature of the incubator is typically maintained at 21° to 24°C with a 75% to 80% relative humidity for dry sausage, and 30° to 37°C with a 75% to 80% relative humidity for semi-dry sausage. The fermentation time, however, is longer for dry sausage (1 to 3 days) than for semi-dry sausage (8 to 20 hours) (Terrell, 1977). For dry sausage production, the fermented meat is placed in a drying room to allow further dehydration and flavor development. As a general recommendation, the temperature of the drying room should be controlled to 7° to 13°C and the relative humidity to 70% to 72%. The air of the drying room should be changed periodically to ensure air quality and prevent moisture buildup on the surface of sausage. The drying time varies considerably, depending on the size (diameter) and type of product. Most dry sausages are aged for somewhere between 10 days to 3 months. Dry sausages are not cooked, and most are not smoked. They do not require refrigeration after manufacture. The low moisture content (aw  0.91) and low pH conditions in the sausage are effective to preserve the product. Semi-dry sausages, however, are generally cooked to an internal temperature of at least 68°C following fermentation. Semi-dry sausages have a relatively high moisture content (aw ~ 0.95) and hence, require refrigeration to prevent microbial spoilage. Most semi-dry sausages are smoked. IX. LUNCHEON MEATS This group encompasses a broad range of processed meats that are manufactured in the form of loaves or slices (Fig. 4). They have been introduced to the retail and convenience store or deli markets as consumer demand has grown for ready-to-eat or ready-to-heat products. Luncheon meats are fully cooked/pasteurized and require refrigeration for storage. Many of the loaves are pre-sliced and packaged for distribution to retail stores. Wholesale loaves, typically in the 3 to 5 kg range, are usually sliced in the deli at the time the customer purchases them. Because these meat products are sold in the deli section, they are sometimes referred to as deli meats. Luncheon meats, as the name indicates, are utilized primarily for lunch sandwich preparation. These products are normally restructured and may be cured or uncured with a variety of flavor profiles to meet the demand of ever-changing

Figure 4 Sliced deli or luncheon meats: restructured ham (left) and pickled loaf (right).

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consumer makeup. Among the popular luncheon meat items manufactured in the United States are sliced ham (e.g., honey ham 98% fat free), bologna- and salami-type loaves, liver cheese (wrapped in a layer of pork fat), ham and cheese loaf, and head cheese (made of pork snout and tongue meat with added water, salt, gelatin, spices, flavorings, monosodium glutamate, dextrose, nitrite, etc.). It must be pointed out that the traditional luncheon meats, which are high-temperature processed and canned, have lost their true meaning, and should be distinguished from the deli-type meats that are consumed as convenient lunch items. There is a great variation in flavor and texture among luncheon meats, and the differences are derived from the specific processing procedures involved as well as the different formulations employed. For bologna-type luncheon meats, essentially the same meat ingredients as for sausages are used, and the same emulsion for making frankfurters can be used for luncheon meat loaves. However, luncheon meats often contain also other condiments to enhance palatability, e.g., pickles, pimentos, and olives. Luncheon meat loaves are stuffed into plastic or cellulose bags inside a stainless steel tube that has a 10 to 12 cm inner diameter. The meat batter is compressed into the tube, capped, and then cooked. Some luncheon meat loaves are prepared by stuffing inside a pan (similar to the bread pan). The open top surface is sometimes brushed with a thin layer of syrup to create a brown appearance after cooking. This type of luncheon meat, after slicing, can easily be used for sandwich preparation. A notable processing change that is made by some luncheon meat manufacturers in recent years is repasteurization of the fully cooked product after it is sliced. This is to assure that psychrotrophic pathogens, particularly Listeria monocytogenes, that could be introduced during slicing and handling are inactivated. Since reheating tends to cause additional water loss, it is important that a proper water-binding agent, such as polyphopshate and polysaccharide gums, be included in the product formulation. Another group of luncheon food product, referred to as Lunchables®, have been developed in the past few years, led by Oscar Mayer Foods Division. This type of product is sealed in a small plastic package that consists of several compartments. Bite-size meat (e.g., ham, franks, and pepperoni) is placed in one compartment while cheese, cookies, crackers, desserts, or other nonmeat items are packed in other compartments (Fig. 5). This new marketing concept was developed to target children, including elementary school pupils, from busy families. It is nevertheless more of an ingenious marketing strategy than it is a true processing innovation.

Figure 5 Lunchables containing ham slices (left) or cooked beef patties (right).

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X. PREPARED DINNER MEATS In the United States today, an estimated 50% of the food dollar is spent on foods prepared away from home. This is especially true for double working households that have little time to spend on meal preparation. According to the U.S. Department of Commerce, the percent of women working outside the home has increased from 42% in 1960 to 70% in 1995. These consumers are looking for options that allow their families to continue to enjoy the mealtime experience as a family without the hassle of extended preparation time. Options available may include eating out at restaurants, having takeout foods, or purchasing ready prepared meals to be reheated at home for the family meal. Taking advantage of this shift in consumer attitudes and lifestyle, many processors have developed new products as home meal replacements and component meals. In essence, the preparation time is shortened for the consumer by preparing the meal at the processing plant. Pre-prepared meat items have been available for years, including frozen dinners, sandwiches, pot pies, as mentioned previously. Today, frozen or refrigerated sandwiches are popular items in many institutional settings and can even be found in convenience vending machines. However, in many cases these items tended to lack consumer appeal, which prompted meat processors to develop new processing strategies. The first of the renewed generation of these products were “home meal replacements” that emerged a few years ago. This concept, similar to frozen dinners, provides all the components of the meal ready for the table. Everything, including the meat, vegetables, and other items, are included in the package (Fig. 6). The consumer only needs to cook the items or in some cases just reheat in a conventional oven or microwave. Although these products have filled a niche, consumers are still looking for other options with greater flexibility. This has led to the development of so-called component meals. These meals allow for an entree, and sometimes a side dish, to be prepared at the processing plant and packaged for refrigeration storage until reheated by the consumer. This allows even greater flexibility by the consumer to add variety to the meal at their own discretion. The sale of prepared refrigerated meals has gained wide popularity and is becoming a substantial market force with both the beef and pork industry. Another group of prepared products that are gaining sizable market share are battered and breaded meats. These types of products (long perfected by the poultry and seafood segments of the muscle foods processing industry) are becoming more commonplace in both the retail and the food service segments of the marketplace. Technologically, breading con-

Figure 6 Prepared meat items: home meal replacement (left) and injected pork roast (right).

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sists of three major steps: predusting—applying a finely ground flour and seasoning mixture to the raw meat; battering—applying a flour/seasoning batter of specific consistency to the predusted meat; and breading—applying a flour mixture with a coarser crumb texture. Not all three steps may be required, depending on the specific products. Sometimes, the raw meat is marinated in water and phosphate solutions before predusting to pick up moisture for improved palatability and product yield. The breaded meat can be fried for a few seconds (raw) or up to a few minutes (fully cooked) to help the batter adhere to the meat, and the final product is either frozen or refrigerated before marketing. Breaded meats are of great interest to the meat industry partly due to profit margins available with these items induced by a lower input cost per unit. In addition, this process allows for creation of greater product variety. Whether the product is restructured or is made from whole muscle, the addition of up to 30% breading allows the processor to meet the consumer demand for convenient products while also targeting both the retail and food service industries. Many of these products are now being used or evaluated as “finger foods” or appetizers. Another type of product that has gained widespread acceptance is that of “enhanced products.” In essence, these are fresh whole muscle products, both beef and pork, that have been subjected to injection or marination to improve sensory acceptability (Fig. 6). These products normally are injected with a combination of water, salt, and phosphate with the addition of seasoning or flavoring if an ethnic flavor profile is desired. In most cases, the products have an addition of less than 10% added ingredients (which are labeled as “x% solution containing ....”). This process not only allows the processor to improve the economic viability of many lower value meats but also enhances consumer acceptance by reducing dissatisfaction with the product that may result from the use of less palatable raw materials or consumer abuse due to overcooking. REFERENCES American Meat Institute (AMI). Meat and Poultry Facts. Washington, DC: American Meat Institute. 1999. Cassens, R.C. Residual nitrite in cured meat. Food Technol 51(2):53–55, 1997. Fennema, O. Freezing preservation. In Principle of Food Science. Part II. Physical Principles of Food Preservation. M. Karel, O.R. Fennema, and D.B. Lund (Eds.), Ch. 6, pp. 173–215, New York: Marcel Dekker, 1975. Frye, C.B. Manufacturing sausage without casings. Proc Recip Meat Conf 49:39–48, 1996. Hinkens, J.C., Faith, N.G., Lorang, T.D., Bailey, P., Buege, D., Kaspar, C.W., and Luchansky, J.B. Validation of pepperoni processes for control of Escherichia coli O157:H7. J Food Prot 9:1260–1266, 1996. Pearson, A.M., and Gillett, T.A. Processed Meats. 3rd ed., New York: Chapman & Hall, 1996. Romans, J.R., Costello, W.J., Carlson, C.W., Greaser, M.L., and Jones, K.W. The Meat We Eat, 13th ed. Danville, IL: Interstate Publishers, 1994. O’Boyle, A.R., Rubin, L.J., Diosady, L.L., Aladin-Kassam, N., Comer, F., and Brightwell, W. A nitrite-free curing system and its application to the production of wieners. Food Technol 44(5):88–104, 1990. Terrell, R.N. Practical manufacturing technology for dry and semi-dry sausage. Proc Recip Meat Conf 30:39–48, 1977. United States Department of Agriculture (USDA). Control of added substances and labeling requirements for cured pork products. Updating provisions. Fed Reg 49:14856–14887, 1984. USDA. Agricultural Statistics. National Agricultural Statistics Service, Washington, DC, 1998. USDA. U.S. Agricultural Trade Update. Monthly Supplement to Foreign Agricultural Trade of the United States, Washington, DC, 1999.

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16 Spices and Flavorings for Meat and Meat Products PATTI C. COGGINS Mississippi State University, Mississippi State, Mississippi

I. INTRODUCTION II. HISTORY OF SPICES A. A Brief History of Spices B. The Spice Industry Today III. SPICES AND HERBS A. Definitions and Differences B. Spices and Herbs in General C. Peppers in General D. Heat in Red Peppers IV. SPICE BLENDS A. Spice Usage in Foods V. INDUSTRIAL SPICES AND HERBS A. Indigenous and Cultivated Spices B. Fresh versus Dried Spices and Herbs C. Whole and Ground Spices D. Spice Grinding VI. SPICE QUALITY AND STANDARDS A. Spice Quality and Evaluation B. Spice Standards VII. OILS, OLEORESINS, AND EXTRACTS A. Spice Oils and Oleoresins B. Manufactures of Oils and Oleoresins C. Flavor Quality of Natural Spices, Oils, Oleoresins, and Extracts D. Natural Spices versus Oils and Oleoresins VIII. STORAGE AND SHELF LIFE A. Spice Storage B. Spice Shelf Life C. Spices and Food Spoilage

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IX. CLEANING AND PROCESSING OF SPICES A. Cleaned and Uncleaned Spices B. How Spices Are Cleaned C. Sterilization of Spices X. PROPERTIES OF SPICES AND HERBS A. Antimicrobial Properties of Spices B. Antioxidants and Spices XI.

SPICE USAGE IN MEATS A. Use of Spices in Meats B. Seasoning Blends C. Basic Meat Blends D. Commonly Used Spices in Meats E. Measurement of Meat for Formulations F. Meat Seasonings and Cures G. Brine Solutions

XII. REGULATIONS AND RECORD KEEPING A. Formulations and Record Keeping B. Regulations of Seasonings and Natural Flavorings XIII. MARKETING AND CONSUMERS OF SPICES A. Marketing of Meat Seasonings B. Consumer Loyalty to Spices XIV.

SUMMARY REFERENCES

I. INTRODUCTION Spices are defined as the aromatic or fragrant vegetable product used for flavoring, seasoning, or imparting aroma to foods; the term applies to the product in the whole or ground form. Herbs are soft-stemmed plants from which the leaves and flowering tops are used in both fresh and dried forms for the seasoning of foods. Spices and herbs come from the following parts of aromatic plants: fruits (capsicum, black pepper), seeds (aniseed, caraway, celery, coriander, cumin, fennel, fenugreek, mustard), rhizomes, roots (ginger, turmeric), leaves (bay, marjoram, parsley, sage, thyme), barks (cinnamon and cassia), floral parts (saffron, cloves), or bulbs (onion, garlic). Spices and herbs are grown in different parts of the world and can be separated according to quality grade. Although spices are cultivated in most of the tropical countries, production for commercial use is confined to only a few regions. A comprehensive nontechnical reference of spices is provided by Rosengarten (16), who gives interesting and reliable information on the history of spices from 2600 BC to the present. This reference is enjoyable to read as well as informative. The fascinating history of spices is a revelation of adventure, excitement, exploration, discovery, and much more. Herbs and spices have been associated with many different religions of the world. Herbs were most notably associated with the Romans and the Greeks. Due to improved worldwide communications over the past decade, the herb and spice map of the world has become more widespread, with the new origins challenging the old ones and producing quality products at lower prices.

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Today, spices are used primarily as condiments in cooking and flavoring foods and beverages, to provide medical use and advantages, to color cosmetics, and to provide aroma to perfumes. These are just a few of the many uses of spices. II. HISTORY OF SPICES A. A Brief History of Spices Spice is a magical word. Names that imply adventure and romance such as Zanzibar, Ceylon, and Java are used to describe varieties of spices that we know and use consistently. It is often the allure of these names or varieties of spices that entice us into flavoring our foods, perfumes, or other concoctions with such exotic additions. The term spice is derived from the Latin word species, which means “fruits of the earth.” Spices were sought after as zealously as gold and were accepted as currency in the late 13th century. Spices inspired nations to compete globally with fervor in search of new trade routes and even to go to war. The search for spices actually was behind the discovery of new continents and the merging of Eastern and Western civilizations (16). The story of spices reaches down through the ages and into the civilizations of the earliest times. The history of spices would engulf a complete volume as a single topic. It will be addressed only briefly in this chapter. Spices have played a major role in shaping world history. They were used in a host of applications, including ingredients of incense, embalming preservatives, perfumes, cosmetics, and medicines. They were also found to change insipid food into more palatable food and were used to preserve some foods. The early uses of spices can be traced back to Egypt, China, Mesopotamia in the Tigris and Euphrates Valley, and India, and to the ancient Romans and Greeks (16). Spices were a valuable commodity, and as a result, the spice trade was disputed over. Spices were used as money and for payment of debt in historical times. Therefore, if a country possessed all of a certain spice, or controlled the cultivation and distribution of such a spice, then there was much power to be had and used. The story of spices and the spice trade is complete with battles, romance, power struggles, conquests, and much more mystery and suspense. It is indeed a blueprint of world history. To study spices is to study the history of the world from the very beginnings of civilization to the present day. B. The Spice Industry Today Toward the end of the 19th century and the beginning of the 20th, immigrants from Europe and Asia began entering the United States in enormous waves. Each group of immigrants brought with them their own unique culture, ethnic food preferences, and habits. Usually, the newcomers continued their traditional culinary habits for one or more generations. To satisfy these demands, they brought with them spices and herbs from the Old Country, many of which were unknown in their new land. These unknown spices and herbs created another virtually unstoppable trading explosion. Due to this explosion in demand for spices, dominance in the ancient spice trade shifted to the United States. Today, the heart of the spice trade is at Wall Street in New York City. Western ports such as San Francisco and Los Angeles also have substantial volumes of spices entering their ports (17). Today, spices account for less that 0.1% of world trade. However, there are countries that still rely heavily on the spice trade. In Tanzania, the production of clove accounts for a large percentage of that country’s economy. Tanzania grows about two-thirds of the

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world’s requirement for cloves. About half of Grenada’s revenue is derived from the sales of nutmeg and mace. Vanilla, which technically is not a spice, is the second largest product in the Malagasy Republic (14). There are 36 different herbs and spices that are generally recognized and commonly used. White and black pepper account for the largest amount of spices used in terms of monetary value. The next most valuable spices consumed are cloves, nutmeg, cardamom, cinnamon, ginger, mace, and allspice, respectively. It is difficult to track an accurate figure for the value of spice imports and exports, because typically spices are not recorded separately from other food products (14). Spices shipped into the United States are usually in the whole form. Spice extracts, the oils and oleoresins, are also imported in large quantities. Imported spices are inspected for wholesomeness and cleanliness by the United States Food and Drug Administration inspectors on the docks. The spices that pass inspection are shipped to the spice “houses,” or companies dealing with spices throughout the country. The spice companies further process the product by cleaning, grinding, extracting, compounding, blending, or repackaging into smaller containers for retail distribution (4). Most of the tropical spices still come from the Eastern Hemisphere, as they have for centuries, but Central and South America, as well as the West Indies, are now supplying high-quality spices to the world market. Large spice plantations have been established in the Americas. Guatemala is known for its quality cardamom, and Grenada produces the finest nutmegs and mace, whereas Brazil produces select black pepper. According to monetary value, Indonesia is a leading producer of spices. However, it can no longer supply the world’s markets with large amounts of cloves as it did in the past. Cloves must be imported from China, Zanzibar, and the Malagasy Republic. This short fall is due to the Indonesian people’s most popular cigarette, which contains one-third ground cloves and two-thirds tobacco (14). Black and white pepper are by far the leading types of imported spices, followed by mustard seed, capsicum, cassia, paprika, coriander, ginger, and oregano. These nine spices represent about 75% of the weight and 63% of the monetary value of the imported spices. The “hot” spices, comprising mustard, pepper capsicum, and ginger, account for 80% of this tonnage and 70% of the value for these nine spices (4). The United States has been involved in the spice trade since the end of the 18th century. It is currently the largest importer in the world. A contributing factor to the Western world’s increasing interest in new flavoring agents is the growing anxiety about the toxicity of synthetic food additives. People have become more interested in new flavors, colors, and texturizing agents that are of natural origin. Today’s consumer is looking for foods with less salt, sugar, and saturated fats, with more emphasis being placed on good nutrition. Americans who have been placed on boring, low-sodium and cholesterol-controlled diets can have flavor in their diet with the use of fresh, high-quality spices or spicy relishes (14). III. SPICES AND HERBS A. Definitions and Differences Spices are aromatic vegetable substances that have been dried. The term spice is used to describe all dried plant products that include “true spices,” herbs, aromatic seeds, and dehydrated vegetables. Spices are natural products and have variations in flavor, strength,

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and quality because of climactic conditions. True spices such as allspice, ginger, nutmeg, and pepper all come from tropical plants. They may represent different parts of the plant, including the root, bud, bark, flower, or fruit. Herbs are often confused with the true spices. Herbs are the leaves of plants grown in both temperate and tropical zones. They are relatively low in oil content. True spices have an oil content that is somewhat high. Examples of herbs include marjoram, sage, and thyme. Aromatic seeds come from plants grown in temperate and tropical areas. Examples of seeds are cumin, anise, and fennel. The word “herb” comes from the Latin herba and means medical plant. The definition of an herb is a nonlasting plant that withers after it blooms without the stems becoming woody. Some edible components can be categorized as spices if the poisonous element can be neutralized. An herb is classified botanically as a perennial plant, but the meaning of a spice is derived from its use in cooking, not its plant classification. A spice should be edible. There are no definitions that clearly distinguish between the meaning of a spice and an herb. A spice can broadly be defined as a compound having a pungent flavor or coloring activity, or one that increases the appetite or enhances the digestion of food. A spice is obtained from buds, leaves, bark, seeds, berries, and roots that mainly grow in tropical and temperate areas. A spice can be explained as follows: Many plants used for spice are grown in tropical or temperate areas. The whole plant is not used as a spice. Only parts of the plant are effective. The effect of a spice is generally distinguished by its stimulating flavor. Herbs and spices are aromatic vegetable materials that enhance the savory characteristics of food and stimulate the appetite. They can be used in almost any food. The use of specific herbs or spices is often indicative of a certain geographical or ethnic origin. For example, oregano and basil are generally found in Italian foods, and cumin and red pepper are popular spices used in Mexican dishes. Herbs and spices are usually distinguished by the fact that herbs are mild and are usually used for delicate flavoring. They are considered to be better when they are fresh. Spices, on the other hand, are more pungent and give a stronger flavor to the food. Herbs are fragrant plants of which the flowers, leaves, stems, seeds, and roots are used to flavor foods. Spices are mostly grown in the tropics and are dried parts of aromatic plants. They include flowers, bark, leaves, seeds, and roots (18). Spices are divided into categories according to the type of flavor and color they give to foods. There are five groups. Hot and pungent spices are cayenne pepper, ginger, horseradish, leek, and onion. The aromatic group contains bay, cinnamon, clove, nutmeg, pimento (allspice), and mace. Herbaceous spices are anise, basil, caraway, cumin, dill, laurel leaf, rosemary, sage, marjoram, thyme, and tarragon. The final category is the spices used for coloring which are paprika, saffron, and turmeric. B. Spices and Herbs in General Individual spices and herbs include the following: Allspice, anise, basil, bay leaves, caraway seed, cardamom, celery seed, chervil, cinnamon, cloves, coriander, cumin seed, dill seed, fennel seed, fenugreek, ginger, horseradish, mace, marjoram, mustard flour, nutmeg, oregano, paprika, parsley, pepper (black), pepper (white), pepper (red), rosemary, saffron, sage, savory, star anise seed, tarragon, thyme, turmeric.

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1. Allspice Allspice is the dried unripe berry of a tree found in Jamaica, Mexico, and Honduras. It gets its name from the fact that it tastes like a blend of cinnamon, nutmeg, and cloves. It is used whole as a pickling spice. In the ground form it is used in desserts and in curry powder. It is used in sausage and meat blends throughout the world. 2. Anise Anise is an annual herb in the parsley family. It has a licorice flavor, and anise oil is used widely in beverages, baked goods, soups, and select sausages. 3. Basil Basil is an herb and is part of the mint family (16). It is used with tomatoes, salads, casseroles, sauces, sausages, and mixed herb blends. 4. Bay Leaves Bay leaves are grown in many geographical locations. The bay tree is indigenous to the eastern Mediterranean. The leaves can be used whole, cut, ground, or as bay leaf oil. It is used in stews, marinades, pickling spice, meats, and vegetables. 5. Caraway Seed Caraway seed is the fruit of an herb in the parsley family (18). It is used whole in rye bread, cheese, cakes, sauces, meats and canned goods. Its main flavoring constituent is carvone. 6. Cardamom Cardamom grows at high altitudes in the tropical forests of India, Guatemala, and Sri Lanka. Its flavor is a combination of sweet, pungent, and aromatic. It is used in curry powders, coffee, and baked goods. 7.

Celery Seed

Celery seed is derived from a plant that is a part of the parsley family (18). The seeds are very small, but they have a very bitter taste. They are used whole in seasonings, or the ground form is mixed with salt to produce celery salt. It is used in pickles, salads, soups, and sauces. 8. Chervil Chervil is an herb that is quite similar to parsley in the shape of the leaf, but the taste is sweeter and more aromatic. It is mostly used as a specialty herb. 9. Cinnamon Cinnamon comes from the peeled bark of a tropical tree. The bark is rolled into quills to form cinnamon sticks. It has a very fine flavor and is commonly used in desserts and wines, bread, cakes, and select meats. Cinnamon is grown in Sri Lanka, the Seychelles and Madagascar (13). Cinnamon provides antimicrobial, preservation, and texturizing properties to select foods. 10. Cloves Cloves are the dried unopened flowers of a tropical tree. They are used in the popular Indonesian cigarette known as a kretek (18). They are very strong in flavor. They are used to

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stud hams when baked and also as an ingredient in curry powders. Cloves are grown in Sri Lanka, Madagascar, Zanzibar, Brazil, and Indonesia. Extractives of cloves are used in many meat and sausage formulations. 11. Coriander The coriander plant produces a spice seed and an herb. The herb is known as cilantro and has a bitter, soapy flavor. It is used in Mexican sauces and yogurt. The spice seed is used in spice mixes, seasonings, pickling spices and select meat formulations. Coriander is primarily grown in Morocco, Egypt, Pakistan, and India. 12. Cumin Seed Cumin seed is the seed of an herb in the parsley family (18). It is similar to the caraway seed, but it has a lighter color and a more uniform size. It is a common ingredient in curry and chili powders. Cumin is used to flavor meat, sausages, casseroles, and soups. Cumin is grown in China, Iran, Turkey, and India. 13. Dill Seed Dill seed comes from the dill plant, which also yields dill weed. The seed is taken from mature seeded flower heads and is used in making dill pickles, mixed spice blends, pastries, sauces, and as a flavorful complement to fish. 14. Fennel Seed Fennel seed is the dried fruit of the fennel plant. It is indigenous to southern Europe and the Mediterranean countries (13). The seed has a green color, and it has a licorice flavor. Fennel seed is used in fish seasonings and select sausages, such as Italian sausage and pepperoni. 15. Fenugreek Fenugreek is the seed of an annual herb, which is a part of the pea and bean family (13). It is better in taste and is used as an ingredient in curry powders. It is often used for flavoring syrups and related condiments. Fenugreek is cultivated in North Africa, India, and the Mediterranean countries. 16. Ginger Ginger is the rhizome of the plant Zingiber officinale (13). It is both sweet and savory, and it has a hot and “lemony” taste. It is used in curry powders, sweet dishes, and many Asian foods. Ginger is harvested widely in the tropics. 17. Horseradish Horseradish is the root of a plant in the mustard family (13). Its flavor is sharp and pungent, and it is often used as a condiment for cold meats, such as roast beef. 18. Mace Nutmeg and mace are produced from the same tree. Mace is the reddish colored, membranous tissue that surrounds the nutmeg. The membranous tissue is dried before use in foods. Mace and nutmeg are used in baked goods, savory foods, select sausages and meats.

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19. Marjoram Marjoram is an herb related to oregano. It is primarily cultivated in Egypt, but it also grown in Eastern Europe. It is sold as rubbed, sifted, or ground. Marjoram is mainly used to flavor meats, casseroles, soups, and as a pizza herb. 20. Mustard Flour Mustard flour is a blend of finely ground endosperms from the seeds of the mustard plant (13). It is used in seafood cocktail sauces, barbecue sauces, hams, meat loaves, salad dressings, and many other dishes (4). 21. Nutmeg Nutmeg comes from the same tree as mace. It is grown in Indonesia and Grenada. It has a warm, aromatic flavor, and it is used in cakes, select processed meats, curry blends, and many other dishes. 22. Oregano Oregano is indigenous to the Mediterranean region, particularly Greece, Italy, and Spain. It is cultivated in Albania, France, Mexico, Turkey, Yugoslavia, and the Mediterranean region. Oregano is part of the mint family. The leaf color is grayish-green and the flowers are purple. Thymol and carvacrol are the primary flavoring ingredients of oregano (3). 23. Paprika Paprika is mainly used as a coloring agent. It has a reddish color and is obtained from the dried pods of select capsicums. It has a mild flavor, and it is popular in Germany, where it is used to give a red color to sausages. It is one of the most widely used spices because it can be used for both added flavor and color. It is available in many different red, orange, and yellow colors. 24. Parsley Parsley comes in many forms: curly leaf, flat leaf, celery leaf, and Hamburg parsley. Flat leaf and curly leaf are the most widely used. The flavor of parsley is very delicate, and it is usually used to give color and appeal to foods. It is used in mixed herb blends, and it is a source of vitamin C and iodine (13). 25. Pepper, Black Black pepper is the dried, green, unripe fruit of a tropical vine. It is strong in flavor and can be used with many foods. It is grown mainly in India, Indonesia, and Brazil. The whole, crushed, ground and extractive forms are widely used in meats, sausages, and many other kinds of food. It is the most popular and widely used spice. 26. Pepper, White White pepper comes from the same vine as black pepper. The berries are picked just before they ripen, then soaked in water, and dried. White pepper is used in foods in which black pepper specks are undesirable. White pepper and extractives of white pepper are widely used in meats.

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27. Pepper, Red Red pepper is grown in Central and South America. The flavor is very pungent and almost overwhelmingly hot. The effect lingers in the mouth and throat. Red pepper is used in many Mexican, Indian, and Italian foods such as hot sauce, pickles, meats, and pizza (4). Red pepper is widely used throughout the world. 28. Rosemary Rosemary is a shrub that is grown in the Mediterranean region, mostly in coastal regions. It is widely used with lamb and in stews, soups, and casseroles. The leaves are similar to pine needles and are not easy to manage in the whole form because of their cylindrical needle-like shape. Rosemary is widely known for its antioxidant properties and because of that is widely used in the meat industry. 29. Saffron Saffron is the dried stigma of the flowering saffron crocus. It is the most expensive of all the spices. To produce 1 kg of saffron takes 70,000 flowers. It is grown mostly in Spain, Greece, Iran, and India. Saffron has a bitter flavor and a yellow color (3). 30. Sage Sage is an evergreen shrub that is a part of the mint family grown in the Mediterranean region. It is used with pork, poultry, sausages, canned foods, and prepared meats. It is the principal flavoring component of fresh pork sausage (13). 31. Savory Savory is an herb that is similar to thyme. It is used in herb blends, and with legumes to add flavor. 32. Star Anise Seed Star anise seed is the seed of the star-shaped fruit of a small tree that is indigenous to southwestern China. Its flavor is similar to anise, but is more subtle. It is widely used in Chinese foods (3). 33. Tarragon Tarragon is a perennial herb indigenous to Europe and western Asia. The two types of tarragon are Russian and French tarragon. French tarragon is the most widely used. It has a flavor that is similar to aniseed. It is used with chicken, herb blends, rubs, soups, and many other foods (3). 34. Thyme Thyme is a shrub-like plant that grows wild in Mediterranean countries. It is used in savory foods, as an ingredient in poultry stuffing blends, and in herbal seasoning blends. 35. Turmeric Turmeric is a dried rhizome. It has a yellow color, and a slight earthy flavor. Turmeric is used in curry powder, and to color prepared mustard, rice, breadcrumbs, and many other foods. It is grown in many countries, but the primary producer is India.

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C. Peppers in General Peppers are used as a condiment, spice, or vegetable. There are two families of peppers: vine peppers and capsicum (red) peppers. Vine peppers are grown in tropical areas and yield black pepper, white pepper, and long pepper. The different kinds of pepper vary in aroma, size, pungency, and peppercorn color. They are often named for their country of origin. The quality of pepper is often distinguished by the price, although sometimes the price is only an indicator of crop yield or availability (18). Black pepper is used in most countries in basic cooking. It contains piperine, a compound that stimulates the flow of gastric juices. The flavor is the result of a nonvolatile resinous substance. The pepper tastes strongest when it is freshly ground, but usually, preground pepper is used in seasonings for convenience. White pepper comes from the same plant as black pepper, but the berries are picked ripe instead of green. White pepper is used mainly in dishes in which black pepper specks are undesirable (18). Capsicum peppers are in the same family as chili peppers, red peppers, sweet bell peppers, cayenne peppers, and paprika. The peppers differ greatly in size and degree of pungency. Chilies and red pepper are used mostly in foods of South India, Mexico, and Asia. Cayenne pepper is very hot and ranges in color from orange to red. Capsicum peppers are known to have a very hot bite, sometimes to the point of being overwhelming. Chili powder is a blend of several peppers and spices. Paprika is usually used for its red color. Sweet bell peppers are larger than chilies and they have no bite. They are mostly used as a vegetable or salad fruit (18). D. Heat in Red Peppers The pungent principles of red pepper are capsaicinoid compounds. The major pungent compound contained in red pepper is capsaicin. The degree of pungency depends on the species, the region in which the harvest was grown, and the geographical and climatic conditions. The pungency of the pepper is also dependent on the maturity of the plant from which it was harvested. Taking into consideration the variables mentioned regarding red pepper, it is usually checked quantitatively to determine the heat units (7). To measure the degree of pungency of red pepper, the Scoville heat value method is used. This method measures pungency by determining a distinct pungent sensation. This is done based on an extraction process in an alcohol system. Dilutions of the alcohol extracts are made and presented to a trained panel, usually containing not less than five expert panelists. When three of the five expert panelists detect heat, the heat level is calculated based on the dilution from which the heat was determined. In spite of this tests’ originating date, early in the twentieth century, it is still one of the best heat measuring techniques used today. It is an official method of the American Spice Trade Association, ASTA. It is a simple test in terms of equipment and training, but does have limitations. It requires the use of a trained panel to measure the heat level of the pepper. The panel must be trained and adjusted frequently to maintain accuracy. This can be time consuming for the technical experts involved. The Scoville heat values for red peppers available for industrial means are usually 20,000, 30,000, 40,000, and 60,000. The Scoville heat values for red peppers range from 10,000 to 120,000. To achieve different heat levels, the defined heat units are blended, usually with paprika, to the desired level of pungency. The extracts of red pepper, oleoresins, have a very concentrated pungency. The oleoresins are available from 200,000 to 1,000,000 Scoville heat units. They are extracted in

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a very concentrated form and are blended down to desired level of heat. Oleoresins can be blended with oils and emulsifying agents. The oleoresin can be decolorized or blended with paprika extracts to achieve a desired color (17). There are several other methods used for measuring the pungency of red peppers. There are colorimetric methods that measure the blue color generated by the interaction of certain test compounds with capsaicin. There are also ultraviolet methods used. One UV method in particular was compared with the Scoville method, and a very close correlation between these two methods was observed (10,17). Many methods based on gas chromatography have been developed as well as high-performance liquid chromatography (HPLC) methods. One HPLC method has been adopted as an ASTA official method. It has been observed that the HPLC methods produce greater accuracy due to their high resolution, especially for capsaicin analysis. However, all of the mentioned methods have the disadvantage of being time consuming because of the extraction process involved (8,17). IV. SPICE BLENDS Spices are often blended together to provide certain characteristics to products. Rarely are they used singularly to flavor foods. Most spices and spice extractives are very potent. In fact, small amounts of spices are sufficient to flavor hundreds of pounds of seasoned meat. The aromatic and pungent aspects that make spices valuable are found in their volatile oils and oleoresins. Seasoning and flavoring foods with spices is a very complicated process that demands much skill and experience. The blending of spices and the careful addition of the spice blend to the food at the proper stage is a necessity. The blend should be compatible with the food that is to be seasoned. The blend should not be dominated by one spice. Bitter and astringent flavors should be suppressed. The production of a well-balanced seasoning for a processed food is a difficult art (9). Seasoning manufacturers have many different seasoning combinations in their library of blends. In general, seasonings may be divided into several categories: ground spice seasoning, ground spices and whole or crushed herbs, whole or crushed herbs, soluble spice seasoning, or a combination of all. Spices are used to give flavor to many different kinds of food either separately or in a combination as a seasoning containing sugar, salt, or other ingredients. Spice seasonings and condiments are complex blends. There are many different definitions of spice seasonings. Spice seasonings can be added to different kinds of foods and can improve the flavor or enhance preference for food. A seasoning is an herb, spice, or salt blend that improves the food flavor. The spice seasoning can also be defined as a mixture that contains one or more additional spices or spice extracts that improves the flavor of the original food. It is added at processing or during cooking. The aromatic ingredients of spices improve the flavor of foods. Some spices are warming and aromatic, with a tangy bitterness that gives a clean taste. Other spices possess a subtle flavor that stimulates the appetite. Some spices have extreme flavor and are very pungent, and strong in aroma. Others have little or no pungency. Today, many companies offer blended spices to the food industry. Natural and synthetic spiced foods and other flavors in industrial quantities can now be produced. Companies sell essences, emulsions, solubles, dispersed spices, encapsulated spices, spices in fats, and the list goes on. Tailor-made blends of spices are also offered for products such as canned meat, sauces, other meat products, and vegetables. Chili powder, curry powder, and pickling spice are examples of spice blends. These blends, which are considered seasonings by

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consumers, are different from the industrial product called seasonings. Industrial seasonings contain one or more spices or spice extracts in addition to many other dissimilar ingredients. They are added during the manufacturing or processing of food and are distinguished from condiments added after the food is served. Industrial seasonings are used widely in meat products such as bologna, frankfurters, sausage, soups, dry gravy mixes, instant sauces, and salad dressings. A spice blender must understand the physical and chemical aspects of powders and liquids and also what effect the different processing methods have on the blends. The technologist specialized in the compounding of seasonings should also have knowledge of governmental regulations that apply to the regions where the seasonings are produced and consumed. The ingredients in the seasonings are not always compatible with each other. The storage temperature, moisture, chemical composition, light, and oxygen should all be considered when developing a seasoning blend (18). The compounding of seasonings is a specialized skill. Dissimilar components such as salt, dairy products, emulsifiers, and preservatives must be added to the complex seasoning mixture, and this process requires a great deal of expertise and experience. The compounding specialist must be aware of restrictions and have a sense of economics when selecting the ingredients. A seasoning can be a single ingredient or a blend of several ingredients. Seasonings may also contain ingredients that have no effect on the food flavor but provide advantages such as anti-microbial preservation, physical stability, or nutritional value. These ingredients are referred to as additives. Spices used in seasonings are available in several forms. A spice extractive is sometimes preferred when extra strength is necessary, or to prevent spice particulates from entering a uniform blend. A plated spice which is an oleoresin or essential oil that is coated on the surface of a sugar or salt carrier might be used for rapid flavor impact and low cost. If the compounding specialist is trying to achieve flavor retention and slow release, a more expensive, more stable encapsulate might be used. A spice blend must be compounded in such a manner that it enhances the natural flavor of the product in which it is incorporated. The seasoning, or spice blend, should never be overwhelming or diminishing. It should blend and round the flavor without having an aftertaste. A. Spice Usage in Foods Spices can be used by themselves or in combination with other spices to enhance the flavor and to color foods. When formulating a blend, it is best to start with low levels of spices, and to build the flavor gradually. The flavor of spices will increase in foods that are kept frozen before use. Spice levels have to be decreased, therefore, to obtain the desired level of flavor (18). Not only are spices used to give flavor to foods, but they also enhance the latent flavors of food. Because of the low sodium content of spices, they are often used as salt substitutes. Some spices contain antioxidants and are regarded as beneficial. Spices such as paprika, turmeric, and saffron can be used as colors in foods and are referred to as spices and colorings. The yellow color of mustard, the red-brown color of barbecue sauce, and the orange-red color of Spanish rice all come from these spices (9). By using the appropriate method of cooking, the coloring effect of spices can be manipulated. Dissolving a spice is the easiest way to use it as coloring. The spice can be used as it is, or by crushing it into a

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paste. The tone of a spice’s color changes according to how it is dissolved in water or oil. For example, acidifying the water can change a spice’s coloration. Spices have different effects when used in foods. They not only give flavor, pungency, and color but also have antioxidant, antimicrobial, pharmaceutical, and nutritional properties. In addition, they have complex or secondary effects. These effects include reduction of salt, reduction of sugar, and improved texture for some foods. As an example of texture improvement, cinnamon will be discussed. Cinnamon has strong antimicrobial activity. It is sometimes used in making bread. Cinnamon adds flavor and also suppresses yeast activity to avoid too much fermentation or rise of the bread. The flavor is a direct effect, and the texture improvement is secondary (18). When cooking or using spices in processed foods, the objectives should be clear. There are four categories summarizing the basic effects of spices. The categories are flavoring, pungency, coloring, and deodorizing/masking. The deodorizing/masking category may actually overlap some of the others, but it is most often desired on its own. Each spice performs at least one of these (18). V. INDUSTRIAL SPICES AND HERBS A. Indigenous and Cultivated Spices Two terms commonly used when referring to spices are indigenous and cultivated. Indigenous refers to the presumed country of origin or where they grow naturally. Cultivated refers to a situation where the spices have been adapted to the climatic and growing conditions of a certain area, and the growth is controlled. Some cultivation practices may modify the plants and in some cases, the plant may even lose its desirable attributes. However, many modern cultivation techniques actually improve flavor and yields. B. Fresh versus Dried Spices and Herbs Herbs and spices are generally cut and dried in the sun or shade. Dried herbs are then sold to a processor who takes the leaves from the stalks. This process is known as sifting, and “rubbing” is the process of sieving to remove stalks and rocks. The best form in which to use herbs is considered by most to be freshly cut. This, however, gives a different end result when compared to dried herbs. These give a more concentrated flavor. In seasoning formulas, most compounding specialists tend to use dried herbs in either the rubbed, sifted, or ground form. Herbs also yield essential oils, which can be used in this way or as oleoresins (18). There are noticeable differences between dried and fresh spices. Drying changes the chemical composition of the spice and thus affects the smell and taste. Although it would be preferable to use fresh spices, these generally are not available. Therefore, dried spices are used commercially. Drying is the most important processing step in post-harvest technology. The objective of drying is to decrease the level of moisture naturally present at the time of harvest to a safe limit. This will decrease the chances of insect and mold infestation. In the producing and developing countries, sun drying is mainly used, although this does promote contamination by microorganisms from the soil. The quality and flavor of sun-dried spices is not usually uniform. When the spices are dried on raised platforms designed to expose the products to the sun, chances for soil contamination are considerably reduced. On the raised platforms, drying is more uniform because of the air passing through the bottom and sides. Mechanical dryers have many more advantages to the platforms.

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They avoid dependence on weather and reduce contamination by microbes and reduce the drying time (18). C. Whole and Ground Spices Whole spices give flavor and aroma, as well as visual and textural effects, that are not attainable by extractives. Usually, the spice’s intact cellular structure and the natural antioxidants present protect important flavoring from volatilization and oxidation. Many whole spices may release flavor too slowly to be of value. Ground spices, on the other hand, are milled to the degree of fineness desired by the processor. When they are compared to the whole spices, they can be incorporated more uniformly into the blend. They also release their flavor more readily because the cells that contain the flavor have been ruptured during the milling process. In spite of these attributes, the finely ground spices have a limited shelf life. They are subject to oxidation, loss of flavor, and degradation during storage. It is recommended that they be stored for no longer than 3 months in manufacturing practices. In industrial applications, ground spices are generally used to provide a pleasing appearance and they are often supplemented with essential oils and oleoresins (17). Spices are available in several forms. Besides the form they occur in naturally, whole or ground spices can be used as spice oil, oleoresins, oleoresin on salt or other carriers, or encapsulated. There are cases where one or more can be used, and there are many instances where one form is preferred over another due to the nature of the desired use. D. Spice Grinding Spice grinding is based on a very simple process. There are many different mills used to grind spices and they are generally designed to crush the spice particles. First, the grinding ruptures many of the glands, or cells, in the spice that contain the volatile oil and this frees the oil for reaction or evaporation. This rupturing presents a problem in grinding. In addition to the volatile oil being exposed, the grinding also generates heat. This heat tends to vaporize this oil leading to a reduction in the strength of the flavor. Most spice mills are designed to pass the spice through quickly and also to minimize the buildup of heat. The choice of mill is often determined by the temperature rise during processing (17). A few processors use liquid nitrogen to keep the temperature low and minimize the amount of oil lost. Although this process is not widespread, there is some value in its use. By freezing the spice and solidifying the oils, the spices grind and sift easier. They also shatter when subjected to a milling operation. Cryogenic grinding retains more of the flavor normally lost in other processes. Cryogenically ground spices contain more volatile components, and the grinding minimizes oxidative deterioration of flavors because of the nitrogen blanket during grinding. A spice ground cryogenically may have a flavor profile that is different from the usual. Since the product has more flavor, a smaller amount of spice can be used to achieve the same level of flavor (17). VI. SPICE QUALITY AND STANDARDS A. Spice Quality and Evaluation Organoleptic properties are the attributes of food that are perceived by the senses; they are difficult to quantify objectively. Flavor, aroma, color, and texture are important organoleptic properties for they are critical in determining the acceptability of foods to consumers and also in promoting repeat purchases. Selecting and combining ingredients for a seasoning

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blend should be controlled to ensure the greatest degree of consistency possible. To ensure that this happens, a detailed quality assurance program is necessary. This requires communication between the ingredient vendor, seasoning manufacturer, and the consumer (18). The seasoning manufacturer should understand as much as possible about the end use of the seasoning. Once the formula is approved by the end user, the seasoning manufacturer must make sure that no deviations are made in the process. Organoleptic testing is generally used to verify the conformity of the seasoning to specification (1). These tests include flavor, taste, texture, aroma, and overall mouthfeel sensation when the product is consumed. The seasonings may be tasted as is, or in a liquid slurry form. A sample of the batch is also kept to compare to future batches to check for consistency. The standard will degrade over time, so it is necessary to obtain a fresh sample occasionally based on the customer’s needs (18). Sensory evaluation of spices, spice oils, and oleoresins initially presented great difficulty. Many of the first oils and oleoresins were very flammable and not easy to work with. They were very viscous and often contained undesirable components. Sensory evaluation was also in its infancy, and desirable testing methods had not yet been devised. Today, many techniques are used to test spice oils and oleoresins. Most, however, are dependent on sophisticated equipment. Sensory panels are used, but when testing oils and oleoresins versus natural spices, it should be kept in mind that the flavor effect of the oils and oleoresins is immediate and complete whereas the flavor cells of the spice are not always foremost (18). B. Spice Standards Standards for spices are now being regulated. The two most important considerations are cleanliness and flavor quality. No molds or insects should be found in the spices, and little or no extraneous or defective material should be found. Irradiation is sometimes used to sterilize spices, but it is not allowed in many countries. These standards are being discussed at national and international levels. Microbial standards for spices are important because it is a matter of cleanliness and hygiene. However, because spices are agricultural products, they will inevitably be exposed to contamination by microorganisms at different stages like any other agricultural product. Because of this, it is hard to decide on microbiological standards. The pathogenic bacteria Bacillus cereus and Clostridium perfringens and the toxic molds Aspergilllus flavus and the Penicillium citrium group and Salmonella are all found on varying spices. Many nations are sterilizing spices using agents such as ethylene or propylene oxide mixed with carbon dioxide. Using spice extractives overcomes the defect, but in many cases, a ground spice is preferred. Also, spices must be processed in hygienic conditions (9). Some spices, unless given antimicrobial treatments such as irradiation, may contain microorganism counts as high as 106–7 per gram. The most important are mold spores. Spices may also harbor toxins. Even though spices are used in small amounts, they can be the source of spoilage and pathogenic microorganisms found in food (9). VII.

OILS, OLEORESINS, AND EXTRACTS

A. Spice Oils and Oleoresins The aspects that make spices valuable are the volatile oils and oleoresins. By using different types of flavoring extracts and spices, the food industry shows value and versatility in

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many ways. By themselves, spices are not very convenient to use in food because they do not yield their flavors to the food readily. Finely ground spices have many disadvantages, such as variable flavor strength and quality, loss of flavor during storage, and discoloration of food, and they are not easily handled in bulk sizes. Because of these disadvantages, many natural spices have been replaced by the appropriate counterpart of essential oils, oleoresins, encapsulated spices, dispersed spices, emulsions, and essences. An oleoresin consists of essential oil, organically soluble resins, and other related materials found in the natural spice. Nonvolatile fatty acids are also present. Spice seeds yield more fatty oils than spice constituents from other parts of the plant. Oleoresins are usually very viscous and often have to be thinned with propylene glycol to obtain flowability. Many flavor companies offer oleoresins that are standardized with mono-, di-, and triglycerides, lecithin, and lactic acid, which are easily dispersible. These oleoresins may be added directly to the formulated blend and do not have to be dispersed onto an edible carrier. An essence is defined as an extraction that is prepared by macerating the ground spice with 70% ethanol. An emulsion is a liquid seasoning prepared by emulsification of essential oils and/or oleoresin with gum arabic or other emulsifiers. Heat-resistant spices are oleoresins and/or essential oils that have been encapsulated with a water-insoluble coating, which extends the product’s shelf life and renders it suitable for baking applications. Fatbased spices are essential oils and/or oleoresins blended with a liquid edible oil or hydrogenated fat, and sold either bulk or as a spray-cooled, fat-encapsulated spice (2). Spice oils are the principal flavoring constituents of spices. Spices depend on their essential oil content for their characteristic aromatic profile (3). Spice oils are obtained by steam or water distillation of ground spices, and in some cases, whole spices. The debris of cellular material contains no flavor value and is left behind, yielding a concentrated flavor. Solvent extraction of ground spices and the removal of the solvent by distillation gives the dark-colored, viscous products known as oleoresins. These represent the total flavoring of the spices in very concentrated form. Spice essences, emulsions, and solubles make oils and oleoresins easier to use. Some countries do not permit the use of solvents, and some users object to their presence. Dispersed spices are produced by mixing bases, such as salt, sugar, flour products, or milk whey, with oils and oleoresins, which makes them more soluble and easier to disperse. Encapsulated spices are made by mixing spice oils and oleoresins with solutions of gums, gelatins, and so forth and are spray dried or precipitated to get flavors that are encapsulated. The processing cost is higher, so the flavors are usually made in tenfold concentration (2,18). B. Manufacture of Oils and Oleoresins There are three different methods used in the industry to capture the flavor and characteristics of spices to prepare all extracts, oleoresins, and essential oils. The first method is infusion or maceration. This method is quite similar to the brewing of tea. The spices are placed directly into a solvent, and they are then allowed to steep until the flavor has been extracted. The entire process can take as little as a day or as much as a month. The length of time depends on factors such as spice particle size, extracting solvent, temperature, and mechanical stirring. The second method is percolation. This method is quite easily described because it is exactly like the percolating of coffee. With spices, the percolator is a large tank. A solvent

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system is placed into the bottom of the tank, and the spices are put in a basket-like container at the top of the tank in cloth bags. The solvent, in the base of the percolator, is forced up into the basket containing the ground spice. The solvent is sprayed over the spices and is then allowed to drip back to the base of the pot to be percolated again and again, until all accumulated flavors have been extracted. The final method of capturing the flavor and characteristics of spices is distillation. The leaves, seeds, and peels are placed in a still and covered with a solvent system. The distillation occurs either atmospherically or under reduced pressure. The flavor can be soluble or insoluble in the distillate, depending on the solvent system. The distillates that result are colorless as compared those produced by with maceration or percolation, which retain the spice color (6). C. Flavor Quality of Natural Spices, Oils, Oleoresins, and Extracts Food and spice companies are now researching ways to preserve the consistency of spices and spice blends. Many different things can influence the abundance of the active components that are responsible for the natural flavor of the spices. In one year, varying climatic conditions can have a negative effect on the flavor of spices. Flavor quality is also affected by variety, geographical origin, processing methods, and storage conditions. Spices grown within the same country, but in an area that is geographically different, can vary in flavor and other characteristics. The food industry must be able to understand these differences and be able to adapt to them to maintain the flavor quality of the spice. The flavor quality is as important as the cleanliness of the spice, because the main purpose of the spice is to improve the flavor of the food. The spice oils and oleoresins, and other extractives, are standardized by their manufacturers to yield the same aroma and flavor of the named spice. Geographical region is also important. Manufacturers are different, and the products they produce are not the same as others. This could be because of the country of origin, or the spice itself. Manufacturers determine the spice equivalency of their liquid version to the ground spice. Spice equivalency of oils and extracts is defined as the number of pounds of oleoresin required to equal 100 lb of freshly ground spice in aroma and flavor characteristics. For a complete listing of spice equivalencies, refer to Farrell (3). In addition to looking for ways to improve flavor quality, spice producers and sellers are researching new ideas to increase the demand for spices. Already a new need has emerged in the field of spices and flavoring extracts. It is possible to formulate new beverages, snacks, fillings, and other similar products. Also, it should also be noted that spices have always been known to have some medicinal uses. New pharmacological properties may be discovered in the future. D. Natural Spices versus Oils and Oleoresins Natural spices are used when the “Old World” look is desired. They do have the disadvantage of causing some discoloration of fresh sausages, but if the discoloration is critical, extractives can be used. When using extractives, they must be handled and stored carefully because the flavors dissipate much more quickly than in natural spices. The spice extracts are much easier to control for quality purposes. The flavor intensity is also much easier to control when using extractives versus the whole or ground spice. If spice visibility is not a must in the product, it is at times more cost and quality effective to use the extractive version of the spice. Much less quantity of spice extractive is used in the final product and the

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quality is built into the product. The meat processor does not have to worry about foreign material or loss of flavor when using spice extractives. These things must be heavily taken into account when using the whole spice or ground spice. Fresh spices have slow flavor release in high-temperature processing. They are also easy to weigh and handle. It is easy to observe if the spice looks less fresh than it should. Also, there are few problems encountered when designing a label when working with fresh spices. However, the meat processor does have to worry about product storage with using real spices versus oils and oleoresins. Spices take up much more room and infestation can be a problem. The flavor quality can also vary from lot to lot of raw spices. This can cause problems with the end product. One of the biggest problems with using raw spices instead of the oils or oleoresins is concern with the microbial count. This is not a problem with the liquid counterparts. One drawback to using liquid spices is that the oils and oleoresins experience volatile loss under high-temperature processing conditions, unlike the raw spices. The water-soluble spice oils and oleoresins have a much lighter flavor, and often more must be used to achieve the same flavor effect. The encapsulated oils and oleoresins are very expensive in comparison with the other varieties and much more must be used to achieve the same flavor as an oil, oleoresin, or raw spice. VIII. STORAGE AND SHELF LIFE A. Spice Storage Spices should be stored in cool and dry conditions away from light. They should be packaged in an airtight container to avoid oxidation. Whole spices have a long shelf life, but when ground, the pungency and flavor can be lost. Ground spices that are stored longer than 6 months can have a major loss in pungency. Herbs will usually store for 6 months to a year. Glass is a good storage container because it is airtight and inert. Some spices will react chemically with other storage materials, causing an off-flavor to develop and degrade the spice. Glass containers should be stored in a cool, dry, and dark location. B. Spice Shelf Life Spices have a specific shelf life that is determined by their form, storage conditions, source, and age. Some spices have naturally occurring antioxidants. Others contain lipase and tannins that promote the degradation of color and oxidation. When purchasing spices, only the amount of spice that will be used should be bought. It is better to replenish spices as needed than to work with an old spice that has lost its flavor and color. Spices and extractives should be stored under cool and dry conditions. They should be protected from sunlight. This prevents caking and fading. It also keeps essential oils from volatizing. Spices used as colorings require even colder storage to retard the oxidation of the pigments. This also helps prevent infestation. All spice containers should be sealed tightly after use to prevent the loss of important flavor and aroma volatiles. Cryogenic milling of certain spices avoids the oxidation, volatilization, and enzymatic damage seen with the conventional techniques of milling. The higher volatile content of these spices provides a better shelf life and flavor perception for products that are low in fat and sugar. It also contributes to the development of flavors that are not damaged in the microwave and have a fresh taste. As the flavor industry becomes more market driven, it will most likely continue to take new approaches to new processing methods and flavor systems (18).

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C. Spices and Food Spoilage Contaminated meat is too often a problem. Spices are known for their contamination and filth. But, when one considers the areas from which they are cultivated and the feat it takes to get them around the world, contamination is to be expected. Bacteria counts may run into the millions per gram on many uncleaned, unsterilized spices. Once cleaned and sterilized, spices contribute in many remarkable ways to retard food spoilage. Recently, it has been found that common spices such as garlic and clove have been proven effective against some strains of E. coli (7). A major problem that is encountered when trying to use spices to stop food spoilage is finding the right mix between the taste of the food and the amounts of spices necessary to inhibit pathogens. The amounts of spices necessary range from one to ten percent. Research is being done regarding recommendations of spice usage levels for manufacturers and consumers. It should be noted that spices are not a substitute for the proper handling of food. While spices can lessen the amounts of E. coli in meat products, they do not totally eliminate the pathogen. This necessitates proper cooking methods. Meats should be cooked at about 160°F. Spices can be added as a protective measure and also to add flavor to the meat. Many people have studied the preservative action of spices and condiments on foods. Many spices have been found to exert a definite inhibiting action against microorganisms, but many others have no significant preservative action. It has been found that mustard, cinnamon, and cloves are among the most potent spices in slowing the growth of molds, yeasts, and bacteria (7). IX. CLEANING AND PROCESSING OF SPICES A. Cleaned and Uncleaned Spices In the United States, spices can be imported as raw uncleaned spice, uncleaned spice, or cleaned spice. The spices can be post-processed to reduce microbial counts. Many times, spice buyers observe only the prices and overlook that the spices are used as food items. There is a large market for spices that have been minimally cleaned directly after harvesting. A spice processor with an adequate spice cleaning facility can easily show the debris that has been collected from “cleaned spice.” Stones, rodent droppings, insects, nails, baling wire, dead rodents, and wood are examples of debris found in uncleaned spices. It is very easy for a spice supplier to transfer a spice from its original package to another form of packaging and say that the spice has been cleaned. Thus, it is an advantage to buy from a reputable spice house or supplier. Oftentimes, buyers may think they are getting a very good deal on the price per pound of a spice. But in actuality, buying some of those “good deals” could cost a company a great deal of money, especially if some of the debris that finds its way to a consumer causes harm. B. How Spices Are Cleaned There are different methods of spice cleaning, and each one makes use of a physical difference between the spice and the foreign material being removed. Most often the physical differences are shape and density. The closer the foreign material is in shape and density to the spice, the harder it is to remove. The cleaning of spices is an expensive process; thus, the spice processor must consider the cost of the cleaning equipment, the labor, and the loss of product that comes with the cleaning. It is impossible to guarantee that no foreign material is present in a batch of cleaned spice (17).

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Every spice cleaning system should have magnets in as many locations as possible. They are protection not only for the end users but also for the processor because magnetic material needs to be removed so it will not damage the milling equipment. There are many different types of magnets, and there is no one magnet that is perfect for every type of system. To be effective, the magnet must come very close to the metal and be designed so that the flow of spice over the magnet cannot brush the piece of metal back off the magnet and into the product again. The magnet must be cleaned often because they are designed to hold a certain amount of metal before the spice flow knocks the metal back into the spice (17). The cleaning operation is a basic use of sifters. Running the spice over a collection of screens, particles both larger and smaller than the spice can be removed. Although the principle sounds simple, the process is generally very difficult. Spices are not uniform in size. They are uneven oval seeds or pieces of leaves. Sifters are not usually used for cleaning. Instead, they are often used for sizing. If the harvester does any cleaning at all, it is usually not more than a simple sifting operation to remove large debris from the spice (17). The air table or gravity separator is the most versatile type of cleaning equipment for spices. A processor usually acquires this piece of machinery first and uses it most often. A wire mesh screen with a stream of air blowing up through it suspends the spice particles over the top of the screen. The lighter pieces are suspended higher than the heavier ones. The lightest pieces are actually blown out of the system. The screen is then tilted and the spice particles move to the bottom end of the screen. A rotational vibration is applied to the screen, causing the heavier particles to move up the screen. The lighter particles are not trapped, and they continue to move toward the lower end. This causes a separation based on size. The process is adjusted so that the cleaned spice migrates to the middle of the screen. The very heavy and very light particles are generally discarded as debris, and the middle particles are collected as clean spice. Often the clean spice is then run through the air table again for a second cleaning operation (17). De-stoners are similar to air tables, but they are generally much smaller. An air table is able to separate the product into many divisions, but a de-stoner removes only the heavier stones and rocks. De-stoners usually have a much smaller screen surface than air tables do, and they remove only the heaviest pieces. With a change in air flow, the incline of the screen, the screen vibration, and the screen type, the stones move up the screen and cause a separation from the lighter material. De-stoners are sometimes used in combination with air tables (17). Air separators are designed in many different ways, but they all are based on the same principle. The principle is that a narrow stream of spice falls through a horizontal air stream. Generally, the heavier particles fall straight down, but the lighter particles are blown to the side. This causes a size separation. Air separators are built in a variety of styles, with some vertical or horizontal airflows (17). Another type of spice cleaning equipment is the indent separator that makes use of the difference between the spice and the foreign material. The spice is fed into one end of a revolving drum. The outside of the drum is lined with cavities of uniform shape. The cavities are sized so that a spice particle of the desired shape will easily fit. The centrifugal force from the rotating drum holds the right-shaped particles in the cavities longer than particles that do not fit well. The rotational force lifts the correctly shaped particles until they eventually fall out. When this happens, they are caught in a trough and moved out of the machine. The wrong-shaped particles eventually fall out of the far side of the rotating drum. Very effective separations, based on size or shape, can be made by varying the shape of the cavities and the drum rotation (17).

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To separate round seeds from non-round foreign material, many processors use spiral separators. The spiral separator is a U-shaped trough that is curved into a downward spiral. Spices are fed into the top of the separator, and the round particles increase in speed as they roll down the chute. As the round particles gain speed, the centrifugal force drives the round particles up the side of the chute. The non-round particles do not roll and are not able to gain momentum. They end up sliding down the center of the chute. At the bottom of the chute a divider separates the round particles that have risen up the side of the chute from the non-round ones that slid down the center. Spiral separators are very simple machines and do not require motors or blowers. The entire process utilizes gravity as the driving force (17). C. Sterilization of Spices When unsterilized spices are incorporated into meat and food products, there is a possible health hazard. Spices may be sterilized with ethylene oxide or propylene oxide. The addition of either of these methods increases the price of the spice by several cents per pound but is well worth it. The simplest type of equipment for sterilizing spices consists of a walled chamber made of steel, about 10 inches deep, 4 ft high, and 4 ft wide. The chamber is fitted with a lock-tight door with a recording temperature and pressure instrument. The chamber must be capable of withstanding at least 28.5 in. of vacuum and 10 to 15 lb of pressure. It is also desirable to have a heating unit within the chamber capable of raising the temperature 10 to 15 degrees above ambient temperature. The other equipment needed is a tank of ethylene oxide fitted with a pressure gauge and a supply line to the chamber. The chamber briefly described is capable of holding ten 200-lb poly-lined fiber drums or about twenty 100-kg sacks of spices (3). After the chamber is loaded, the door is locked and the chamber evacuated to at least 28.5 in. of vacuum. The vacuum is held for about 10 minutes and released, replacing the chamber with ethylene oxide gas up to a pressure of 5 to 10 lb. Heat of 90° F is held, along with the vacuum, for 8 to 10 hours. After the required conditions are met, the ethylene oxide is shut off and the gas within the chamber penetrates the gas-permeable packaging materials and kills the microorganisms present. This process destroys 95% to 100% of the total microbes present. All pathogenic organisms, molds, yeast, insect eggs, insects, and other living matter are also destroyed (3). Ethylene oxide usage in regard to safety for humans has been questioned by many. Ethylene oxide is chronically toxic at levels not detected by smell. It is a skin irritant producing erythema and edema with a potential for sensitization. White (20) discusses the toxic effects of ethylene oxide (3). Propylene oxide is used like ethylene oxide, but its penetrating ability is weaker. It is a microbiocidal. It requires a higher temperature or longer treatment to accomplish the same microbial kill as ethylene oxide (3). The only known effective and safe alternative technique is the use of radiation (12). Industry has not responded very well to the use of radiation. Food irradiation employs an energy form termed ionizing radiation. There are several particular attributes of ionizing radiation that make it useful for treating foods. Certain kinds of ionizing radiation have the ability to penetrate into the depth of a food. Through physical effects they interact with the atoms and molecules that make up the food and also those of food contaminants such as bacteria, molds, yeast, and insects, causing chemical and biological consequences that can be utilized in beneficial ways. Although ionizing radiation frequently is referred to as high-

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energy radiation, the total quantity of energy needed to secure the beneficial effects with foods is relatively small, and gross changes in a meat or food that could affect its acceptability usually do not occur (19). The Food Safety and Inspection Service (FSIS) has proposed an amendment to its regulations to permit the use of ionizing radiation for treating refrigerated or frozen uncooked meat, meat by-products, and certain other food products. The FSIS proposal allows the specified food products to be treated with ionizing irradiation at dosages of up to 4.5 kiloGrays (kGy) for refrigerated products and up to 7 kGy for frozen products, with no minimum dosage (5). Inactivation of all spoilage microorganisms present is sterilization, and when radiation is used, the process is termed radappertization. Radappertization is the treatment of food with a dose of ionizing radiation sufficient to reduce the number and/or activity of viable microorganisms to such a level that very few, if any, are detectable by any recognized bacteriological or mycological testing method applied to the treated food. The treatment must be such that no spoilage or toxicity of microbial origin is detectable regardless of how long or under what conditions the food is stored after treatment, provided it is not recontaminated. Radappertization does not include inactivation of viruses, bacterial toxins, mycotoxins, or enzymes (19). Most spices are irradiated with a dose of 6.5 or 10 kGy. Spices normally are used at the level of 0.1% to 1% in meat products, and untreated spices can cause a product contamination level of as many as 105 to 106 bacteria per gram (19). The low moisture content of spices and seasonings limits chemical change resulting from irradiation. As might be expected, the effects of radiation are different for different spices. The threshold doses required for flavor changes are not the same for all spices. The yield of essential oils from spices, important in spice quality, can be changed by irradiation. Irradiation has proved to be superior to chemical sterilization with ethylene oxide and propylene oxide. Irradiation inactivates molds more efficiently than the chemical sterilization techniques. Irradiation is easier to use than chemical sterilization and a shorter exposure time is required. Most importantly, there are no residues remaining after the process is complete. Also, the safety of operating personnel is not a problem with irradiation. Adequate product volume, however, is essential for satisfactory economic feasibility (19). X. PROPERTIES OF SPICES AND HERBS A. Antimicrobial Properties of Spices In many areas of the food industry, especially the meat industry, microorganisms play a major role. Many spices, as mentioned earlier, have antimicrobial and or antifungal properties. The antimicrobial properties of spices have been known for hundreds of years. For example, cinnamon, thyme, and cumin were used in mummification in ancient Egypt. Spices were also used in ancient India and China to preserve foods and also as medicine. In ancient Rome and Greece, coriander was used to prolong the preservation period for meat, and mint was used to prevent the spoilage of milk. In the 1880s, research on the antimicrobial properties of spices was begun, and mustard, clove, and cinnamon were all found to have antimicrobial characteristics. Since the beginning of the twentieth century, research has been broadened to include both spice extracts and the essential oils of spices. Rosemary and sage have antioxidant properties, but marjoram does not. Marjoram has the opposite effect and is known as a prooxidant. If microbial contamination is of concern, the spice equivalent in the form of a spice extractive might solve the prob-

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lem. Spice extracts result in fewer microbial contamination problems because the oilbased extracts do not provide a source of spoilage microorganisms like their natural counterparts. Also, the extracts are easier to store and present less of a problem with infestation (7,16). B. Antioxidants and Spices Spices have been used to cover spoilage or off-flavors in meats for hundreds of years, without knowledge of how the spices were being effective. All that was known was that the spices made the food taste more desirable and that they helped maintain its quality. It is now known that many spices such as rosemary and sage act as antioxidants that help preserve the quality and color of the meats as well as providing health benefits to consumers. The antioxidant quality of the spices is partly due to the reduction in the degree of oxidation of fats present. Foods deteriorate gradually during storage for several reasons. One major reason is the oxidation of oil or fat found in the foods. Antioxidants such as spices can extend the shelf life of many kinds of foods and could actually make it possible to sell many new food products. The fats in meat are known to react with the oxygen in the air, generating peroxides, which are then further oxidized and degrade into low-molecular-weight alcohol and aldehyde compounds. This process results in rancidity. These “free radicals” are believed to damage DNA in the human body and to promote cancer and aging (14). The problem of oxidation in packaged foods can be addressed by replacing oxygen in the container with an inert gas. The most commonly used method, however, is to use antioxidants in the foods. Synthetic or natural antioxidants such as spices can be used. Although synthetic antioxidants seem to work quite well, they are easily decomposed at high temperatures, and there is some concern of possible liver and lung toxicity. Tocopherol is a natural antioxidant that has been used widely in the food industry. Its antioxidant effect is still inferior to the synthetic antioxidants. From the many studies that have been conducted on the antioxidant qualities of spices, it can be concluded that sage and rosemary are by far the most effective in retarding fat or oil oxidation. It is also known that other leafy spices such as oregano and thyme have stronger antioxidant activities than most of the other spices (14). Two of the most effective germicidal spices are cinnamon and clove. Cinnamon contains cinnamic aldehyde and cloves contain eugenol. XI. SPICE USAGE IN MEATS A. Use of Spices in Meat Of the ten markets for spices, the largest is the food industry and household sector. Spices are used in many categories of the food industry, such as meat, fish, vegetable products, bakery products, snack foods, and others. Of these categories, the meat industry is the largest consumer of spices (9). The household sector generally uses only a few spices, such as pepper, nutmeg, cinnamon, paprika, and vanilla. Food industries tend to demand many of the less known spices such as turmeric, coriander, mace, ginger, chilies, and cardamom. Spices have been used for centuries to flavor food across the world. Spice usage differs considerably in each culture and country. The number of spices used in meats has increased. Spices provide micronutrients and are used to increase food palatability. Spices also to help cleanse foods of pathogens and thereby contribute to the health of people who enjoy the flavor of spices (15).

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Many different spices, seasonings, and flavorings are used in meat products. The amount of spices, seasonings, and flavorings used is dictated by product identity standards and flavor preferences. Combining different levels of the various spices, seasonings, and flavorings available creates an almost infinite variety in the meat supply (15). Spice blends and seasonings are often unique to the type of meat they are designed to flavor. There is no one particular known spice blend that will flavor all types of meat or meat products to a desirable degree. The species of animal from which the meat was taken, the fat level of the final product, the physical and chemical components, just to name a few, must be carefully evaluated and taken into consideration when using seasoning blends or spices in meats. For instance, when a seasoning blend or individual spice is used to flavor low-fat meat products, less quantity must be used. Without fat as a carrier for the flavor system much less of the seasoning blend or spice is needed in the product. For example, if the proper amount of a seasoning blend was used in full-fat bologna, and the same amount was used in fat-free bologna, the fat-free product would have an overwhelming spice effect and leave an undesirable astringent aftertaste in the mouth of the consumer. B. Seasoning Blends “Seasonings are compounds, containing one or more spices, or spice extractives, which when added to a food, either during its manufacture or in its preparation, before it is served, enhances the natural flavor of the food and thereby increases its acceptance by the consumer” (3). Seasonings are added to a food before it is ready for serving, as opposed to condiments, which are added after the food is served. Some compounds can serve as a seasoning or a condiment. An example of this is tomato catsup. It is normally used as a condiment, but if it were to be added to beef stew during its cooking, it would be considered a seasoning (3). There are many different formulas for seasoning different types of meats. Some companies are quite secretive about their mixes, others are not. Not all seasoning formulas are complete. Formulations can be adjusted to suit the flavor profile desired. Spices and flavorings are used to season ham and other cured-meat products. This gives these products their unique flavor characteristics. The soluble spices, usually on a salt or dextrose carrier, can be dissolved in the pumping pickle to flavor the cured meat product. Some hams are rubbed with spices on the surface instead. The spices commonly used to flavor ham are pepper, cinnamon, nutmeg, allspice, and clove (15). C. Basic Meat Blends 1. Frankfurters The basic frankfurter seasoning is quite simple, containing approximately four ounces of pepper and about one ounce of nutmeg as the major ingredients with the basics of salt, cure, and so on (3). There are many different and more complex formulas that contain the basic spices but also contain other spices that are complementary. Some formulas have almost three times the amount of spices as the basic formula, but the blend is not overpowering. In fact, it is considered to be at the right level for a high-quality flavored frankfurter. Common ingredients often found in frankfurter blends are salt, corn syrup solids, sodium erythorbate (a cure accelerator), white pepper, nutmeg, onion powder, garlic powder, ginger, coriander, mace, cardamom, paprika, dextrose, oleoresins of paprika, ginger, pepper and coriander, oils of mace or cardamom, mustard, and select anti-caking agents. Various heat levels can

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be obtained with varying amounts of capsicum. There are products with added capsicum that are marketed as “hot” in flavor. 2. Bologna Bologna seasonings are very similar to those of frankfurters. Depending on the preference of the consumers, garlic may or may not be added. To round out the flavor of the bologna, monosodium glutamate may be added as a flavor enhancer. Today’s consumer often prefers to shy away from this ingredient, however. Allspice and cloves give the product a sweeter, spicy flavor. Cardamom can be substituted for the allspice and cloves in certain blends. Cardamom is used in small amounts because it is a very flavorful and aromatic spice. Some common ingredients found in traditional bologna seasonings are salt, dextrose, sodium erythorbate (a cure accelerator), monosodium glutamate, garlic or onion powder, mustard, white pepper, paprika, coriander, mace, allspice, cloves, oleoresins of pepper, paprika, and coriander, oils of mace, allspice, and cloves, and select anti-caking agents. 3. Sausage Sausage is prepared from comminuted and seasoned meat. It is usually shaped into a symmetrical form. Placing the sausage in natural casings, made from the gastrointestinal tract of animals, was done for the convenience of packaging the sausage. Sausage seasonings are made up of mixtures of various spices. In addition to giving flavors and aromas to the sausage, some spices have antioxidant properties (11). Manufacturing fresh pork sausage is one of the easier processes in the preparation of processed meats. It is also the most widely manufactured fresh sausage. It is made from comminuted and seasoned pork. Special attention must be paid to the cleanliness of the spices, equipment used and the surrounding work areas. The basic seasoning of pork sausage contains salt, ground white or black pepper, ground sage, ground mace or nutmeg, and sugar, dextrose, or corn syrup solids, monosodium glutamate, a preservative agent (citric acid), oleoresins of sage, pepper, ginger, paprika, and capsicum, and oils of mace, nutmeg, or marjoram. Optional spices added to this formula are ground ginger, ground marjoram, and ground cayenne pepper. A superior blend of spices calls for approximately five times the usual amount of oil of sage, slightly more sweetness, and a capsicum blend to give a fresh pink color before cooking. The unit package weight is increased about five ounces, but this also adds to the weight of the finished product. Although ground sage is used in some sausage seasonings, oil of sage has mostly replaced it because the product has a more appealing appearance and does not have the green specks that are caused by the fresh spice (11). Some meat processors make sausage from freshly slaughtered pork carcasses. They grind and season the meat while it is still warm. Once the sausage is made, it is then chilled quickly or frozen to retain maximum flavor. This sausage is called “hot boned” or “hot processed” pork sausage (11). Although the names sound similar, Italian pork sausage differs considerably from fresh pork sausage. They differ in the seasonings and also in the size of the ground meat particles. A formula for a basic sweet Italian sausage is salt, ground white pepper, whole seed fennel, and ground paprika. A formula which is more typical of what is found on the market contains salt, sugar, ground white pepper, ground paprika, ground nutmeg, ground mace, ground coriander, ground anise, ground clove, and ground cinnamon. Red pepper can be added to various seasoning blends to make a hotter sausage. Usually, one ounce of corn oil or another vegetable oil is added to the seasoning blend to aid in producing the blend. Another type of sausage is the Kielbasa or Polish sausage, which is not cooked but

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smoked. It is highly seasoned with garlic, pepper, coriander, nutmeg, dextrose or corn syrup solids, and oleoresins of the mentioned spices (11). 4. Meat Marinades Another use for spices is meat marinades. A typical fajita marinade recipe includes salt, monosodium glutamate, dextrose, grill flavor, hydrolyzed vegetable protein, smoked malt powder, lime juice solids, caramel color, onion powder, celery, and a free-flow agent. All ingredients are added and mixed until uniform. Water is added to the mix. The meat is either marinated or tumbled to allow the marinade to thoroughly penetrate the meat. 5. Gravies Spices are also used in gravies, which enhance the flavor of meats. An example is meat gravy mix. A general recipe includes salt, lactose, monosodium glutamate, dextrose or corn syrup solids, oleoresin turmeric, wheat flour, cornstarch, powdered meat, yeast, meat fat, non-fat milk solids, onion and garlic powder, meat flavoring or hydrolyzed vegetable protein, yeast extract, dried meat fat, dehydrated meat, free-flow agent, celery seed, white pepper, and paprika. D. Commonly Used Spices in Meats The following is a listing of spices and the meats that they commonly season: Allspice Anise seed Basil Bay leaves Caraway seed Celery flakes Celery seed Chili powder Cinnamon Cloves Coriander seed Cumin seed Dill seed Fennel seed Garlic, dried Ginger Mace Marjoram Mustard Nutmeg Onions, dried Oregano Paprika Pepper, black

Bologna, pork sausage, frankfurters, hamburgers, mincemeat Dry sausage: pepperoni Pizza sausage, certain poultry products Pickling spice for corned beef, beef tongue, lamb tongue, pork tongue, and pigs’ feet Polish sausage Chicken and turkey products Beef stews, meat loaf, chicken and turkey products Chili con carne, taco meat, some Spanish and Mexican sausages Ham loaf, other pork loaves, pastrami rub, bologna, and blood sausage Bologna, frankfurters, head cheese, liver sausage, corned beef, and pastrami; whole cloves can be stuck into baking hams Frankfurters, bologna, knackwurst, Polish sausage, other cooked sausages Chorizo, chili con carne, other Mexican and Italian sausages Head cheese, souse, jellied tongue loaf Italian sausages, pizza sausage, pizza, pepperoni, salami Polish sausage, beef sausage, salami, bologna, frankfurters Pork sausage, frankfurters, knackwurst, other sausages Bologna, bratwurst & other sausages Braunschweiger, liverwurst, head cheese, Polish sausage Bologna, frankfurters, salami, summer sausage Frankfurters, bologna, knackwurst, minced ham sausages, liver sausage, and head cheese Braunschweiger, liver sausage, head cheese, baked luncheon loaves Most Mexican and Spanish sausages, fresh Italian sausage, frankfurters, bologna Frankfurters, bologna, fresh Italian sausage Frankfurters, bologna, pork sausage, summer sausage, salami, liver sausage, loaf products, most other sausages

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Chorizo, smoked country sausage, Italian sausage, pepperoni, fresh pork sausage Used when black pepper specks are not wanted; pork sausage and deviled ham Chicken stews, some poultry products Used mostly for color, and in some sausages Pork sausage, pizza sausage, breakfast sausage, and old-fashioned loaf Pork sausage, other sausages Pork sausage, liver sausage, headcheese, and bratwurst Used mostly for color; curry powder

Source: Ref. 15.

E. Measurement of Meat for Formulations The amount of meat used in a formula is called a meat block. The amounts of seasonings and other ingredients added are based on the meat block, typically in 100 lb increments. An example would read: “Use 6.5 lbs seasoning per 100 lbs meat block” (17). A meat processor makes items in 100 lb increments such as 500 lb, 700 lb, or 1000 lb. The weight of the items depends on the equipment used. Other ingredients, including water, are added based on the 100 lb increments. Often, manufacturers will be provided the seasonings in individual containers, formulated for the amount of product the meat processor is making. In a 500 lb block, the seasoning is packed into a 32.5 lb bag. “Restricted ingredients are usually based on the amount per 100 lbs meat block” (17). Many processors work with the ingredients in a controlled-access room where the temperature and humidity are carefully monitored for measuring the critical ingredient. The critical components are weighed, packaged, and assembled for batch productions. A checklist must be prepared and verified in order to make sure that all of the materials are accounted for. F. Meat Seasonings and Cures The product that is used to treat meat to give it a longer shelf life and a characteristic pink color and cured flavor is called a cure. Some examples of cured meats are ham, bologna, bacon, wieners, and corned beef. Meat-curing ingredients (sodium nitrite) increase the product shelf life and inhibit the growth of Clostridium botulinum. This organism can grow and form its deadly toxin in meat items that are vacuum packed. Cures contain salt, sodium nitrite and, occasionally, sodium nitrate, sugar, and an anticaking agent. The amount of the nitrate is limited because it is associated with increased formation of nitrosamines during cooking, which have been found to be carcinogenic. Cures that give the pink color contain FD&C Red Dye #3. The coloring is added to eliminate confusion between cure and salt in the processing plant. Most cures contain 6.25% sodium nitrate, and others contain 12.5%. Although other levels can work, the two above are most common. Because many seasonings contain amines and are able to combine with nitrites and nitrates to form nitrosamines, cures are never included with seasonings. Cures are sold separately or in batch sizes. In the 500 lb meat block example, 1.25 lb of cure would be placed in a separate bag and put inside or attached to the outside of the seasoning bag. Then, the operator would only have to add a bag of each product to the formula without extra weighing of the seasoning ingredients (3). To formulate for meats, it is practical to formulate based on the ingredient weight, which is necessary for the amount of meat block. It is much less practical to work

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with percentages. When making a product such as a seasoning for wieners, the manufacturer must work in weight of seasoning per 100 lb of meat. It can then be easily converted to percentages and the seasoning formula can be determined. For meat blocks that may not be in 100 lb increments, simply figure the percentage usage of spice per pound and then multiply by the pounds desired to find the amount of spice to use. G. Brine Solutions Brine is a water-soluble solution containing seasonings, salt, sugar, sodium erythorbate (a curing accelerator), phosphates, and cure. It is pumped or injected into a meat item. All of the flavoring materials should be water-soluble, but small levels of micro-mesh powder are possible because of the small particle size. Many times, oleoresins are used with polysorbate 80 or another emulsifier. The brine is formulated so that if the product is pumped at 20%, the restricted ingredients are at the correct level. Pumping 20% means that 120 lb of finished product will result from 100 lb of meat if it is pumped with that solution. The term pickup is used mostly in the poultry industry. If the manufacturer wants a 15% pickup, then the poultry is marinated, vacuum tumbled, or injected so that 100 lb of the finished product weighs 115 lb. This is very important when formulating the seasoning so the strength of the flavor, salt, and phosphate are kept at the appropriate levels. The seasonings that are marinated can utilize some particulate ingredients that are not soluble (18). Brines are used in many products to give flavor and also to cure the product. The two products most often made in the United States are corned beef and ham. Ham is usually cured and flavored with brine that is soluble because it is injected or pumped to give an evenly cured product. Ham seasonings generally contain spice extractives on a sugar or salt carrier, garlic, and sodium erythorbate. Corned beef is usually soaked in a pickle seasoning. The seasoning may or may not be soluble. Many times, a whole pickling spice is used. XII.

REGULATIONS AND RECORD KEEPING

A. Formulations and Record Keeping Food processors keep records on the formulas of seasonings. The formulas are designed so that costs can be easily calculated and updated as needed. Each spice blender may have a special way of recording formulas. As was mentioned previously, some are quite secretive, and others are not. However, given time, a good seasoning blender can duplicate a competitor’s blend, so there is really no need for secrecy. There is an understanding between the blender and the customer that the blender will not divulge the secrets of one customer’s blend to another customer. Some standard items found on blending or formulation forms are laboratory and plant code numbers, the ingredients, the weight of the individual ingredients in one seasoning unit, and the percent of each ingredient in the batch. For some ingredients such as oils and oleoresins, coded labels are affixed to containers to prevent visitors and even employees from knowing the exact formula of the seasoning blend. These forms and codes are especially helpful in meat processing plants to get the correct seasoning formula for whatever meat is being processed, such as frankfurters or sausage (18).

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B. Regulations of Seasonings and Natural Flavorings There are flavoring regulations that apply to the manufacturers of seasonings. In March 1991, FSIS issued a flavoring regulation that defined “flavoring,” “natural flavoring,” and “natural flavor” as follows: Essential oils, oleoresins, essences, extractives, distillates or any product of roasting or heating which contains the flavoring constituents derived from any spice, fruit, or fruit juice, vegetable or vegetable juice, edible yeast, herb, bud, root, bark, leaf, or other edible portion of a plant. Spices, onion powder, garlic powder and celery powder, oleoresin black pepper, ginger, and garlic oil are examples (FSIS NOTICE USDA, U.S. Government Printing Office: 1990262-858:20312/FSIS). Any other item not listed above must: 1. Be listed by common or usual name or 2. Common or usual name with a sub-listing of ingredients or 3. Subject to USDA approval for acceptable labeling All proteinaceous materials are not flavoring, and must be listed by their common and usual name including source, such as hydrolysates and autolysates of animal, plant, dairy, and yeast sources.

This new legislation mandates that almost all ingredients be broken down into their component ingredients. For example, previously, a flavor blend that replaced MSG might have been labeled as a natural flavor. Currently, it must be labeled with all components, which may include maltodextrin, salt, autolyzed yeast extract, hydrolyzed vegetable protein, citric acid, and natural flavor. As one can see, this regulation has made labeling products extremely complicated. Another problem that has been encountered is that reaction flavors are produced. The treatment of amino acids of other proteins along with sugar under heat to produce meat flavors can produce the reaction flavors (18). XIII. MARKETING AND CONSUMERS OF SPICES A. Marketing of Meat Seasonings Meat seasonings are generally low-margin items. Many companies will often change suppliers to save only a few cents per pound. For example, when formulating sausage seasonings, it is necessary to have an economical source of ground mustard seeds. Sausage seasonings have a high amount of mustard. Unless the manufacturer grinds his own, it is very hard to be competitive with other companies. Many seasoning companies are known in the industry to primarily be suppliers of meat seasonings. This is generally because of two reasons. First, many of the smaller seasoning companies were initially formed by large meat companies to provide seasonings to their processing plants, and then they were expanded or sold. Second, some of the seasoning houses were initially involved in meat seasonings because of their technical expertise. In the past, many of the small meat companies relied on technical support from the seasoning company to help formulate their meat items and also to instruct them on how to produce the product. Some of the seasoning companies provided the seasoning, the smoke flavors, the sausage casings, and other ingredients, and the technical support to produce the processed meat items such as summer sausage and wieners. Even the relatively large companies used the seasoning companies as technical reference much more in the past than they do today. Processed meat manufacturers now usually have their own laboratory and research personnel. Seasoning companies provide seasonings for many items in the red meat industry such as fresh, smoked, dried, and cured sausages, nonspecific items including meat loaves

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and luncheon loaves, ham brines, chili, taco meat, corned beef pickles, and roast beef rubs. They also provide seasonings for poultry companies, including pumps and basting blends; sausage seasonings, such as turkey bologna, ham, and breakfast sausage; glazes and marinades. Thousands of flavor combinations are possible for each type of product. The flavor of the product even varies by the region in the United States. For example, chorizo that is purchased in different geographic areas will be extremely different. It can be dry, semidry, hot, mild, fresh, cooked, cured, red to pale orange, or fine or coarse grind. There are also an infinite number of flavor variations available. B. Consumer Loyalty to Spices The United States is consuming more red pepper than it ever has. It seems that the American public is becoming more adventuresome as to what they like to eat. The people are consuming many more hot foods, and normally bland foods are becoming spicier. The hot taste in spices such as pepper and ginger is due to specific compounds found in the spice. While the public may consider this degree of heat pleasing, most people consider the heat from the spice capsaicin to be extremely hot and undesirable. Mild spices are also on the increase. This is partially attributed to the restriction of salt in diets. It is expected that the use of spices will continue to increase (14). One of the most important methods of determining the success of a product is repeat purchase. Most consumers will buy a product once, or they might even buy it twice. However, if the product varies each time they purchase it, they will avoid the product completely. Product consistency is very important. Changes in the food product are the most important factor, but changes in price, package, and availability also affect the frequency of repeat purchase. Achieving this consistency is not an easy task. Agricultural products such as spices vary due to climatic conditions and harvesting conditions. The more ingredients used, the greater the chance for errors and inconsistencies. To avoid some of these inconsistencies, currently, quality management is the responsibility of the supplier (18). Consumer tastes and expectations are increasing in sophistication, and therefore, the food and flavor industries have been presented with a challenge to produce more “healthy” foods that contain less salt, sugar, and fat. These ingredients are very important to the overall flavor of products. In addition, there is a need to develop flavors that can withstand preparation in a microwave oven. In response to these challenges, the flavor industry has implemented more efficient methods of synthesis that will have a major impact on the production of less expensive synthetic flavors. Flavor compounds that can elicit specific mood sensations can be used on food to change the mood of the consumer. Research has shown that peppermint, orange, and eucalyptus odors are relaxing and soothing, whereas sage and rosemary are more stimulating (18). The spice market offers many different types of products, from whole spices to extractives that gives a variety of tastes, from spicy heat to aromatic, savory, or bitter. By using the imagination, spices can add distinctive touches to food, whether it is to perk up the flavor, add textural contrast, or carry out an ethnic theme. Just as they were once treasured as much as gold, spices still hold value in the eyes of a food technologist or compound specialist. XIV. SUMMARY Spices are incorporated into meat products to add or enhance flavor and to ultimately achieve a desired level of flavor. Spices impart varying levels of flavors and tastes to meats.

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A meat seasoning must be compounded in such a way as to potentiate the natural flavor of the meat in which it is to be incorporated. It should not be overwhelming or diminish the product’s flavor but “balance” the product with a blended, well-rounded flavor with no perceptible, undesirable aftertaste. Proper spice selection can make the difference between a successful meat product, either new to the marketplace or existing, and an unsuccessful one. The quality of the meat incorporated into a meat item can be of the highest available, but if the blend is not seasoned properly, the quality of the meat used becomes less of an important factor. This chapter has briefly touched the surface of the overwhelmingly large topic of spices and their usage in meats. It is to be used more as a basic guideline to assist the processed meat formulator, or those working in the area of product development of meat items, meat seasoning blends, or other related areas. This chapter provides a brief history of spices, a general discussion of spices, a detailed listing of standard cleaning procedures relating to spices, and so forth that pertain to the vast world of spices. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Desrosier, N. (Ed.). Reitz Master Food Guide, Westport, Conn: AVI Publishing Co., 1978. Dziezak, J. Spices. Food Technology, 43, (1); 1989. Farrell K.T. Spices, Condiments, and Seasonings. New York: Van Nostrand Reinhold Company, 1985. Farrell K.T. Spices, Condiments, and Seasonings. 2nd ed. New York: Van Nostrand Reinhold Company 1990. Federal Register. 1999. Food Safety and Inspection Service. 9 CFR Parts 381 and 424. Vol. 64, No. 246. Glazer, M. 1989. “A Flavor Odyssey”, Food Technology, Vol. 43, No. 7. Hirasa, K., et al. Spice Science & Technology, New York: Marcel Dekker, 1998. Hollo, J., E. Kurucz, and J. Bodor. Lebensm Wiss Technol 2:19, 1969. Lewis, Y.S. Spices and Herbs for the Food Industry. Orpington, England: Food Trade Press, 1984. Mori, K., Y. Yomamoto, and S. Komai. Shokuhin Kogyo Gakkaishi 21:466, 1974. Price, J.F., and B.S. Schweigert. The Science of Meat and Meat Products. 3rd ed. Westport, Conn:. Food & Nutrition Press, Inc, 1987. Proctor, B.E., S.A. Goldblith, and H. Fram. Effect of supervoltage cathode rays on bacterial flora of spices and other dry food materials. Food Res 15:440, 1950. Pruthi, J.S. Spices and Condiments: Chemistry, Microbiology, Technology. New York: Academic Press, 1980. Risch, S.J., and C.T. Ho. 1997. Spices Flavor Chemistry and Antioxidant Properties. American Chemical Society. Washington, D. C. Romans, J.R., W.J. Costello, C.W. Carlson, M.L. Greaser, and K.W. Jones. Romans. The Meat We Eat. Danville, Ill: Interstate Publishers, Inc. 1994. Rosengarten, F. The Book of Spices. Wynnewood, PA: Livingston Publishing Company, 1969. Tainter, D.R., and A.T. Grenis. Spices and Seasonings A Food and Technology Handbook. New York: VCH Publishers Inc, 1993. Underriner, E.W., and I.R. Hume. Handbook of Industrial Seasonings. Bishopbriggs, Glasgow: Blackie Academic & Professional, 1994. Urbain, W.M. Food Irradiation. Orlando, FL: Academic Press, Inc. 1986. White, J.D. Standard aeration for gas sterilized plastics. J Hygiene Cambridge 79, 225–232, 1977.

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17 Intermediate-Moisture Meat and Dehydrated Meat TZOU-CHI HUANG National Pingtung University of Science and Technology, Pingtung, Taiwan WAI-KIT NIP University of Hawaii at Manoa, Honolulu, Hawaii

I. INTERMEDIATE-MOISTURE MEATS AND DEHYDRATED MEATS A. Hams and Meat Chunks B. Slabs, Sheets, and Slices C. Strips D. Pieces E. Floss or Shreds F. Powder G. Sausages II. STABILITY OF INTERMEDIATE-MOISTURE MEATS A. Microbial Stability and Safety B. Chemical Stability III. STRUCTURAL COMPONENTS OF MUSCLE TISSUE RESPONSIBLE FOR PHYSICOCHEMICAL PROPERTIES OF INTERMEDIATE-MOISTURE MEAT PRODUCTS A. Gross Meat Structure IV. EFFECT OF HEATING ON PHYSICOCHEMICAL PROPERTY CHANGES DURING INTERMEDIATE-MOISTURE MEAT PROCESSING A. Proteins B. Sausages V. PARAMETERS CONTROLLING THE STABILITY OF INTERMEDIATE-MOISTURE MEATS A. Heating B. Salt C. Sucrose

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Chemicals Intact Muscle IM Meat Sausages Innovative Water Activity Control Technology

VI. CONCLUSIONS REFERENCES

I. INTERMEDIATE-MOISTURE MEATS AND DEHYDRATED MEATS Intermediate-moisture (IM) meat contains 15% to 50% moisture, and dried meat products contain even less moisture. They have a water activity (aw) value of 0.60 to 0.92. IM meat products are shelf-stable without refrigeration or thermal processing, and some can be eaten raw without rehydration or cooking. These dehydrated meat products usually are resistant to bacterial spoilage because of their low water activity and fairly high salt content. Some are fairly tough, with poor texture. However, some are highly palatable and are even considered delicacies. Freeze-dried meat products are very porous in nature and can be rehydrated to almost the original shape. Intermediate moisture meats and dried meats can be categorized into two groups: intact muscle–based product and the emulsion-type sausage. Each group can be further subcategorized by the form because many are similar products produced in different cultures. Table 1 is a summary of selected dried or intermediate-moisture meat products. Dehydrated and freeze-dried raw or cooked meats are resistant to bacterial spoilage but have poor texture and are sometimes expensive to prepare, such as freeze-dried meat products. This section will cover some of the traditional IM and dried meat products and their production processes, as well as the production of freeze-dried meat products. A. Hams and Meat Chunks This group of IM meat products usually are made by using chunks of meat with or without the shank bone. Traditionally they are dry-cured or pickle-cured. The dry-curing process is especially time consuming and labor intensive because the curing agents have to be rubbed onto the raw meat chunks, which are then turned a few times. Pickle-cured products also have to be turned a few times during the curing period. For dry- or pickle-cured products, enough time must be given for the curing ingredients to penetrate to the center of the chunk of meat. They are then washed or soaked occasionally to remove some of the salt. With advances in technology, cured meats can now be produced by stitch-injection cure to save time. The drying process can be accomplished usually in several days by smoking or heating in a chamber. Some products are partially dried first, and the drying process is completed by gradually evaporating the moisture from the chunk of meat in a cool environment. At the same time, fermentation can proceed to give the product a typical flavor. Some products will take over 6 months to a year to produce. The dry cured hams of continental Europe (such as French Bayonne ham, German Westphalian ham, and Italian Parma ham) continued to be produced in a traditional manner and have retained their original organoleptic qualities. 1. Italian Parma Ham The Parma ham is made from 1-year-old pigs weighing roughly 180 kg raised in the northern Italian region around Parma. The pigs must be fed exclusively on corn (maize), oats,

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Table 1 Examples of Dried and Intermediate-Moisture Meat Products Form / type Ham (chunk)

Slab, sheet, slice

Strip

Pieces

Floss

Powder Sausage

Examples

References

Parma ham (Italian) Westphalian ham (German) Scotch ham Old time Virginia ham (American) Virginia ham (Smithfield, American) Chinese ham Dried beef Bundlnerfleisch, Rohschinken, Coppa, Speck, Bindenfleish (Swiss) Bacon Charque (Brazilian) Beef jerky (American Indian) Tasajo (Cuban) Kilishi (Nigerian) Balanqu (African) Dendeng (Indonesian) Spiced beef slices (Malaysian, Chinese) Ruogan, ruopu (Chinese) Shafu (Chinese) Jirge (African) Dried beef cecina (Spanish), cecina (Mexican) Biltong (African) Pemmican (American Indian) Ndariko (African) Basterma/Patirma (Egyptian/Turkish) Chinese-style bacon Tsire/Suya (African) Banda (African) Freeze-dried meat pieces Rou song (Chinese) Zousoon (Taiwanese) Sambal daging (Malaysian) Sharmoot (Sudanese) Bologna (Lebanon) Salami, pepperoni, Genoa (Italian) Salchichon (Spanish) Cervelet (European) Lup cheong (Chinese)

1 1 1 1 1 2–4 5 5–7 8 9–11 12–14 15 16–17 18 19–22 3,20,21 3 2,3 18 23–24 14,18,25 26 18 27–28 2 18 18 29–30 31–32 33–34 20 35 32 5 36 37–38 2–3

and rinds from Parmesan cheese (1). After slaughter, the carcasses are deep-frozen for 24 hr and are pared of outer fat into a drumstick shape, salted, seasoned, and frozen for 8 days. On the ninth day, they come out to the kneading bench for the first of 30 to 40 massages to squeeze out the juice and tenderize the meat. They are salted again, put in deep freeze for 18 days, hauled out into fresh air for a day or two, then have a 30-day chill at exactly the freezing point, and spend 3 months in the drying chamber. Then they are scrubbed with warm water, sandpapered to an attractive outer finish, and put into ventilated storage until time to sell. The total curing time lasts from 10 to 15 months.

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2. German Westphalian Ham Westphalian hams are sliced and eaten raw (1). They are prepared by rubbing the ham with a mixture of 16 lb (7.26 kg) of salt and 1 oz (28.4 g) of saltpeter per 100 lb (45.36 kg) of pork. After being placed on shelves or stacked on concrete floors and allowed to cure for 2 weeks, they are then placed in a 22% brine solution (90° pickle) and allowed to cure for 18 more days. At the end of this period, they are taken out from the brine and packed one above another in a cool, dry cellar for 4 weeks of ripening period. Then the hams are cleaned with a stiff brush in lukewarm water and allowed to soak in fresh water for 12 hours. They are then ready for smoking. Only beechwood is used, with the exception of juniper twigs and berries constantly thrown on the fire. Beechwood sawdust is strewed over the fire in case it becomes too hot. The smoking process continues for a period of 7 to 8 days. 3. Scotch Hams Scotch hams are made from fresh-skinned and deboned hams; most of the fat is removed, after which they are given a mild cure according to a formula. The cured ham is then rolled, tied and placed in a cellulose casing but it is not smoked (1). 4. American Virginia Ham (old-time) The animals used in the production of old-time Virginia ham were slaughtered when the points of the new moon were up. Fine table salt was rubbed into each ham in one long thorough rubbing (1). Then the hock end was packed with salt, and an extra layer of salt was spread over the entire ham. They were packed in barrels and left to cure for several weeks in a dry, cool place. At the end of 7 weeks, the hams were rubbed with a mixture of New Orleans molasses, brown sugar, black pepper, cayenne pepper, and saltpeter. These ingredients were mixed in the following amounts to be rubbed on 100 pounds (45.4 kg) of ham: 1/2 lb (22.7 g) black pepper, 1 qt (0.951) of New Orleans molasses, 1 lb (45.4 g) of brown sugar, 1 oz (28.4 g) saltpeter, and 1 oz (28.4 g) cayenne pepper. The hams to be rubbed were brushed of all visible salt and then rubbed with the mixture and left to lie in a cool room for another 2 weeks. At the end of this second period, they were hung in the meat house with the hocks hanging downward. They were not smoked but were allowed to age for another 30 days. 5. Virginia Ham (Smithfield, Virginia) Smithfield (Virginia) hams must be processed in Smithfield and come from hogs grown in the peanut belt of Virginia and North Carolina (1). The hams are sprinkled with saltpeter, using 4 lb (1.81 kg) per 1,000 lb (454.6 kg) ham, and are then given a rubbing of fine salt. Three to five days later, they are given a second rubbing of salt and stacked in a curing room to cure for one day per pound (454 gm) of ham. After the cure, they are washed and given a cool smoke (26.6° to 29.4°C) for 7 to 10 days, using hickory wood and smothering the blaze with applewood sawdust. After smoking, they are rubbed with pepper and hung in aging rooms for a period of 7 to 18 months. The shrinkage from green weight is around 25%. The dryness and saltiness of the hams require the consumer to soak them for 24 hours and simmer them 4 to 6 hours. 6. Chinese Ham The production of Chinese-style ham is similar to the production of country-cured (dry cured) hams in the United States and can be divided into three phases. First the curing pe-

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riod, during which the curing ingredients [a mixture of 8 lb (3.63 kg) salt, 3 lb (1.36 kg) sugar, and 3 oz (85 g) sodium nitrate] are rubbed on the ham surface. Approximately 1 1/4 oz (35.5 g) of the curing mix is used per lb (454 g) of ham. The total amount of curing mix should be applied at three intervals and this will allow more uniform salt penetration. During this time, generally about 30 to 40 days depending on the size of the hams, the products should be maintained under refrigeration. During the second phase, hams are hung in the smokehouse and are subject to a cool smoke but are not cooked. The smokehouse temperature is kept between 70°F (21°C) and 90°F (32.2°C) and the initial temperature of the ham usually is approximately 10°F (5.5°C) lower than the smokehouse temperature. The smoke is applied continuously for 2 to 3 days until the hams obtain amber or mahogany color. After smoking, the third phase, which is the aging process, begins. Hams are normally aged for 6 to 9 months, and this is usually not accomplished under refrigeration. During this time, the full flavor characteristic of the ham develops, probably as a result of enzymatic reactions. To encourage enzymatic activity, hams are usually aged between 70° and 85°F (21 and 29.4°C) (2–4). 7. Dried Beef Dried beef is cured, dried, and sometimes smoked, but not cooked. It is low in moisture, containing 25% to 35%. Either the dry- or pickle-cure procedure can be used, although most commercially produced dried beef is cured in pickle with salt, sugar, nitrate, and nitrite. If the meat is dry-cured, 1 oz (28.4 g) of curing mix per pound (454 g) of meat should be applied to the meat surface. The curing mix should be applied to the meat surface. If the pickle cure is used, the cure ingredients are dissolved in water, enough to cover the raw meat. The length of cure depends on the size of cuts. Generally, muscles of the round—top, bottom, and knuckle—are used for manufacture of dried beef, but shoulder clods are also used. As a rule of thumb, dry- or pickle-cured meat should remain in cure of 2 to 3 days per pound (454 gm) of meat. After being removed from cure, the meat is rinsed with cold water and allowed to dry. When produced in a commercial establishment, the beef is generally dried in a smokehouse for 2 to 3 days. Smokehouse temperatures range from 90° to 100°F. Depending on the desires of the individual processor, smoke is applied during part of the drying period (5). 8. Swiss Bundlnerfleisch, Rohschinken, Coppa, and Speck Bundnerfleish is prepared from the choice cuts of lean quarter beef. It is dry-salted with a curing period of 3 to 4 weeks, followed by a fermenting, drying and maturing period of 3 to 4 months. During processing the total weight loss is about 40%. The final product has approximate 34% protein, 56% to 59% moisture, 4% to 6% fat, and 3% to 4% salt. The resultant products appear hard and mummified with most of their surface covered by a white mold. Prior to consumption, this outer casing has to be trimmed off and the dark red inner meat is usually sliced very thinly and eaten raw. The pork products (Rohschinken, Coppa, and Speck) are prepared in a similar manner (5–7). B. Slabs, Sheets, and Slices Slab-type, sheets, or slices of IMM are produced in a similar manner. Meat is cut into slabs, sheets, or slices, cured for the appropriate periods, and dried by heating or smoking. However, the water activity of the final products may be different because of cultural habits and consumer preference.

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1. Bacon Bacon can be made by dry cure or pickle cure. In the case of dry cure bacon, a mixture of curing ingredients is rubbed on all surfaces of the green bellies. Bellies are placed in a cooler to cure for 10 to 14 days before cooking and smoking. Most commercial bacon is pickle-cured by the stitch-injection method. Bacon is usually cooked in the smokehouse according to a one-step or three-step cook schedule, reaching a final internal temperatures of 130° to 140°F (54.4° to 60°C). The bacon is then cooled to an internal temperature of 26° to 28°F (2.2° to 3.3°C) before slicing and vacuum packaging. Bacon is generally stored under refrigeration (8). Canadian bacon is made from the large muscle of pork loins, the strip or sirloin muscle. Wilshire bacon is made from selected hogs with special cutting procedures of the carcasses. This product is lightly matured and can be smoked (8). 2. Jerked Beef [Charque (Spanish, South America), Jerkey (English) or Xarque (Portuguese, Brazil)] (9–14) Charque is a beef product that should not contain more than 45% moisture within the intramuscular portion and not more than 15% of mineral residue, with aw of 0.70 to 0.75. Manufacturing cuts for charque must be carefully prepared after deboning to produce muscle pieces of uniform thickness. Muscle blocks greater than 5 cm thick are not suitable. Where flank and rib pieces are used, little further work is required apart from small incisions to facilitate salt penetration. In the case of hind and forequarter cuts, more extensive opening of the muscle blocks is necessary to ensure uniform salt penetration and consistent drying times. The wet salting or brining operations is carried out in pickled vats of approximately 80 cm depth, of a size depending upon the capacity of the plant. The interior of the tank should be lined with impermeable fine screened concrete or with high quality glazed tiles. A brine strength near 100° salometer as possible, but never less than 95, should be maintained constantly during the wet salting stage. The brine should be maintained as far as possible at 15° to 20°C. The immersed meat pieces are agitated vigorously, by the use of paddles, for a period of 50 minutes, after which they are removed from the tank. The meat acquires a blue tone during this phase of the processing. Dry salting is initiated, after the meat is removed from the brine tank, on a concrete floor covered with a 1 cm layer of salt. The floor should be slightly inclined, falling away into lateral channels to carry away the meat juice expressed during salting. The meat pieces are stacked into piles, separated from each other by layers of coarse marine salt (1 mm thick). The salt should be shoveled over the meat as a fine shower from several directions to ensure even penetration into meat cuts and openings. The height and dimension of the pile are likely to be governed by the scale of production but should not be allowed to exceed 1.5 meters for fear of exaggerating the pressure on the lower meat layers, thus causing excessive weight loss. Each pile when completed should be capped with a 2 cm covering of salt. The pile is restacked after 8 hours in order to equalize pressure throughout. Thus, the uppermost pieces are repositioned on the bottom of the new piles. As in the initial salting step, thick layers of coarse salt are placed between each successive meat layer and also on the top of the pile. The meat remains in the second pile for a period of 16 hours before being repositioned into its original order. The piles are remade every 24 hours in order to ensure even salt penetration and water loss and microbial contamination. The number of tumbling varies according to pile size and weather conditions but should be 4 to 5 days.

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Before initiating drying, the meat pieces are subject to a rapid washing with acidified water (pH 5.5) to remove excess salt adhering to the surface. If weather conditions are favorable, charque is usually dried directly in the sun on wooden rails (varales) positioned parallel in runs oriented north-south. This orientation permits an even solar coverage. The distances between these runs should be sufficient to allow movement of barrows used to transport the meat from the washing to the open drying area. The meat pieces are extended over the rails with the muscle layer uppermost in order to limit undesirable changes to the fat caused by direct exposure to the solar rays. The initial drying, directly in the sun, is limited to a maximum period of 4 to 6 hours. This period of exposure may be subsequently lengthened to a maximum of 8 hours. Temperatures in excess of 40°C on the meat surface should be avoided. The meat pieces are exposed to the sun each day over a period of 4 to 5 days. After each period of exposure, the pieces are recollected, stacked in piles on concrete plinths and covered with an impermeable cloth or tarpaulin to protect them against rain and wind and to hold the heat absorbed by the sun. The top of the pile is usually built with a crown to facilitate purging and, in the case of rain, water will run off. During times of the year where there is constant rain or cloud cover, or in regions where weather patterns are uncertain, the process of winter pillage is used. Meat pieces are stacked in piles up to 3 meters in height on special concrete plinths immediately after the third or fourth tumbling and without prior washing. Piles should be built with a 10% slope from center to sides to allow free draining. The piles may be erected in a covered area adjacent to the dry salting room or in a refrigerated store, if this is available. Fine salt (less than 2 mm grain size) should be positioned between layers at the moment of putting into piles. While piles are kept at ambient temperatures, they must be carefully maintained to obviate microbial spoilage. This is achieved by covering the whole pile with a thick layer of marine salt, treated with sodium hypochlorite solution (0.4%) up to a thickness of 20 to 30 cm. Alternately, salted offals such as lungs, hearts, and kidneys may be used to cover the meat pieces. Air penetration into the pile should be prevented as far as possible. A heavy hessian cover moistened with 50% sterilized brine is usually employed to prevent desiccation of the upper meat layers. This is tied down using strong cord. The fermentation process takes place under strictly anaerobic conditions. The piles may be maintained in this state for up to 4 months, after which the meat is washed using acidified water, and dried in the sun according to procedures described earlier. Winter-pillaged charque is usually subject to 2- to 3-day sun drying. 3. Cuban Tasajo Tasajo is a traditional Cuban intermediate-moisture product made from beef sheets that are soaked in brine, drained, rubbed in salt and layered with salt in vats for several days, washed, and sun-dried (15). 4. Nigerian Kilishi Kilishi is a traditional means of preserving meat used by the Fulani and Hausen herdsmen. Kilishi is mainly prepared from the hindquarters of beef carcasses, but mutton and goat meat are also sometimes used. From the choice, lean cuts, thin sheets of fresh meat of about 17 to 20 mm thickness and an average length of 140 cm are cut manually. These are first sun-dried at 30° to 36°C, and 21% to 26.5% relative humidity on a raised bed for 4 hr. Then the slices are immersed in a slurry of groundnut cake powder and seasonings for one hr and redried in the sun under similar conditions for 2 to 3 hr to a low moisture content (20% to 25%). Depending on the quantity of product and the climatic conditions, this drying may

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take from 5 to 12 hr or even more. The product is finally roasted over a fire for 3 to 5 min with final moisture of 12% to 15%. There are some concerns about the carcinogenic polycyclic aromatic hydrocarbons (PAH) in these smoked products (16,17). 5. African Balanqu Balanqu may be defined as boneless slabs of lean/organ meat seasoned with salt, spices, groundnut flour, and oil, and roasted over a low-burning/glowing smokeless fire. It is prepared mostly from beef, but also occasionally from mutton and goat meat. The meat is boned and cut into chunks that are sliced with a curved knife into slabs not less than 1 cm thick, most commonly 4-6 cm or more. The meat slabs are dusted on both surfaces with a seasoning mixture of salt, spices, groundnut flour, and oil, and then placed on a wire mesh over an oven of low-burning/glowing smokeless fire to roast slowly until done. Roasting takes 30 to 60 min depending on meat thickness and fire intensity. As for tsire/suya (see below), the product receives no further stabilizing treatment. It is similar in moisture content (20% to 30%) and composition to tsire/suya, and its shelf life is also about 24 hr under ambient conditions (18). 6. Indonesia Dendeng Dendeng is a traditional Indonesian IM meat product containing coconut sugar, salt, and spices. It is prepared from meat in the form of thin slices (dendeng sayat) or from minced meat (dendeng giling). It may be prepared from beef, chicken, pork, or fish, but dried beef (dendeng sapi) is the major product found in the markets. Dendeng has a sweet taste due to its high sugar content, and together with strong flavor of the spices and the dried meat gives dendeng a characteristic flavor that differentiates it from other traditional IM meats, (e.g., jerky, biltong). Fresh meat is sliced to about 3 mm thick and soaked for up to 12 hours at ambient temperature in a mixture of coconut or palm sugar, salt, and spices (coriander, tamarind, garlic, and the root of the greater galangal, which can also contain onion, pepper, and other spices). The cured meat slices are put on trays to be sundried (dendeng sayat) or mechanically dried to aw of 0.52 to 0.67. For dendeng giling, the minced meat is mixed with ground spices, coconut sugar, and salt, and then rolled into thin sheets 3 mm thick, and put on bamboo trays to be sun-dried. Typical dendeng giling has aw of 0.62 to 0.66. The approximate composition of dendeng is pH 5.6, moisture 26%, protein 35%, fat 10%, salt 8%, and sugar 35% (dry wt. basis). Dendeng becomes tough and less elastic during storage for 3 months at 50°C (18). Preservation of meat in Africa is conducted mainly by control of the internal aqueous environment in relation to product quality and stability (19–22). 7. Malaysian Spiced Beef Slices (bak kua, rou pu) In Malaysia, this product is usually made from pork. The product thickness is 2 to 3 mm with a fairly high sugar content and it is sometimes known as dried sweet meat or barbecued dried meat. Traditionally this product is made only from pork, usually using ham or shoulder meat. The meat is partially frozen and sliced to the required thickness along the muscle grain using a meat slicer. The sliced meat is then soaked in a curing mixture made up of salt, sugar, soy sauce, monosodium glutamate, spices, and permitted colors. After curing for about 2 hr, the slices are placed on slightly oiled bamboo trays at 40° to 70°C for several hr until a moisture content of about 30% to 40% is reached. After drying, the slices are stacked up and cut into squares. The product is then packed and kept in the chill room. For longer storage, a freezer is used. The product is usually sold in ready-to-eat form by grilling partially dried slices over a charcoal fire until brownish in color. Maltose (5%)

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Intermediate-Moisture Meat and Dehydrated Meat

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can be added to give the product a wet and bright appearance. Rou pu has an aw of 0.69 (3,20,21). 8. Chinese Style Dried Meat Slices (Chinese Rougan or Roupu) Chinese rougan (Chinese slice-cured pork) has been described. The procedure utilizes paper-thin (0.2 cm) slices of lean meat cut parallel to the grain. The meat is cured with a mixture of sugar, salt, soy sauce, monosodium glutamate, and spices. The traditional cure does not use nitrite or nitrate. The meat is cured for 24 to 36 hours at 4°C and then the thin strips are placed side by side (slightly overlapping) on an oiled bamboo basket or wire rack and dried at 50° to 60°C, until they lose approximately 50% of their original weight. The meat is removed from the containers and further dried by roasting over charcoal or deep fat fried to give a final aw of about 0.6 (3). In another similar process, the whole muscle is boiled for 40 to 45 minutes, after which it is cut into cubes or pieces (5  5  10 cm) (2,4). Roupu is a product made very similarly to rougan except that more sugar is added to the curing mixture to give the final product a sweet taste (2,4). 9. Chinese Shafu Shafu is an improved rougan with a softer texture and lighter color; it is less sweet. The aw of Shafu is about 0.79, and it is storable without refrigeration. Shafu is produced with nitrite curing salt, sugar, and salt. The finished product is vacuum packaged (2,3). C. Strips Strip-type intermediate moisture meat or dried meat is produced by dry curing or pickle cure. This step can be short (a few days) as the strips of meat are much thinner than the hamtype products. They are then sun-dried, smoked, or mechanically dried. 1. African Jirge Jirge is the Hausa name for the sour sun-dried meat strips prepared and consumed by Shuwa tribesman. It is a fermented, sun-dried meat prepared mostly from beef and occasionally mutton or goat meat. Jirge is like nkarki (see below) in all respects except that it is fermented prior to tearing into strips and drying. Meat for jirge is boned and cut into chunks that are left until the meat starts to ferment to desired sour taste. It is then torn into strips not more than 2 cm thick and sun-dried with or without addition of salt and spices for seasoning. Drying in the sun is by hanging strips over sticks, ropes, and galvanized/barb wire or by spreading over a mat. Drying takes about 5 days, depending on the weather, and the meat strips are turned daily to ensure uniform drying. Storage is as for ndakiko, in sacks, pots, and metal cans, and product shelf-life is up to 6 months or more (18). 2. Spanish (Dried Beef) Cecina Spanish cecina resembles South American charqui (pronounced sharkey) and carne seca, and European Bundnerfleisch and bresaola. Spanish cecina is a salted, dried, and smoked beef meat product manufactured almost exclusively in the province of Leon (northwest Spain). It has an aw of 0.86, posses excellent microbial stability, and can be stored without refrigeration. The processing steps are as follows: Fresh beef pieces, make up of biceps femoris and semi-membraneous muscles, weighing 4 to 6 kg each, are covered with a mixture of coarse salt (ca. 0.15 kg per kg) and sodium nitrite (50 to 60 ppm) and held at 2° to

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5°C and 75% to 95% R. H. for 72 hours. They are then washed to remove excess salt and held under the same conditions for 30 days at 2° to 6°C and 80% to 90% R.H. This is the post-salting or salt equalization stage. The meat is then placed in a smokehouse set at 12° to 15°C and is smoked for about 20 days. The smoking process is achieved by burning both oak and beechwood inside the room. They are then dried for 40 days at 10° to 12°C and 75% to 80% R. H. in a conditioning room. Finally, the meat is aged in a cellar for several weeks (more than 60 days in a cold, dry environment) (23). 3. Mexican Cecina The production method of Mexican cecina varies by location (24). It is generally prepared from lean hindquarters, cutting it into long slices 52 mm thick; the length can reach 1 m and width varies from 10 to 25 cm. Cutting is performed in the same direction as the muscle fiber. Addition of salt and other ingredients is not controlled. Experience determines the amounts to be added in each case. Cecinas so prepared stand unfolded on tables or are hung from 4 to 24 hours. Some cecinas are covered with a thin coat of safflower oil, and the cecinas are folded for distribution. Cecina is also allowed to stand for a time, followed by folding and distribution. Vinegar is added after standing, and then moisture is reduced by a light sun-drying process. The cecina is refrigerated and fat is added. Finally, it is also folded and distributed. 4. African Biltong Biltong can be made from the fillet steak (Binne biltong or ouma se biltong), eye muscle (garing biltong), or more commonly the hindquarter muscle of a young animal. In the manufacture of biltong, the selected muscles are dissected along their seams and cut into strips resembling tongues (hence biltong) 25 to 30 cm long and 2 to 10 cm in diameter. A comparatively lean well-fleshed “buttock” (i.e., hindquarter) will yield 70% biltong, 12% trim pieces, and 18% bone. During the drying process, 60% of the mass of the meat will be lost, so that a whole 6-kg cut of rump steak would yield only about 2.5 kg. Once the raw biltong has been dissected and prepared, it is hung to dry suspended by strings or wire hooks. In dry weather or at a time of year when there are no flies, the strips may be hung in the sun on the first day. Thereafter, the biltong should be hung in the shade. Mildew may form unless drying is rapid. If biltong is dried indoors, an electric fan may be used to keep the flies away. The period of drying and the stage of dryness at which the biltong is considered suitable for further processing both appear to be flexible and a matter for the processor’s own judgement. The meat is salted either by immersion in brine or, more usually, by packing in dry salt. The longer the biltong is left to salt, the more salt is absorbed. Biltong that contains a lot of fat takes longer to absorb salt than lean biltong. The biltong also becomes more salty the longer it is left to dry out. As a result of these factors it is difficult to determine the qualities exactly since personal taste also plays an important role. One or other of various spices such as aniseed, coriander, allspice, or garlic may be mixed with pepper and added to the salt. Other optional ingredients are sugar, saltpeter (to promote a red color), and sodium bicarbonate (to counteract mold). Shin and Leitner (1983) found 25 biltong samples had aw values ranging from 0.36 to 0.93, with most samples falling between 0.65 and 0.85. The pH varied from 4.8 to 5.8, with most being around 5.5. Salt levels ranged from 5% to 15%, with an average of 7%. The final product when packed away should be absolutely dry. Although the fat may become rancid, biltong will keep its qualities for many years if it is stored in a dry place. It may also be vacuum-packed in plastic film and placed in frozen storage where it will keep indefinitely (14,18,25).

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5. Pemmican (American Indian, especially the Cree) Pemmican is the cold-environment equivalent of biltong. The original pemmican was invented by the American Indians, specifically the Cree. It is a dried meat product made from buffalo, caribou, or deer, and later beef, which was packed in melted fat into specially made rawhide bags. The meat was dried in the sun and pounded or shredded prior to being mixed with the melted fat. This preserving method is based on the air exclusion provided by the fat, which not only reduces oxidative changes but diminishes microbial growth. This is accomplished not only by suppressing the growth of aerobic bacteria, but because the combination of a fat medium and dry conditions deprives the microorganisms of the water indispensable to metabolic functions. Mostly, the pemmican was flavored and partially preserved by the addition of dried, acid berries (26). 6. African Ndariko Ndariko is a Fufulde (i.e., Fulani) and Hausa name for sun-dried meat with or without salt and spices. It is prepared mostly from beef and occasionally mutton and goat meat. The meat is boned and the flesh torn into strips no more than 2 cm thick. The best products are obtained by tearing the muscle to pieces so that a group of muscle fibers can be dried as a unit. Salt, if applied, is only at a seasoning, rather than a preservative, level; so also is the use of spices. The meat strips and cleaned intestines, with or without salt seasoning, are hung out in the sun on sticks, ropes, and galvanized/barb wire or spread on grass mats to dry. Drying takes usually 6 to 7 days depending mainly on the weather and to some extent the nature and size of meat strips. During drying the meat strips are turned daily, especially if spread on mats, to ensure uniform dehydration. The fully dried product is stored in sacks, pots or metal cans and has a shelf life of 3 to 6 months under ambient conditions (18). 7. Egyptian Basterma or Turkish Patirma Basterma is one of the most popular cured and dried meat products in many Islamic countries. It is produced mainly from beef and requires simple equipment and little energy for preparation. Three steps are involved in the manufacture of Egyptian basterma: curing, drying, and covering. The curing agents (common salts and potassium nitrate at 200 ppm) are rubbed dry over the surface of the beef (mainly Longissimus dorsi muscles) after incisions are made on the meat surface. The meat is then layered to a thickness of up to one meter. The meat is left to stand for one day, after which more curing salts are added and the layers are shifted. The meat is then left to stand for a second day, then removed from the curing box and hung at room temperature for about 2 to 3 days. It is arranged in layers up to 30 cm in length and put under one ton of pressure for 12 hr. The meat is then covered with paste of 35% garlic, 20% fenugreek (Trsgnnella foenum graecum), 5% to 6% red paprika and cumin, and 38% to 40% water. The dry meat is covered with paste (1 to 2 mm) and stored at 5°C for one day. It is stored at room temperature with good ventilation and sold after ripening (nearly one month) (27,28). 8. Chinese-Style Bacon (La rou or smoke meat) Chinese-style bacon is made from pork. It was usually made in the winter months when the weather is cold and dry. Nowadays, it can be made using refrigerated facilities and temperature-humidity controlled drying chambers. The cleaned meat with the skin on is cut into long strips of about 0.6 kg each. Salt (6% to 12% of green weight of meat) and a small amount of wild pepper (xanthoxylon seeds or fajou in Chinese) are stirr fried with low heat

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to remove moisture, sanitize the seasoning ingredients, and develop the flavor of fajou. Three percent saltpeter is added and mixed well. This curing mix is set cooled and rubbed on the meat surface. The meat is then stacked in a container with layers of curing mix and pressed with a weight (stone) for 3 days. The position of meat strips is reversed and pressed again for 3 days. The cured raw meat is hung in the sun to dry for 4 to 5 days, then held in a cool and ventilated place indoors. This cured meat is stable for over a year. The cured meat can also be surface treated with soy sauce three times to improve the flavor. The cured meat can also be smoked with oak and orange peels (2,4). The drying process can also be accomplished by drying in electrical or gas-heated chambers. Vacuum packaging of these products to preserve quality is also practiced nowadays. D. Pieces These products are usually in small pieces. The meat is cut into small pieces, precooked, and then smoke-dried or freeze-dried. 1. African Tsire (or Suya) Suya is an Hausa word meaning roasted or fire-treated meat. It is in this sense a generic term for partly or fully roasted meat products such as kilishi, tsire, and balanqu. Of these, however, the single product most commonly called suya in the trade is tsire, and the two names are used synonymously. Tsire or suya is defined as boneless meat pieces (mostly beef, but also mutton and goat meat) staked, smeared with a mixture of salt, spices, groundnut flour, and oils, roasted over around a low-burning or glowing smokeless fire. In the production of tsire or suya, meat is boned and cut into chunks about 10 cm long and 8 cm wide. The chunks are later sliced with a curved knife into slices about 1 cm thick. The meat slices are stacked on a slender wooden sticks about 30 cm long and dusted with a mixture of salt, spices, groundnut flour, and groundnut oil for seasoning. The meat stacks are then pinned, or incline on a wire mesh on top of an oven containing low-burning or glowing firewood. Hardwood, low in resins, is used. Roasting takes place for 20 to 40 minutes with occasional turning of the meat stakes. The product is displayed unpacked for sale and wrapped with paper for procurement. It receives no further treatment to enhance stability and its shelf life is only about 24 hours. Under ambient conditions, product moisture is about 20% to 25% (18). 2. African Banda Banda consists of hard-smoked pieces of meat mostly from reject cattle, discarded transport beast such as donkeys, horses, asses, camels, buffalo, and elephants as well as wildlife. Banda is the most commonly produced traditional African dried meat. The animal, after slaughter, is eviscerated and butchered and the large bones are removed. Virtually all of the carcass, including the lean meat, organs, neckbones, ribs, and legs as well as hides/skins and intestines, are used up. These are cut into pieces about 3 to 6 cm and cooked with a small amount of water in half-drums for 15 to 30 min with intermittent stirring. When done, the meat pieces are shovelled out and spread on the floor or mat or on wire mesh over the smoking pit or oven to drain-dry. The meat pieces are then smoked with fire generated from burning hardwood in the smoking pit or earthen oven directly below the meat pieces. This involves high-temperature (hot) smoking leading to further cooking, drying, and shrinkage. Smoking lasts for 18 to 30 hours, depending on meat size and fire intensity, during which time the meat pieces are turned periodically to ensure uniform smoke-drying. A mat is used

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to cover the meat pieces to trap the smoke while the fire intensity is controlled by regulating the quantity of burning wood so as to prevent meat charring. At the end of smoking, the fire is quenched and the meat pieces allowed to cool down to ambient temperature before storage or packaging in sacks and jute/mat bags. The product is dark in color with a stonedry texture and 5% to 16% moisture. It is a very stable product with a shelf life of 6 to 12 months or even up to 2 years under ambient temperature (18). 3. Freeze-dried Meat Pieces With the popularity of instant noodles and instant soup mixes, the demand for freeze-dried meat pieces has increased considerably in recent years. These products are usually produced by cutting or grinding the raw meat into small pieces, precooking the meat pieces, freezing the cooked meat, and finally freeze-drying to very low moisture content (29,30). E. Floss or Shreds Meat floss or shreds are unique Chinese products. They are usually produced by cooking the meat to tender followed by shredding the meat into bundles of meat fibers before cooking in the seasoning liquid to a dried product. However, the final water activity may vary depending on the kinds of product. The texture can be a little bit chewy to very crispy. 1. Meat Floss (rou song) The method of producing meat floss is quite similar to that of shredded beef (see below). Lean pork or chicken meat is cut along the fiber and cooked in water until it is very soft. The broth is then drained from the meat and kept for later use. The meat is mashed manually into fibrous strips. While the broth is concentrated, the shredded meat is added. The meat mass is then heated at low heat and continuously stirred manually until the desired dryness is achieved. At the final stages of drying, the temperature can be raised to speed up the drying process. The aw of beef floss is 0.60–0.62 (31,32). 2. Taiwanese Zousoon (pork floss) Zousoon is a semi-dry pork product that has a aw of 0.60 to 0.65. In preparation, the prerigor muscle is cut into pieces parallel to the direction of the muscle fibers to allow heat penetration and yet still maintain the integrity of the long muscle fibers (33). The raw prerigor muscle is boiled in water in a large shallow gas-fired kettle for about 80 minutes to solubilize the collagenous fibers and to evaporate some of the moisture until the leachedout solids are reabsorbed by the muscle. The heated muscle is then manually pressed with a paddle to loosen the fibers from the large muscle bundles, in order to facilitate drying and to yield the long fibers. The partially disintegrated bundles of fibers are transferred to a gasfired frypan equipped with a continuous mechanical scraper to aid in concomitant drying and disintegration of the muscle bundles into long fibers. Sucrose and salt are added to the muscle at a ratio of raw lean meat:sucrose:salt of 1000:100:15 while heating. Addition of the sucrose and salt too early during heating decreases the efficiency of moisture removal. Dehydrated starch cells are added at a ratio of 0 to 40 of the original raw meat at a moisture level of about 42%. This lowers the average moisture content of the mixture while occupying the void space and aiding in maintaining a loosened texture. Once the desired consistency and water activity are achieved, the predried mixture can be stored at either ambient or cooler temperature until it is removed and finished. The finished product is prepared by adding the predried fibers back to the scraping-frypan and heating. Hot melted

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lard (at a ratio of 20 to 120) is added and the material is heated until the long fibers reach aw of 0.60 to 0.65. The finished product has a sweet-meaty flavor, a chewy texture, and a light brown color. There are commercially available two types of meat floss, which differ in texture. Although both have cotton-like appearance, one is more crispy. This is made by adding some vegetable oil to the normal product and a short drying period at a lower temperature is given to achieve the crispy texture. In general, ingredients used are soy sauce, five spice powder, pepper, sugar, and monosodium glutamate (33). If the rate of heating or drying is too fast or the temperature gets too high, the fibers collapse and become short (about 1 to 2 cm) and have a darker color and poorer flavor. In this case, it is referred to as the ‘short-fibered’ or the ‘dry roasted’ product, and has aw of 0.40 or below. 3. Malaysian Sambal Daging (shredded spiced beef) In the production of sambal daging, coarse fiber meat such as rump is normally used. The meat is cut into pieces of about 5  5  10 cm. The pieces are then boiled in water until they are quite tender, then are drained and allowed to cool. When sufficiently cooled, the fibers of cooked meat are pulled apart manually to obtain strands of meat. While the meat is being shredded, the sauce is prepared. The broth obtained earlier is used in the blending of the ingredients, which include coconut milk, sugar, onion, garlic, ginger, salt, tamarind, dried chili, and coriander. The type and amounts of ingredients used may vary from manufacturer to manufacturer depending on consumer taste preferences. All the ingredients are poured into an oval-shaped saucepan and heat-concentrated with constant stirring to prevent charring. When concentrated to about 50% of its original volume, the shredded meat is poured into the saucepan and stirred continuously until the desired dryness of the product is achieved. Usually toward the later part of cooking, the intensity of the heat is lowered to prevent charring of the product. The manufacturing process could take up to 5 hours, a greater part of this period requiring continuous stirring. The aw is 0.48 to 0.61 (20). F. Powder Meat powder such as Sudanese sharmoot is another unique product made by first drying the meat strips in the sun or in drying chambers. The dried meat is then ground to a powder form for storage and usage. Sudanese Sharmoot. Sudanese Sharmoot is a powdered dried meat product prepared traditionally by cutting the meat into thin strips, hanging it in the sun for 3 to 5 days until it is dry, then grinding it into a fine powder. An improved procedure is to grind the meat, precook it, and then dry at 65.5°C with forced air for 3 hours before grinding into powder (34). G. Sausages Sausages are usually produced by grinding or cutting the meat, followed by mixing it with curing and seasoning agents. The mixture is then stuffed into natural or artificial casings. The size of the sausage may vary depending on the origin and culture. The raw sausage is then subject to drying, smoking, or both. Some products are dried to the desirable water activity in a short time, and some are dried slowly with a mild fermentation. Intermediatemoisture sausages can be classified by particle size or degree of chopping into three categories: emulsion-type, coarse ground, and muscle cube. Most of the sausages processed in Mid-East, European, and American countries belong to the category of emulsion-type products. Usually the lean meat is first comminuted

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in the presence of salt to extract the highest concentration of myofibrillar proteins (actin, myosin, and actomyosin) to stabilize the batter. As the fat component is added and minced, the fat particles are encapsulated by the extracted protein. The extracted proteins facilitate surrounding and interacting with the fat particles or droplets with their emulsification capability. A sausage batter is actually a very complex mixture consisting of suspended particles (collagen fibers, myofibrils, cellular organelles), immobilized and free water, lipid droplets and particles, and hydrated, solubilized, and nonsolubilized myofibrillar proteins. 1. Lebanon Bologna Lebanon bologna is a semidry type, coarse-ground, fermented product requiring knowledge of the art. It can be prepared using starter cultures, or it can be held under specific conditions that preferentially promote the growth of organisms that impart flavor, texture, and preservative qualities. Whole-carcass cow meat is salted with 2% salt and held for 8 to 10 days at 34° to 38°F. The beef is ground through a 1/2 inch plate and mixed in a ribbon mixer with the salt, sugar, spices, and sodium nitrate. The mixture is then passed through an 1/8 inch plate and stuffed into No. 8 fibrous casings. The filled casings are tied and stockinetted for support. The product is transferred to a smokehouse for a 4- to 7-day cold smoke, usually 4 days in the summer months and 7 days in the late fall and winter months. Lebanon bologna is traditionally made under conditions that call for little or no refrigeration. The finished product is extremely stable, even though the moisture content may be as high as 55% to 58%. The salt content of the finished product is usually 4.5% to 5.0% and the pH ranges from 4.7 to 5.0 (32). 2. Dry Sausages (beef and pork Genoa, hard salami, pepperoni) The manufacture of dry sausages is steeped in art. However, the art is slowly yielding to the advance of science. Usually the meat is ground and mixed with the curing mix consisting of salt sugar, spices, and nitrate. Sometimes wine is also added as in Genoa. The mixture is briefly mixed until a good distribution of the fat and lean is apparent. The mixture is then stuffed into casings. The stuffed product is held under controlled temperature and humidity conditions until the desirable degrees of desiccation and fermentation are reached. Hot smoking may also be applied. Safety of these products are usually accomplished by overcoming the hurdles in the manufacturing processes (5). 3. Spanish Salchichon Salchichon is a popular Spanish-style sausage; the Spanish name means large sausage. It can be made from lean beef, lean pork, or a combination of both. Pork backfat is added along with seasoning (1% to 4% salt, nitrate, and white pepper) and sugar 1% of a 1:1 mixture of sucrose and dextrose). It is normally held at a temperature of 25° to 30°C and a R. H. of 80% to 90% for 5 days, after which it is held for an additional 60 days at 30° to 37°C and 70% to 80% R. H. for maturation. This reduces the moisture content from about 50% to 60% to 26% to 35%. The final aw is 0.80 to 0.87, and the final pH is 4.6 to 4.8 (36). The production and physicochemical characteristics of salchichon, which was made from lean ground cow 40% beef, 30% lean pork and 30% pork fat. It contains about 3% salt, 1% sucrose, 1% dextrose, 0.5% wine, 0.25% ground pepper, whole pepper, potassium nitrate, and sodium nitrite, which were ground and mixed together. Incubated at 20°C and 80% R. H. for 5 days after stuffing in large casings (natural or artificial), salchichon then undergoes maturation at about 12°C at 70% R. H. for an additional 60 days. Salchichon

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made this way has a final composition of about 25% protein, 27% moisture and 43% fat, and 5% ash. It has a final pH 5.90 and aw 0.85 (37). 4. Cervelet Cervelet is a dried, smoked, cooked sausage. An improved processing procedure includes hot meat freeze-dried with curing salt and starter culture, rehydrated in cutter to 100% with 80% ice chips  20% water, frozen to 42°C in a plate freezer, comminuted in a frozenmeat-mincer, added to the cutter before the other ingredients and processed in the usual way (37,38). 5. Chinese Sausage (Lup Cheong) Several grades of Chinese sausages are available commercially. The quality of grade depends on the meat to fat ratio. For liver sausage, the meat is replaced by liver. Pork is usually used for the manufacture of Chinese sausage, although chicken or turkey meat can also be used. The meat used in the manufacture of Chinese sausage is usually ham meat and the fat is from backfat. The meat is manually cut into short strips and the fat is cut into 10 mm cubes. The meat and fat are then mixed thoroughly with the seasoning mixture and are left to marinate for several hours before filling is carried out. The casing used for Chinese sausage is usually derived from the small intestines of the pig. This type of casing is usually in the dry form and must be soaked in water before use. With the help of a funnel, the mixture of meat and fat is forced into the casing, which is tied at one end. The casing is punctured with needles to allow air trapped during the filling process to escape. After being knotted into short sections and given a water spray to remove any adhering ingredients, the sausage is allowed to drip for a while before it is dried in a drying cabinet. The heat source is usually burning charcoal. This drying process can continue for up to 3 days, depending on the temperature used. The aw of lup cheong is 0.75 (2,4). Instead of slicing and cutting the meat, it can be minced through an 8 mm plat. Frozen slabs of backfat that have previously been mixed with salt to remove some water are passed through the machine again with the strips fed across the series of parallel rotating blades to obtain cubes. The minced meat and fat are then mixed thoroughly in the seasoning mixture before filling into the casing. The filling process is carried out using an automatic filler with automatic linking facility that can control the weight of each sausage. Pig intestine casing is also replaced by collagen casing. The amount of heat applied can also be regulated more precisely by modern convection type temperature-controlled drying cabinets equipped with condensers to reduce the drying time from 72 hour to 35 to 48 hour (2,4). One suggested seasoning mixture consists of ascorbic acid, Chinese wine, light soy sauce, antioxidant, water, and curing mixture (0.25% sodium nitrite, 0.5% sodium nitrate and 99% salt) (2,4). II. STABILITY OF INTERMEDIATE-MOISTURE MEATS Microbial issues, enzymatic deterioration, chemical deterioration, nutritional and sensory stability, and packaging issues will be discussed in this section. A. Microbial Stability and Safety Although fresh meat is an ideal medium for bacterial growth and subject to rapid spoilage, the interior of the animal is virtually free of organisms except for the lymph nodes and excluding the gastrointestinal and respiratory tracts (39). Environmental conditions prior to

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slaughter and during processing affect the degree of contamination of the surfaces of meat. Microbial spoilage of meat generally may be attributed to the activity of the surface microflora. Deep muscle penetration and growth are not significant (40,41). A different group of microflora is found on cured, processed meats as compared to those in fresh meats. Bacteria such as Staphylococcus, Micrococcus, Pediococcus, Streptococcus, Lactobacillus, Clostridium, and Bacillus have been isolated from cured meat. Yeasts and molds are not usually associated with freshly made cured meats, but after aging, sausages and countrycured hams may show growth of these fungi. In general, yeasts and especially molds are the major spoilage factors of traditional intermediate-moisture meat products. Recent outbreaks of foodborne illness due to Salmonella spp. in beef jerky and Escherichia coli O157:H7 in venison jerky raise great concern over the safety of intermediate-moisture meat products made in the home. The potential of injured bacterial cells to regain the ability to cause illness is a particular threat with pathogens such as E. coli O157:H7, which is believed to have a low infectious dose (42). Micrococci and streptococci, which predominate on cured meat, resist salt and nitrite but are affected by pH in the practical range. 1. Minimum Water Activity for Microorganisms The aw of food influences the multiplication and metabolic activity (including toxin production) of microorganisms, also their survival and resistance. In the aw range of IMM (0.60 to 0.90) some bacteria (Pediococcus, Streptococcus, Micrococcus, Lactobacillus, Vibrio, Staphylococcus), yeast (Hansenula, Candida, Hanseniaspora, Torulopsis, Debaryomyces, Saccharomyces) and molds (Cladosporium, Paecilomyces, Penicillium, Aspergillus, Emericella, Eremascus, Wallemia, Eurotium, Chrysosporium, Monascus) may multiply. Most of these organisms cause spoilage; some produce toxins (Staphylococcus, Paecilomyces, Penicillium, Aspergillus, Emericella, Eurotium). Yersinia enterocolitia has been isolated from meat foods and has become of increasing concern as a cause of gastroenteritis and other syndromes in humans (43). The aw requirements of some microorganisms have been reported by different authors. The generally accepted minimum aw for some important meat pathogen are listed in Table 2. Among them, Staphylococcus aureusi is the lowest aw tolerating bacteria, which under anaerobic conditions is inhibited at an aw lower than 0.90 but aerobically at an aw 0.86. It was noted that the mean growth rates of Staphylococcus aureusi are higher at an aw level of 0.995 and 0.990 than at 0.999. Below these optimal aw levels, growth rates decrease as a function of aw. Slow growth was observed at 0.86 aw, with total growth suppression at 0.84 aw (44,45). Table 2 Minimal aw for Multiplication of Major Microorganisms Present in IMM Bacteria Clostridium Salmonella Escherichia coli Bacillus Microbacterium Staphylococci

IMM

Minimum aw

Pork sausage Beef jerky Venison jerky Sharmoot Cecina Basterma

0.97 0.95 0.95 0.95 0.94 0.86

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Of the gram-negative rods, Salmonella and Escherichia coli are the representatives. The growth and toxin production of Clostridium botulilum type C is inhibited below an aw of 0.98 (46) of Clostridium botulilum type E  0.97 (47), and Clostridium botulilum type A and B (48), and Cl. Perfringens 0.95 (49). 2. Microflora in Traditional Intermediate-Moisture Meat Products Obviously, environmental factors such as temperature of processing, curing salts, sugars and other ingredients, smoking, and storage conditions all affect the microflora that develops on IM meats. Microbiological quality of 125 samples of the most popular Egyptian meat products [(75 samples of Egyptian fresh sausage (EFS) and 50 samples of basterma] was determined (27). The aerobic plate count (APC) and Lactobacillaceae count of basterma ranged from 1  104 to 9  106 cfu/g, respectively. Enterobacteriaceae, fungal, and yeast counts for basterma were similar (1  102 cfu/g). Twenty samples each of (a) minced meat, (b) luncheon meat, and (c) basterma (a dried meat product) were obtained from the Assiut City market and analyzed mycologically (50). The genera and number of species identified were Debaryomyces, 5 species; Candida, 7 species; Saccharomyces, 3 species; Torulopsis, 3 species; Rhodotorula, 2 species; Trichosporon, 1 species. Predominant mold genera were Penicillium and Aspergillus; predominant yeast genera were Debaryomyces and Trichosporon, which in addition to Debaryomyces frequently contained Candida and Rhodotorula (50). Twenty one samples of cecina was examined microbiologically, both on their surfaces and internally, for salt-tolerant microflora, Micrococcaceae, lactic acid bacteria, and yeasts (23). Throughout curing, the dominant flora on the product (both internally and externally) were micrococci; surface microccoci were present at a level of 107 cfu/g, whereas levels in the deep tissue of the product were approximately 103 to 104 cfu/g. Levels of yeasts and lactic acid bacteria were also high in both locations. Of 159 isolates belonging to the family Micrococcaceae, 81% were Staphylococcus (with S. equorum, S. xylosus, S. saprophyticus and S. simulans being the most abundant species). The remainder were Micrococcus spp. (11%) or were unknown (8%). In finished biltong, which is an unheated meat product, total counts of 107/g and 6 10 /g molds and yeasts respectively have been reported (51). The dominant component of the microflora of the final products is gram-positive, halotolerant staphylococci (coagulasenegative) and micrococci (51). Stable biltong had a total count of 103 to 106/g, and contained lactobacilli and Micrococaeae in relatively high numbers (52). Twenty genera of molds were isolated from different kinds of meats products including bacon, hams (particularly country-cured hams), salami, and dry sausages. Among the molds isolated, Aspergillus and Penicillium species predominate (53). It was concluded that fungi are important in retention of moisture and color of aged hams and sausages. Penicillia and aspergilli grow on hams after the most loss about 25% to 30% of the original weight as moisture, and the aw become reduced (54). A total of 670 strains of fungi were isolated in the cured and aged meats (fermented sausages, country-cured hams, and country-cured bacon) that were collected from the United States and Europe (55). These strains included 456 molds and 214 yeast, which were considered to be typical of the microflora from the products. Yeast was more commonly associated with fermented sausages than with country-cured hams, and were found mainly on the surface of products; they contributed to spoilage when present in high numbers.

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From the 42 stable imported commercial Chinese IMMs from Taiwan (20 samples), Singapore (18 samples), and Hong Kong (4 samples), rarely was recovered more than 104 microorganisms/g, and most samples were in the range 102 to 103 g. This microbial stability was contributed to the hurdles of water activity and heat treatment, with little contribution from the pH hurdle. It was concluded that Chinese IMMs are microbiologically safe products because the heat treatment eliminates most organisms present in the raw material. Further, the survivors and microorganisms that recontaminate the product are inhibited and inactivated by the critical aw of 0.69 (56). 3. Pathogens in Traditional Intermediate-Moisture Meat Products It was reported that commercial pork sausage had an incidence of C. perfringens of approximately 39% (57). In a survey of retail stores, the possible causes of outbreaks of staphylococcal intoxication following consumption of any fermented sausage such as Genoa salami was reviewed. Microorganisms, particularly staphylococci, may grow in products that are not smoked or cooked and are held at temperature 18°C before drying (58). Botulism is a rare food toxemia caused by anaerobic, spore-forming bacillus. Most botulism outbreaks are caused by home-preserved foods. Nitrite and salts as well as refrigeration play important roles in controlling botulism in cured meat; inadequate thermal processing was a contributory factor in some instance (59). The safety of basturma, an intermediate-moisture meat product, from salmonella was investigated and it was found that the product is generally safe (60). The identification of staphylococci and micrococci isolated from basturma was studied. Of the 120 isolates, 92.5% were classified as staphylococci and 7.5% as micrococci. Differentiation of the species revealed 42% Staphylococcus epidermidis, 32% Staphylococcus saprophyticus, 12% Staphylococcus simulans, 4% Staphylococcus carnosus, 2% Staphylococcus hyicus subsp. hyicus and 7.5% Micrococcus varians (61). Microbial stability and potential for survival and growth of certain food poisoning microorganisms in basturma were evaluated (61) by inoculating the freshly pasted product with two common pathogens, Staphylococcus aureus and S. abortusovis, and following their counts during storage. Both pathogens survived the final dehydration stage but their counts remained fairly steady during 60 days of refrigerated storage. Enterobacteriaceae, salmonellae, S. aureus, yeasts and molds were not detected in commercial samples (62). The survival of salmonellae, anthrax bacilli, and pathogenic clostridia in pastirma was investigated and this product was found virtually free of these organisms (63). Staphylococcs aureus and its enterotoxins are not of much concerns in biltong with low aw (52). However, the recovery of Salmonella spp. from biltong has been reported by Van den Heever (65). Salmonellae survive for a long time in biltong, especially in the product made from the muscle of diseased animals. Biltong with such endogenous infection has caused salmonellosis in humans (66). Care in the selection of meat as well as good hygiene in the processing of biltong must be emphasized. Dry sausages have often been implicated in outbreaks of staphylococcal food poisoning and are frequently contaminated by Salmonella and coliforms (67). Both groups of microorganism are widely distributed in meats and may grow during the first part of the fermentation process. 4. Artificial Inoculation Tests in Traditional Intermediate-Moisture Meat Products Artificially contaminated slices of basterma plus garlic paste showed that the paste inhibited growth of Salmonella typhimurium (27). APC and Enterobacteriaceae counts of EFS

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(Egyptian fresh sausage) ranged from 1.1  104 to 1  108 and from 1  102 to 1  107 cfu/g, respectively. Nearly 26% and 29% of EFS were positive for Clostridium perfringens and coagulase-positive Staphylococcus aureus, respectively. Salmonella was not detected in any samples. The fate of pathogens inoculated onto ready-to-eat cured meats has been examined (68,69). The data show that Clostridium perfringins was incapable of growing on sliced ham, chopped ham or bologna at abusive holding temperature. Intermediate moisture meat products were collected in Taiwan, Hong Kong, and Singapore, and evaluated (64) for physicochemical and microbial composition, as affecting stability. The reproduced Chinese IMM showed that salmonellae, pathogenic staphylococci, yeasts, and molds are eliminated during the usual processing by heat applied. Enterococci may survive the process, but die during storage of the product. Spores of bacilli and clostridia decrease during processing and storage too but do not disappear completely (64). Three different procedures used in China for preparation of dried meats, as well as that for preparation of Chinese sausage, were studied (4). They had on receipt water activities in the range of 0.785 to 0.200, and pH values of 6.21 to 5.27. Thirty-five of the products proved microbiologically stable after inoculation with xerotolerant molds of the Aspergillus glaucus group and storage for 3 months at 25°C. These stable products contained 15% to 35% sucrose, 3% to 5% NaCl, and 10% to 20% moisture; some, more drastically treated, had 2% to 12% moisture. The sausage produced in the laboratory using traditional recipes contained 10% sucrose and 2% NaCl; Salmonellae did not multiply in it, but Staphylococcus aureus did, hence heating before consumption was necessary. Whole dry-cured (country-style) hams from six manufacturers were sliced and inoculated with approximately 105 cfu/g of ham of Escherichia coli O157:H7, Listeria monocytogenes, a mixture of three Salmonella spp. (Salmonella typhimurium, Salmonella enteritidis, and Salmonella choleraesuis), or Staphylococcus aureus. All ham slices were vacuum-packaged and stored at 25°C or 2°C. S. aureus was detected in 2 of 60 control slices, Salmonella in 2 of 120, L. monocytogenes in 4 of 120, and E. coli O157:H7 was not detected in any of the 120 control ham slices analyzed before or after storage. The extent of the decreases in populations of the inoculated pathogens during storage of vacuum-packaged dry-cured ham slices. Decreases in Salmonella spp. and E. coli O157:H7 populations were greater in slices stored at 25°C than at 2°C, whereas decreases in L. monocytogenes were similar at both storage temperatures. Staphylococcus aureus enterotoxin was not detected in either S. aureus–inoculated or control ham slices after storage for 28 days. Survival of these pathogens in vacuum-packaged dry-cured ham slices suggests that contaminated hams may pose a safety risk to consumers if eaten without adequate cooking (70). A total of 420 samples of smoke-dried meats (smoked meat products, ham, pork sausage, bacon) was collected over 3 years from different regions of Croatia and analyzed for the presence of aflatoxigenic strains of Aspergillus. Strains of A. flavus (69 samples) and A. parasiticus (6 samples) were found in 17.8% of samples. Eight of these 75 strains produced aflatoxins. A. flavus strains produced mainly aflatoxin B1 (1.4 to 3.12 mg/kg). Some strains of A. parasiticus produced aflatoxins B1, B2, G1, and G2; others produced aflatoxins B1 and G1 only, at concentration of 0.1 to 450 mg/kg (68). Aflatoxins are unlikely to be encountered in biltong with aw 0.80, even though A. flavus quite frequently occurs on the product (25). Similar results were reported by other researchers (72): They stated that although Aspergillus flavus is frequently isolated from biltong, aflatoxins are not normally found in biltong. The decrease of aw during charqui processing promoted a gradual inhibition of microbial contamination (10). Investigators claimed that it is possible to produce charqui with

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Intermediate-Moisture Meat and Dehydrated Meat

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low counts of microorganisms when adequate hygienic conditions are maintained during processing and storage. The high level of contamination observed in charqui sample purchased from a retail market were a consequence of mishandling the products. The Sudanese dried meat, Sharmoot is a major food product in East Africa (73). The product was chemically and microbiologically stable for at least 4 months without refrigeration. Although staphylococci and Enterobacteriaceae were the most common major bacterial groups isolated from dried meat samples at the beginning, micrococci and bacilli predominated during the last stages of storage. Microbiological data (total, spore, yeast, and mold; Staphylococcus aureus counts; and Clostridium perfringens detection) indicated that the product was microbiologically more acceptable than the comparable product made traditionally in the Sudan. The potential exists for large-scale production of Sharmoot. An inoculation study was conducted to determine the efficiency of glycerol in reducing known bacterial loads in intermediate moisture pork products (74). Cooked samples of pork tenderloin were equilibrated in glycerol solutions at both high (55°C) and low (5°C) temperatures and the preservative effect of intermediate-moisture conditions in meat was investigated. A mixed spoilage culture containing aerobes, pseudomonads, and coagulasepositive staphylococci was obtained from a pork sample kept at 5°C for 5 days. The results indicated that the glycerol infusion equilibrium process appeared to be successful in preservation of meat stored at both room temperature and refrigeration temperature for a period of 3 weeks. This finding is in good agreement with that published by others (75). These IM systems studied were microbiologically stable for up to 6 months storage (76). In summary, intermediate-moisture beef, pork and chicken products have been extensively examined, and processing methods included salting, drying, smoking, immersing in oil, and casing. The survival ability of various organisms on meat is at low aw (approx. 0.6) and, generally, bacilli and streptococci survived best and yeast worst. B. Chemical Stability Although intermediate-moisture products are microbially more stable than raw or cooked meat, they are still subject to deterioration through chemical and physical processes including oxidation, protein denaturation, cross-linkage and browning, which can reduce their nutritive value and eating quality (77). Dry sausages made with a normal recipe have a fat content of around 40% to 50% (78). Pork back fat used in fermented sausages has above 60% unsaturated fatty acid. The large majority of IMMs are stored in air-permeable packaging material. This permits the oxidation of unsaturated fatty acid, especially those in the finely comminuted meat products (79). A close relationship between moisture, protein, and ash values was found (10), suggesting the possibility of using the resulting charqui aw value as a parameter to define the product instead of the official moisture and mineral residue contents. TBA determination, which expresses the state of lipid oxidation, rapidly reached a maximum value, corresponding to previous observations on the pro-oxidant role of salt, and then decreased gradually. Kilishi stored for 60 wk under ambient conditions in cellophane bags was analyzed for lipid and fatty acid profiles and oxidative stability. Total lipid content was 25%, of which 89.42% was triglyceride (TG) and 10.57% phospholipid (PL). TBA number had reached 2.01 in 60 weeks (in mg malonaldehyde/1000 g meat). The major TBA-reactive substance in kilishi distillates was not malonaldehyde, as in beef or chicken, but an unidentified aldehydic browning product with a spectrophotometric peak at about 440 nm (17). It was re-

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ported that TBA numbers of dendeng decrease during the early stages of storage but increase during prolonged storage (e.g., 6 months at 37°C) (19). The oxidation of lipid and cholesterol in Chinese-style sausage stored for 5 months was investigated (80). The 2-thiobarbitaric acid active substance and peroxide value of sausage stored at 15°C were significantly greater than at 4°C. The polyunsaturated fatty acids in sausage decreased with storage, and the content of cholesterol decreased significantly after 3 months of storage. 7-Beta-hydroxycholesterol, 7-keto cholesterol, and 22-keto cholesterol were the major cholesterol oxidation products, but there was no detectable 25-hydroxycholesterol or cholesterol. Biochemical changes in cecina-like meat were evaluated during storage (81). Samples of cecina from different states of Mexico were tested for aw, color, texture, fat, protein, moisture, and chloride content. Based on the low activation energy for hematin complex degradation (around 1 Kcal/mol), the manufacturing of cecina is regarded as a low-energy biochemical process. Chemical analyses of kundi (normally beef-based) showed that, apart from a high level of carcinogenic benzo(a)pyrene (10.5 to 66.9 ng/g), eight other polycyclic aromatic hydrocarbons (PAH were present at various concentrations (82). The high levels of PAH in kundi were due to the high glow and smoking temperature, averaging 926°C and 191.5°C, respectively. Column chromatography was used for PAH extraction, with propylene carbonate as the eluting chemical; TLC on acetylated cellulose layer plates was used for separation, and the determination was performed using spectrophotofluorimetry. The public health implications of PAH as one of the possible carcinogenic factors in the high incidence of primary liver and stomach cancer reported in Nigeria are highlighted. III. STRUCTURAL COMPONENTS OF MUSCLE TISSUE RESPONSIBLE FOR PHYSICOCHEMICAL PROPERTIES OF INTERMEDIATE-MOISTURE MEAT PRODUCTS In order to fully understand the structural components of muscle and their involvement in the production of IMM, the gross structure of muscle will be described first, followed by a description of some of the ultrastructural elements involved. A. Gross Meat Structure The bodies of meat-producing animals are composed of about 300 anatomically distinct structural units that differ greatly in size, shape, and appearance (83). Muscle tissue contains numerous structural components; among them connective tissue, myofibrillar proteins, muscle membrane, and water are significant contributors to the physical properties of intermediate moisture meat products (84). The intact muscle is composed of numerous fibers that generally parallel the long axis of the muscle. These fibers, representing muscle cells, are between 50 and 100 m in diameter and vary in length from a few millimeters up to several centimeters. Muscle fibers are described as cylindrical. The fibers themselves contain about one thousand myofibrils about 1 to 2 m in diameter that run through the whole length of the muscle fiber. The myofibrils consist of alternating thick and thin filaments. The segment between two consecutive Z-lines is called the sarcomere (85). 1. Muscle Membrane and Connective Tissue The lipoprotein nature of cell membrane precludes a primary function for this material in the physical properties of muscle. The entire surface of the muscle fiber is covered by a unit

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membrane called the plasmalemma (86). Another membrane system, the sarcoplasmic reticulum (SR) is found in the sarcoplasm that separates myofibrils. Endomysium has at least three roles: (a) muscle fiber-muscle-fiber connections (b) muscle-fiber-capillary connection, and (c) a weave network of collagen intimately associated with the basal laminae of the muscle fibers (87). The plasma membrane of the muscle fiber has several functions: (a) it forms a selective barrier that regulates transport of ions and molecules in and out of the cell, (b) it has the ability to transmit an action potential generated by a nerve, and (c) it is involved in the transmission of force produced by the contractile apparatus within the muscle cell (88). On the examination of muscle tissue that is used widely in the traditional IM meat products in cross-section (as shown in Figs. 1 and 2), there is a fairly thick envelope of connective tissue surrounding each individual muscle. This thick layer of connective tissue around an entire muscle is known as the epimysium, whereas the smaller layer of connective tissue dividing the muscle into bundles or fasciculi is called the perimysium. A still finer connective tissue layer extends from the perimysium into the muscle fiber bundles and encircles each muscle fiber. This thin layer of connective tissue consists of reticular fiber carrying the blood capillaries and is called the endomysium. The epimysium is generally easily separated from the body of the muscle, whereas perimysium and endomysium are not practically separated from meat (89).

Figure 1 Schematic diagram of disintegration of muscle tissue into muscle fibers during the preparation of an intermediate moisture meat product such as Zousoon.

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A

B

C

Figure 2 Disintegration of intact pork muscle fiber bundles (A) into separated bundles (B) and then single pork muscle fiber (C) during preparation steps in the production of Zousoon.

The connective tissue layer is composed of two proteins, collagen and elastin. Both of these proteins have their own characteristic amino acid composition. Collagen is the more abundant of these two proteins, representing about 2% to 6% or more of the dry weight of muscle. Collagen is usually found in the fascia surrounding the epimysium, perimysium, and endomysium (90). At least 15 genetically distinct types of collagen have been characterized (91). The major type of collagen present in muscle have been characterized. Among them, intramuscular collagen deserves special interest. These collagen are located in the

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endomysial basement membranes by immunofluorescent staining. A specific distribution of genetically distinct forms of collagen within epi-, peri-, and endomysial connective tissues was demonstrated by the presence of type I collagen in the epimysium, type I and III in the perimysium, and type III, IV, and V in the endomysium from bovin muscle (92). 2. Myofibrillar Proteins Among the 200 distinguishable protein complements, only a few appear to be directly involved in dictating the physical properties of intermediate-moisture meat products (93). 3. Contractile Proteins The muscle myofibril has been found to be composed of a number of distinct elements. The thick filament consists mainly of myosin (55% of the myofibrillar proteins) and C protein (2%). The components of the thin filament are actin (23%), tropomyosin (6%), and troponin (6%). Other myofibrillar proteins are present in lower concentrations (94). As a consequence of postmortem depletion of adenosine triphosphate (ATP), actin (located in the I band and the major constituent of the thin filament) combines through cross-bridges with myosin (located in the A band and the major constituent of the thick filament) to form actomyosin. The filaments are built up by the so-called myofibrillar proteins (94). The protein matrix in muscle has a marked effect upon its functionality and properties. The contractile myofibrillar proteins are recognized as essential for the characteristic textural properties of emulsion-type sausage such as bologna (95). Two major protein components, actin and myosin, which together account for about 70% of the weight of the myofibril, are those involved in the gelation of processed meat products (96). 4. Water Water is the major constituent of muscle; it accounts for at least 75% by weight of fresh tissue. The bulk phase water in muscle tissue is located (a) within the filament, (b) in the interfilamental spaces, and (c) in the extracellular space. According to the form of binding, water in meat is classified as strongly bound (hydrational), immobilized, and “free” (97,98). Of more importance than the total amount of water present is the water holding capacity (WHC) of the tissue or the ability to retain its own water during processing. a. Bound Water. The water within the filament is primary a result of its association with myofibrillar protein through hydrogen bonding. It has long been appreciated that the binding of water to the surfaces of protein molecules is too small to account for the observed changes in water content (99). The weight fraction of protein in meat is about 20%, and proteins are commonly believed to bind water only to the extent of the order of 0.5 gram of hydration water per gram of protein (97). A relatively small part of the tissue water (4% to 5%) is tightly bound on the surface of the protein molecules as hydration water. Water actually bound to protein molecules therefore represents a small fraction of the total water present (98). It is estimated that only a very small part of the muscle water is present as “constitutional water” (about 0.3 g H2O/100 g protein, i.e. 0.1% of the total tissue water) which is located within the protein molecule (100). b. Immobilized Water. In addition to the bound water, it was postulated that intrafiber water may possess the ability to reduce adhesive force between the myofibrils. Capillary water, which is entrapped in myofibrils within muscle membrane, deserves special interest for intermediate-moisture meat products. Changes in WHC are closely related to pH and to variations in muscle proteins (101). It is reasonable to suppose that water is

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held in muscle by capillarity, mostly in the interfilamental spaces within the myofibrils, but that a substantial part also exists in the spaces between the myofibrilar and in the extracellular space. Bulk-phase water is immobilized within the microstructure of the intact or comminuted tissue (102). The muscle protein molecules in an aqueous solution interact with water, and when it moves through the solvent it carries some water with it. Part of this bound water is believed to be hydrogen-bonded to the surface of the protein molecule, while most of them may be present in clefts or pockets (103). It is generally accepted that a further part of tissue water (5% to 15% of the total water) shows a relatively restricted mobility (94). About 10% of the total water in the living muscle must be associated with the extracellular space. It is supposed that about 80% of the water in the muscle fiber would be in the myofibrillar space and about 20% in the sarcoplasm. The muscle fiber is covered by the sarcolema, the cell membrane. The membranes of the sarcolemma and the sarcoplasmic reticulum consist of proteins of the connective tissue type (104). Among physical stabilization factors in meat mixtures, the heated protein gel matrix is generally recognized as the predominant factor controlling water retention. Gelation of meat proteins during heating takes place to some degree in all products. It first involves unfolding, then the interlinking of muscle proteins to form a three-dimensional continuous network (105). The myofibrillar proteins, myosin and actin, are the building components in a three-dimensional network. Within the three-dimensional protein network a large amount of water is absorbed (106,107). The moisture mobility in frankfurter emulsions during cooking. They suggested that the bulk of unbound moisture is fixed in the gel matrix (108). A large amount of water is retained within the three-dimensional protein network of manufactured sausage batters (109). 5. Isotherm Isotherms appear to be related to different modes of water binding (110). IM meats are multi-component systems. The isotherms of the meat or meat mixture show a relationship of averaged moisture content and the common water activity (33). In reconsideration of the aw measuring techniques, it was suggested that measuring the water vapor pressure (i.e. isotherm) is a better index of water binding by muscle. Of great importance in IM meats are the sorption properties of the muscle components (33). The possibility of using the Guggenheim-Anderson-De Boer (GAB) equation to delineate thermodynamic aspects of moisture sorption behaviour in basturma, a traditional Mediterranean intermediate-moisture meat product, was studied (28). For aw values between 0.4 and 0.9 and with mean relative deviation modules values of 5%, the GAB equation gave satisfactory goodness of fit. Moisture sorption characteristics of spiced and nonspiced kilishi (a Nigerian sun-dried meat product prepared from beef, mutton, or goat meat) were also investigated. A BET type III behavior between aw of 0.10 and 0.96 was described, and hysteresis was exhibited. Four sorption models (Oswin, Halsey, Iglesias and Chirife, and Guggenheim-Anderson-de Boer) used to predict the sorption characteristics of kilishi were found not to be as suitable as an exponential model in predictive ability (28,110). GAB monolayer moisture contents and the constants in the exponential model were temperature-dependent. Spiced products had higher equilibrium moisture contents and their monolayer values were higher. The practical importance of the sorption properties of kilishi is noted. These equations can be used to determine the proper extent of product dehydration for safe storage, increased

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product yield, and improved sensory quality. Sorption isotherm was used to predict the critical moisture content for storage of biltong (111). The Halsey equation gave higher mean relative deviation modules values, indicating that this isotherm model has limited ability to describe moisture sorption behavior of the specific product. The utilization of the sorption data for predicting dehydration requirements and product stability of commercially produced basterma has been reported (28). Moisture sorption isotherms of thin slices of basturma were determined using the static method with saturated salt solutions. Desorption isotherm data were used to predict the extent of dehydration required to reach the desired aw. The soluble solids in the amorphous state absorb more water than that of the muscle solids within a water activity range of IM meats. In production of IM meats, in addition to selecting raw muscle with good sorption properties, one should try to modify the shape of the isotherm in the meat by formulation (adding sugar and/or salt). Addition of these nonmeat ingredients such as sugar and salt changes the sorption phenomena of the meat solid in Zousoon processing (33). In the range of aw 0.60 to 0.90, which is equivalent to an open storage relative humidity of 60% to 90%, the meat product has a higher moisture content than that of the muscle alone. This phenomenon was attributed to the altering structure of the solid matrix, such as developing a gel or capillary structure. It was further pointed out that it is the intact muscle cell with membrane with its selective barrier characteristics that retained the water. IV. EFFECT OF HEATING ON PHYSICOCHEMICAL PROPERTY CHANGES DURING INTERMEDIATE-MOISTURE MEAT PROCESSING A. Proteins For isolated muscle protein, Differential Scanning Calorimetry (DSC) has long been used to procure calorimetric data under dynamic conditions because the sample is heated at a precise rate. This technique has been used to investigate meat proteins as influenced by processing (96,112). The three thermic peaks at 60°, 67°, and 80°C, through the use of the purified proteins, were found to correspond to thermal denaturation of myosin, sarcoplasmic proteins, and actin, respectively. It was postulated that the second peak also includes the contribution of the thermal transition of collagen (112). Protein denaturation was followed by DSC analyzing peaks for myosin (I and II), ] sarcoplasmatic proteins and collagen and actin (113). Three endotherms were observed for post-rigor bovine semimembranous meat (Tmax at 57°, 66°, and 80°C) (114). The temperature range of maximum changes in the solubility of myofibrillar proteins seems to be about the same in the intact muscle fiber as in the isolated state (114). Changes of tenderness, rigidity, and water-holding capacity of beef muscle on heating occurs in two phases: the first phase is between 30° and 50°C and the second is between 60° and 90°C. In the temperature range between 50° and 55°C, negligible changes occur. Changes in the first phase are due to heat coagulation of the actomyosin system. The second phase seems to be due to denaturation of the collageneous system (shrinking and solubilization of collagen) and/or to the formation of new stable cross-linkings within the coagulated actomyosin system. In the temperature range between 50° and 55°C, the coagulation of myofibrillar proteins is almost completed, but remarkable changes in connective tissue protein have not yet started (115).

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B. Sausages The gel formation of myofibrillar proteins (mainly actomyosin) is of particular importance in emulsion-style sausage. A protein matrix is necessary for the desired texture and aw level of fermented sausages. The formation of this network is predominantly induced by myosin and actin proteins (116). After comminuting, the cell wall (sarcolemma) of the muscle fibers is, in great part, destroyed (117). A large exudate issues from the cell after rupture of the plasma membrane. During chopping, with simultaneous release of meat proteins, the salt brings about a change in the original structure of proteins by swelling and partial solution of myofibrils (100). The dissolved proteins are transformed into a thin fluid colloidal transition state. As a matter of fact, a meat batter is not considered a true emulsion. A meat batter is actually a very complex mixture consisting of suspended particles (collagen fibers, myofibrils, cellular organelles), immobilized and free water, lipid droplets and particles, and hydrated, solubilized and non-solubilized myofibrillar proteins. The decreases in solubility of myofibrillar proteins between 30° and 60°C is accompanied by an unfolding of the protein chain. Probably an association of the unfolded peptide chain causes protein coagulation (115). A thin coating adhesive substance of meat proteins may cover the surfaces of all particles in the meat batter and binds them (118). During sausage ripening, as a result of denaturation by lactic acid and due to gradual loss of water (drying), the unstable bonds are replaced by condensation bonds and lead to a gel formation (116). The gradual drying during ripening of fermented sausage result in a gel solidification at the edge of the particles. The water in a sausage is postulated to be associated with the myofibrillar protein molecules, or more precisely be entrapped in the actomyosin gel (119). For a stable intermediate-moisture sausage, a low heating or smoking temperature is recommended. Heat coagulation of myofibrillar proteins may reduce the water-holding capacity. The greatest decrease in the solubility of myofibrillar proteins during heating of meat occurs at temperatures between 40° and 60°C; above 60°C, these proteins become almost insoluble (120). V. PARAMETERS CONTROLLING THE STABILITY OF INTERMEDIATE-MOISTURE MEATS Intermediate-moisture foods normally range in aw from 0.7 to 0.9 and in water content from 20% to 50% (121). The aim of aw controlling in IMM is to reduce the aw of a meat product to a range in which most bacteria in foods will no longer grow (76). As pointed out previously, bacteria, other than halophiles, will not grow at 0.83 aw or below, and most are inhibited markedly at 0.90 aw or less. Intermediate-moisture meats are stabilized by lowering their aw to a level insufficient to support bacterial growth, typically about 0.85. Molds and yeasts are able to grow at these aw values and it is usual to add an antimycotic such as sorbate to ensure microbial stability (120,122). Sausages, such as salamis, have a relatively high aw of 0.85 to 0.95 (123). These products rely on a combination of factors for their microbial stability—e.g., nitrite, reduced pH, addition salt, reduction of redox potential by vacuum packaging, and sometimes a heat treatment during manufacturing. However, dried meats made from whole muscle, such as biltong and Chinese dried meat products, rely primarily on reduction of aw for their stability (56).

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A. Heating The thermal process is among the most important factors controlling the microbial content and stability of the finished product (124). It seems clear that humans first learned the drying and smoking of meat. The preservation of foods by drying allowed early humankind to survive in times of food scarcity and to separate themselves from their food sources during periods of migration. It is a widespread art practiced in pre-Columbian, ancient Eskimo, Mediterranean, Oriental, and African cultures. Sun drying is probably the oldest dehydration technique known and is still practiced in various parts of the world. Heating may result in a significant structural change of muscle tissue. This procedure requires that drying be sufficiently rapid to reduce aw to a level at which microbial growth will not occur before drying is complete. Accordingly, microbial destruction that might occur during the smoking process would be due to antimicrobial properties of the smoke and further dehydration at the surface of the meat. Later, the use of salt in conjunction with drying and smoking of meat as a preservative was discovered. B. Salt Several solutes serve as safe and effective agents for reducing aw levels in IMM. Sodium chloride and sucrose have been the most widely used for this purpose. The preservation of food by salting is nearly as old as sun drying. An adequate aw for preservation in aerobic conditions is 0.7, achieved preferably by sugar or salt addition and dehydration. Criteria for producing shelf-stable and intermediate moisture meat products by using hurdle technology are surveyed with the aid of examples (biltong, Chinese dried meat) by Tannahill (125). Commerce in salt by ancient Egyptian, Chinese, and Phoenecian culture is well documented (126). Salt has long been used not only as a preservative but also as flavor enhancer. Sodium chloride is used mainly to reduce aw in meat products for human consumption, such as salt-cured hams, bacon, and some types of sausages. Brining continues to be an important method of curing hams. The relatively high NaCl content and drying had a major effect on spread of bacteria in pasterma. The manufacture of ‘charqui’ (a salted dried meat consumed in Chile and other South American countries) from low-quality beef by hot air drying was studied (127). Beef neck muscles were pretreated with 15% or 25% brine, immersion time 16 or 24 h. Investigators found the only important parameter was brine concentration, which produced a significant increase in weight at the lower concentration. Total weight loss during drying was 51% to 56% (fresh meat basis) or 47% to 52% (salted meat basis) at 30°C, 63% to 66% and 61% to 65%, respectively, at 50°C. Final moisture content was 21.2% for the product dried at 30°C, 5.7% at 50°C. The NaCl content of ham-curing brines normally ranges between 60% and 70% of saturation (0.87 to 0.82 aw) (128). Brines usually are pumped into the vascular system of the ham and along the bone, followed by total immersion into the brine prior to trimming and smoking. Bacon (129) is normally processed in a similar manner. Considering foods with aw from 0.60 to 0.91 to be intermediate-moisture foods, aw, pH, and some chemical parameters (moisture, protein, fat, ash, NaCl, and non-protein N) were determined for 70 samples of 17 types of Spanish intermediate-moisture meat products. It was concluded that NaCl is the main aw depressor (35).

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C. Sucrose The microbial stability of Chinese IM meats is due mainly to the rapid reduction of aw (55). This is aided by the addition of a significant amount of sugar (15% to 35% dry basis). Production technologies for several Malaysian low- and intermediate-moisture traditional meat products (shredded sliced beef, spiced beef slices, Chinese sausage, meat floss) and problems related to keeping them under Malaysian conditions (mold growth and rancidity development) were investigated (20). These IM meats technically are similar to those Chinese-style meat products. Traditional Indonesian dendeng has a moisture content and aw low enough to prevent microbiological spoilage. This microbial stability could be attributed to its high sugar content, which reduces the water activity of the finished products. Higher-moisture dendeng, however, can spoil due to mold growth (19). In addition to its water activity–lowering effect, amorphous sucrose contributes to the plasticized texture in Chinese IM meat (55). It was reported that sucrose (added to sliced pork) contributed to the softening of dried pork by drying the tissue uniformly, due to reduction of its drying rate, as well as by preventing muscle protein from excessive aggregation. Stiffness of the dried pork decreased with increasing sugar concentration; the decrease was significant if sucrose was added (130). Newly developed IM meats are often not sufficiently palatable, contain too many additives (“chemical overloading” of the food), and pose legal problems because of the need to obtain approval of new additives (131). D. Chemicals Bacteriologically, Egyptian fresh sausage might pose a potential health hazard, making it imperative to investigate sanitary measures during its production and sale (27). Dry cured hams produced commercially in North America have a good record of safety. The major cause of spoilage of finished dry cured products is yeast and mold growth during storage in conditions of high humidity. The microbiological stability of finished dry cured meats is the result of their low water activity and the presence of nitrite (132). The microbial stability of short-ripened products depends primarily on a combination of a low pH (below 5.3) and relatively low aw (0.95); in fermented sausages with a long ripening time, the aw is low (0.80 to 0.90) and the pH is relatively high (5.7 to 6.5) (133). E. Intact-Muscle IM Meat For some IM meat products such as Chinese ruogan and Indonesian dendeng, the gel formation of the solubilized collagen for structure and water activity is important. The gelation process occurs in two steps. In the first step, more or less collagen is transformed to soluble gelatin; then, during heating, a new helicoidal structure is formed (134). Denaturation of muscle collagen occurs at higher temperatures than the denaturation of myofibrillar proteins. The soluble gelatin seems to bind the muscle fiber together to form an intact structure. Muscle structure by SEM showed shorter sarcomeres, reflected in increased toughness (84). The structure of meat treated with brine was examined under light and electron microscopes, and only a few muscle cells at the surface of the meat were found altered (135). The process used in the transformation of raw animal tissue into a traditional Chinese IM meat was found to preserve to a great degree the initial cellular structure of the material. The material can be considered made of a matrix, usually water insoluble, intact cell membrane, and a complex aqueous solution of sugars, amino acids, proteins, salts, and lipids. The cell membranes play a major role in the water retention in IM meats (135). Collagen

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fiber in cooked bovine M. sternimandibularis muscle undergoes a thermal contraction when heated to 65°C (136). This is brought about by a collapse of the tertiary structure of the collagen molecules, converting them from highly organized triple helices to random coil configuration. The perimysium contains mainly Type III collagen (137), which has a shrink temperature of 64°C (138). The binding of the myofibrils in cooked fibers were observed (139). Initially, they part as intact fibers with interfiber strands of collagen fibers, which then rupture on further extension. These strands are the weak points in the structure. Recent studies (140) have indicated they are composed mainly of denatured collagen (i.e., gelatin). The partial solubilization of the endomysial collagen of bovine muscle is initiated at about 60°C and completed at approximately 70°C, whereas perimysial connective tissue solubilizes at a higher temperature. Effects of heat-drying on properties of myofibrils from pork were examined, together with the relationship between myofibril characteristics and pork texture (141). It is suggested that contractile protein starts to denature very early in drying, and then moisture begins to continuously decrease. Results imply that dried pork would be tender if denaturation of contractile protein could be prevented during drying. The temperature employed for drying and smoking must be sufficiently low to retain the raw texture of the meat and not inactivate the inherent enzymes involved in flavor development during aging. The muscle fiber is cover by the sarcolema, the cell membrane. The membranes of the sarcolemma and the sarcoplasmic reticulum consist of proteins of the connective tissue type. Thus the plasma membrane of the muscle fiber is easily perforated by freezing and thawing (88). Electrical stunning of hogs causes fragmentation and breakage of the muscle fibers (34); this can explain why for all Chinese dried meat processes, hot-boned meat is preferred (33,142). A cellular food, such as intact animal tissue, has a definitive structure and some rigidity at drying temperatures (143). The thermal contraction of the peri- and endomysial collagen during cooking results in compression and loss of water from the denatured actomyosin fiber. Light microscopy showed considerable shrinkage of muscle cells and formation of fluid channels (9). The area occupied by muscle cells in jerked beef was 30% to 40% less than in fresh beef. At the ultrastructural level, A-bands (including the M-line) disappeared, indicating that proteins were lost during processing. Z-lines appeared to be fragmented. In the enlarged extracellular spaces, banding patterns of collagen fibers were retained; empty spaces surrounded these fibers. It was concluded that denaturation of myofibrillar proteins during processing and the osmotic pressure caused by salting create conditions for water movement from the myofibrillar compartments to the intermyofibrillar space, then to the extracellular matrix, and ultimately to the meat surface (139). F. Sausages The growth of microorganism that predominate on fresh meat is readily inhibited by pH 5.5 and by salt concentrations in the range of 5% to 8%, and the combined effects that occur in processed meats are even greater than either condition alone (144). Factors affecting growth in cured meats and some of the consequences of their growth was reviewed (141). Salmonellae, Clostrdium botulinum, C. perfringens, and Staphylococus aureus have been recognized as major types of pathogens involved in foodborne disease, with Streptocococci, Bacillus cereus, Vibrio spp., enteropathogenic Escherichia coli, and other organism are also of concern. Attention also has been given to the newer potential pathogens, Yersinia enterocolitica and Campylobacter species (144).

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Studies were conducted on the possibility of reduction of the quantity of NaCl used in the manufacture of raw dry ham and dry sausage, and hence the sodium content of the final products. For ham, data are given showing the relation of aw and temperature to growth of spoilage bacteria (Proteus vulgaris, Enterobacter agglomerans, Serratia liquefaciens and Clostridium botulinum). These results showed that if reduced levels of NaCl are to be used, NaCl distribution within the ham must be uniform, maturation temperature must be sufficiently low to prevent bacterial growth at the NaCl concentration used, and moisture loss from the product during curing and so forth must be increased above current levels. Results for dry sausages show that reduction of NaCl content in combination with acetic acid and KCl may give acceptable results; consistency of the product may, however, be affected (142). In order to secure the expected stability and safety, the aw of those IM meat products that are not heated to a higher temperature during the processing and are consumed raw has to be low, in the range of 0.84 to 0.94, depending on the drying process used and the actual pH value. The use of starters in the manufacture of meat products, particularly fermented sausages, is discussed with reference to new developments (146). Comminution of meats used in fermented sausages improves distribution and diffusion of starters in comparison to whole cured meats (e.g., dried ham). Contributions made by microorganisms to the cured meat product are considered and include the following: (a) nitrate reduction by Micrococcaceae; aroma formation by Micrococcaceae, Streptomyces griseus, and yeasts; (b) visual appeal due to presence of surface molds (non–mycotoxin forming), e.g. Penicillium chrysogenum and P. nalgiovensis; and (c) color development and product consistency due to Lactobacillus and Pediococcus spp. New developments concerned with the prevention of spoilage in meat products include optimization of starters, improving product resistance to spoilage, and the use of bacteriocin-producing microorganisms. The use of starters in dried meat is less effective than in sausages because of high salt levels and low distribution. New developments for this industry include selection of salt-resistant strains of microorganisms and improved enzyme systems that ensure effective microorganism concentration. G. Innovative Water Activity Control Technology Previous study has shown that microbially stable glycerol/salt desorbed meat can be easily and cheaply prepared (147). Traditionally, in Nigeria, fresh meat is precooked in water without adding any ingredients, spices, or preservatives and hot-smoked to yield hard, desiccated, and sometimes brittle products. It was demonstrated that smoked meat products can be made by incorporating glycerol and other preservatives into products. The organoleptic quality of the traditional Nigeria smoked meat products can be improved (148). There is a developing consumer demand for softer, lighter-colored, less sweet dried meats. A new Chinese dried meat product, shafu, was developed to meet these requirements (3). Meat (beef, pork or mutton) is cut into 200 g pieces, cured with a special mixture, steam-cooked for 40 to 60 minutes to a core temperature of 80° to 85°C, cooled, cut into strips 0.3 cm thick, and dried at 85° to 90°C to a moisture content of less than 30%. Water activity is less than 0.79, generally between 0.74 and 0.76. If vacuum packaged, it may be stored without refrigeration. Sensory, physicochemical, and microbiological properties are compared with those of traditional products. Factors of importance for microbiological stability of shafu are discussed in relation to the hurdle technology concept.

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Glycerol reduced aw and moisture levels of IM meat and increased water holding capacity; hardness, springiness, and chewiness were reduced by glycerol, and color was affected. Glycerol increased levels of Ca-activated factor activity in IM meats (149). A processing procedure was standardized by infusion soaking of buffalo meat samples in a solution of humectants: glycerol (2.0%) and sodium chloride (10.0%), chemical preservatives: trisodium citrate (2.0%) and sodium benzoate (0.2%) followed by milk heat treatment and air drying. Mean % net yield of IM product was 49.67. Samples revealed pH in the range of 5.72 to 5.79, aw of 0.84 to 0.88, % moisture of 47.52 to 42.73, and % residual salt content of 10.97 to 9.58. Sensory evaluation indicated no spoilage in the IM meat samples during ambient temperature storage for 2 months, with acceptable palatability (150). An intermediate-moisture meat product (aw  0.7) is prepared by cutting meat into small pieces and treating it with an edible humectant (e.g., a sorbitol/NaCl preparation), optionally at approximately 50°C. The resulting meat product may be used as a constituent or filling in bakery products (151). Functions of soluble and insoluble solids as aw depressants in alginate restructured beef heart meat (BHM) were evaluated (152). Water activity, pH, bind and moisture of alginate restructured BHM were evaluated using a 25-factorial design based on combinations of beef heart meal and glycerol (10%, 20%), and dextrose, bone meal, and glycine. The effects of these components were significant (P  0.05). Use of 20% glycerol in the formulation enhanced overall quality by reducing pH, depressing aw, enhancing bind, and reducing moisture content compared to products containing 10% glycerol. However, 20% to 30% glycerol was reported to impart a bitter flavor. Glycine lowered aw and moisture content but weakened binding in alginate-restructured BHM. Dextrose and dried BHM reduced aw and did not affect bind. Bone meal strengthened bind and reduced moisture content in intermediate-moisture meat products. The aw of the BHM control was 0.94, whereas aw for 32 treatments ranged from 0.66 to 0.90. It was concluded that an intermediate-moisture BHM product could be formulated using the hurdle concept and the alginate system for restructuring meat with incorporation of selected soluble (glycerol, dextrose) and insoluble (beef heart meal, bone meal) components. Glycerol, glycerol-nitrite and nitrite-based intermediate moisture (IM) buffalo meats were prepared by 24 hr equilibration in their respective infusion solutions, then ovenheated and air-dried (153). Meat patties were prepared from minced meat, with the addition of NaCl and potassium sorbate, and dried at 75°C for 11 hrs. Results showed that dried meat patties were stable (154): No detectable changes were observed in the product during the first 2 months, according to chemical and bacteriological results. Organoleptic evaluation showed that the product was acceptable. Reports have been presented on meat products with extended shelf life (due to reduced water activity values) as manufactured primarily in Europe (such products as salami, landjaeger, and buendnerfleisch) and more recently in the United States. The use of additional preserving agents, especially sorbic acid, is being considered as well as the traditional use of low pH and aw values and smoking. Preparation of intermediate-moisture meat (beef, pork, chicken, rabbit) by osmotic dehydration at 4° or 25°C with a Shona Denko contact dehydration sheet was developed (155). Osmotic dehydration at 4°C gave better retention of the native state of proteins, and hence better quality, than drying at 25°C. Dehydration at 4°C resulted in maintenance of much of the original muscle structure; isolated myofibrils retained contractible activity. Myosin could easily be extracted after 10 days storage. Species differed little in response to osmotic dehydration. Dehydration with osmotic dehydration sheets at low

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temperature permits production of a high-quality, low-salt intermediate-moisture meat product (155). A gradual decrease in microorganism count during processing and storage of charqui was observed. These results indicate the feasibility of obtaining a final product with a low microbial count when raw materials of good quality, and adequate handling conditions, are used for charqui production (10). Use of the ingredient optimization program allowed production of a satisfactory Cervelat sausage by improved economical means (36). Recently, the procedure for processing traditional dried beef into Kilishi was optimized (156). Wheat flour was added to the traditional sauce (a mixture of ground paste, water, and spices) to improve sauce adhesion to the dried meat. The coated beef was grilled for 10 min (5 min each side such as traditionally done in Niger) using a household grill. A stable end-product with good keeping qualities of kilishi was obtained. VI. CONCLUSIONS Intermediate-moisture and dried meat products have been produced by various methods in different cultures through the centuries. It is interesting to know that these methods are fairly similar. By controlling the amount of salt, sugar, nitrate/nitrite, and other ingredients, as well as the curing, dehydration, and maturation times, and proper packaging and storage conditions, these products can be highly acceptable, fairly stable, and safe. The principles behind these techniques are being revealed by the various scientific studies on the effect of ingredients and processing methodology used in the preparation of these products. The safety of these products had been investigated extensively. The term “hurdle technology” probably describes these principles best nowadays. Consumers always demand better, safer, and more convenient products; scientists are being challenged to develop the innovative technologies to meet these demands. REFERENCES 1. AH Varnam, JP Sutherland. Meat and Meat Products. New York: Chapman & Hall, 1995, pp 167–412. 2. AM Jiang, YH Yuan, XJ Lian, ZM Liu. Methodology for classification of meat products. Meat Res 3:27–30, 1997. 3. W Wang, L Leistner, L. Shafu. A novel Chinese dried meat product based on hurdle technology. Fleischwirt 73 (8):867–869, 1993. 4. L Leistner, HK Shin, H Hechelmann, SY Lin. Microbiology and technology of Chinese meat products. Proceedings of the European Meeting of Meat Research Workers, No. 30, 6:11, 280–281, 1984. 5. DR Smith. Effects of rigor state, salt level and storage time on chemical and shelf-stable intermediate moisture meat products. Food Res Report, CSIRO, 44 (1):12–19, 1984. 6. DA Ledward. Intermediate moisture meats. In RA Rowrie, ed. Development in Meat Science—2. London: Applied Sciences, 1981, pp 159–194. 7. C Kellar, P Ossent, E Schlapfer. Damaged meat sections in production of dried meat products. Effects, significance and causes. Fleischwirtschaft 68 (1):36–40, 70, 1988 8. JR Romans, PT Zielgler. The Preservation and Storage of Meat. In: TR Romas, PT Zielgler, ed. The Meat We Eat. Danville: The Interstate Printer & Publishers, Inc, 1997, pp 559–611. 9. TMB Biscontini, M Shimokomaki, SF Oliveira, TMT Zorn. An ultrastructural observation on charquis, salted and intermediate moisture meat products. Meat Sci 43 (3/4):351–358, 1966. 10. EAFS Torres, M Shimokomaki, BDGM Franco, M Landgraf, BC Carvalho Jr., JC Santos. Parameters determining the quality of charqui, an intermediate moisture meat product. Meat Sci 38 (2):229–234, 1994.

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VL Durance, DL Restall, H Beard, FJ Bourne, AJ Bailey. The location of three collagen types in skeletal Muscle. FEBS Letters 79: 248, 1977. V Mohr, JR Bendall. Constitution and physical chemical properties of intramuscular connective tissue. Nature 222: 404–405, 1969. DG Siegel, DM Theno, GR Schmidt. Meat massaging: the effect of salt, phosphate and massaging on the presence of specific skeletal muscle proteins in the exudate of a sectioned and formed ham. J Food Sci 43: 327–330, 1978. TJ Sims, AJ Bailey. Structural aspects of cooked meat. In: DA Leward, DE Johnston, MK Knight, ed. The Chemistry of Muscle-based Foods. Cambridge: Royal Soc Chem, 1992, pp 106–112. M Matsuura, H Negishi, S Yoshikawa. Effect of changes in biochemical properties of myofibrillar proteins during heat-drying on texture of dried meat. Nippon Shokuhin Kogyo Gakkaishi 38 (3): 196–201, 1991. L Leistner. Hurdle technology applied to meat products of the shelf stable product and intermediate moisture food types. In: D Sinatos, JL Multon, ed. Properties of Water in Foods: in Relation to Quality and Stability Dardrecht: M. Nijhoff, 1985, pp 309–329. JG Kapsalis. The influence of water on textural parameters in foods at intermediate moisture levels. In: RB Duckworth, ed. Water Relations of Foods. New York: Academic Press, 1975, pp 627–653. H Riemann. Food Borne Infections and Intoxications. New York: Academic Press. 1969. P Baldini, M Campanini, G Pezzani, F Palmia, F. Reduction de la quantite de chlorure de sodium employe dans less produits seches. Viandes et Produits Carnes 5 (3): 83–88, 1984. D Krotje. Starter cultures. New developments in meat products. Int Food Ingredients 6: 14–18, 1992. ZA Obanu, DA Ledward, RA Lawrie. Lipid-protein interactions as agents of quality deterioration in intermediate moisture meats: an appraisal. Meat Sci 4: 79–88, 1980. TM Okonkwo, ZA Obanu, DA Ledward. Characteristics of some intermediate moisture smoked meats. Meat Sci 31: 135–145, 1992. AA Ibrahim, HT El-Zanfaly, AR Abd-El-Latif. Keeping quality studies on dried meat patties. Chemie Mikrobiologie Technologie der Lebensmittel 9 (3): 76–80, 1985. SM Kim, SK Sung. Effects of level of glycerol addition on physicochemical characteristics of intermediate moisture meat. Korean J Animal Sci 31 (5): 342–352, 1989. K Prabhakar, R Ramamurthi. A study on the preparation of intermediate moisture meat. J Food Sci Technol (India) 27 (3): 162–164, 1990. G Rohns, HD Lechte. Shelf-stable organoleptically acceptable meat product without chemical preservatives, and process for its manufacture. German Federal Republic Patent Application PI DE 3630131 A1 1988. EAE Boyle, JN Sofos, GR Schmidt, G. R. Depression of aw by soluble and insoluble solids in alginate restructured beef heart meat. J Food Sci 58 (5): 959–962, 967, 1993. K Prabhakar, CV Govindarajan, VR Kosalaraman, AM Shanmugam. Colour and related quality changes in intermediate moisture meats. J Food Sci Technol (India) 29 (1) 65–67, 1992. M Muguruma, K Katayama, M Nakamura, M Yamaguchi. Low-temperature osmotic dehydration improves the quality of intermediate moisture meats. Meat Sci 21 (2): 99–109, 1987. S Kalilou, A Collignan, N Zakhia. Optimizing the traditional processing of beef into kilishi. Meat Sci 50: 21–32, 1998.

137. 138.

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143. 144. 145. 146. 147. 148. 149. 150. 151.

152. 153. 154. 155.

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18 Manufacturing of Reduced-Fat, Low-Fat, and Fat-Free Emulsion Sausage ROBERT W. ROGERS Mississippi State University, Mississippi State, Mississippi

I. INTRODUCTION II. SELECTION OF RAW MEAT MATERIALS III. SELECTION OF NONMEAT INGREDIENTS A. Major Additives B. Miscellaneous Additives IV. PROCESSING TECHNIQUES V. SUMMARY REFERENCES

I. INTRODUCTION For several years, Americans have been advised to choose leaner (meats) and lower fat foods as these are perceived, at least by many, to be “better for you” than many traditional foods that contain higher levels of fat. Emulsion-type sausage products (e.g., bologna, frankfurters) have been very popular meat items in America, and in many other countries, and have traditionally contained relatively high levels of fat. For example, USDA regulations for many years have allowed these types of products to contain up to 30% fat. Until the recent (12) mandated use of the Nutrition Facts label, it was not evident to most consumers that these products contained fat. This is because the fat was not visible to the naked eye: it was the dispersed phase (fat droplets) in a complex continuous phase composed of water, solubilized proteins, cellular components, and miscellaneous spices and seasonings (60). These finely comminuted sausage batters are commonly called emulsions, although by strict definition they are not true emulsions. A true emulsion is a heterogeneous mixture of two immiscible liquids, stabilized by an emulsifying material, such as protein. Meat bat-

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ters, or emulsions, may be more properly called a three-phase dispersion (solid, liquid, and [gas] air). Because of the common usage of the term “emulsion sausage” in the literature and by industry personnel, the word emulsion will be used throughout this chapter as if it were technically correct, and to differentiate these products from sausages that have obvious particle definition and are commonly referred to as coarse ground, such as fresh pork sausage or cured smoked sausage. The specific requirements of foods labeled with any type of wording to indicate that the fat content has been reduced in relation to a similar food with normal fat levels are published in the U. S. Code of Federal Regulations. Most of those regulations apply to all foods, not just sausage products. Part 100 of Title 21 of the CFR (14) has the specific requirements for foods in general, and Title 9 (13) (part 317) has those requirements that are specific to meat and (part 381.4) poultry products. Basically, those regulations require specific fat levels for each different descriptor used to identify each product. For example, the reduced-fat descriptor requires a minimum of 25% reduction in fat compared to an equivalent product. Low-fat labeling requires that the fat level for a normal serving would provide 3 grams or less of fat, that saturated fatty acids make up no more than 40% of the total fatty acid content, and that the serving supply less than 95 mg cholesterol. Fat-free labeling requires that a normal serving would supply less than 0.5 gram of fat, and 100% fat-free labeling requires products to meet the same standards as fat-free and also that they contain 0.5 gram of fat or less per 100 grams with no added fat. X% fat-free labeling (e.g., 98% Fat-Free) is allowed if the product meets the requirements for low-fat labeling. Meat and poultry products labeled as “lean” are allowed to have up to 10% fat; those labeled as “extra lean” are allowed to contain only up to 5% fat. These products also have specific requirements about the percentage of saturated fatty acids (maximum of 45% saturated fatty acids for “lean” and 40% saturated fatty acids for “extra lean”) and cholesterol ( 95 mg) in a serving. There are several good references (56,58,60,61) on the subject of emulsion sausage manufacturing. These publications detail the specific requirements for raw material selection, least-cost formulation, nonmeat additives, particle size reduction, casing selection, stuffing, linking, cooking and smoking, packaging, defects, and troubleshooting, of all the various types of sausage products. These specific variables will not be duplicated here in detail except where they have a significant influence on production of the lower-fat versions of emulsion sausage. The yearly per capita consumption of sausage in the United States is about 32 lb. Thus, the U.S. population consumes about 8.8 billion lb of sausage products yearly. Emulsion-type products make up about 50% of total U.S. sausage consumption (58) or about 4.4 billion lb annually. The lower-fat versions of these products account for about 20% of total hot dog and 2% of total bologna sales (4). These products are generally becoming more widely accepted because of improvements in formulation and processing that have yielded products quite similar to traditional products of this type; and many consumers feel that these products are “better for you” than the traditional full-fat items. This chapter will primarily address the materials published in the literature relative to factors associated with the successful production of lower-fat varieties of emulsion sausage as well as to some personal experiences of the author, who, along with graduate student Mike Martin, actually first reported on the production of “low-fat” or “fat-free” (74% reduction in calories) frankfurters (71). This original research was published in a Master’s thesis (46); the products were commercialized as “Lean Franks” by Bryan Foods Inc. of West Point, MS, in 1989; and the data were published in the Journal of Food Science in 1991 (47).

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There are others who report they were the first to develop low-fat products of this type (74). A major meat processing company introduced frankfurters and bologna containing approximately 20% fat in 1969, but they were discontinued within a few months because of lack of sales (9). Oscar Mayer first introduced “Light Line” (85% fat-free) in 1990, Healthy Favorites products in 1992 (97% fat-free), and Oscar Mayer Free in 1995 (3). The success of low-fat food products requires that palatability attributes not be significantly reduced from those of traditional products and that costs will be similar (9). Most fat-free meat products average about 25% higher per unit cost (49), yet approximately 50% of the new red meat items introduced in 1995 contained one of three reduced fat claims (39). In 1994, only 12.3% of new meat items contained such claims. Several processors (Hygrade, Eckrich, Oscar Mayer, ConAgra Inc., Bryan Foods, Hillshire Farms, Kahns, Hormel, etc.) currently offer different varieties of reduced-fat emulsion sausage products to both food service and retail outlets. Retail sales of “fat-reduced” hot dogs alone have grown from practically none in 1990 to approximately 100 million lb. in 1995 (39). The development of acceptable reduced-fat emulsion sausage products was stymied by USDA regulations, which permitted only 10% added water until they were modified to allow up to a total of 40% fat and added water, with fat restricted to a maximum of 30% of the finished product (2). This regulatory change was essential so the proteins could be diluted, preferably with a low-cost item that allowed for modified production procedures to yield a product acceptable in skin toughness, bite, purge, processing yields, flavor, juiciness, texture, and other similar traits. Because federal regulations prohibited the production of “low-fat” or “fat-free” sausage items, there was not much written about the process until the late 1980s. Because all or most of the fat has been removed from “low-fat” or “fatfree” emulsion sausage products, the problems of producing such items have changed drastically from those of producing conventional products. It will be primarily those changes in production techniques and additives that will be discussed in this chapter. II. SELECTION OF RAW MEAT MATERIALS To produce emulsion sausage products, the first matter to consider is what “fat reduction claim” will be made because this mandates the fat level in the raw meat used in the formulation. In traditional emulsion products, the level and type of proteins present in the raw meat are of paramount concern to ensure adequate quantities of salt-soluble, heat-coagulable proteins (SSHCP) to stabilize the emulsion or dispersion. In addition to the quantity of these proteins, their solubility is also of major concern. Lower protein solubilities of frozen meat, not using preblended meat, the use of post-rigor meats vs. pre-rigor meats, using meats with high microbial counts, the use of rancid meat, and other similar traits affect the actual amount of functional proteins available to stabilize the emulsion by coating the fat droplets and binding water. Almedia (1) reported the use of pre-rigor beef resulted in firmer, juicier products than when post-rigor beef was used to produce “lite” frankfurters, but it contained more package purge than the post-rigor product. He also stated that preblending did not affect the products produced, but long-term frozen storage of beef used to make the products caused them to receive lower flavor scores, retain less added water, produce lower yields, and be darker in color. The “lite” product produced by Almedia (1) had only a 25% reduction in fat content compared with the conventional product. Park et al. (54) reported that the addition of polydextrose and phosphates to pre-rigor beef resulted in a product that was basically equal to fresh pre-rigor beef even though it was held in the frozen state for 5 mo. They

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also concluded that the effect was almost totally attributable to the addition of phosphates. According to Honkavaara (32), pork trimmings held at 29°F (34°C) lost a significant amount of their water-holding capacity over time. The mean values for water-holding capacity declined from 35% after 1 month to 17% after 6 months of storage, although fatholding capacity was not affected by freezer storage. Likewise, the species of product used (i.e., beef or pork) can affect the color of the finished product (47). In most cases, when “lean,” “extra lean,” “low-fat,” or “fat-free” products are made, there is a considerable excess of the SSHCP materials present because very lean meat must be used in the formulation. If frozen meats are to be used, the amount of water added to the mixture needs to be reduced by 1% to 2% in order to minimize purge in the packaged product caused by reduced protein solubility and water-binding capacity. Some processors have shown concern about using beef from cows that have been treated with recombinant bovine somatotropin (rbST) because of the lack of information relative to the possible effects of rbST on the processing characteristics of these meats. However, Ellis et al. (24), Hariri (28), and Healey (30) all have shown that rbST treatment to cattle does not affect processing characteristics of the meat for making emulsion sausage. Chemical modification of meat proteins can affect the texture and gelation properties of finely comminuted beef (5). Scientists reported that urea which disrupts noncovalent bonds, increased rigidity and hardness values. Oxidation of free sulfydryl groups by H2O2 resulted in lower rigidity but hardness values similar to those of control products. Treatment of the meat with beta-mercaptoethanol produced higher values for hardness, gumminess, and chewiness, and ethylenediamine tetra acetic acid (EDTA) caused excessive cooking losses, low rigidity values, and hard texture. Polyoxyethylene sorbitan mono-oleate (Tween 80) addition did not have any effect on the textural properties of the product but resulted in lower rigidity values than the control product. Except for EDTA, none of the additives affected the pH values of the products. The use of this wide range of different products, although most are either not practical or non-approved additives for meat products, does illustrate the importance of different functional groups and bonding mechanisms in meat emulsion systems. The term “very lean” meat should not be construed, however, to mean just the absence of fat, because very lean meat having an abundance of connective tissue (collagen) can cause the production of “jelly pockets” in the finished product as collagen begins to gelatinize at about 140 to 145°F (60° to 62°C). These are considered major product defects. Depending on the fat level desired in the finished product, some meats used in the formula may not normally be considered lean or very lean, but by limiting the use of those products, the correct amount of fat might be achieved by using large amounts of meats that have practically no fat. Generally, cost, availability, and actual fat content of both the leaner meats and the fatter meats will determine the exact amount of each to use for a targeted fat level in the finished product. Because of the many possible combinations of raw materials that may be satisfactory for use in making correct emulsion formulations, the use of “leastcost formulation” procedures with tight constraints on fat and collagen content is generally recommended. Because the fat present will be diluted with the addition of water, seasoning, extenders, and so forth the level of these additives used must also be considered when determining the maximum level of fat allowed in the raw meat materials to meet a particular fat level in the finished product. In conjunction with the amount of diluents used, the amount of processing losses during cooking and chilling will also significantly affect the finished product fat level. Therefore, one cannot only consider the level of fat in the raw meat materials

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Table 1 Illustration of How Variances in Different Factors Can Affect Product Labeling

Meat block (lb) 100 100 100 100 100

Fat in meat block (%)

Additives (lb)

Processing loss (%)

Chill loss (%)

Final prod. wt.

Fat in fin. prod. (%)

15 14 7 4 1.5

60 55 65 70 70

8 4 6 5 5

2 1 1 2 1

144.00 147.25 153.45 158.10 159.80

10.41 9.50 4.56 2.53 0.94

Grams of fat in a typical 56-gram serving 5.80 5.32 2.55 1.41 0.52b

Legal label for a typical serving of 56 g Light Lean Extra-lean Low-fat a Fat-freeb

a

Must also meet requirements for cholesterol level and ratio of saturated to unsaturated fatty acids. This designation would be allowed for a 53 gram, or less, serving since one serving would supply less than 0.5 gram of fat. b

but must also consider all factors of production that can affect the ratio of fat to total product weight in the finished product (Table 1). Of course, serving size designation of the product must also be considered at the time of formulation in order to comply with the regulations concerning the Nutrition Facts Label. Because of the higher cost of “very lean meats,” most processors choose to use multiple species sources (i.e., beef, pork, turkey, chicken, etc.) of meat materials to formulate reduced-fat emulsion sausage. This practice is also a good one to follow to minimize regulatory problems with cross-contamination of the product with meat from a species that is not in the ingredients statement. III. SELECTION OF NONMEAT INGREDIENTS A. Major Additives In making reduced-fat emulsion sausage products, the amount, quantity, and quality of nonmeat ingredients used can be of equal or greater importance than the raw meat materials in determining final product characteristics. This is generally not the case in making traditional products, although additives are always important in the manufacturing of any sausage product. When selecting nonmeat ingredients for their functional purposes, one should also consider the possible connotation that a particular additive might give to consumers about the product if they discover its presence in the ingredient list. For example, one might choose not to use monosodium glutamate (MSG). Although it has a very important functional role as a flavor enhancer, many consumers avoid buying products containing MSG because of some adverse publicity about this material; however, one might choose to replace MSG with yeast hydrolysates as they perform similarly for flavor enhancement and increased mouth feel (56). A review of scientific literature reveals that many scientists have elaborated on the topic of additives applicable to the production of reduced-fat, low-fat, and fat-free emulsion sausage items. These include such additives as carrageenan, native starch, modified starch, phosphates, milk protein, soy protein, chloride salts, modified beef connective tissue, fat, gums, applesauce, rice, potatoes, beans, olive oil, defatted soy flour, pre-emulsified corn oil, konjac flour, wheat germ proteins, corn germ protein flour, high-protein oat

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flour, vital wheat gluten, high-methoxyl pectin, locust bean gum, flaked sinew, collagen fibers, gellan gum, sunflower oil, hydroxypropyl methylcellulose (HPMC), sodium lactate, acid enzyme deheated mustard, and oat bran. From the exhaustive list of additives evaluated, it is obvious that researchers have been looking for a “magic bullet” to use as a fat replacer. To this date, none has been found and it is doubtful if one will ever be discovered. As in most cases, the successful production of mimicked items requires the incorporation of various items to replace essential components used in an original product. Some of the most popular additives, in addition to water and seasonings, that have been evaluated in reduced-fat, lean, extra-lean, low-fat, and fat-free emulsion sausage will be discussed in the following paragraphs. The use of nitrite, ascorbate, salt, and so forth in the formulations are the same as for traditional emulsion sausage. 1. Seasonings An important factor relative to nonmeat additives is the amount or level of seasoning used in the formulation. When adding seasonings to full-fat formulations, a greater quantity of spices or seasonings are generally used compared to the reduced fat versions. The exact amount of reduction depends on the fat level, but many formulations need only contain about one-third the amount of seasoning. Excess seasoning in low-fat products causes a very unpleasant “metallic” or “astringent” taste to linger in the back of the mouth and throat of the consumer. Using a model system of water, oil, and an emulsifier (Tween-80), Schirle-Keller et al. (64) demonstrated that protein fat replacers behaved more like fat than did carbohydratebased products relative to various flavor compounds. Because fat contributes much to meat flavor, its absence demands careful selection (item and amount) of flavorings, fillers, binders, seasonings, and spices to produce a product with sensory attributes similar to products containing higher levels of fat. The actual process used to add some components could likewise be very important. For example, liquid smoke should be added at the very end of the mix cycle after all free water has been absorbed or it will clump and produce an uneven blend of this material; it can also cause the production of black specks in the finished product where there was a lack of uniform mixing due to clumping. Also, the addition of agents such as sodium lactate should be done some time well into the mix cycle because they tend to stick to the mixer walls and never become incorporated into the batter. Care should be taken to ensure the proper blending or mixing of all additives such as salt, nitrite, flavorings, seasonings, starches, and soy proteins because of the importance of each additive in reduced-fat sausage products. For more detail on the effects of seasonings, see Chapter 16 on seasonings and flavorings. 2. Water Water is the most abundant additive in these products and becomes very prominent in the ingredient list. To avoid such prominence, items such as (species) broth might be used to serve the function of water without using its name. Although water is important in the production of reduced-fat sausage products, excessive amounts that are not bound in the final product generally cause significant problems with purge in the package, especially in retail packages. Purge not only makes the package look unattractive, it also provides abundant moisture, which will in turn support excess microbial growth and drastically shorten the product’s shelf life. In addition to problems with purge, the excess water used in reducedfat products also lowers the brine concentration, possibly shortening shelf life. This can be

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overcome by adding extra salt, but extremely high sodium products may have negative connotations to many consumers. There is a very thorough review of the literature concerning nonmeat ingredients for all low/no-fat processed meats in the Proceedings of the 49th Reciprocal Meats Conference (37). Therefore, my comments will be more focused on specific areas of emulsion sausage research that relates to reduced-fat products. The early research examined replacing fat with brine solutions (20%, 40%, or 60% addition) in frankfurters (46,47) or with water alone (16) in bologna. Researchers reported that many sensory or physical properties of emulsion sausage were affected by substituting water for fat. Among the traits affected were springiness, firmness, cohesiveness, fracturability, hardness, color, purge, cooking loss, and skin toughness. The addition of extra water (generally from 50% to 80% of meat block weight) to replace fat appears to be universally accepted as one component that is essential to produce lower-fat versions of emulsion sausage. Generally speaking, the lower the fat content in the finished product the more water is needed in the product. Because water is inexpensive, has no calories, and increases the bulk of the meat batter, it plays an important role in maintaining the meaty characteristics of the product along with reducing costs, increasing yields, solubilizing proteins, and imparting favorable sensory traits to the product. However, water does not have the properties of fat and this must be considered in relation to other factors that affect end product characteristics, such as mouth feel, spice intensity, and purge. The quality of potable water used is also important because water containing an abundance of heavy metal ions, excess chlorine, and other impurities could be responsible for the production of substandard or unacceptable product. 3. Phosphates As is the case in the production of full-fat emulsion sausage, phosphates play an important role in the successful production of reduced-fat versions of emulsion sausage. The major role of the alkaline phosphates in emulsion sausage manufacturing is to assist in holding or binding free water to reduce cooking and chilling losses and reduce purge in the packaged product (22,54). The maximum amount allowed by the USDA is 0.5% in the formulation. 4. Nonfat Dry Milk Various forms of dried whey, dried skim milk, calcium-reduced nonfat dry milk, and so forth are allowed in limited amounts in various meat products for the purposes of extending and binding (13). Keeton et al. (35) reported that calcium-reduced nonfat dry milk was a viable additive to frankfurters and assisted in the development of a desirable flavor in regular full-fat products. Personal experiences have shown that products of this type offer some advantages to the sensory properties of low fat-frankfurters and bologna. They seem to reduce some of the harshness in the taste of fat-free products as well as improve the mouthfeel and help “round out” the flavor. 5. Carrageenan The three common types of carrageenan evaluated are kappa, iota, and lambda. These materials are all officially classified as gums but are being reported separately because of their common use. The kappa and iota versions are gel-forming materials, whereas the lambda version is typically used only to thicken liquids as it will not set to a gel. Trius et al. (68) evaluated all three of the carrageenans in conjunction with pH differences with and without sodium tripolyphosphate (STP) in beaker sausage. They reported lambda carrageenan

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at a high pH produced product with the softest texture and lower cooking losses, whereas the kappa and iota versions were superior for water retention at low pHs. In the presence of K cations, the lambda version was the most effective in reducing cooking losses. The different carrageenan products also had different structural effects on the finished products. Trius et al. (69) evaluated carrageenan in bologna and stated that none of the carrageenansalt combinations reduced cooking losses, and that the lambda product reduced firmness at both high- and low-fat concentrations. Earlier, Dexter et al. (17) reported that kappa carrageenan added late in the chopping process along with starch reduced cooked product hardness; but if added before chopping, it increased cooked product bind values in bologna. Purge was decreased by the addition of carrageenan to frankfurters as were texture profiles and sensory values when increased from 0.5% to 1.0% but no more if increased to 1.5% (73). Carrageenan does not produce the same results under different conditions relative to amounts and kinds of salts present. Trius et al. (70), studying a model system containing carrageenan, stated that when NaCl was reduced from 2% to 1%, cooking losses and firmness both increased, but increasing salt levels from 2% to 3% did not show any improvement in yields; kappa and iota carrageenan did cause reductions in cooking losses. They also reported that when CaCl2 was used as the salt, poor water retention and instability of the finished product occurred whereas KCl reduced cooking losses when used with kappa and iota carrageenan and increased water-binding values for the product containing iota carrageenan. Duda et al. (18) also reported that carrageenan caused an increase in waterholding capacity (WHC) of scalded sausage and that the color and color stability of the sausage was equal to the control product. Carrageenan does show promise as one material that may be used to at least partially replace fat in emulsion sausage, but one must consider which form (kappa, lambda, or iota) to use, with other additives such as the type and amount of salt (NaCl, Kcl, CaCl2), pH, and so forth. Kappa carrageenan is currently the most commonly used version of carrageenan in reduced-fat sausage products. Bloukas et al. (8) concluded that iota carrageenan, at 0.5% to 1.0% of finished weight, appeared to be more suitable in low-fat frankfurters than kappa or kappa plus iota carrageenan. I have seen no compelling evidence in the literature to make me think that carrageenan can be used alone to replace fat and achieve successful results on all major factors of product quality and yields. 6. Gums Different gums (i.e., gellon, xanthan, locust bean, locust bean plus xanthan, and other polysaccharide gums) have been evaluated for effectiveness as fat replacers in emulsion sausage. Some gums (xanthan, dextran) are obtained from microbial sources whereas others (locust bean, sodium alginate) are derived from plant seeds, trees, seaweed, and similar materials. Pettitt et al. (57) published that xanthan gum plus propylene glycol alginate caused an increase in the viscosity of emulsions (batters); Lopes da Silva (44) stated that it caused a shear thinning behavior on the emulsion, which was dependent on the pH and ionic strength of the emulsion. Duda et al. (18) also reported that gellon gum affected yields, thermal drip, firmness, hardness, fracturability, external color, and WHC of scalded sausage. Xiong et al. (72) evaluated alginate, locust bean gum, locust bean gum plus xanthan gum, and the three carrageenans in a low-fat beef sausage and obtained mixed results. In general, alginate resulted in higher cooking yields at different pH and salt levels. The differences were of less magnitude as the salt content increased from 1% to

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2.5%. At the 1% levels and at a pH value of 5.6, the control and other treatments yielded only 65 to 70% while the alginate product yielded 80%. At 2.5% salt and pH values of 5.3, 5.6, and 6.2, cooking yields generally increased as pH level increased for all treatments, with the control product the only one not reaching the cooking yield level of most other treatments. The control product was not different (P  .05) from the kappa carrageenan and the locust bean gum products for this trait. At the low salt level, the sausage structure was markedly weakened, making it practically impossible to measure sample texture with the Instron device. The products at pH 6.2 were harder, and the kappa and iota products were again the most effective. Sensory panel scores for tenderness and juiciness were higher for the alginate, locust bean gum plus xanthan gum products that contained 1% salt. In general, the products containing 2.5% salt received higher sensory scores than those containing 1% salt; however, their results showed the products to receive scores only at the midpoint or lower range of a five-point scale. These results agree with those of other scientists who have stated that the exact effects of these types of additives are very dependent on the actual salt(s) present, level of salt present, pH of the emulsion, and other factors and cannot be expected to yield consistent results unless all significant processing factors are controlled. 7. Pectin Pectin has been touted in the popular press as a natural fat replacer for many foods, including sausage (19). One such product is “Slendid”™ marketed by Hercules Inc. It is reported to be suitable for use in emulsion sausage products when made into a gel and chopped with the batter. However, two groups of researchers—Halloran et al. (27) and Lopes da Silva et al. (44)—both reported that the use of high-methoxyl pectin as an additive to reduced-fat versions of emulsion sausage products caused major problems. Halloran revealed that pectin totally destroyed firmness of the emulsion and the product was basically a paste, although it had the configuration of a frankfurter. Halloran’s work was actually performed in 1992 and 1993, and Lopes da Silva’s work was reported in 1992, but both obtained similar results. Halloran stated that the emulsion was totally destroyed, and Lopes da Silva reported that the low molecular weight of pectin could possibly be the reason for emulsion failure. Their postulation was that the low molecular weight of the pectin prevented both macromolecules from developing intermolecular associations. In any case, the use of pectin powder in the emulsion-making process destroys the integrity of the finished product and is totally unsuitable for use in products of this nature, although purge was reduced by its use. 8. Connective Tissue Various researchers have reported on the effects of different connective tissue materials used in processing lower-fat emulsion sausage. Eilbert et al. (20) stated that thermal processing yields increased by 2% to 3% when 20% modified connective tissue (MCT) was used and Kramer Shear values were higher as MCT levels increased. However, frankfurter cohesiveness values declined with increasing amounts of MCT and color values were lighter. Consumer panels rated the MCT product equal to the high-fat control product and the color values and yields were similar. These same researchers, Eilbert et al. (21), also reported that MCT caused increased resistance to flow of the emulsion and higher emulsion temperatures during chopping along with more collagen solubilization and higher thermal processing yields as the amount of MCT was increased. They concluded that MCT may be a viable material to use in the production of lower-fat sausage products and that its use could cause an increase in the value of MCT for harvesters of MCT products. Calhoun et al. (10) stated that

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preblending MCT with alkaline phosphates creates sausage products similar to controls and that MCT offers desinewing operations a potential market for their materials. Letelier et al. (43) reported that flaked sinew caused reduction in yields and decreased purge along with increased color intensity values in low-fat salami. They also concluded that large-particlesize sinew increased peak force values more than small-particle-size material. Products containing flaked sinew received palatability scores equal to high-fat control products, thus making this material a potentially suitable low-cost ingredient. Eilert et al. (23) stated that connective tissue addition to lean frankfurters did not react the same when used in conjunction with different (acidic, neutral, or alkaline) phosphates. For example, color intensity increased when acidic phosphates were used. Additionally, when 20% connective tissue was used cohesiveness was decreased but there were no effects on the microbiological stability of the products. Calhoun et al. (11) evaluated MCT in the production of lean franks along with the use of sodium tripolyphosphate (STP) with preblended and non-preblended materials. Preblending produced no major effects except to reduce color intensity of the finished products. They concluded that MCT and STP added separately might be feasible additives for similar products if the color issues associated with them can be resolved. Bologna firmness was affected (increased) due to the addition of collagen fibers to the mixture as was the stability (decreased) of the batter (50). The authors speculated that the batter instability was due to shrinkage of the muscle proteins and collagen aggregation, with islets formation. Chicken frankfurters made with collagen fibers (2%) and high amounts of added water (~20%) were not affected for the traits of hardness, springiness, and juiciness. But when collagen fiber levels were increased above the 2% level, the values for hardness, springiness, juiciness, and flavor were adversely affected (48). Modified connective tissue prepared by freezing, grinding, and flaking was capable of producing gels that would bind from 100% to 600% added water, and when used to make reduced-fat bologna gave only positive results (52). Low-fat versions of emulsion sausage do not need to have as much muscle protein (myosin) to coat fat droplets as required in high-fat products, so limited use of various collagen coating materials as fillers in low-fat emulsion products may be of great value, provided that emulsion stability and sensory traits are not compromised. 9. Soy Proteins Lecomte et al. (42) evaluated soy flour, concentrate, and isolate in reduced-fat franks as pre-emulsified fat (PEF) and dry powders and reported the following results. When soy protein was used in PEF, specific soybean off-flavors (beany and bitterness) and aroma were reduced and water-holding capacity and yields were improved. Color was not affected. The addition of soy isolate and soy concentrate caused different effects on emulsion microstructure (average fat droplet area) because of differences in their water capacity, water sorption rate, and gel-forming ability, indicating that these two proteins could produce different results on end product characteristics (45). Kazemekaityte and Simkeviciene (34) showed that a particular soy protein (soy isolate—Supro 500 E) had a water-binding capacity of 1,550% whereas a host of other proteins evaluated (sunflower protein, sodium caseinate, whey protein, blood protein, and blood protein plus skimmed milk) showed water-binding capacities of only ~600%. Similar, but not as drastic, differences were observed for these same materials for fat-binding capacity. The investigators concluded that the functional properties of protein additives were highly dependent on the origin of the material and on the method of processing that material into an additive.

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10. Starches Other than cellulose, starch is the most widely distributed, naturally occurring organic compound known. The manufacturing treatments performed on starch serve to classify this material. There are native (as extracted) starches, modified (chemically) starches, and pre-gelatinized starches. There are several problems associated with using native starches in cooked meat items. For example, they will hydrate rapidly, causing a sharp increase in batter viscosity; but upon prolonged cooking the granules rupture, leading to a very sharp decrease in viscosity, which causes undesirable textures, poor stability, gel pocket formation, and loss of water. These are undesirable traits for an additive to possess for use in cooked emulsions. There are two modifications of great importance to meat applications. These modifications are cross-linking and stabilization. These products (crosslinked) yield granules with increased resistance to overcooking, acids, and shear (e.g., high-speed mixing, emulsifiers, colloid mill exposures, homogenization) and are made by using small amounts of chemical compounds to “reinforce” the natural hydrogen bonding within the granule. Stabilization is the process whereby “blocking” groups are attached to the starch polymer to inhibit retrogradation or “set back,” the major problem associated with the use of native starch. This process also imparts textural and freeze-thaw stability to food products, which is very important to refrigerated storage of food products because retrogradation of starch is accelerated at low temperatures and allows for syneresis (water loss) to occur. Pregelatinization of starch allows for instant viscosity development, an undesirable trait for meat emulsions. The selection of a particular starch will depend on a variety of circumstances. For example, if the emulsion is to be made in a chopper, a starch with less cross-linking would be suitable, but if a mixer-emulsifier is to be used, a starch with more cross-linking would be superior because of their resistance to the excess shear of the mixer-emulsifier unit. The temperature of gelatinization is also of great importance in selecting the appropriate starch. In most typical situations, meat emulsions are cooked to internal temperatures of 160° to 170°F (71° to 77°C), so the starch selected should gelatinize within this range (or other ranges if cooking temperatures are different). Also, to prevent excessive cooking shrinkage (moisture loss), a part of the starch used in a formula should gelatinize at a lower temperature (i.e., 128° to 130°F or 53°C). Different starches gelatinize at different temperatures. For example, unmodified potato starch typically gelatinizes at 147°F (64°C), waxy starch gelatinizes at 165°F (73°C), rice starch gelatinizes at 178°F (81°C), and wheat starch gelatinizes at 171°F (77°C). Different methods and levels of modification will produce different gelatinization temperatures of the various starches and this should be known before selecting a starch to use. Often, the starch selected can be a carrier for the seasoning/spice blend because it should be added to the formulation at the same time the seasoning is normally added. The starch selected for use in reduced-fat emulsion products should not increase batter viscosity and should become functional during the cooking process to bind water during cooking and reduce purge during storage (38). Starches are used in many food products and appear to be accepted by most consumers. Rogers et al. (59) evaluated 10 modified starches to determine their effects on various characteristics of fat-free bologna. All starch-containing products displayed less purge; other attributes (color, fracturability, hardness, coeshiveness, gumminess, juiciness, firmness, and chemical composition) were affected in different ways by different starches.

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Other attributes (batter viscosity, final yields, cooler shrink, cooked product diameter, flavor, texture, and overall satisfaction scores) were not affected by starches. It is also feasible to use more than one starch, based on gelation temperature, to trap as much of the free water as possible while temperatures are increasing during the cooking process to maximize cook yields and minimize purge. B. Miscellaneous Additives The literature contains several references to other additives that have been evaluated in some form of reduced-fat emulsion sausage. A brief summary of each of these items follows. 1. Oat Flour Sausages containing high-protein oat flour formed an external gel-like layer under the casing during processing, probably due to the low pH of the flour. These products were also judged to be less firm and juicy than the controls. The “cleanness” of odor and flavor was also distinctively diminished. These authors, Lapvetelainen et al. (41), also concluded that cereal products should be considered as the primary products for utilization of high-protein oat flour, not meat products. 2. Oat Bran Oat bran has been promoted by many as one of the most beneficial foods available. Therefore, it is not surprising to see it evaluated for use in low-fat sausage products. Because of its “grainy” or “gritty” texture, oat bran should be limited to approximately 2% in low-fat frankfurters. Although it reduces the amount of expressible water, it requires more force to shear these same products, and this is usually not a desirable trait (15). 3. Wheat Germ Wheat germ protein flour (WGPF), at 3.5% of the complete mixture, has been shown to affect protein matrix density in reduced-fat franks and to produce a more uniform interfacial protein film (IPF) with a slight increase in thickness. At higher levels (5.0% and 7.0%), increased water retention occurred and there were fewer fat globules, some with incomplete IPFs (26). 4. Applesauce The addition of applesauce to low-fat bologna was evaluated by Sanchez-Escalante et al. (63), who concluded that applesauce added at 15% of the formula had no effect on cooking yields or texture traits. They stated, however, that it did affect water, fat, protein, and color values of bologna. There were no sensory data included in this report so it cannot be ascertained how those attributes might have been affected. 5. Konjac Flour Konjac flour is obtained from the tuber of the Amorphophallus konjac plant. It is an ingredient commonly used in traditional Asian foods, as well as in other foods, and is capable of producing a gel that is stable in boiling water. The molecular weight of the glucomannan in this product is approximately 300,000 daltons. It has acetyl groups scattered randomly along the essentially linear molecule with an occurrence of about 1 per 19 glucose/mannose units. These acetyl units impart water solubility in an otherwise amylose-like molecule

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(55). Konjac flour is “generally regarded as safe” (GRAS). It has been used to produce gels that have been reduced by grinding/chopping to act as a substitute for fat particles in coarsely ground sausage where particle definition is essential (51). This material (gel produced from the konjac flour) may be seasoned and colored during the gel-making process to avoid seasoning and color voids in the sausage product (36). Konjac flour has also been shown to produce certain advantages in surimi gels (55). In emulsion products, however, the gel particles are not desirable, so the flour must be used in the powdered form to function as a water-binding agent without producing visible gel particles. One can find konjac flour listed in the ingredients statement of fat-free sausage products along with other additives that are combined in order to produce products acceptable to consumers as replacement foods for the traditional fatter ones. The usage level may vary, but when used in combination with other bulking agents and water-binding materials, it is generally used at relatively low levels (i.e., 0.25% to 0.50% of meat block weight). The legal limit of use is 3.5% of formula weight. 6. Lean Finely Textured Tissue Emulsion sausage products are an appropriate means by which to use low-cost protein sources (40). Two such materials that have been available for only a relatively short period of time are lean finely textured beef (LFTB) and lean finely textured pork (LFTP). They are obtained by low-temperature recovery of protein from trimmed beef and pork tissue. He and Sebranek (29) stated that the functionality of these materials is less than for their lean meat counterparts, and other additives (i.e., 2.5% salt, 0.25% STP, 0.5% kappa carrageenan) are needed to improve the overall quality of emulsion sausage made with these materials at high levels (i.e., 50% substitution for regular muscle meats). Both types of tissue used in low-fat frankfurters resulted in less functionality, lower yields, and a softer texture. The softer texture could be an advantage because excessive toughness or firmness are problems in some fat-reduced products. He and Sebranek also reported that the use of 2% isolated soy protein improved product stability and firmness, but lowered sensory scores. 7. Enzyme-Deactivated Mustard Enzyme-deactivated mustard, at 3.5% of finished weight, was evaluated along with various other additives in lean frankfurters (27). It proved to be quite effective for improving the following traits: (a) processing yields, (b) flavor scores, (c) texture scores, and (d) mouth feel scores. It was detrimental, however, to penetrometer scores (skin toughness) and juiciness scores for lean frankfurters. If this material is used, it must be listed as some type of modified mustard (i.e., enzyme-deactivated mustard, de-heated mustard, de-flavored mustard, de-characterized mustard, etc.), and may not be listed as mustard or spices, and the amount to use is limited to 3.0% of the formulation unless larger amounts are justified and approved by the USDA, FSIS. 8. Sodium Lactate Halloran et al. (27) also evaluated sodium lactate at 3% of formula weight with a 60% lactate-containing material in lean frankfurters. It proved to be the most effective additive studied by this group for increasing processing yields, but it also had the highest purge value (~7.5% after 60 days vacuum storage) of all treatments, even the control. It had no effect on total plate counts compared with several other additives or with the control product.

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9. Oils The substitution of vegetable oils (olive, corn, sunflower, and soy) for animal fats in reduced-fat emulsion sausage has also been investigated (6,7,53). Researchers reported that pre-emulsifying the fat or oil decreased the firmness in bologna and that purge increased with its use. They also stated that the addition of olive oil produced products with similar flavor to the controls, but had lower yields and overall palatability scores. Other work, where olive, corn, sunflower, or soybean oils were used, produced firmer products with a darker red color, lower yields, more purge, and lower juiciness scores than controls. Type of oil used had no effect on those characteristics, but did affect fatty acid composition of the finished products. 10. Conclusions About Nonmeat Additives The preceding paragraphs about nonmeat additives to low-fat emulsion sausage products reveal varying research results with a variety of additives. Several combinations of additives (fat replacers or fat substitutes) probably will prove useful in the production of lowfat emulsion sausage, but there does not seem to be any particular combination that will be 100% successful in replacing all characteristics possessed by full-fat frankfurters and bologna. Of course, that assessment is one of a personal nature and not universal to all processors or consumers. Water appears to be the only universally accepted additive that all agree must be used. Many researchers who have published on this subject also agree that one or more additives, in addition to water, salt, nitrite, phosphate, erythorbate, sugar, seasonings, flavorings, and so forth, are essential for the successful production of reduced-fat emulsion sausage products. IV. PROCESSING TECHNIQUES The processing techniques used to manufacture reduced-fat versions of emulsion sausage usually employ the same procedures as those used for manufacturing any emulsion sausage product. These are grinding, chopping, additive addition, stuffing, linking, cooking, peeling, slicing, and packaging. Some modifications are needed in some of the procedures to successfully produce lower fat emulsion products. The extent of the changes required can be a function of the target fat level in the final product. For example, a reduced-fat version could be manufactured in a similar fashion to a regular product, whereas a fat-free product would require some significant changes in the manufacturing process. The reduced-fat version would still require adequate chopping and particle size reduction to “emulsify” the materials. A fat-free product would only need to be ground and chopped to reduce the muscle particles enough for adequate protein extraction and to produce the smoothness required in this type of product—there would be no “fatting out” or “shorting out” of the product during cooking because of either inadequate fat particle size reduction or excessive fat particle size reduction. Mixing, chopping, or emulsifying raw materials causes a temperature rise of the meat batter. Extended mixing (30 min) has minimal affects on yield, purge, and texture of reduced-fat (15%) franks. Mixing temperature of 36°F (2°C) was superior for producing firmer franks, but this temperature had no affect on other properties when compared with mixing at 60°F (15°C). The timing of addition of the water is important to allow a good mixture of all ingredients and enhance protein extraction (67). The extent of the rise in temperature is a function of chopping speed, time, plate opening size, number of plates, and

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temperature of the meat block, but would normally be about 10° to 20°F (5° to 10°C). Hensley and Hand (31) and De Lopez and Hand (25) reported that a chopping temperature endpoint of about 50° to 54°F (10° to 12°C) produced the best results in low-fat, high-moisture emulsion products made from beef. Rogers et al. (59) reported that end-point-chopping temperatures of 64° to 65°F (18°C) produced very acceptable products. Sutton et al. (66) reported chopping end-point temperatures of 59°F (15°C) produced products superior to those produced at higher end-point chopping temperatures for beef franks. Temperatures in the ranges reported here are superior because they are the temperatures for optimum protein extraction. Traditionally, emulsion sausage containing a significant amount of beef fat has been chopped to about 70°F (21°C) to improve the texture of the emulsion. However, if the fat source was primarily from pork or poultry, lower end-point temperatures were used. In low-fat, high-moisture products, the difference in fat attributed to species appears to be much less meaningful. Particle size reduction (i.e., chopping or emulsifying) may also be of significance to end-product characteristics (65). It was reported that product made from materials passed through a 2.0 mm plate were harder and required more energy to shear than similar products passed through a 1.4 mm plate. They also concluded that particle size was of more importance than mixing time in low-fat, high-moisture pork frankfurters. Traditionally, sausage emulsions are vacuum chopped/stuffed to reduce voids, remove oxygen, and control density for sensory properties of the products as well as to control size/weight variables in order to more easily comply with the net weight provisions of USDA regulations. When full-fat products (e.g., frankfurters) are produced with the use of vacuum chopping and stuffing, the density is increased but the specific gravity is low enough to allow them to float in water while being cooked. However, unless specific provisions are taken to extend the emulsion in low-fat products with bulking materials (starches, dextrose, etc.) and reduce the vacuum, these products sink when placed in water for cooking—unlike routine products and cause of some consumer resistance to using these types of products. A small amount of inert gas (i.e., nitrogen) can also be injected into the batter after it has been exposed to a vacuum, to “fluff” it before stuffing. This decreases the density and allows the product to float when cooked in water. The gelation process of the various proteins is an important aspect of the ultimate rheological properties of reduced-fat emulsion sausage. This process affects the penetration force (PF) or the force exerted at the point of gel rupture and work of penetration (WP) or gel strength (33). Pork and poultry proteins, however, seem to act similarly and produce gels with maximum rheological values at near the same temperature (140°F)(60°C) and there is no gel formation at 113°F (45°C). Early work on heating of fat-free emulsions by Martin (46) and Martin and Rogers (47) centered on using a rapid, moist system (live steam) to set the gel early and not dry out the material with long-term exposure to dry heat. This system works well for cooking the products and obtaining excellent “bite” properties, whereas conventional cook-smoke schedules produced finished products with a very tough skin surface. The live steam process also allowed for the entire cooking process of a smoke house of frankfurters to be done within a few minutes (8 to 10). However, surface color development was not as acceptable as desired for commercially produced products. Therefore, modified cook-smoke schedules, using a short drying phase and a long moist heat (very high humidity) cook-smoke phase, were developed to produce products with acceptable exterior color, skin toughness, and internal rheological properties (1,27,28,30,59). These schedules also minimized cook-

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ing losses by minimizing evaporative losses. Because smokehouses and weather conditions vary so much, each processor needs to develop his own appropriate cook-smoke schedules for specific products (i.e., fat-free, low-fat, extra-lean, lean, and light). These schedules of high temperature and humidity are in contrast to recommended schedules for full-fat product because these conditions produce adverse effects on emulsion stability, texture, and color (62). With cook-smoke schedules of high temperature and high humidity, some products such as bologna can be cooked to 170°F (76°C) without having significant cooking losses or shrinkage in diameter of the finished product, traits very important when the product is sliced and vacuum packaged. The packaging, especially vacuum packaging, of low-fat emulsion products is also a processing factor needing special attention. Because of the high moisture content of these products, purge or free water in the package presents a special problem relative to consumer acceptability and shelf life. Some factors that can reduce purge were discussed earlier in this chapter, but two factors that relate specifically to packaging are correct cup or cavity size and film integrity. The cup must fit the product and the film must have enough integrity to hold the product in place in order to minimize the problem of having soft, irregular packages where the purge causes serious visual and microbiological problems. Of course, good seal integrity is required in all vacuum-packaged meats. V. SUMMARY The functionality of the system for emulsion-type sausage products is greatly affected by the reduction or elimination of fat in the system as well as by the addition of fat replacers (substitutes), other additives, and processing variables. The major factors affected by the reduction or elimination of fat from emulsion sausage are skin toughness, internal product characteristics (i.e., hardness, fracturability, cohesiveness, gumminess) sensory attributes, chemical composition, and purge. Product color is also affected, but to a lesser degree. The success of production of these altered-fat-level products depends heavily on the target fat level, the type(s) and amount(s) of fat replacer(s), the level and kind of spices and flavorings, the technique used in stuffing the batter into the casing, the cooking/smoking process used to reach a target finished product temperature, and the packaging techniques used in the manufacturing process. Of course, initial meat quality, species of origin, and composition (chemical and physical) can have significant effects on end-product characteristics, just as they do on any type of sausage product. REFERENCES 1.

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Osburn, W. N., and J. T. Keeton. 1994. Konjac flour gel as fat substitute in low-fat prerigor fresh pork sausage. J Food Sci 59, (3): 484–489. Osburn, W. N., C. M. Calhoun, and R. W. Mandigo. 1996. Modifications of connective tissue proteins for low-fat processing applications. 49th Annual Reciprocal Meat Conf., p. 193, Chicago, IL. Paneras, E. D., and J. G. Bloukas. 1994. Vegetable oils replace pork backfat for low-fat frankfurters. J Food Sci 59 (4): 725–728, 733. Park, J. W., T. C. Lanier, and D. H. Pilkington. 1993. Cryostabilization of functional properties of pre-rigor and post-rigor beef by dextrose polymer and/or phosphates. J Food Sci 58 (3): 467–472. Park, J. W. 1996. Temperature-tolerant fish protein gels using konjac flour. J Muscle Foods 7:165–174. Pearson, A. M., and T. A. Gillett. 1996. Processed Meats, 3rd ed. Chapman and Hall, New York, NY. Pettitt, D. J., J. E. B. Wayne, J. J. R. Nantz, and C. F. Shoemaker. 1995. Rheological properties of solutions and emulsions stabilized with xanthan gum and propylene glycol alginate. J Food Sci 60 (3): 528–531, 550. Price, J. F., and B. S. Schweigert. 1987. The Science of Meat and Meat Products, 3rd ed. Food and Nutrition Press, Inc., Westport, CT. Rogers, R. W., T. Healey, T. Armstrong, P. Coggins, F. Hairi, M. Martin, and B. Williams. 1996. Effects of various starches on the characteristics of fat-free bologna. Proc. 49th Annual Recip. Meat Conf., p. 196, Chicago, IL. Romans, J. R., K. W. Junes, W. J. Costello, C. W. Carlson, and P. T. Ziegler. 1994. The Meat We Eat, 13th ed., The Interstate Printers and Publishers, Inc. Danville, IL. Rust, R. E. 1976. Sausage and processed meats manufacturing. AMI Center for Continuing Education. American Meat Institute. Saffle, R. L., J. A. Carpenter, and S. B. Zirkle. 1967. Rapid method to determine stability of sausage emulsion and effect of processing temperature and humidities. Food Technology 21:784. Sanchez-Escalante, A., N. Gonzalez-Mendez, J. P. Camou, M. N. Ballesteros, and G. Torrescano. 1995. Usage of applesauce (malus pumila) for preparing a low fat bologna. 41st Annual Int. Congress of Meat Science and Technology. Vol. 2, pp. 425–426. San Antonio, TX. Schirle-Keller, J. P., G. A. Reineccius, and L. C. Hatchwell. 1994. Flavor interactions with fat replacers: Effect of oil level. J Food Sci 59 (4): 813–875. Small, A. D., J. R. Claus, H. Wang, and N. G. Marriott. 1995. Particle size and mixing time effects on sensory and physical properties of low-fat, high-moisture pork frankfurters. J Food Sci 60 (1): 40–41, 54. Sutton, D. S., L. W. Hand, and K. A. Newkirk. 1995. Reduced fat, high moisture beef frankfurters as affected by chopping temperature. J Food Sci 60 (3): 580–582, 586. Sylvia, S. F., J. R. Claus, N. G. Marriott, and W. N. Eigel. 1994. Low-fat, high-moisture frankfurters: Effects of temperature and water during extended mixing. J Food Sci 59 (5): 937–940, 945. Trius, A., J. G. Sebranek, R. E. Rust, and J. M. Carr. 1994a. Carrageenans in beaker sausage as affected by pH and sodium tripolyphosphate. J Food Sci 59 (5): 946–951. Trius, A., J. G. Sebranek, R. E. Rust, and J. M. Carr. 1994b. Low-fat bologna and beaker sausage: Effects of carrageenans and chloride salts. J Food Sci 59 (5): 941–945. Trius, A., J. G. Sebranek, R. E. Rust, and J. M. Carr. 1995. Ionic strength and chloride salt effects on the performance of carrageenans in a model system sausage. J Muscle Foods 6:227–242. Wright, L. H. 1988. Hot dog! At last, a low-fat sausage. The Clarion-Ledger Daily News, Jackson, MS. July 21, 1988, pp. 1F-2F.

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Xiong, Y. L., D. C. Noel, and W. G. Moody. 1995. Textural and sensory evaluation of salted low-fat beef sausage with added water and gums. 41st Annual Int. Congress of Meat Science and Technology, Vol. 2, pp. 439–440. San Antonio, TX. 73. Yang, A., G. R. Trout, and B. J. Shay. 1995. Evaluation of carrageenan, isolated soy protein and a modified starch in low-fat frankfurters. 41st Annual Int. Congress of Meat Science and Technology, Vol. 2, pp. 435–436. San Antonio, TX. 74. Yarbrough, N. 1993. Low-fat hot dogs in the works. Birmingham Post-Herald, Birmingham, AL. August 25, 1993. pp. C1-C2.

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19 Meat Packaging: Protection, Preservation, and Presentation

R. GRAHAM BELL MIRINZ Centre AgResearch, Hamilton, New Zealand

I. INTRODUCTION II. MEAT AS A MICROBIAL SUBSTRATE A. Substrate Composition B. Substrate pH C. Water Activity D. Initial Contaminating Microflora E. Microbial Spoilage F. Temperature G. Gaseous Environment III. FUNCTIONAL REQUIREMENTS OF MEAT PACKAGING A. Containment B. Protection C. Preservation D. Apportionment E. Unitization F. Convenience G. Communication IV. THE PACKAGING ENVIRONMENTS A. Physical Environment B. Climatic Environment C. Human Environment V. PRODUCT PACKAGING ENVIRONMENT INTERACTIONS VI. PACKAGING METHODS A. Nonpreservative Packaging B. Preservative Packaging C. Two-Phase Packaging

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VII. PACKAGING FOR CHILLED STORAGE A. Vacuum Packaging B. Saturated Carbon Dioxide Atmosphere Packaging VIII. PRODUCT-LIFE EXPECTATIONS FOR CHILLED PRESERVATIVELY PACKAGED FRESH MEATS A. Product Safety IX. PACKAGING FOR RETAIL DISPLAY A. Overwrapped Trays B. Modified Atmosphere Packaging X. TWO-PHASE PACKAGING A. Mother Packs B. Gas-Exchange Packs C. Removable Top Web Packs XI. CHILLED MEAT PACKAGING REQUIREMENTS XII. FROZEN MEAT PACKAGING A. Types of Packaging for Frozen Meat XIII. PRODUCT DETERIORATION DURING FROZEN STORAGE A. Gross Carton Deformation and Breakdown B. Freezer Burn C. Frost Formation D. Recrystallization E. Freeze-Thaw Cycling F. Rancidity G. Nutrient Loss XIV. STORAGE LIFE EXPECTATIONS FOR FROZEN MEAT XV. SUMMARY REFERENCES

I. INTRODUCTION Although fresh meat is highly perishable, its packaging has, until relatively recently, been a matter of minor concern to meat traders, health officials, and the public. Unwrapped fresh or frozen carcasses have long been the currency of the wholesale meat trade. Small meat species such as rabbits and poultry are, in many parts of the world, still traded commercially in natural packaging, their own skins. This is also the primary packaging choice of most commercial and recreational hunters. At retail, the grease-proof and wrapping paper package has given way to the overwrapped polystyrene tray typical of self-service merchandising of meat. The very obvious difference between full-service and self-service packaging is not simply a matter of modernity but is a reflection of different functional requirements of the two retailing systems. This chapter will consider the functional requirements and principles of packaging in relation to its various and changing roles in the protection, preservation, and presentation of meat. Those interested in the chemistry and functional characteristics of individual polymers and polymer combinations used in proprietary packaging films should consult manufacturers or specialist texts such as Brown (1992) and Brandrup et al. (1998). Packaging is not simply the materials immediately surrounding a product but is the synthesis of product, processing, labor, and machines, for addressing specific functional and/or marketing requirements. These functions relate to all aspects of distribution, storage,

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and merchandising: containment, protection, preservation, apportionment, unitization, convenience, and communication. With meat and meat products, packaging should provide users—whether intermediate traders, processors, or the end consumers—with appropriately portioned product in a safe and wholesome condition. Meat packaging has to function in a physical environment that threatens product damage and loss of pack integrity; a climatic environment presenting temperature, moisture, light, oxygen, and microbiological challenges; and a complex human environment that includes functional, psychological, and legislative elements. The functional requirements of meat packaging systems are dictated by the required marketing performance. Obviously the packaging requirements for international trade in chilled meat differ from those for domestic supply. The overriding performance requirement is, however, the same in both cases: adequate storage life ensuring product resilience to meet customer expectations. With chilled products, the onset of microbial spoilage, considered fully in Chapter 10, is the usual determinant of the end of practical storage life. Hence, storage life of chilled meat and microbial growth limitation are inextricably linked. Microbial spoilage can be easily prevented if meat is frozen to temperatures too low for microbial growth to occur. However, even frozen meat is subject to deterioration through desiccation and chemical changes leading to changes in color and palatability. It is these quality changes that packaging of frozen product must minimize or prevent. Successful meat packaging and storage denies, or limits, the opportunity for microbial growth. Consequently, it is appropriate to preface this discussion of packaging with a brief overview of meat as a substrate for microbial growth. II. MEAT AS A MICROBIAL SUBSTRATE Fresh meat is an oxygen-sensitive chemical entity that provides an excellent substrate for microbial growth, allowing contaminating bacteria to proliferate rapidly while conditions, particularly temperature and the gaseous environment, remain favorable. Such microbial growth will eventually cause spoilage and can also pose a hazard to health. In normal healthy slaughter animals, the tissues destined for the table are generally sterile. However, during slaughter and dressing, microorganisms contaminate the surface of carcasses and meat cuts. Microbial growth is limited to surface sites until spoilage is well advanced. A. Substrate Composition All meat spoilage bacteria utilize low-molecular-weight, soluble components of muscle tissue for their growth, particularly glucose and amino acids. Generally, glucose is the preferred substrate and as long as it is available at the meat surface other substrates are not significantly degraded. When bacterial numbers at the meat surface are high enough that the organisms consume glucose more rapidly than it can diffuse from within the tissue, the organisms begin to attack amino acids, releasing large amounts of ammonia and smaller amounts of malodorous organic sulfides and amines. Consequently, the glucose content of meat is a critical factor determining the relationship between spoilage flora development and the time to spoilage onset. The glucose concentration in normal pH meat varies between 100 and 1000 gg1, being low in beef, intermediate in mutton, and high in pork (Newton and Gill, 1978). Off odors begin to develop when bacterial numbers reach about 108 cm2 where the initial muscle glucose concentration is about 100 gg1. When the initial muscle glucose con-

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centration approaches 1000 gg1, odor development and slime formation may occur simultaneously when bacterial numbers reach 109 cm2 (Gill, 1976). By contrast, high ultimate pH meat, also known as dark firm dry (DFD) meat, is low in glucose, and spoilage may occur when bacterial numbers approach 106 cm2 (Newton and Gill, 1978). B. Substrate pH Growth of the major aerobic and anaerobic spoilage organisms, Pseudomonas spp. and lactic acid bacteria, respectively, is insensitive to pH within the fresh meat range (5.3 to 7.0). Meat pH has little effect on the onset of aerobic spoilage but is important under anaerobic conditions because it affects the composition of the developing microflora. C. Water Activity For most meat spoilage microorganisms, water activity (aw) values above 0.98 are optimal for growth. As fresh meat has an aw above this value, inhibition of microbial growth by aw reduction needs to be engineered either by drying and/or—in cured products—by the addition of solutes such as salt or sugar. The spoilage bacteria of chilled meats are generally very susceptible to lowering of the aw at product surfaces. However, when product is packaged to prevent moisture loss, surface drying can play no part in controlling microbial spoilage. D. Initial Contaminating Microflora The major sources of microbial contamination are the slaughter animals themselves, the process workers, and the processing environment (Empey and Scott, 1939). Most microorganisms present in the initial contaminating microflora are unable to grow at chill temperatures. (These are termed mesophilic; cold-tolerant microorganisms are termed psychrotolerant or psychrophilic.) However, a few psychrotolerant organisms will also be present and will grow at chill temperatures, causing spoilage. In temperate regions, the proportion of psychrotolerant bacteria present in the initial contaminating microflora ranges from approximately 1% in summer to 10% in winter (Newton et al., 1978). E. Microbial Spoilage It is remarkable that so many societies independently came up with the same solutions to the problem of microbial spoilage of fresh meat. These classical preservation methods include drying, smoking, salting, curing, pickling, fermenting and, in colder latitudes, icing or freezing. These processes all make meat a less desirable substrate for microbes by either reducing its aw, changing its acidity, adding toxic substances, or lowering its temperature. However, these processes, excepting cooling, produce products with sensory characteristics that differ markedly from those of fresh meat. The secret to meat preservation without sensory compromise lies primarily in the control of storage temperature. F. Temperature Microbial spoilage of meat can be prevented if meat is frozen to temperatures too low for microbial growth. Without resort to freezing, the effective product life of fresh meat can be greatly extended through chilling. Chilled storage of meat at between 2 and 5°C prevents the growth of mesophilic pathogens and delays the onset of spoilage. Spoilage microflora

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developing on chilled meat are composed of psychrotolerant bacteria carried on the hides of slaughter animals and contaminating carcasses during dressing. Microbial spoilage is influenced by the size and composition of the initial contaminating microflora. The rate at which the psychrotolerant component of that microflora proliferates determines the time to spoilage onset. At chill temperatures, meat spoils most rapidly through microfloras dominated by Pseudomonas spp. (Ayres, 1960). These microorganisms are described as having a high spoilage potential because they produce offensive metabolic by-products as they grow on meat. G. Gaseous Environment The gases that have the most marked influence on meat spoilage microflora development are oxygen, carbon dioxide, and nitrogen. Oxygen is the universal electron acceptor necessary for the growth of strict aerobes. Consequently, establishment of an oxygen-free environment will prevent the growth of the meat spoilage pseudomonads. Facultative anaerobes such as Shewanella putrefaciens also use oxygen as an electron acceptor, but in its absence are capable of fermentative metabolism. When the meat pH is greater than 6.0, anaerobic growth of Shewanella putrefaciens is accompanied by copious H2S production, resulting in a green discoloration due to sulfmyoglobin. Brochothrix thermosphacta will accelerate spoilage of anoxic meat above pH 5.8, as will some psychrotolerant enterobacteria (Grau, 1980, 1981). Carbon dioxide inhibits the growth of many microorganisms, but because all organisms are not equally sensitive (Enfors et al., 1979), increased concentrations of this gas within a package change the microflora composition. Pseudomonas spp. are particularly sensitive, whereas lactic acid bacteria are largely unaffected (Coyne, 1933). By increasing the carbon dioxide concentration in a packaging atmosphere, the microflora is shifted from high to low spoilage potential as lactic acid bacteria progressively displace the pseudomonads. Under aerobic conditions, there is an initial large decrease in pseudomonad growth rate as the carbon dioxide concentration is increased to 20% but with little additional inhibition at higher concentrations (Gill and Tan, 1980). In anaerobic systems, by contrast, the inhibitory effect on facultative anaerobes increases progressively, with the maximum effect being achieved with a pure carbon dioxide atmosphere (Gill and Penney, 1988). Perhaps most important of all, from a preservative perspective, is that at any concentration of carbon dioxide, its inhibitory effect increases as storage temperature decreases (Adams and Huffman, 1972). Nitrogen and other inert gases do not in themselves inhibit bacterial growth. However, by displacing the oxygen fraction in air they influence microbial growth by lowering the in-pack oxygen concentration. Nitrogen is also often included in pack atmospheres to prevent pack collapse as carbon dioxide used in meat packaging is absorbed by the packaged product. III. FUNCTIONAL REQUIREMENTS OF MEAT PACKAGING A. Containment This basic function of packaging is so obvious that it is often overlooked. With the possible exception of carcasses, meat and meat products must be contained before they can be

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moved from one place to another. The containment function of meat packaging extends from the packing plant to the consumer’s refrigerator. Imagine the response, at retail, of customers offered a pile of pork chops but no containers in which to take their selection to checkout. B. Protection Packaging isolates its contents from environmental effects such as dust, microorganisms, water, chemicals, gases, odors, shocks, and compressive forces. Conversely, packaging also protects the environment from its contents. Consider the pile of pork chops at the meat counter. Although paper bags would satisfy the containment function, plastic bags would afford both containment and protection. C. Preservation For meat and most other perishable products, the major cause of deterioration is microbial spoilage (discussed above and in Chapter 10). Other causes of product deterioration include moisture loss (desiccation), color change, and oxidative rancidity. To accomplish the preservative function, packaging must restrict microbial growth, prevent moisture loss, and control gaseous exchange between the package and ambient atmospheres. Loss of water from fresh meat reduces the weight of meat available for sale and in extreme cases renders the product unsaleable because of its unsightly appearance. However, enclosing meat within a water-impermeable film, while preventing moisture loss, will accelerate the onset of microbial growth because water activity remains high. The color of raw meats is determined by the oxidation state of the muscle pigment, myoglobin (Chapter 5). When no oxygen is present, myoglobin is in its oxygen-free form (deoxymyoglobin), which gives meat in anoxic packs its characteristic purple-red color. Oxygenated myoglobin (oxymyoglobin) is bright red, a color consumers associate with freshness. The deoxymyoglobin/oxymyoglobin reaction is reversible and occurs in response to changes in the partial pressure of oxygen surrounding the meat. Oxidized myoglobin (metmyoglobin) is brown, a color consumers associate with staleness and the loss of nutritional and sensory quality. Metmyoglobin forms most rapidly at low oxygen concentrations, around 0.5%. The relationship between oxygen concentration and the chemical state of myoglobin (Forrest et al., 1975) shows that metmyoglobin formation is minimal in atmospheres containing less than 0.1% or more than 15% oxygen. Therefore, either a highly aerobic or a completely anoxic environment is required to prevent browning. However, as the consumer associates a bright red meat color with freshness, the packaging requirements for retail display and extended storage are unlikely to be the same. The reaction of fats with oxygen is responsible for the development of rancid odors and flavors. Oxidation takes place with atmospheric oxygen and is often accelerated by heat, light, high energy radiation, and various oxidation catalysts. In meats, the development of rancidity results primarily from a free radical chain reaction. This means that an anoxic packaging environment may slow but will not prevent the development of rancidity if product is packaged after the initial oxygen–fat reaction has occurred.

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D. Apportionment This function of packaging is to reduce industrial output (i.e., dressed carcasses) to an appropriate size for further processing or consumer use. E. Unitization Unitization describes the function by which primary packages are consolidated for shipment. Primary packages are unitized into secondary packages, for example by placement inside a cardboard carton. The secondary packages in turn are unitized into a tertiary package; for example, a stretch wrapped pallet that may, in turn, form part of a quaternary package—a shipping container or truck load. Unitization allows optimization of materials handling by minimizing the number of discrete packages that need to be handled. On delivery, the process is reversed from distributor to consumer so the latter is presented with a primary package and (fortunately) not a container load. F. Convenience In modern industrialized societies the time spent by the consumer in obtaining and preparing food continues to fall. Although meats are not in the forefront of the convenience market, microwaveable packs and meat-based whole meals are appearing on supermarket shelves. G. Communication There is an old packaging adage that says “a package must protect what it sells and sell what it protects.” In the case of meat, “sell what it protects” requires that product be presented in an attractive manner and also that the product be clearly recognizable. The latter means branding or other distinctive labeling that will identify the product to the consumer. Currently this is a grossly underdeveloped aspect of meat retailing. IV. THE PACKAGING ENVIRONMENTS A. Physical Environment The physical environment is the environment in which physical damage can be caused directly to the product or indirectly to the product through damage to the package. Such damage includes shocks from drops, falls or dumps, vibration, compression, and crushing. Loss of pack integrity—for example, puncture of a vacuum pack—will compromise the preservative function of that package. B. Climatic Environment The climatic environment can cause meat deterioration as a result of contact with gases, (especially oxygen), water or water vapor, exposure to light (particularly ultraviolet wavelengths), and the effects of heat and cold. Packaging acts as a barrier separating the ambient environment from the package’s internal environment. The water and gas barrier properties of the packaging material define an internal environment that may differ markedly from the ambient external atmosphere. Consequently, it is essential that barrier and other functional properties do not change over the expected storage period and range of external and internal environmental conditions.

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Figure 1 Influence of temperature on the oxygen transmission rates of a nylon and a vinylidenebased laminated film used for vacuum-packaging of meat. (Data from Lambden et al., 1985.)

Temperature is probably the most important climatic factor because of its influence on the functional properties of packaging materials. Most meat primary packagings have no effect on the temperature of the packaged product, which tends toward that of the external environment. However, secondary and tertiary packaging can have a marked effect on the rate of initial product cooling and on product temperature changes during storage and distribution. Among other parameters, temperature changes influence oxygen permeability, microbial growth and color deterioration rates. The permeability of packaging films to oxygen decreases with temperature. However, permeability is particularly affected around the freezing point of water. At sub-zero storage temperatures, the difference in the permeability of different films is dramatically reduced (see Fig. 1), perhaps arising from the freezing of water associated with the film (Lambden et al., 1985). In practical terms, oxygen transmission rates at subzero temperatures cannot be accurately predicted from data obtained at higher temperatures (Table 7). A small decrease in temperature below 0°C can alter both the absolute and relative permeabilities of packaging films. At chill temperatures, the rate of growth of psychrotolerant bacteria is inversely related to temperature. Consequently, the optimum storage temperature for chilled meat in respect to length of storage life is the lowest temperature that can be maintained without freezing. Within the chill temperature range (2.0 to 5.0°C), the storage life of packaged meat reduces by approximately 10% for every 1°C that the product temperature exceeds the 1.5°C optimum. Deterioration of meat color is extremely temperature sensitive, with the rate doubling for every 5°C rise in temperature (Greer and Jeremiah, 1981). The effect of temperature on these oxygen-mediated changes (see Chapter 5) is critically important in successful retailing of fresh meat. Exposure of meat, in particular cured meat, to light can increase the rate of color deterioration. Cured meat products tend to fade when exposed to light.

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C. Human Environment This is the environment in which the package interacts with people, including regulatory bureaucrats and the consuming public. The most easily understood interactions are those associated with regulatory requirements, such as the use of approved packaging materials and appropriate labeling. Provided these and functional requirements are adequately satisfied, the interaction with the consuming public enters the realm of psychology and includes experience, perception, recognition and preference. V. PRODUCT PACKAGING ENVIRONMENT INTERACTIONS In the preceding sections, an attempt has been made to separate product properties, packaging functions, and environmental factors. Such a separation is arbitrary as these elements interact to produce the conditions experienced by the packaged product. In some cases those conditions differ little from those found in the external environment (e.g., meat wrapped in greaseproof paper) whereas in others the difference is profound (e.g., meat held in a modified atmosphere pack). The more important factors contributing to the synthesis of the in-pack environment are presented in Table 1. The term intrinsic refers to variables associated with the product at the time of packaging. Extrinsic, on the other hand, describes environmental and packaging-associated variables. The interaction of the intrinsic and extrinsic factors determines the in-pack conditions presented to the packaged product by any given packaging system. The in-pack conditions determine product performance characteristics. Selection of a packaging system requires matching of performance capabilities with specific functional and/or marketing requirements. VI. PACKAGING METHODS Both chilled and frozen raw meats are generally protected by flexible plastic packaging. However, consumer packs can take a variety of forms which for convenience can be grouped into rigid (glass jars, cans, etc.), semi-rigid (plastic trays, boxes, etc.) and flexible

Table 1 Intrinsic and Extrinsic Factors That Determine In-Pack Conditions and Consequent Product Performance Extrinsic Environmental factors

Packaging factors

In-pack factors

Temperature

Temperature Air

Insulation Gas barrier Gaseous environment Vapor barrier Opacity

Temperature

Water activity (aw) Composition Acidity (pH) Redox Potential Preservatives Initial microflora

Relative humidity (RH) Light

Pack atmosphere aw and RH Light Substrate

Contaminants

Protection

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Microflora

Product performance

Intrinsic factors

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(plastic bags, pouches, etc.) packagings. The characteristics of the most common types of packagings used to contain, protect, and preserve raw meats are detailed below. A. Nonpreservative Packaging This type of packaging contains and protects the product from contamination and water loss without creating in-pack conditions very different from ambient. Consequently, unless microbial growth is prevented by freezing or retarded by chilling, product in such packs is highly perishable—i.e., has a very short product-life. Wrappers are the simplest type of flexible package, in which a sheet material is used to enclose a quantity of product. Examples of this type of packaging relevant to meat include greaseproof paper and plastic cling films. Such packages are generally not sealed by plastic welding. Overwrapped trays are widely used in supermarkets for fresh meat and poultry. Fresh meats are placed on a semi-rigid plastic tray that is overwrapped with a plastic cling film of high oxygen permeability. As with wrappers, overwrapped trays are not sealed and because of the high oxygen permeability of the films used, provide aerobic conditions around the product. Loose-fitting plastic bags and pouches are not generally used at retail because of unattractive product presentation. However, such packagings are widely used as primary packaging for fresh carcasses and for frozen bulk or individually wrapped cuts and offals. Bags and pouches can be heat sealed, in which case the degree of product protection is determined by the permeability of the packaging material to water vapor and gases. B. Preservative Packaging This group of packagings is characterized by an ability to extend product life by modifying or restricting microbial growth. This is achieved by creating and maintaining in-pack conditions that differ markedly from those of the ambient environment. In vacuum packaging product is placed into a bag or pouch that is evacuated and heat sealed. The packaging material must have a low permeability to oxygen so that the anoxic in-pack environment is maintained. Vacuum packaging is widely used as a primary transport and storage packaging for larger (primal) cuts. To date, its use at retail has been limited because of the unattractive presentation of product: a purple-red color due to deoxymyoglobin, squashed appearance, and drip accumulation. However, vacuum packaging is widely used for sliced processed meats, for which the color is produced by the nitrite cure and is protected by anoxia. Moreover, the rigidity and superior water binding of processed meats mitigate deformation and drip problems. In modified-atmosphere packaging (MAP), the gaseous environment around the product is modified before heat sealing, and then gradually changes as a result of the interaction between product and packaging. With meats, the in-pack gaseous environment is usually altered by evacuation followed by gas flushing with the desired gas mixture. Subsequent changes in the composition of the in-pack atmosphere are determined by the gas barrier properties of the packaging and the metabolic activities of the product and its microflora. Respiratory activity of meat contributes markedly to changing the in-pack atmosphere if meat is packed before rigor attainment. As with MAP, in controlled-atmosphere packaging (CAP) the gaseous environment around the product is altered but is then maintained at a specified composition regardless of product or microbial respiration, temperature, or other environmental changes. The prin-

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cipal difference with fresh meat between MAP and CAP is in the gas permeability of the packaging. As plastic materials are not absolutely impermeable to gases, the composition of the in-pack atmosphere will change, although very slowly. With CAP, gas-impermeable packagings such as plastic aluminum foil laminates or metallized films have to be used. For most contemporary packaging systems, the term “controlled atmosphere” would be a misnomer because it is not possible to control the atmosphere within a pack once it is sealed. However, with the development of active packaging systems, the distinction between MAP and CAP is becoming less clear. As well as acting as a barrier between the in-pack and external atmospheres, active packaging modifies the in-pack atmosphere. With meat, active packagings include oxygen scavengers and carbon dioxide generators. Active packaging systems may also include oxygen indicators and time-temperature indicators. C. Two-Phase Packaging Two-phase or double-phase primary packs combine extended product life and a retail display potential. The general principle is to change the gaseous environment surrounding the meat between the storage and display phases. This can be achieved either by gaseous exchange or by removal of part of the package to allow replacement of the carbon dioxide preservative atmosphere with air. This process will result in the meat color changing from purple-red (deoxymyoglobin) to the more desirable bright red (oxymyglobin) color for retail display. VII.

PACKAGING FOR CHILLED STORAGE

Packaging systems that allow national and international trade in chilled meat must, while maintaining sensory quality, prevent the growth of high spoilage potential strict aerobes, and of high spoilage potential facultative and obligate anaerobes. Simultaneously, the packaging must retard the growth of low spoilage potential anaerobes. The growth characteristics of the major spoilage microorganisms of chilled meat are presented in Table 2. Table 2 Growth Characteristics of the Major Groups of Chilled Meat Spoilage Microorganisms Spoilage organisms

Oxygen requirement

Pseudomonas Acinetobacter /Moraxella Enterobacteriaceae

Strict aerobe Strict aerobe Facultative anaerobe

Brochothrix thermosphacta

Facultative anaerobe

Shewanella putrefaciens

Facultative anaerobe

Lactic acid bacteria Psychrotolerant Clostridia

Aerotolerent anaerobe Strict anaerobe

a b

pH requirement None None No anaerobic growth below pH 5.8 No anaerobic growth below pH 5.8 No growth below pH 6.0 None None

Aerobic environment. Aerobic or anaerobic environment.

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CO2 sensitivity

Spoilage potential

Higha High a Moderateb

High Low High

Moderateb

High

Moderate

Very high

Lowb Lowb

Low High

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The role of psychrotolerant clostridia as agents of chilled meat spoilage was recognized when a causal relationship was established between psychrotolerant clostridia and “blown pack” spoilage (Dainty et al., 1989; Kalchayanand et al, 1989). Blown pack spoilage is characterized by gas production in non–temperature-abused vacuum packs, leading to gross pack distension during storage Drip in blown packs usually contains large numbers of psychrotolerant clostridia (Broda et al., 1996). By contrast, blown pack spoilage in temperature-abused vacuum packs is usually associated with the growth of psychrotolerant enterobacteria (Hanna et al., 1979). The extension of the storage life of chilled meat achieved by removing oxygen and/or increasing carbon dioxide concentration was recognized in the 1930s (Killifer, 1930). However, for almost thirty years there was little commercial interest in modified-atmosphere preservation of meat. In the 1960s, following developments in the plastics industry, vacuum packaging became a practical, preservative packaging technology for extending the storage life of chilled meat. A. Vacuum Packaging In vacuum packs, the preservative effect is achieved by maintaining an oxygen-deficient environment around the product. Therefore, when considering the product-life extension achieved by this well-established packaging technology, parameters that have an impact on the establishment, and maintenance, of that oxygen-deficient environment are important. As the vacuum is drawn, flexible packaging collapses around the product, squeezing most of the air from the pack. Any residual air in the pack is largely trapped in film folds that restrict its contact with the meat. The vacuum applied is not critical in successful vacuum packaging because atmospheric pressure collapses the film tightly to the meat surface. The application of a vacuum to 0.8 to 0.9 atmospheres is generally sufficient to produce satisfactory vacuum packs. Close contact between the product and the packaging film is enhanced by heat shrinking after pack sealing. Packaged product is passed through a shrink tunnel that subjects the films to a temperature of about 90°C for 2 to 3 seconds. However, if flexible packaging cannot conform closely to the product surface, or if the product contains voids such as the body cavity of a lamb carcass, residual air remains within the pack, resulting in an “evacuated” rather than a vacuum pack. Evacuated packs are less effective than vacuum packs in extending chilled meat storage life. The development of vacuum skin packaging has improved oxygen removal by effectively eliminating the void volume around, but not within, irregularly shaped products. In this thermoforming variant of chamber vacuum packaging, the “skin” is formed by drawing a high vacuum on the inner and outer sides of the film, and subsequently venting the upper side to atmosphere, forcing the film tightly over the product. The upper film material is softened by heating, so when it is applied under vacuum to the lower film (the plastic tray), the soft film molds itself to the shape of the meat to produce a skin-tight package. Unfortunately, all transparent plastic barrier films in commercial use today have low, but measurable, permeabilities to both oxygen and carbon dioxide. Therefore, the concentration of oxygen within a vacuum pack is determined by the equilibrium established between the rate of oxygen diffusion through the film and its utilization by the respiring meat and developing spoilage microflora. Measurements of oxygen in vacuum packs have usually yielded a figure of about 1%, a value more than adequate to allow rapid growth of aerobic spoilage bacteria. However, such high measured oxygen concen-

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Table 3 Relationship Between Film Oxygen Permeability of Vacuum Packs and the Storage Life of Normal-pH Beef at 0°C Film permeability (ml.m2.atm1.day1 at 25°C)

Storage life (days)

0 190 290 532 818 920

105 105 77–105 42–63 24–42 14–28

Source: Data from Newton and Rigg (1979).

trations are sampling-induced artefacts, because they are inconsistent with observed microbial growth and meat color. Product life of vacuum-packed meat at a given storage temperature is inversely related to the oxygen permeability of the barrier film used (Table 3). The influence of differences in barrier film permeability on product life is markedly reduced at subzero storage temperatures (Fig. 1). Therefore, a slight rise in storage temperature from below 0°C to above 0°C will have an effect on product storage life that is disproportionate to the actual temperature rise. Any residual oxygen trapped in vacuum packages at sealing will be converted into carbon dioxide by the respiratory activity of fresh meat. Within a day or two of packaging, the minuscule residual in-pack atmosphere will be predominantly nitrogen but may contain up to 20% carbon dioxide. This accumulation of carbon dioxide inside a vacuum pack as a result of respiration of product contributes to the preservative effect achieved in that pack (Table 4). The reduced preservative performance of polyvinylidene chloride (PVDC) packs compared to foil laminate packs reflects the poorer gas barrier properties of the former packaging film in respect to both oxygen ingress and carbon dioxide egress. Plastic films are approximately four times more permeable to carbon dioxide than oxygen. Comparison of foil Table 4 Influence of Packaging Film, Meat pH, and Carbon Dioxide Accumulation on Spoilage Flora Development (Aerobic Plate Count, 25°C) on Vacuum-Packed Beef during Storage at 1°C Storage time (weeks) Normal pH (5.5–5.7) 7 10 12 High pH  6.0 7 10

Spoilage microflora (log10 count.cm2) PVDC

Foil laminate

Foil laminate  CO2 absorber

6.59 7.46 7.36S

6.32 6.53 6.96

6.45 6.66 7.38

7.30 P N.D.

7.11P 8.30 P

6.74P 8.43P

N.D.
Not determined; S
sour odor; P
putrid odor. Source: Adapted from Gill and Penney, 1988.

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laminate with foil laminate plus carbon dioxide absorber shows the inhibitory effect of carbon dioxide accumulation. Vacuum packaging can be regarded as a form of MAP with elevated carbon dioxide. However, that gas, because of its high solubility in meat and fat, is not evident within the packs, unlike the situation with saturated carbon dioxide packaging. B. Saturated Carbon Dioxide Atmosphere Packaging Saturated carbon dioxide controlled atmosphere packaging (Gill, 1989) for the prolonged chilled storage of red meats may be more correctly described as a modified-atmosphere packaging system. Although the use of gas-impermeable films will prevent gaseous exchange between the ambient and pack atmospheres, no control is extended over the latter in respect to its composition or volume after pack sealing. Microbial growth control is achieved by a combination of an oxygen-deficient environment and the antimicrobial properties of a high partial pressure of carbon dioxide. A high (100%) carbon dioxide packaging system was once believed to be impractical because in packages where air has been largely replaced with carbon dioxide, red meats discolored rapidly (Ledward, 1970). This discoloration was in fact oxygen mediated, the result of metmyoglobin formation (Chapter 5). The relationship between oxygen concentration and the chemical state of myoblobin indicates that the formation of brown metmyoglobin would be minimal in atmospheres containing less than 0.1% oxygen. Achieving oxygen concentrations at packaging as low as 0.1% lay beyond the capability of most of the packaging machines available in the early 1980s. The essential elements of generic saturated carbon dioxide packaging systems are as follow: a packaging film that is totally impermeable to gases; a gas replacement system producing a pack atmosphere containing less than 0.1% oxygen; and a pack content of carbon dioxide sufficient to saturate the product at the anticipated storage temperature. How these requirements are met is now discussed. At present the requirement for a total gas barrier can be fulfilled only by plastic laminates containing a layer of aluminum foil (foil laminates) or by double metallized films. Both these barrier films have the silver appearance of aluminum metal, making observation of product within a sealed pack impossible. In the future, new “glass” films that incorporate a layer of silica may allow product visibility without compromising gas barrier requirements. Currently several atmosphere-replacement packaging machines capable of achieving pack atmosphere containing less than 0.1% oxygen are available commercially. The suckand-blow action of so-called snorkel-type atmosphere replacement machines imposes flexing stresses that can compromise the gas impermeability properties of foil laminate and metallized films. Also, consistent results have been found to be difficult to achieve using snorkel-type machines. These performance problems are particularly severe when packaging irregular shaped product (e.g., a lamb carcass) or when multiple units are aggregated in a single pack. Chamber machines are preferable not only because they do not stress the packaging film but also because they produce consistent pack atmospheres. The residual oxygen concentration at packaging achievable with state-of-the-art chamber packaging machines approaches that of the claimed oxygen-free carbon dioxide supply. As the oxygen concentration cannot be less than that in the oxygen-free carbon dioxide, the quality of the gas is critical in saturated carbon dioxide packaging. Oxygen absorbers can be used to compensate for poor oxygen removal or oxygen entry occurring when packaging films with inferior barrier properties are used. Oxygen ab-

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sorbers active in high carbon dioxide atmospheres are now available. Their use with clear plastic high barrier films (i.e., an active packaging system) is proving comparable to the use of gas-impermeable aluminum foil laminates or double metallized films. It must be emphasized that the practical acceptability of oxygen absorber systems and the use of modern clear plastic ultra high barrier films in place of metallized films will be determined by their ability to satisfy marketing requirements. Carbon dioxide is very soluble in both muscle and fat tissue (Gill, 1988). The inhibitory effects of carbon dioxide on bacterial growth increase with the equilibrium partial pressure of that gas (Gill and Penney, 1988). If the amount of carbon dioxide added to a pack is insufficient to saturate the meat, then absorption will continue until essentially all is absorbed. In this case, the equilibrium partial pressure of carbon dioxide within the pack will be less than atmospheric. Approximately 1.3 to 1.5 liters of gas per kilogram of meat are required for saturation. Failure to introduce sufficient carbon dioxide will result in reduced inhibition of bacterial growth (Fig. 2). This figure also shows that for a given carbon dioxide volume to meat weight ratio, growth inhibition is less effective in high-pH than in

Figure 2 Effect of initial carbon dioxide volume to meat weight ratio, meat pH, and storage time (weeks) on spoilage microflora development (aerobic plate count, 25°C) in beef stored at 1°C in foil laminate packs. (Data from Gill and Penney, 1988.)

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normal-pH meat. The pH dependency of inhibition efficacy by carbon dioxide occurs with meat-borne pathogens as well as spoilage organisms. The technical requirement that the meat be saturated with carbon dioxide can create material handling problems, as the packs are initially distended by the carbon dioxide they contain. Subsequent absorption of the gas by the meat can produce change in a pack volume approaching 50%. At chiller temperatures, gas absorption is sufficiently complete after 24 hours to allow packs to be cartoned. As with vacuum packaging, the product storage life achieved by saturated carbon dioxide packaging is similarly inversely related to the storage temperature (Adams and Huffman, 1972). Further, the storage-life differential achieved by saturated carbon dioxide over vacuum packaging decreases to become insignificant at temperature above 12°C to 15°C (Gill and De Lacy, 1991). With any chill packaging, the importance of storage temperature as the principal determinant of effective product life cannot be overemphasized. VIII. PRODUCT-LIFE EXPECTATIONS FOR CHILLED PRESERVATIVELY PACKAGED FRESH MEATS The chilled product life achieved using preservative packaging, be it vacuum, modified, or controlled atmosphere, is determined by the interaction of product, packaging, and storage parameters. Moreover, a product-life estimate varies with the criteria used for assessment. In the early development of saturated carbon dioxide packaging, the principal criterion for product-life assessment was product appearance and the absence of off-odor (microbial spoilage) at pack opening. However, meat that is visually and microbiologically acceptable, but with a retail display life of less than a day and a liver flavor, is not marketable. Consequently, extreme caution must be exercised when using research results to set commercial product-life specifications. Consumers demand product that consistently meets high standards of eating quality and appearance during retail display. If a displayed product does not look good, the customer will look elsewhere. Current specifications often demand 7 days product life for retail distribution, display, and home storage from preservative pack opening. Consequently, buyers are now imposing retail specifications that limit the acceptability of chilled product to well below the longevity claimed for state-of-the-art packaging systems. In the market place, preservative packaging systems are being judged not on their ultimate microbiological product life, but on their capability to preserve quality attributes pertinent to specific clients, and thereby satisfy marketing requirements. The extension of chilled storage life afforded by the use of saturated carbon dioxide packaging over that achieved by vacuum packaging is highly temperature dependent. The storage life of meat at 1.5°C in a saturated carbon dioxide atmosphere is doubled compared with equivalent meat in vacuum packaging, but above 10°C there is little improvement in storage life. In commercial practice with a cold chain nominally operating between 1.0°C to 0°C, the increase in storage life over simple vacuum pack is around 50%. Comparable storage-life expectations for beef, lamb, pork, and chicken are given in Table 5. Also given in Table 5 are the retail display-life expectations for the four meat species. A. Product Safety As product safety is the subject of Chapter 17, discussion will be limited to changes in product safety effected by meat storage and packaging regimens. The majority of meat-borne

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Table 5 Expected Storage Life for Hygienically Produced Beef, Lamb, Pork and Chicken Held at 1°C  0.5°C and Displayed at 4°C; Storage Life is Defined by the Product Attribute That First Fails to Satisfy Market Needs (Microbiological, Eating Quality, or Retail Display Requirements) Assured product-life (days) Packaging system Transport /storage packaging Vacuum Carbon dioxide Display packaging Cling film overwrap (fresh meat) (stored meat) High oxygen MAP (fresh meat) (stored meat)

Beef

Lamb

Pork

Chicken

84 126

60 90

28 63

25 70

3–5 1–3 7–10 2–6

3–5 1–3 7–10 2–6

3–5 1–3 7–10 2–6

2–4 1–2 4–8 2–4

pathogens are mesophilic (e.g., Salmonella, E. coli O157:H7) and require temperatures above about 7°C for growth. Therefore, the health hazard they pose is not increased during chilled or frozen storage. Psychrotolerant pathogens, on the other hand, may be capable of growth during chilled storage. Health concerns have been raised, particularly for preservative packagings that suppress the growth of competing spoilage microorganisms. In this case, it is feared that psychrotolerant pathogens could reach high numbers in product that neither looks, smells, nor tastes spoiled. The four psychrotolerant pathogens of concern are Aeromonas hydrophila, non-proteolytic strains of Clostridium botulinum, Listeria monocytogenes, and Yersinia enterocolitica, whose growth characteristics are summarized in Table 6. Adequate refrigeration will control the growth of all four pathogens; however, other variables such as substrate pH and gaseous environment markedly influence the minimum temperature at which they can grow. Generally, the minimum temperature for growth increases as carbon dioxide concentration and substrate acidity increases (pH falls).

Table 6 Growth Characteristics on Chilled Meat of Psychrotolerant Meatborne Pathogens Psychrotolerant pathogen

Oxygen requirement

pH requirement

Aeromonas hydrophila

Facultative anaerobe

Clostridium botulinum

Obligate anaerobe

Sensitive to pH below 6.0 No growth below pH 4.8 Sensitive to pH below 5.0 No anaerobic growth below pH 5.8

Listeria monocytogenes

Facultative anaerobe

Yersinia enterocolitica

Facultative anaerobe

a b

Strain and substrate dependent. Toxin production reported at 2°C (Moorhead and Bell, 1999).

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CO2 sensitivity

Minimum growth temperature

High

4–0°Ca

Low

3–3°Ca,b

Moderate

4–0°Ca

High

4–0°Ca

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IX. PACKAGING FOR RETAIL DISPLAY The trend to self-service merchandising of fresh meat requires a high standard of product presentation. As consumers relate the bright red color of oxymyoglobin with freshness, retail packaging of meat normally provides an aerobic environment. Under aerobic conditions the meat surface is fully oxygenated, with the concentration of that gas decreasing with depth below the surface (Chapter 5). About 8 mm below the surface, the oxygen concentration will favor the development of the brown pigment metmyoglobin. This layer advances with time toward the meat surface, bringing about the meat color deterioration sequence from bright-red through tired/dull red to brown or even green. A. Overwrapped Trays An aerobic display requirement is satisfied by placing meat on a semi-rigid plastic tray and overwrapping with highly oxygen permeable cling film. This system does not restrict the growth of aerobic spoilage microorganisms, in particular the high spoilage potential pseudomonads. Consequently, the effective display life may be determined either by the onset of microbial spoilage or by color deterioration. Which occurs first depends on the interaction of various parameters, including microbial load, time in storage, and display temperature. Typical display times for overwrapped trays are given in Table 5. The retail performance of meat after prolonged chilled storage is shorter than that of fresh meat. There are two reasons for this: during storage, metmyoglobin reductase activity eventually stops, and a greater proportion of the myoglobin in the oxidized state (metmyoglobin). Activity of metmyoglobin reductase requires a biochemical reductant. During prolonged chilled storage, reductant reserves become exhausted and the enzyme is no longer able to reverse the accumulation of metmyoglobin. The result is more rapid browning during retail display. During prolonged chilled storage, meat in vacuum packs will react with the traces of oxygen that permeate the barrier. Progressively during storage, this reaction converts an increasing proportion of the deoxymyoglobin into brown metmyoglobin. On exposure to the air the meat will bloom (i.e., become bright red) but that meat already contains a significant proportion of metmyoglobin. This being the case, browning will occur more rapidly in stored meat, as less oxymyoglobin has to be transformed to metmyoglobin. Saturated carbon dioxide packaging with its associated oxygen-impermeable films affords a display life advantage over vacuum packaging in respect to browning. Greening of the original outside surfaces of preservatively packaged primals stored for long periods, or retail cuts derived from them, may occur during aerobic retail display. This type of discoloration results from the reaction of myoglobin with hydrogen peroxide, the latter generated by high surface lactic acid bacteria populations on exposure to oxygen. This problem, green choleglobin formation, is not usually seen on fresh cut surfaces but is evident on the edges of cut steaks. It can be particular severe with the first and last steaks cut from a strip loin, as in both cases one side is an original outside surface carrying a high lactic population. Another problem is the greening or graying of fat (Bell et al., 1996), arising from drip that stains fat during prolonged storage. Initially such fat has an acceptable but pinky hue. With time, the staining myoglobin/haemoglobin pigment is oxidized to the brown met forms, which appear green or gray. The color is green or gray and not brown because perceived fat color is determined by the combination of light reflected from, and absorbed by,

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the fat surface and immediate subsurface layers. Saturated carbon dioxide packaging does not offer a fat color advantage over vacuum packaging (Bell et al., 1996). B. Modified Atmosphere Packaging For red meats, high-oxygen MAP systems utilize atmospheres containing approximately 20% to 30% carbon dioxide, 60% to 80% oxygen, and up to 20% nitrogen. The elevated oxygen concentration enhances the bright red meat color and the elevated carbon dioxide concentration inhibits the growth of aerobic spoilage microorganisms. High oxygen concentrations in display packs enhance meat color by increasing the thickness of the oxymyoglobin surface layer. At the same time the metmyoglobin layer lies deeper in the meat. The time taken for that layer to reach the surface is increased, so display life is extended. Unlike overwrapped trays, high oxygen display packs use a film with high gas barrier properties, to prevent the gases equilibrating with the ambient atmosphere. The modified atmosphere display packs consist of deep high barrier trays that are gas flushed before an upper high barrier film or lid is sealed in place. However, the pack atmosphere tends to change during display because the oxygen is lost to respiration and carbon dioxide is highly soluble in meat. The absorption of carbon dioxide can lead to pack collapse. The pack atmosphere remains reasonably stable and the pack shape is maintained when the ratio of pack volume to meat volume exceeds approximately 3 to 1. The use of high oxygen with high carbon dioxide effectively doubles the color stability and time to spoilage over that achieved using ambient atmosphere overwrapped packs (Table 5). High-oxygen MAP, which provides a chilled product life of only 5 to 10 days, is not suitable for prolonged storage of meat. Its suitability for display packaging is determined as much by commercial merchandising strategies as by the preservative capability of the packaging. The excessive space occupied by deep tray packs, compared to net weight of meat sold, tends to restrict MAP packaging to high value products catering to the upper end of the market. As discussed previously, the rate of discoloration is inversely related to temperature, so the importance of display cabinet temperature management cannot be overemphasised. X. TWO-PHASE PACKAGING In two (double)-phase packs, the gaseous environment surrounding the meat is changed between storage and display to combine the long storage life of saturated carbon dioxide packaging with the retail display requirement for oxygen-rich atmospheres. Such systems are ideal for centralized preparation of product for retail display. A. Mother Packs These are the simplest two-phase packaging systems, consisting of retail-ready packs inside an outer preservative pack. The retail-ready packs may be overwrapped trays or lidded packs. In both cases, retail films must be highly gas permeable, first, to allow the meat contact with the carbon dioxide preservative atmosphere and later, on removal from the mother pack, to allow atmospheric oxygen to bloom the meat. While a simple outer bag could be used to contain the carbon dioxide atmosphere, the inner packs would be free to move within the pack. Such movement could damage both inner and outer packs. Consequently,

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most proprietary mother pack systems employ a semi-rigid outer container to protect and restrain the inner packs. B. Gas-Exchange Packs In gas-exchange packs, the storage carbon dioxide atmosphere is replaced by one enriched in oxygen before product is put on display (Ho et al., 1995). The two-phase packs consist of deep-lidded gas-impermeable packs fitted with some means (valve or injection septum) for exchanging gas atmospheres. For storage, the packs are gas flushed with carbon dioxide and sealed. To prepare the pack for retail display, the carbon dioxide atmosphere is replaced with a high-oxygen carbon dioxide atmosphere, for example 65% oxygen, 35% carbon dioxide. The disadvantage of gas-exchange systems is that each retailer must have gassing equipment. C. Removable Top Web Packs For display, part or all of the top web (film) is removed from a gas-flushed deep-tray pack to allow the high carbon dioxide atmosphere to escape and oxygen (in air) to contact the product, which is often secured to the bottom of the deep tray by an oxygen-permeable vacuum skin (Boghossian et al., 1995). The use of the inner vacuum skin pack not only holds the product firmly within the relatively large outer pack but also helps to contain drip. An active-packaging variant of this system, which reduces the overall pack size, employs an oxygen absorption/carbon dioxide generation system to establish and maintain the carbon dioxide storage atmosphere. Such systems offer advantages over gas flush systems as they allow the volume of the preservative atmosphere to be reduced without compromising bacterial growth inhibition. As carbon dioxide is absorbed by the meat, more is generated to maintain the saturated state. Furthermore, the consistent attainment of extremely low residual oxygen concentrations ensures that meat color degradation during chilled storage is minimized. At display, the upper gas-impermeable web with the gas-absorption/generation sachet attached is removed, and the meat in the oxygen-permeable inner pack is able to bloom. XI. CHILLED MEAT PACKAGING REQUIREMENTS The gas barrier properties of packaging films used in the various preservative and display packs are summarized in Table 7. The information given in Table 7 is for guidance only. Specific customized, or proprietary, systems may achieve performance specifications using films of higher or lower oxygen transmission rates. With storage packagings, higher oxygen transmission rates reduce effective product life (Table 3). For display packagings that use atmospheric oxygen to bloom the meat, the use of lower permeability films may compromise display life by accelerating metmyoglobin formation. XII.

FROZEN MEAT PACKAGING

At commercial frozen meat storage temperatures (less than 12°C), microbial spoilage is completely stopped but meat is still subject to deterioration through desiccation and chemical changes, such as oxidative rancidity and deterioration in color. Apart from protecting frozen product from physical damage and contamination, the major requirement of frozen packaging is control of water loss. Frozen meat, particularly individual table cuts,

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Table 7 Oxygen Permeability of Packaging Film Required for Storage and Display of Chilled Meats Packaging type

Oxygen permeability

Oxygen transmission (ml.m2.atm1.day1 at 23°C)a

Storage Vacuum

Low

40 to 15

Very low

Not measurable

High

10,000 to 20,000

Low

15

Carbon dioxide

Display Overwrap

Modified atmosphere

Comments The lower the transmission the better to retard metmyoglobin formation Requires oxygen-impermeable metallized film or active packaging that includes and oxygen scavenger system and ultra high barrier plastic films Oxygenation of myoglobin to bloom meat requires ingress of atmospheric oxygen; very high permeability films are used with ground meat Oxygenation of myoglobin and inhibition of bacterial growth are achieved by atmosphere retained within the pack

a

At chill storage temperatures, oxygen transmission rates will be lower and very much lower at temperatures below 0°C.

can be sold frozen in their transportation packs, so that packaging must also be effective in presenting the product to the consumer. A. Types of Packaging for Frozen Meat Boneless beef and lower-value cuts of other species destined for further processing are bulk packed in 27 kg cardboard cartons within low-density polyethylene liner bags. This packaging is cheap, provides good moisture barrier properties, is rugged, and still retains film flexibility at temperatures approaching 40°C. However, the high oxygen permeability of the packaging film results in short quality product life because of color deterioration and oxidative rancidity. Polyethylene bags may also be used for frozen carcasses. Traditionally, lamb carcasses were frozen in stockinet bags. Demands by regulatory authorities for better hygienic protection saw stockinet bags replaced by polyethylene bags. This created product handling problems, because cold polyethylene is slippery, making handling and stacking difficult, or

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even dangerous. The initial solution was to develop special rough-surface polyethylene bags. This solved the stacking/handling problem but proved too expensive. The final solution was polyethylene bags with stockinet outers. (Removal of stockinet in the cause of improved hygiene was unpopular because customers had used stockinet for a variety of purposes, including wash cloths for automobiles.) Another method employs a plastic film that is heat shrunk onto the product in a heat tunnel. Shrink wrapping is cheaper than vacuum packaging but because of its poor appearance is more suitable for product destined for further processing, such as frozen cutting. Vacuum packaging of meat for frozen storage does not differ markedly from that for chilled storage. While oxygen-impermeable films are the norm, oxygen permeable films are sometimes used so that the product will bloom. These combined storage and retail ready packs can have a high standard of visual display, provided storage time is short. Whether gas-permeable or barrier films are used is often determined by the customers’ product color preference. XIII. PRODUCT DETERIORATION DURING FROZEN STORAGE A. Gross Carton Deformation and Breakdown The unattractive or damaged appearance of cartons of frozen meat, including offals, may result from deficiencies in one or more of five areas: handling before freezing, type of packaging used, freezing regimen, materials handling, and temperature control (Turczyn, 1980). Not only are these five problem areas equally important to carton condition, but their effects are interrelated: a deficiency in one area will adversely affect how the others provide essential product protection. Frozen product within the carton contributes to the stacking strength and impact resistance of a shipping carton. If product in a carton stack is not completely frozen or thaws during shipment, the cartons will deform unless extra-strong (more expensive) cartons have been used. Similarly, underfilling will compromise stacking strength and impact resistance. Underfilling also contributes to slow freezing and frost formation, as air gaps slow heat transfer and are sites for frost accumulation. If subsequent thawing occurs, the humid conditions caused by melting frost will soften and weaken the cardboard carton. Shipping cartons must be strong enough to prevent side and bottom bulge when they are filled with unfrozen product prior to freezing. Bulges set during freezing and prevent square and secure stacking of cartons. Improper handling will also cause carton damage. Frozen meat or offal cartons are handled several times and may experience two or three transport modes between freezing and final thawing for use. Cartoned product can experience impact, vibration, fluctuating temperatures and humidities, and large stacking forces. Even strong, well-designed cartons containing fully frozen product will break down when exposed to rough handling, and to inadequate blocking, bracing and unitization during transit. Palletization and the use of mechanical handling equipment such as forklift trucks will eliminate much rough handling. B. Freezer Burn During prolonged frozen storage, moisture can be lost. Products lose moisture as a consequence of vapor pressure gradients within the product and between the product and the external environment. These gradients are caused by temperature fluctuations during storage (Jul, 1969; Anon., 1986). When the product surface is warmer than the core, moisture

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moves toward the surface. Unfortunately, when the temperature gradient is reversed during cooling, moisture is not easily transferred back to its original location (Reid, 1993). When the air temperature is higher than the product temperature, moisture moves from the product into the air, resulting in surface desiccation known as freezer burn. Freezer burn, characterized by changes in the surface appearance of frozen meat or offal, is the result of sublimation of ice from product surfaces during storage. The desiccation resulting from this sublimation appears as grey patches on the product surface. Fluctuating storage temperatures accelerate the onset of freezer burn. Freezer burn occurs most rapidly at higher storage temperatures when the vapor pressure of the ice in frozen product exceeds the relative humidity of the air in a freezer. Freezer burn can lead to accelerated lipid oxidation. The open structure at severely desiccated surfaces provides a large surface area for interaction with oxygen. As well, water loss causes reactants to be concentrated in the remaining free water (Varnam and Sutherland, 1995). Freezer burn can be reduced if product is suitably packaged in a tight-fitting film that is impermeable to water and vapor or if the cold store is maintained at a high relative humidity, so-called storage over ice. With meat cuts and offals, freezer burn is most commonly associated with damaged packaging. Loss of pack integrity allows areas of the product surface to be exposed to the external environment. The use of loose packaging can lead to an intra-package freezer burn condition manifest by frost or “snow” within the pack. C. Frost Formation The mechanism of intra-package frosting is the same as freezer burn except that the moisture moves from the product to the inside surface of the packaging. Intra-pack snow formation cannot occur when a water- and vapor-impermeable film is tightly applied to the product surface. However, should a space exist between the product and its packaging, moisture will move into the space and then condense on the inside of the package to produce snow. In a consumer pack, this snow will prevent the buyer from seeing the product. If the product temperature is lower than the environment, then condensation occurs on the product (Anon., 1986), to appear as surface ice. Consumers may erroneously, but understandably, construe surface ice as an underhand method of adding weight to the product. Extensive frost formation can to account for up to 20% product weight losses (Anon., 1986). As with freezer burn, water movement within a pack during frozen storage is associated with deleterious changes in product quality. Such changes are minimized if a low, constant storage temperature is maintained. D. Recrystallization Recrystallization is a physical phenomenon whereby ice crystals in frozen foods increase in size but diminish in number during storage. The formation of large ice crystals can cause considerable tissue damage (Mazur, 1960), leading to increased drip losses on thawing (Martino and Zaritzky, 1988). Recrystallization is temperature dependent. The rate of recrystallization decreases abruptly at temperatures below about 7°C (Sy and Fennema, 1973), and few cases of ice recrystallization have been observed at temperatures below 12°C (Bevilacqua and Zaritzky, 1980). The most common type of recrystallization occurring in frozen meat—migratory recrystallization—is associated with temperature fluctuation during storage. Small ice crystals melt as the temperature increases, and the water is then transferred to the surface of larger crystals during the cooling phase. In other words,

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the unfrozen water does not renucleate to form new ice crystals (Reid, 1993; Sahagian and Goff, 1996). E. Freeze-Thaw Cycling Today, one would expect freeze-thaw cycling problems to be a matter of only historical interest. Unfortunately, such temperature abuse can still occur as a result of plant failures or where a lack of national or international infrastructures compromises cold chain integrity. Cyclical freezing and thawing results in considerable cell damage and tissue disruption, including membrane fragmentation; movement of water, solutes, and small particulates in the tissue; and compaction of cell organelles such as mitochondria (Strange et al., 1985a, b; Jones et al., 1986). F. Rancidity Frozen storage slows but does not prevent the onset of oxidative rancidity. Generally, the lower the storage temperature, the slower the rate of rancidity onset. Oxidative rancidity occurs most rapidly at 2°C to 4°C and virtually ceases below 30°C (Varman and Sutherland, 1995). Pork is very susceptible to rancid spoilage and must be stored at 18°C or below if good eating quality is to be retained for more than about 6 months. Cured pork products are especially prone to rancid spoilage, because the salt component of the cure acts as a catalyst for lipid oxidation. With poultry, the onset of rancidity is typically slowed by the use of oxygen-barrier packaging. The susceptibility of animal fat to oxidation relates to the degree of unsaturation of the fatty acids, which is higher in poultry and pigs than in ruminants. G. Nutrient Loss Of the principal macronutrients, proteins and fats can readily undergo chemical change during frozen storage. Changes to proteins result in loss of solubility, meat toughening, and changes in meat water holding capacity. With fats, oxidation leads to off-flavor development. However, changes determined by chemical or physicochemical methods are of limited value in estimating the residual nutritional value of these constituents. In practice, during storage at temperatures below 18°C for periods of a year or more, the nutritional value of macronutrients is not reduced (Anon., 1986). For meats, losses of vitamins during frozen storage are product dependent. At temperatures between 18°C and 12°C, between 10% and 40% of the total vitamins can be lost during storage, with thiamine losses being the most prominent (Anon., 1986). Perhaps of more practical importance is the loss of soluble nutrients, including vitamins, associated with drip loss during thawing. It is important that any judgment on the significance of nutrient loss associated with prolonged frozen storage be made in the context of the relative contribution that the food item makes to the consumers’ daily intake of the nutrient in question. XIV. STORAGE LIFE EXPECTATIONS FOR FROZEN MEAT The International Institute of Refrigeration (Anon., 1986) define the Practical Storage Life (PSL) of a product as “. . . the period of frozen storage after freezing during which the product retains its characteristic properties and remains suitable for consumption or the intended

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Table 8 Practical Storage Life for Frozen Meat and Meat Products Practical storage life (months) Temperature (°C) Product

12

18

23

Beef Chopped beef Pork Pork sausages Lamb Veal

4 to 12 3 to 4 2 to 6 1 to 2 3 to 8 3 to 4

6 to 18 4 to 6 4 to 12 2 to 6 6 to 16 4 to 14

12 to 24 8 8 to 15 3 12 to 18 8 to 15

Source: Adapted from ASHRAE Refrigeration Handbook (Anon., 1994).

process.” With frozen meats and offals, the recommended length of storage remains controversial because of the influence of packaging, storage temperature, relative humidity, moisture loss during freezing, and variation in the products themselves (Anon., 1994). The lack of any international agreement either on the method of PSL assessment or the end point has resulted in different PSLs being reported by different researchers for the same products. These differences, distinct from those that can reasonably be attributed to time/temperature/tolerance or product/processing/packaging factors (the so-called TTT and PPP factors), reflect differences in assessment protocols. Because of the variability of taste panels, the use of physical, chemical, or other objective tests for assessing quality attributes appears attractive. However, it must not be forgotten that acceptability of the product to the consumer and the value the consumer will attach to that product is what is important (Jul, 1984). The range of indicative practical storage lives of frozen meat and meat products is given in Table 8. As discussed previously, temperature fluctuation can have a greater deleterious effect on storage life than the use of a higher but constant temperature. From Table 22.8 it can be seen that there is an inherent species difference in respect to frozen storage stability. Susceptibility to oxidative rancidity is a major determinant of effective storage life. Another determinant is the degree of processing, chopping, or grinding. Each reduces effective storage life, almost certainly by increasing cellular damage and increasing the surface area exposed to the deleterious effects of oxygen. XV. SUMMARY Fresh meat is a highly perishable commodity whose prolonged storage requires chill (0° to 1.5°C) or freezing (less than 12°C) temperatures. At chill storage temperatures, effective storage life is determined by the onset of microbial spoilage, whereas desiccation and chemical changes limit frozen storage life. Storage life of chilled product is increased through the use of systems that provide an oxygen-free environment within the package. Increasing the carbon dioxide concentration within an anoxic package further enhances storage life. Both vacuum and carbon dioxide packaging systems demand packaging materials with high oxygen and carbon dioxide barrier properties. Under anoxic conditions, meat becomes purple-red, which is unattractive to most retail-level customers. Consequently, retail display packaging systems must provide an aerobic environment to allow the meat to bloom. Aerobic environments can be provided

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by the use of oxygen-permeable films, allowing the ingress of atmospheric oxygen, or the use of oxygen-impermeable films to prevent oxygen egress where oxygen enriched modified atmospheres are used. To maximize chilled storage and display life, product should be held at the lowest temperature that can be sustained without freezing. Chilled storage life reduces by approximately 10% for every 1°C that the storage temperature exceeds the optimum of 1.5°C, and color stability during retail display is halved for every 5°C increase in product temperature. At first sight, frozen storage appears less sensitive to packaging and temperature than chilled storage. In relative terms this is not the case, given the expectation of much longer practical storage life. The deleterious effects of oxygen on product color and palatability can be limited through the use of high oxygen barrier packagings or avoided by judicious selection of storage temperature and relatively short practical storage lives. Similarly, product desiccation can be prevented if closely applied moisture-impermeable packagings are used. Selection of packaging materials and storage temperature is dictated by the intrinsic stability of the product and the longevity required for marketing that product. In general, product stability is maximized by the use of packaging materials with high gas and water barrier properties and as low a storage temperature as can be consistently maintained. For effective frozen storage, it is more important that the storage temperature is kept constant than allowed to fluctuate even if the average fluctuating temperature is lower than the constant storage temperature. There is no best packaging/storage system; rather, there are variously effective packaging storage solutions. Judicious selection of an appropriate solution requires integration of functional requirements and the marketing performance that must be delivered. The overriding performance requirement is that the selected system will, in a cost-effective manner, deliver product to the user in a safe and wholesome condition with the resilience to satisfy expectations at each step in the processing distribution chain. REFERENCES Adams, J.R., and D.L. Huffman. Effect of controlled atmosphere and temperature on quality of packaged pork. J Food Sci 37:869–872, 1972. Anon. Recommendations for the processing and handling of frozen foods. 3rd ed. International Institute of Refrigeration, Paris, France, 1986. Anon. 1994 ASHRAE Handbook Refrigeration SI Edition. American Society of Heating, Refrigeration and Air Conditioning Engineers Inc. Atlanta, USA, 1994. Ayres, J.C. The relationship of organisms of the genus Pseudomonas to the spoilage of meat, poultry and eggs. Journal of Applied Bacteriology 23:471–486, 1960. Bell, R.G., N. Penney, K.V. Gilbert, S.M. Moorhead, and S.M. Scott. The chilled storage life and retail display performance of vacuum and carbon dioxide packed hot deboned beef striploins. Meat Sci 42:371–386, 1996. Bevilacqua, A.E., and N.E. Zaritzky. Ice morphology in frozen beef. Food Tech (UK). 15:589–597, 1980. Boghossian, V., R.F. Mawson, M.J. Country, P. Drew, and M.W. Hickey. Commercially prepared retail cuts of beef and lamb with extended shelf life. Proc. 41st Int. Congress Meat Sci. & Technol., Vol. 2, 312–313, 1995. Brandrup, J., E.H. Immergut, and E. Gulke. Polymer Handbook. 4th ed. John Wiley & Sons, New York, 1998. Broda, D.M., K.M. De Lacy, R.G. Bell, T.J. Braggins, and R.L. Cook. Psychrotrophic Clostridium spp. associated with “blown pack” spoilage of chilled vacuum-packed red meats and dog rolls in gas-impermeable plastic casings. Int J Food Microbiol 29:335–352, 1996.

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Brown, W.E. Plastics in food packaging—properties, design and fabrication. Marcel Dekker, New York, 1992. Coyne, F.P. The effect of carbon dioxide on bacterial growth. Proc Roy Soc Series B 113:196–217, 1933. Dainty, R.H., R.A. Edwards, and C.M. Hibbard. Spoilage of vacuum-packed beef by a Clostridium sp. J Sci Food Agric 49:473–486, 1989. Empey, W.A., and W.J. Scott. Investigations on chilled beef 1. Microbial contamination acquired in meat works. Bulletin 120, Council for Scientific and Industrial Research, Australia, 1939. Enfors, S.O., G. Molin, and A. Ternstrom. Effect of packaging under carbon dioxide, nitrogen or air on the microbial flora of pork stored at 4°C. J Appl Bacteriol 47:197–208, 1979. Forrest, J.C., E.D. Aberle, H.B. Hedrich, M.D. Judge, and R.A. Menkel. Principles of Meat Science. W.H. Freeman, San Franciso, p 182, 1975. Gill, C.O. Substrate limitation of bacterial growth at meat surfaces. J Appl Bacteriol 41:401–410, 1976. Gill, C.O. The solubility of carbon dioxide in meat. Meat Sci 22:65–71, 1988. Gill, C.O. Packaging meat for prolonged chilled storage: the Captech process. Br Food J 91(7):11–15, 1989. Gill, C.O., and K.M. De Lacy. Growth of Escherichia coli and Salmonella typhimurium on high-pH beef packed under vacuum or carbon dioxide. Int J Food Microbiol 13:21–30, 1991. Gill, C.O., and N. Penney. The effect of the initial gas volume to meat weight ratio on the storage life of chilled beef packaged under carbon dioxide. Meat Sci 22:53–63, 1988. Gill, C.O., and K.H. Tan. Effect of carbon dioxide on meat spoilage bacteria. Appl Environ Microbiol 39:317–319, 1980. Grau, F.H. Inhibition of the anaerobic growth of Brochothrix thermosphacta by lactic acid. Appl Environ Microbiol 40:433–436, 1980. Grau, F.H. Role of pH, lactate and anaerobiosis in controlling the growth of some fermentative gramnegative bacteria on beef. Appl Environ Microbiol 42:1043–1049, 1981. Greer, G.G., and L.E. Jeremiah. Proper control of retail case temperature improves beef shelf life. J Food Prot 44:297–299, 1981. Hanna, M.O., G.G. Smith, L.C. Hall, and C. Vanderzant. Role of Hafnia alvei and a lactobacillus in the spoilage of vacuum-packaged striploin steaks. J Food Protect 42:569–571, 1979. Ho, C.P., K.W., McMillan, and N-Y. Huang. Effects of distribution and display gas mixture on shelflife of ground beef in dynamic gas exchange modified atmosphere packaging systems. Proc. 41st Int. Congress Meat Sci. & Technol., Vol. 2, 319–320, 1995. Jones, S.B., E.D. Strange, and B.E. Maleef. Ultrastructure of pork liver after freeze-thaw cycling and refrigerated storage. J Food Sci 51:761–765, 1986. Jul, M. Quality and stability of frozen meats. In: Quality and Stability in Frozen Foods. (Eds. W.B. Van Arsdel, M.J. Copley and R.L. Olsen), Wiley-Interscience, New York, pp 191–216, 1969. Jul, M. Quality changes during freezer storage. In: The Quality of Frozen Foods. Academic Press, London, pp 44–80, 1984. Kalchayanand, N., B. Ray, and M.C. Johnson. Spoilage of vacuum-pack refrigerated beef by Clostridium. J Food Protect 52:424–426, 1989. Killifer, D.H. Carbon dioxide preservation of meat and fish. Ind Eng Chem 22:140–143, 1930. Lambden, A.E., D. Chadwick, and C.O. Gill. Technical note: oxygen permeability at sub-zero temperatures of plastic film used for vacuum-packaging meat. J Food Technol 20:281–283, 1985. Ledward, D.A. Metmyoglobin formation in beef stored in carbon dioxide enriched and oxygen depleted atmosphere. J Food Sci 35:33–37, 1970. Martino, M.N., and N.E. Zaritzky. Ice crystal size modification during frozen beef storage. J Food Sci 53:1631–1637, 1988. Mazur, P. Physical factors implicated in the death of microorganisms at sub-zero temperatures. Anal NY Acad Sci 85:610–629, 1960.

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Moorhead, S.M., and R.G. Bell. Psychrotrophic clostridia mediated gas and botulinal toxin production in vacuum-packed chilled meat. Letts Appl Microbiol 28:108–112, 1999. Newton, K.G., and C.O. Gill. Storage quality of dark, firm, dry meat. Appl Environ Microbiol 36:375–376, 1978. Newton, K.G., J.C.L. Harrison, and A.M. Wauters. Sources of psychrotrophic bacteria on meat at the abattoir. J Appl Bacteriol 45:75–82, 1978. Newton, K.G., and W.J. Rigg. The effect of film permeability on the storage life and microbiology of vacuum-packaged meat. J Appl Bacteriol 47:433–441, 1979. Reid, D.S. Basic physical phenomena in the freezing and thawing of meat and animal tissue. In: Frozen Food Technology. (Ed. C.C. Mallet), Blackie Academic and Professional, London, pp 1–13, 1993. Sahagian, M.E., and H.D. Goff. Fundamental aspects of the freezing process. In: Freezing Effects on Food Quality. (Ed. L.E. Jeremiah), Marcel Dekker, New York, pp 1–50, 1996. Strange, D.E., M.P. Dahms, R.C. Benedict, and J.H. Woychik. Changes in connective tissue histology in freeze-thaw cycled and refrigerated pork liver. J Food Sci 50:1484–1485, 1985a. Strange, D.E., S.B. Jones, and R.C. Benedict. Damage to pork liver caused by repeated freeze-thaw cycling and refrigerated storage. J Food Sci 50:289–294, 1985b. Sy, S.H., and O. Fennema. Rates of recrystallization in beef liver. In: Proc. 13th Int. Congress Refrig., Washington D.C. Vol. 1, pp 199–204, 1973. Turczyn, M.T. Guidelines for packaging frozen edible offal for export. Marketing Research Report Number 1115, US Department of Agriculture, 1980. Varnam, A.H., and J.P. Sutherland. Frozen meat and meat products—chemical and physical processes. In: Meat and Meat Products—Technology, Chemistry and Microbiology. Chapman & Hall, London, pp 382–383, 1995.

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20 Meat Curing Technology MIKE MARTIN Bryan Foods, Inc., West Point, Mississippi

I. INTRODUCTION II. PRINCIPLES A. Meat Curing Ingredients B. Chemistry of Nitrite C. Curing Adjuncts D. Added Substances III. APPLICATIONS A. Methods of Curing B. Curing Pickle Injection C. Massaging, Tumbling, and Mixing IV. SUMMARY REFERENCES

I. INTRODUCTION The origin of meat curing can be traced back to the third century BC, when Cato recorded careful instructions for the dry curing of hams. From this historical perspective, meat curing may be defined as the addition of salt to meats for the purpose of preservation. This allowed for carryover of meat from times of plenty to times of scarcity. As the centuries passed, production of salted meats flourished. It is probable that during this time humans discovered, quite by accident, the effects on meat color of saltpeter (nitrate) as a result of its presence as an impurity in salt. Therefore, a more modern definition of meat curing could be the addition of salt and nitrate/nitrite to meat that produces the color and flavor we associate with cured meats. The first dry cured meat products were probably inferior by today’s standards. Scientific principles of curing meats were not applied until the latter part of the nineteenth century when the growing meat packing industry began to search for ways to improve quality. With the successful advent of refrigeration, curing of meats evolved from being a means of

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preserving meat to a fantastically complex and diverse segment of the meat industry, which should continue to provide a unique and diversified variety in the food supply. II. PRINCIPLES A. Meat Curing Ingredients 1. Salt Salt is the most basic ingredient in all curing brines and dry mixes. Without the inclusion of salt the curing process would be impossible. This ingredient provides one of the basic flavors and is essential in solubilizing muscle proteins. Granulated or grain salt was commonly called “corn” (from the old Norse korn, meaning “grain”), thus the term corned beef (55). Due to its undesirably harsh flavor, salt is often used in conjunction with various sweeteners to provide a milder flavor. Additionally, salt improves yields and influences textural characteristics (48). Food-grade salt that is of the highest purity should be used in meat curing practices. Impurities such as metals (copper, iron, and chromium) accelerate the development of lipid oxidation and concomitant rancidity in cured meats. Although salt may be of very high purity, it nonetheless contributes to meat lipid oxidation (3). Nitrite and phosphates, which will be discussed later, help retard this effect. The amount of salt used in brines and dry mixtures can vary considerably. Usually the level is self-limiting. Extreme levels of salt make the product too salty, and too little salt can result in inadequate protein extraction. Most curing brines range from 35° to 85° salometer. Brine strength is checked by use of the salometer, a hydrometer that is graduated to show the extent of saturation of brines (55). 2. Sweeteners There are several types of sugars that may be used in formulating curing brines and dry mixtures. Some examples of commonly used sugars are sucrose, dextrose, and corn syrup. These constitute the most commonly used sweeteners by the meat industry today. Sweeteners function to counteract the harshness of salt, and provide roundness and enrichment of flavor. Sucrose also functions as a preservative, but the levels required to provide this effect would probably render most cured meats too sweet. The types and usage levels usually depend on the product. Dextrose is about 70% as intense as sucrose, whereas most corn syrups are about 42% as sweet as sugar. Corn syrup was formerly restricted to 2% solids on a dry weight basis of the product. However, its usage is now unrestricted and, as with other sweeteners, its usage is typically self-limiting. Generally, economics play some role in determining which type of sweetener to use. The degree of browning imparted to the finished product should also be a consideration. The Maillard browning reaction may become too pronounced with some sugars and excessive carmalization and burned flavors are the result. It is common industry practice to purchase liquid dextrose and corn syrup in bulk quantities. This allows the respective sweetener to be pumped directly to brine formulation stations or instantly into mixers. This eliminates the need for valuable warehouse space to store bulk quantities of bagged dry product. 3. Nitrate and Nitrite Through attempts to preserve and extend the available meat supply, early humans used salt as a means of preservation. Nitrate impurities in salt used for this purpose led to the obser-

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vation of reddish pink colors in meat (7). This observation lead to the direct addition of nitrate to preserved meat in order to maintain a uniform color. During the past century, research demonstrated that nitrate added to meats is reduced to nitrite upon degradation by bacteria. Hence, it was deduced that nitrite was responsible for the fixation of cured meat color through its reaction with the water-soluble proteins myoglobin and hemoglobin (51). The Bureau of Animal Industry of the United States Department of Agriculture permitted the official use of nitrate in meat curing in 1908. Additional research resulted in the USDA allowing the direct addition of nitrite to meat products as part of the curing recipe (30). The properties nitrite contributes to cured meat are indeed unique. The original function of nitrite in meat curing was the production of the cured meat color. In addition to color, nitrite works as an antibacterial agent, it retards the progression of oxidative rancidity, and it has a profound influence on the flavor of cured meats. No other known chemical additive can accomplish these things to preserve cured meat products. There are no current usable substitutes for nitrite as a preservative (4). B. Chemistry of Nitrite Sodium nitrite is the salt of a relatively weak acid and a strong base. It is a pale yellow crystalline substance that is readily water-soluble. Aqueous solutions are highly ionized and slightly alkaline. The nitrite ion is considered to be a highly reactive ion and can serve as both a reducing and an oxidizing agent (40). The word nitrite is used generically to signify both the anion NO 2 , and the neutral nitrous acid HNO2, but it is the latter which forms nitrosating compounds (47). Very low nitrous acid concentrations can generate extremely reactive nitrosating species (40). The initial step in the reaction sequence beginning with nitrous acid, is the generation of a nitrosating species of the neutral radical nitrous oxide (NO). The strongest nitrosating species are a form of a positively charged (eletrophillic) nitrogen oxide, either in its simplest form the nitrosonium ion, NO, or as part of a larger molecule. In strong acid two  species are formed, the nitrous acidium (H2NO 2 ) and nitrosonium (NO ) ions.  HNO2  H → H2NO 2 → H2O  NO

In biological systems the Van Slyke reaction is a classic example in which gaseous nitrogen can be liberated from the alpha-hydroxyl acid (47): RCHNH2COOH  HONO → RCHOHCOOH  N2  H2O This reaction is used in the estimation of the amount of free alpha-amino groups in biological systems. In meat curing, some of the added nitrite may disappear as a result of this reaction. When cured meats are placed in highly acidic solutions, nitrite disappears rapidly. The Van Slyke reaction appears to be the major pathway for its depletion (40). Another important form of nitrite is nitric oxide (NO). The nitrogen is reduced by one electron from nitrite-nitrogen and may be formed from dismutation or reducing reactions. Nitric oxide is an electron-pair donor and forms very stable complexes with the transition metals. The coordinate-covalent complex of nitric oxide with the heme pigments of meat (nitrosylmyoglobin, nitrosylhemoglobin, and dinitrosylhemochrome) form the pink and red color of cured meats (18). Nitric oxide in the presence of air is rapidly oxidized. This accounts, at least partially, for the instability of cured meat color when exposed to air: 2NO  O2 → 2NO2

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This reaction may also explain the antioxidant properties of nitrite because it is very fast. Furthermore, nitric oxide is a radical chain terminator (10), which would explain why it is so effective in retarding the development of lipid oxidation. Additionally, research has proposed three cooperative mechanisms for the antioxidant properties nitrite imparts to cured meats. These include (a) the formation of nitrosylmyoglobin (MbNO), which has antioxidant properties per se; (b) on heating, MbNO forms a stable complex, nitrosylhemochrome, that blocks the catalytic activity of heme iron and also prevents release of heme iron as nonheme iron, which is a very effective catalyst; and (c) nitrite appears to chelate non-heme iron, and possibly copper and cobalt, forming a stable complex, thus inhibiting catalytic activity (37). These findings are in agreement with Kanner et al. (27), who reported that nitric oxide, liganded to ferrous complexes, acts to prevent the prooxidative reaction of these complexes with hydrogen peroxide to ultimately produce “warmed-over flavor.” 1. Cured Meat Color The color of cured meats is generally dependent on three factors: (a) the concentration of myoglobin in the muscle tissues, (b) the degree of conversion to the nitrosyl pigment, and (c) the state of the muscle proteins. Unlike fresh meats, cured meats retain their redness upon heating. It is inferred from this that the curing process changes the chemical nature of the chief muscle pigment myoglobin. Classical early work conducted by Haldane (21) and Hoaglund (24) suggested this difference in reaction of nitric oxide derivatives of the meat pigment toward heat is due to the formation of nitric oxide derivatives of the meat pigment as a result of the chemical interaction of the pigment with nitrite, either added directly in the curing brine or mixture, or obtained through microbial reduction of nitrate to nitrite (56). After the work of Haldane (21) and Hoaglund (24), the existence of a red pigment different from the blood pigment hemoglobin was demonstrated in muscle tissue by Kennedy and Whipple (29). Haldane and Hoaglund assumed the pigment of meat to be identical with the blood pigment. Determination of amounts of muscle hemoglobin or myohemoglobin, by Shenk, et al. (49), occurring in the muscle tissue of different animals indicated that these pigments make up nearly all of the muscle tissue pigment. The slaughter process includes a bleeding period, which removes substantial portions of blood. Thus, it was rationalized that the pigments of cured meats are essentially nitric oxide myoglobin and nitrosyl-hemochrome. Depending upon the type of meat product involved, there are several different methods used to cure meats. Usually they include: 1.

Brine cure. The meat is soaked in a solution containing salt and cure plus adjuncts. The period of cure is dictated by the size of the meat pieces, with adequate time being allocated to allow the complete diffusion of cure into the center of the meat. After this process, the meat pieces are usually cooked and smoked. 2. Pump cure. In this type of cure, multiple-needle pumping machines usually inject the brine solution throughout the piece of meat, thereby reducing the time required to fully distribute the curing solution into the meat pieces (2 pounds nitrite to 100 gallons pickle at 10% pump level “ingoing”). 3. Dry salt curing. This is accomplished by rubbing the meat with a mixture of salt and cure and allowing sufficient time for the mixture to penetrate the product. Again, this is usually dependent on the size of the meat pieces. Smoking or other processing usually follows this type of curing.

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495

Cure for comminuted meats. Coarse ground meat products that are to be cured involve the incorporation of the curing agents by direct mixing. Sodium nitrite may be included in coarse ground meats at levels up to 156 parts per million which equals one-fourth ounce per 100 lb of meat.

All of these processes eventually lead to the formation of nitric oxide myoglobin. The general reaction is: Myoglobin (purplish-red)  NO → Nitrosomyoglobin (dark red)  heat → Nitrosohemachrome (light pink, typical “cured meat color”). All of these reactions are nonreversible. Again, the source of the NO (nitric oxide) is nitrite. Nitrosomyoglobin is then the first pigment of cured meat. Upon the application of heat, it is converted to nitrosohemochrome, which is a denatured protein, and exhibits the characteristic pinkish-red color of cured meats. 2. Bacterial Inhibition Nitrite addition to meat is responsible for the traditional and distinct color and flavor of products such as ham, bacon, bologna, and frankfurters (50). An additional major benefit is the retardation of Clostridium botulinum growth and toxin production. Botulism is a rare but serious neuroparalytic disease affecting humans and animals. It is caused by ingestion of a heat-labile protein neurotoxin produced by the vegetative cells of the microorganism C. botulinum (53). Botulism may also be caused by wound infection. The disease has been recognized as a foodborne illness for over 1000 years (11). The incidence of the disease is worldwide. It can result from the consumption of a variety of foods, including canned meats, vegetables, fruits, and fish. Botulism was first reported in Europe as a disease caused by the consumption of sausage products. The word “botulism” was derived from the Latin botulus, meaning sausage (50). Current research efforts have suggested possible inhibitory mechanism(s) by which nitrite inhibits C. botulinum. These include (a) formation of an inhibitory substance from nitrite and other meat components, (b) nitrite or intermediates acting as an oxidant or reductant on intracellular enzymes or nucleic acids, (c) restriction of iron or other metals essential to C. botulinum by nitrite, thereby interfering with the organism’s metabolism or biological repair system, and (d) reaction of nitrite with cell membranes to limit metabolic exchanges or substrate transport. It is quite possible that more than one mechanism exists in a complex biological system such as meat (50). Metal-sequestering agents such as EDTA have been demonstrated to enhance the inhibitory activity of nitrite whereas excess iron caused a decreased inhibition (54). Research has suggested that nitrite, probably via nitric oxide, reacts with iron of ferredoxin in germinated cells (62). Inhibition of Clostridium sporogenes by reaction of nitric oxide with the non-heme iron of pyruvate:ferredoxin has been reported (41). Another theory is the conversion of extracellular iron, which is essential to C. botulinum, to an unavailable form after reaction with nitric oxide (54). 3. Flavor Nitrite commonly used in the production of cured meats, is a major contributor toward development of the characteristic cured meat flavor (35). This function of nitrite in cured meats has not been linked to any specific flavor components (46). Sodium nitrite alone is a very potent flavor enhancer in cured meat (58). Salt is a catalyst for increased fat oxidation and rancidity (42). Claims have been made that salt is the major factor responsible for cured

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meat flavor, rather than nitrite or the absence of oxidative rancidity. It should be noted, however, that salt pork and bacon have quite different flavors, suggesting a positive role of nitrite in flavor formation. Bailey and Swain (4) reviewed a very limited literature base and reported on the interaction of nitrite and meat constituents that influence flavor. Most studies reported on the effect of nitrite on meat flavors are either of processing and sensory evaluation, or of chemical analyses of the reactions between nitrite and meat components (50). Wasserman (57) presented a review of these studies, but the lack of sufficient information prevented a complete discussion of the role of nitrite on cured flavor development. Cho and Bratzler (8) and Wasserman and Talley (58) reported results indicating that nitrite-containing products (longissimus dorsi muscle and frankfurters, respectively) possessed a more intense cured flavor. Improved color and sensory properties were reported by Kemp et al. (28) in packages of sliced dry-cured hams containing nitrate and/or nitrite. Hustad et al. (26) reported that as little as 25 to 50 g of sodium nitrite per gram was adequate to give the typical color and flavor of cured meats in wieners. 4. Lipid Oxidation Research on rancidity retardation by nitrite has been presented by Cross and Ziegler (9), Watts (59), and Herring (23). The effect of nitrite on retarding development of rancidity is probably due to the same reaction that is responsible for color development. The heme compounds of muscle contain iron ions that are quite active as catalysts of lipid oxidation. When nitrite reacts to form cured pigments, iron is retained in the heme, usually in the reduced (Fe2) form, making it unavailable as a catalyst for lipid oxidation. This reaction therefore probably accounts for the prevention of “warmed-over” flavor in cured meats (40). 5. Nitrite Intake and Nitrosamines Concern over nitrate and nitrite intake and its effect on human health dates back to the beginning of the twentieth century. Richardson (43) stated that most of the nitrite ingested was from vegetables. Nitrite is considered toxic at high concentrations, and it is also implicated in carcinogenic nitrosamine formation. Used under the existing regulations, nitrite is not considered a health hazard. There are reported rare toxic episodes, mostly due to accidental overuse. The lethal nitrite dose is 300 mg/kg of body weight (52). Nitrite can react as a vasodilator and hypotensive agent (44). It can reduce the storage of vitamin A in the liver and it can interfere with thyroid function (13). It is firmly established that nitrite can oxidize hemoglobin to methemoglobin, lowering the blood’s ability to transport oxygen. This anomaly is called methemoglobinemia. It can be fatal and most commonly occurs in infants. The FDA-USDA announced on February 5, 1972, that nitrosopyrrolidine, a nitrosamine, was formed in retail-purchased and fried bacon. The fried product contained levels of this nitrosamine ranging from 30 to 106 parts per billion (ppb). Conversely, raw bacon was found to be free of nitrosopyrrolidine (23). Many different nitrosamines have been found to be carcinogenic (32). Nitrosamines can be produced by a combination reaction of nitrite or nitrous acid and secondary or tertiary amines (46). Cured meats contain both nitrite and amines, thus there is potential for nitrosamine formation under appropriate reaction conditions. Fried bacon is the cured meat product in which nitrosamines (mostly nitrosopyrrolidine) have been found. Bacon is high in the amino acids proline and hydroxyproline, and

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other nitrosopyrrolidine precursors. The most likely pathway for nitrosopyrrolidine formation is that proline is first nitrosated and then decarboxylated (16). In other cured meat products, nitrosamines have not been found or are only occasionally encountered. Researchers have found nitrosopyrrolidine present in bacon but not in other cured meat products. Hustad et al. (26) tested wiener samples for 14 nitrosamines at the 10 ppb level and no positive results were obtained. Under controlled laboratory and normal heating conditions, no nitrosamines were formed in frankfurters formulated with nitrite levels up to 750 g/g. A nitrite concentration of 1500 g/g was necessary for dimethylnitrosamine formation at levels of 10 to 11 ppb. Research concluded that frankfurters conforming to U.S. federal regulations contained insufficient nitrite for nitrosamine formation (15). Based upon the occurrence of nitrosamines, cured meat products can generally be divided into two groups. Bacon (mandatory ingoing nitrite at 120 parts per million), and ham, wieners, bologna, and similar products, which are less involved with nitrosamines. In bacon the nitrosopyrrolidine concentration has been found to be higher in the fat cookout than in the cooked edible portion (14,16). Nitrosamine formation in bacon is affected by ingoing nitrite concentration, cooking conditions, and post-processing age of bacon. Several substances (ascorbate, ascorbyl palmitate, cysteine, glutathione, hydroquinone, alpha-tocopherol and tertiary butyhydroquinone) have been shown to decrease nitrosamine formation in cured meat products (20,36). Cured meats contribute little to the total human intake of nitrate and nitrite. Rubin (45) reported some human nitrite intake comes from cured meats, and the rest comes from saliva, where it is formed from nitrate by the microflora of the mouth. Some common leafy vegetables such as spinach and lettuce and some root vegetables such as beets and radish are major nitrate sources. Additionally, some drinking waters are high in nitrate (61). Furthermore, White (61) indicated that 10% of the ingested nitrate and 21% of the nitrite comes from cured meats whereas vegetables contribute 86% of the nitrate and saliva 77% of the nitrite. Therefore, Friedman (19) noted, “Any possible risk to human health which could conceivably arise from the use of nitrite in food processing must be balanced against its value as an essential ingredient in the production of a host of well known traditional food products.” C. Curing Adjuncts In addition to the commonly accepted curing agents, the meat processor in the curing process commonly includes several other adjuncts (ascorbates and erythorbates, phosphates, starches, and hydrocolloids). Problems over the decades associated with color and yields have been the primary motivation for inclusion of these ingredients. 1. Ascorbates and Erythorbates Ascorbic acid C6H8O6, termed vitamin C, a water-soluble vitamin, is often used as a curing accelerator. That is, it helps speed the conversion of nitrite to nitric oxide to hasten the development of cured color in rapidly processed cured meats. One part of ascorbic acid is equivalent to one part erythorbic acid. Bauernfeind et al. (5) reported these benefits of using ascorbic acid in cured processed meats: (a) curing time can be substantially reduced, (b) a more uniform color will result throughout the product, and (c) better color and flavor can be maintained during storage, distribution, and display. Similar results were reported by Watts and Lehmann (60) by demonstrating that the addition of ascorbic acid with nitrite

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causes rapid development of the cured meat pigment. Additionally, salt, low acidity levels, high temperature, and freezing accelerated color fixation (25). Mirvish et al. (36) reported another possible benefit of using ascorbate: the blocking effect the compound appeared to have on the formation of N-nitroso compounds. However, the blocking effect depended on the compound nitrosated and the experimental conditions. The USDA, for curing purposes, approves the following forms of ascorbates: ascorbic acid, sodium ascorbate, erythorbic acid, and sodium erythorbate. Sodium erythorbate is the most widely used form of ascorbate, due to its lower cost. Federal regulations permit the addition of 550 parts per million (ppm) of ascorbic acid or erythorbic acid; or the equivalent molar level of sodium ascorbate or sodium erythorbate in each 100 gallons of curing pickle. For pumping pickle, this is 75 or 87.5 ounces per 100 gallons when the pickle will be used at 10% of green weight. If the pickle is to be used in excess of 10%, appropriate reductions in the quantity of such substances is required. 2. Phosphates The use of alkaline phosphates in meat curing is widely employed by the meat industry. Although claims have been made with regard to color retention, the various phosphates used by the meat industry have been used primarily to decrease shrinkage and the degree of purge in processed meats. Phosphates appear to present a mode of action that is twofold: (a) elevating the pH of the meat and (b) solubilization of muscle proteins. Polyphosphates contribute to the ionic strength of meat fluids, thus increasing protein hydration without increasing the apparent saltiness of the product (2). Bendall (6) postulated that pyrophosphate is effective in splitting actomyosin into its component parts, thus aiding extraction of more salt-soluble protein. At the isoelectric point of meat, pH 5 to 5.5, fluid retention is at a minimum. Thus any additive that significantly elevates the meat pH should increase the fluid retention of isoelectric meat. Early research suggested that large increases in pH favor fluid retention in meat (33). However, the addition of 0.2 to 0.5% sodium tripolyphosphate to meat only increases the pH by about 0.1 to 0.3 units. It is doubtful that by itself, such small increases in pH account for the pronounced effect of certain phosphates on the fluid retention of processed meats. Bendall (6) pointed to differences between the volume of ground meat treated with polyphosphates alone and meat treated with polyphosphates and salt. He suggested the meat volume was a function of total ionic strength since the pH of both meat samples was essentially the same. Mahon (33) presented research that explained the important synergistic role sodium chloride and sodium tripolyphosphate collectively play in water binding in cured meat systems. His conclusions were: (a) salt concentration and not pH adjustment is the key to maximum cured meat volume, (b) low salt concentrations of the order of 0.5% are detrimental to cured meat volume, (c) salt and tripolyphosphate act synergistically to increase cured meat volume, (d) if used alone, high volumes of tripolyphosphate are required to induce maximum cured meat volume (well above the maximum limit of 0.5%), and (e) as salt concentration decreases, tripolyphosphate concentration must increase accordingly if cured meat volume is to be maintained. Phosphates are essentially the salt forms of phosphoric acid. There are two recognized classes of phosphates: (a) the ortho (simple) phosphates containing a single phosphate anion and (b) the poly (condensed) phosphates containing two or more phosphate anions.

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All phosphates perform three basic functions in foods: (a) they provide some degree of buffering capacity, (b) they sequester metal ions (an important antioxidant effect), and (c) they act as polyanions to increase the ionic strength of solutions (34). These chemical functions of phosphate perform important functions in cured meat systems. Their beneficial functions include increased yields, retardation in oxidative rancidity, and color stabilization. The following phosphates are approved for use in curing pickles: disodium phosphate, monosodium phosphate, sodium metaphosphate, sodium polyphosphate glassy, sodium tripolyphosphate, sodium pyrophosphate, sodium acid pyrophosphate, sodium hexametaphosphate, dipotassium phosphate, monopotassium phosphate, potassium tripolyphosphate and potassium pyrophosphate. Tripolyphosphate and its blends with hexametaphosphate are the most commonly used phosphates for cured meats because, in most cases, these exhibit the most beneficial combination of properties. The use of certain approved phosphates presents specific challenges that the meat processor must be aware of. Sodium tripolyphosphate, perhaps the most widely used phosphate in meat curing, must be the first component added to the curing pickle. It is practically insoluble once salt has been added. Phosphates are generally corrosive in nature, thus mandating the use of stainless steel or approved plastic equipment in handing brines and pumped meat. Another situation sometimes encountered in using phosphates has been the appearance of white crystals on the surface of cured products after processing. Commonly called “snow” or “whiskers,” this condition most often appears on product that has been allowed to dry out in holding coolers. These crystals have been identified as disodium phosphate and are believed to be caused by hydrolysis of the polyphosphates by the naturally occurring phosphatases in meat. This problem can be minimized by reducing the level of phosphate in the cure (maximum level in the finished product is 0.5%), and providing proper protection of the product during cooler storage. Recognizing the fact that meat does contain phosphatases has brought some debate over the merits of using phosphates in pre-blends. Research has shown the addition of phosphate to pre-blends increased soluble protein extraction by 15% to 20% and cooked stability (retained water) by 25% to 30%. There was no loss of protein functionality with time, even though phosphate hydrolysis was occurring. Therefore, phosphates constitute an important part of meat preblends (12). 3. Starches and Hydrocolloids Starch is very abundant in nature, serving as a storage carbohydrate in many plants. Therefore, a wide variety of carbohydrates are available to meat processors. The challenge to the processor is to figure out what type of product is to be manufactured, what quality characteristics it is to possess, and what very specific benefit a particular starch or combination of starches should contribute to the product. Starch is a multifunctional ingredient manifesting properties that can be applied to numerous food products. Starches contribute texture enhancement, binding properties (usually water), and improved mouth feel to meat products. All of the characteristics are dependent on the origin of the starch, and its performance capabilities. In its native form, starch is present as discrete granules that are insoluble in cold water due to hydrogen bonding existing between individual starch molecules, or directly through water bridges. Chemically, starch is composed of two glucose polymers, amylose and amylopectin. The relative proportions of these two polymers will differ depending on the source of the starch. Native starches exhibit optical polarity and display a “Maltese

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cross” diffraction pattern when viewed under plane polarized light. When starch granules are heated in the presence of sufficient water, the cross pattern fades as starch granule swelling is initiated. This phenomenon is termed gelatinization. The initial swelling takes place in the amorphous (noncrystalline) regions of the granule, disrupting hydrogen bonds and then hydrating them. The viscosity proceeds to a maximum, which corresponds to the largest hydrated volume of the granules, which is then its peak viscosity. As heating, agitation/shear continue, the swollen granules rupture and collapse, thereby giving a viscous, colloidal dispersion of hydrated starch aggregates and dissolved molecules. The most common starches used by meat processors are extracted either from cereals (corn, wheat, and rice), or roots and tubers (potato, konjac, and tapioca). Each type of starch has inherent characteristics that can be utilized to produce specific textural and sensory properties in a cured meat system. The contrast in physical properties of starch pastes can be attributed to the chemical composition and physical differences that exist between the various starch sources. Differences in raw material sources play a vital role on the flavor profiles and swelling characteristics of starches. Almost without exception, meat processors must use a starch that is absolutely bland in flavor but is also capable of binding water and holding it throughout the product’s commercial distribution. Again, the specific role the starch plays in a food product will dictate what type of starch the processor chooses. Starch manufacturers are able to offer starches that will be stable under various conditions of pH, shear, heat, and freeze-thaw cycles. These characteristics are imparted to starches by three important modifications: (a) pregelationization, (b) crosslinking, and (c) stabilization. Pregelationization involves cooking/drying the starch in heated drums or spray dryers to make it cold water swellable. This eliminates the necessity of the processor to precook the starch to achieve viscosity. Cross-linking is a method by which the starch granules are strengthened by the introduction of chemical bridges that link the hydroxyl groups on two different starch polymer molecules. This bridging of starch molecules controls the swelling of the starch and imparts stability to the starch to undergo further processing in high or low pH mediums and high shear or temperature processes. Additionally, it has the effect of increasing the gelationization temperature. Stabilization decreases the extent of gelationization of starch and thus increases its water holding capacity. Chemical groups such as acetyl or hydroxypropyl groups increase the polarity of the starch, making it ionic, or by means of steric hindrance cause repulsion of the polymer chains. This opens up the granules to improve clarity and lowers the gelation temperature and increases the water holding capacity. The tendency to retrograde or breakdown is reduced with a concomitant improvement of freeze-thaw stability (17). Carrageenan is a hydrocolloid produced from certain types of red seaweed. This product is particularly useful in its ability to bind and hold water. There are three types of carrageenan and they are classified by their gelling characteristics. Kappa and iota carrageenans are the gelling types, and they are most often used in meat processing. Lambda is a nongelling carrageenan and is not commonly used by meat processors. Carrageenans are mostly used at levels below 1% and need to be heated to become fully functional. Kappa will form a firm gel whereas iota will produce a more elastic gel (31). Konjac flour is refined from the root of the elephant yam (Amorphophallus konjac). It has tremendous water-binding capabilities and is used in relatively low levels in processed meats. Konjac is unique in its ability to form a heat-stable gel when treated with an

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alkali to remove acetyl side groups (31). Konjac also provides enhanced textural improvement in low fat/fat-free sausage products. D. Added Substances Federal regulations pertaining to pickle-cured pork products, including hams, shoulders, picnics, butts, and loins, mandate they comply with the minimum meat protein fat free (PFF) percentage requirements. These standards and labeling requirements for cured pork products are based on the minimum percentage of meat protein on a fat-free basis present in the finished pork product. The percentage is calculated as follows: (% meat protein/100  % fat  100). For example, if the meat protein is 16.8 and the fat is 12.2, the resulting PFF is 19.13. Labels are based on the style and type of product (e.g., bone-in ham), and the minimum PFF percentage determines just what the product can be labeled. For instance, cooked bone-in ham must have a minimum protein fat-free percentage of 20.5%; cooked ham with natural juices, 18.5%; cooked ham, water added, 17.0%; and cooked ham and water product—”X% of weight is added ingredients,”  17% PFF. Shoulders, picnics, and butts are allowed one-half percentage less in PFF values for each category than is required for ham and loin items. This program allows for a broad range of cured pork products to be marketed if they meet the proper standards and are accurately labeled (40). III. APPLICATIONS A. Methods of Curing The meat industry employs several methods of meat curing. However, they are all modifications of two basic procedures: dry salt curing and pickle curing. Dry salt curing is no doubt the oldest method. Evolution of the curing art eventually led up to the more modern method of pickle curing. 1. Dry Salt Curing Dry salt curing is a method dating back to prehistoric times and was the first curing method practiced by humans. The process uses salt alone, or sometimes in conjunction with nitrite or nitrate. The moisture is drawn out of the meat by the curing agents and drains off, leaving the meats drier and harder and leaving the flavor brackish. The product is usually laid skin side down, and all areas of exposed lean are plastered with the curing mix (22). This process is commonly used for fatty cuts such as jowls and fat backs. 2. Dry “Country Style” Curing Curing ingredients employed by dry curing generally are salt, sugar, nitrate, and nitrite. The common practice is to rub the mixture into the surface of the product (usually hams and bellies) and place the products on shelves in a curing room held at about 36° to 38°F. A general rule of thumb is 11⁄2 to 2 days per pound of ham; bellies up to 2 inches thick will cure in 14 days. It is also prudent to remove and “overhaul” or recoat the products with curing mixture half way through this process. Dry curing may also be used in conjunction with brine injection for some products. The product is usually pumped with about 10% of saturated brine, and the balance of the curing ingredients are applied as a dry rub. According to Robert W. Rogers (personal communication, 1999), if dry-cured products are to be smoked, the smokehouse temperature should not exceed 100° F in order to prevent nitrate burn (green spots) in the products. Also, if these types of products are to be aged, care

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should be taken to prevent damage to the products from insects such as cheese skippers, mites, red-legged ham beetles, and larder beetles. 3. Brine Soaking Brine soaking probably followed dry curing and was often used commercially for several years. Meat pieces are placed in curing brine, and the cure is allowed to penetrate the entire portion. This process is relatively slow, and spoilage may develop before the process is completed. Some items such as corned beef briskets and tongues may still be processed this way; however, the practice is becoming less frequent. B. Curing Pickle Injection The industry standard today is the injection of curing pickle directly into meat pieces. Internal injection of curing ingredients enhances efficiency and promotes a more rapid and uniform distribution of the cure throughout the product. There are three basic methods used to accomplish pickle injection: (a) artery pumping, (b) stitch pumping, and (c) multiple needle injection pumping. Smaller establishments may use artery and stitch pumping because these techniques are slow and labor intensive. Larger establishments processing thousands of pounds of product daily use multiple needle injection. 1. Artery Pumping This technique was believed to have been developed by a New Zealand undertaker (39). This method introduces curing pickle through the arterial system of the product. Hams are usually the only products cured in this manner. The single needle is inserted into the femoral artery, and the cure is pumped into the ham. Great care must be taken during fabrication to preserve intact the femoral artery. The process does not lend itself well to highspeed, high-volume production and is therefore seldom used today. 2. Stitch Pumping Stitch pumping utilizes a single needle that has several openings. The operator inserts the needle into the meat piece in many different locations to deliver the appropriate amount of pickle. An experienced operator is required to evenly distribute the curing solution. After pumping, an equilibration period is often required to allow the cure to evenly diffuse throughout the product. 3. Multiple Needle Injection Curing This method is widely used by the meat industry today. Due to its speed and effective distribution of curing agents throughout the tissues, it enables processors to manufacture large quantities of product daily (Fig. 1). These injectors may be configured to pump either bone-in or boneless product of various sizes and shapes. Pumping speed and the volume of pickle injected may be adjusted as needed. Pump head pressure must always be closely monitored. This should prevent excessive tissue disruption and formation of pickle pockets in the tissues (Fig. 2). C. Massaging, Tumbling, and Mixing After the curing pickle has been introduced into the meat pieces, some type of mechanical energy is usually applied. Massaging, tumbling, or mixing the meat pieces for various lengths of time, usually under some degree of vacuum, may carry out this process. These

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Figure 1 Multiple-needle curing pickle injector. (Photograph courtesy of Koch Supplies Inc.) physical processes are employed to extract salt-soluble protein and improve and accelerate the distribution of cure throughout the product. 1. Massaging Massaging involves frictional energy resulting from meat pieces rubbing together (1). Meat massagers are vats that contain a mechanism for the slow stirring of meat pieces.

Figure 2 Ham injection. (Photograph courtesy of Nu-Meat Technology, Inc.)

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The stirring arms or paddles made are set to various configurations. The agitation time can vary depending on the product but generally ranges between 3 and 6 hours. The massaging process is a gentler form of mechanical energy input and works well with softertextured product. This is especially important if the product is to retain a whole-muscle appearance. 2. Tumbling Conversely, tumbling is a more severe type of physical treatment. Tumbling involves the use of impact energy resulting from meat pieces falling and striking baffles or paddles contained in a rotating drum. Tumbling is usually carried out under vacuum to offset the potential problem of incorporating air into the protein exudate. Most products are loaded into tumblers immediately after injection with curing pickle and tumbled from 3 to 6 hours. Longer cycles have been investigated, but due to severe limitations of time in commercial environments, this time frame works best for maximum efficiency and product quality. As with massaging, tumbling is carried out to extract protein for binding, to enhance tenderness and juiciness, and to increase product yields (38) (Fig. 3). 3. Mixing Mixers generally have some type of paddles or ribbons that rotate around a metal shaft. They may or may not be equipped to hold vacuum, and they impart rather vigorous mechanical energy to the product. Short mixing times are usually the rule because longer mixes tend to both tear up whole-muscle product and smear coarse-ground product. Mixers are most often used in the manufacture of sausage products, but some whole-muscle products are processed in this manner (Fig. 4).

Figure 3 Meat tumbler. (Photograph courtesy of Nu-Meat Technology, Inc.)

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Figure 4 Meat mixers. (Photograph courtesy of Koch Supplies Inc.) IV. SUMMARY Successful meat curing is a complex art of scientific principles and good manufacturing practices. The constant emergence of new technologies, ingredients, and equipment has enhanced the meat processor’s ability to produce a wide variety of products. Consumer demand for these products will continue to be driven by quality and consistency. Proper and consistent application of sound manufacturing techniques is of great importance in assuring these truly unique products remain an integral part of the food supply. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

Addis, P. B., and E. S. Schanus. 1979. Massaging and tumbling in the manufacture of meat products. Food Technol 33:36–40. American Meat Institute Foundation. 1960. The Science of Meat and Meat Products. W. H. Freeman and Company. San Francisco, CA. Andersen, H. J., and L. H. Skibsted. 1991. Oxidative stability of frozen pork patties. Effect of light and added salt. J Food Sci 56:1182–1184. Bailey, M. E., and J. W. Swain. 1973. Influence of nitrite on meat flavor. Proc Meat Ind Res Conf, Amer Meat Inst Found, Chicago, IL. pp. 29–45. Bauernfeind, J. C., E. G. Smith, and G. K. Parman. 1954. Ascorbic acid betters color, flavor of meats. Food Eng 125:80–82. Bendall, J. R. 1954. The swelling effect on polyphosphates on lean meat. J Sci Food Agric 5:468–475. Binkerd, E. F., and O. E. Kolari. 1975. The history and use of nitrate and nitrite in the curing of meat. Food Cosmet Toxicol 13:655–661. Cho, I. C., and L. J. Bratzler. 1970. Effect of sodium nitrite on flavor of cured pork. J Food Sci 35:668–670. Cross, C. K., and P. Ziegler. 1965. Comparison of the volatile fractions from cured and uncured meat. J Food Sci 30:610–614.

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McDougall, D. B., D. S. Mottram, and D. N. Rhodes. 1975. Contribution of nitrite and nitrate to the color and flavor of cured meats. J Sci Food Agric 26:1743–1754. Mirvish, S. S., L. Wallcave, M. Eagen, and P. Shubik. 1972. Ascorbate-nitrite reaction: Possible means of blocking the formation of carcinogenic N-nitroso compounds. Science 177:65–67. Morrissey, P. A., and J. Z. Tichivangana. 1985. The antioxidant activities of nitrite and nitrosylmyoglobin in cooked meats. Meat Sci 14:175–190. Ockerman, H. W., and C. S. Organisciak. 1978. Diffusion of curing brine in tumbled and nontumbled procine tissue. J Food Protect 41:178–181. Pearson, A. M., and F. W. Tauber. 1984. Processed Meats. 2nd ed. Van Nostrand Reinhold Company, New York, NY. Price, J. F., and B. S. Schweigert. 1987. The Science of Meat and Meat Products. 3rd ed. Food and Nutrition Press. Westport, CN. Reddy, D., J. R. Lancaster Jr., D. P. Cornforth. 1983. Nitrite inhibition of Clostridium botulinum: electron spin resonance detection of iron-nitric oxide complexes. Science 221:769–770. Rhee, K. S., R. N. Terrell, M. Quintanilla, and C. Vanderzant. 1983. Effect of addition of chloride salts on rancidity of ground pork inoculated with Moraella or a Lactobacillus species. J Food Sci 48:302–303. Richardson, W. D. 1907. The occurrance of nitrates in vegetable foods, in cured meats and elsewhere. J Amer Chem Soc 29:1757–1767. Rubin, A. A., L. Zitowitz, and L. Hausker. 1963. Acute circulatory effects of diazoxide and sodium nitrite. J Pharmacol Exp Ther 140:46. Rubin, L. J. 1977. Nitrites and nitrosoamines in perspective. J Can Inst Food Sci Technol 10:A11–A13.23–28. Sebranek, J. G., and R. G. Cassens. 1973. Nitrosamines: A review. J Milk Food Technol. 36:76–91. Sebranek, J. G., and J. B. Fox, Jr. 1985. A review of nitrite and chloride chemistry: interactions and implications for cured meats. J Sci Food Agric 36:1169–1182. Shackelford, S. D. 1989. Effects of blade tenderization, vacuum massage time and salt levels on chemical, textural and sensory characteristics of precooked chuck roasts. J Food Sci 54:843–845, 905. Shenk, J. H., J. L. Hall, and H. H. King. 1934. Spectrophotometric characteristics of hemoglobins. I. Beef blood and muscle hemoglobins. J Biol Chem 105:741–752. Sofos, J. N., F. F. Busta, and C. E. Allen. 1979. Botulism control by nitrite and sorbate in cured meats: A review. J Food Protect 42:739–770. Sofos, J. N. 1981. Nitrite, sorbate and pH interactions in cured meat products. Proc Recip Meat Conf. of American Meat Science Association 34:104–120. Tannenbaum, S. R. 1976. Relative risk of nitrate and nitrite ingestion. Proc Meat Ind Res Conf, Amer Meat Inst Found, Chicago, IL. pp. 25–33. Tompkin, R. B., and L. N. Christiansen. 1976. Clostridium botulinum pp. 156–169. In M. P. Defigueirdeo, and D. F. Splitterstoesser (eds.) Food microbiology: Public health and spoilage aspects. The AVI Publishing Company, Inc. Westport, CN. Tompkin, R. B. 1978. The role and mechanism of the inhibition of C. botulinum by nitrite—is a replacement available? Proc Recip Meat Conf of American Meat Science Association. pp. 135–147. Townsend, W. E., and D. G. Olson. 1987. Cured meats and cured meat products processing. pp. 431–456. In J. F. Price and B. S. Schweigert (eds), The Science of Meat and Meat Products. 3rd Ed. Food and Nutrition Press, Inc. Westport, CN.23–29. Urbain, W. B., and L. B. Jensen. 1940. The heme pigments of cured meats—Preparation of nitric oxide hemoglobin and stability of the compound. Food Res 5:593. Wasserman, A. E. 1974. Nitrite and the flavor of cured meat. pp. 173–178. In B. Krol and B. J. Tinbergen (eds.) Proc. Intern Symp on Nitrite in Meat Products. Wageningen, The Netherlands.

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508 58. 59. 60. 61. 62.

Martin Wasserman, A. E., and F. Talley. 1972. The effect of sodium nitrite on the flavor of frankfurters. J Food Sci 37:536–538. Watts, B. M. 1954. Oxidative rancidity and discoloration in meats. Food Res 5:1–52. Watts, B. M., and B. T. Lehmann. 1952. The effect of ascorbic acid on the oxidation of hemoglobin and the formation of nitric oxide hemoglobin. Food Res 17:100–108. White, J. W., Jr. 1975. Relative significance of dietary sources of nitrate and nitrite. J Agric Food Chem 23:886–891 (Correction 24:202). Woods, L. F. J., J. M. Wood, and P. A. Gibbs. 1981. The involvement of nitric oxide in the inhibition of the phosphoroclastic system in Clostridium sporogenes by sodium nitrite. J Gen Microbiol 125:399–406.

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21 Meat Smoking Technology DOUGLAS F. ELLIS Bryan Foods, Inc., West Point, Mississippi

I. INTRODUCTION II. IMPORTANT WOOD COMPONENTS AND COMPOUNDS A. Wood Types B. Major Constituents of Wood III. SMOKE EFFECTS A. Color and Flavor B. Antioxidant Properties C. Antimicrobial Properties IV. SMOKE GENERATION A. Friction B. Smoldering C. Steam D. Phases E. Liquid Smoke V. SMOKEHOUSE FUNCTION AND DESIGN A. Size and Types B. Hot versus Cold Smoking C. Airflow D. Humidity E. Balance of Smokehouse Functions VI. SUMMARY REFERENCES

I. INTRODUCTION Smoking of meat and other foods goes back to prehistoric times. Early hunters found that meat lasted longer and acquired a preferred flavor if smoke vapors were allowed to penetrate the surface. This practice probably began by hunters hanging the meat from the daily hunt near the smoke vent of a tent or from the roof of a cave dwelling. Even though the pri-

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mary purpose of the fire was for warmth and protection, this secondary effect on meat was very desirable. Down through time the smoking process has matured. People have found that smoking gives a drying effect to the meat, imparts a desirable taste, gives a pleasant odor, brings out the color of the meat, and helps keep the meat from going rancid and from spoiling (16,22). Smoking of foods as we know it today is the addition of either traditional vaporous or liquid smoke to meat or other food products. The development of liquid smoke has given the meat industry the option of presenting smoke to a product from an internal direction as well as external. At the current time, most food products that are given a smoke flavor have the smoke applied from an external direction, but with the convenience of liquid smoke, more and more foods contain internal smoke flavorings. Smoke is an additive to the food processing system as is salt, sugar, water, and spices. Just as the other additives have a special purpose in the system, so does smoking or the addition of smoke to a product. Early smoking practices were done primarily for reasons of preservation of the meat product, with flavor and color enhancement being very positive secondary factors. As the technology in the areas of canning, freezing, and refrigeration have been perfected, the dependency on smoking for food preservation has become less. Due to the advances in the ways we keep our food safe, the role of smoking has switched its primary importance from preservation to one of flavor and color. II. IMPORTANT WOOD COMPONENTS AND COMPOUNDS A. Wood Types The woods used for smoking meats are in the group referred to as hardwoods. Oak, hickory, pecan, cherry, and maple are a few of the more frequently used hardwoods. As civilization developed its smoking processes, it was determined that the hard wood varieties produced better smoke than the soft wood varieties. This can be attributed to the higher level of resin acids in softwood as compared to hard wood. Although the smoke from pyrolysis of softwood produces good color, the resin acids in the smoke produce an unacceptable flavor in meat and other food products (21). B. Major Constituents of Wood The three major constituents of wood are cellulose, hemicellulose, and lignin, in a respective ratio of 2:1:1. The pyrolysis of the hemicellulose portion takes place at 200°C to 260°C and produces alphatic carboxylic acids and carbonyls, of which the carbonyls are very important in smoke color. Love and Bratzler (17) identified 21 different carbonyls from the pyrolysis of hemicellulose. Some of the major cabonyls are glycolic aldehyde, methylglyoxal, formaldehyde, and acetol. The burning or pyrloysis of the cellulose portion takes place at 260°C to 310°C and produces primarily organic acids and carbonyl compounds. Ham and Saffle (12) found the major acids to be acetic, proprionic, iso-caproic, iso-valeric, n-caproic, butyric, and n-valeric. The pyrolysis of the lignin portion takes place at 310°C to 500°C and produces phenols and phenolic compounds, which are integral elements of smoke flavor. The three major phenolics attributed to smoky flavor and odor are guaiacol, 4-methy guiacol, and syringol. There are many minor compounds such as volatile oils, terpenes, fatty acids, carbohydrates, and alcohols that are referred to as wood extractives. These extractives play

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an important role in smoke generation in that they contribute to the characteristic odor and color of certain woods. During the aging process of harvested wood, the extractive content changes, thus giving a difference in flavor/odor and color from the smoke of green versus aged wood (4,5,13,18). When wood is burned and smoke is generated, there are two phases of the smoke: gaseous and particulate. About 90% of the total volume of smoke is in the particulate phase, which contains many of the undesirable characteristic components of smoke and gives smoke its cloudy appearance. The remaining 10% of smoke volume is made up of the gaseous phase and contains the desirable components for flavor and color (7,13). Because the major components of wood thermally decompose at significantly different temperatures, the quality of the smoke varies with the temperature at which the wood is burned. Therefore, it is important to note the dryness of the wood to be burned and also the temperature at which it is burned. These factors will affect the resulting smoked product, and even though they are relatively minor, they should not be overlooked. III. SMOKE EFFECTS A. Color and Flavor The effects of smoke application to meat can be primarily categorized as flavor, color, antimicrobial, and antioxidant (19). The original purpose of smoking meat gave high priority to preservation, but today the importance of smoke is in giving flavor and color. The pyrolysis of cellulose and hemicellulose produces carbonyls. These carbonyl compounds play an important role in color development of meat when smoke is applied. This is in contrast to early thoughts on color development, which had to do with the amount of tars and resins applied to the meat surface from the smoke. It is currently thought that the process of color development in smoked meat begins with the carbonyls being absorbed into the surface of the meat. The carbonyls then react with amino groups in the meat and follow a similar path of reactions as in the Maillard browning reaction. This group of reactions is enhanced as the temperature and dryness of the product are increased (11,13,18). The rate of color development is important, and Ruiter (23) found that as the temperature increased, color formation increased. The explanation for this can be viewed from two paths. The first is that as temperature increases, the chemical reactions involved begin to accelerate due to increased energy input. The second is a more applied answer in that the rate of surface drying increases with increased temperature, the result being the reddish golden brown to brown color of smoked meat. Ruiter (23) conducted a test using collagen casings to verify that the amino groups play a role in color development. Collagen casings were put through a smoking process with half of the casings having the NH groups chemically removed and the other half having no removal of the NH groups. The treated casings had a light yellow color that was easily removed, but the untreated group had a brown color that was difficult to remove. Increased drying increases the concentration of reactable compounds on the surface of the meat and is controlled by the dry and wet bulb of the smokehouse. Increasing the temperature of the surface only works to a point because once the temperature and moisture of the meat surface reach a particular level, important compounds become either bound or volatilized. This fine line of temperature and humidity is what has made the smoking of meats an art. In the past, smokehouses had more inconsistencies than they do today. This is due in a large part in today’s smokehouse builders having the technical resources avail-

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able to more accurately control heat, air flow, and moisture going into the smokehouse. This technology has then allowed the smokehouse operator to much more consistently obtain the desired color. In the past, with house inconsistencies, all operators had their own procedures for optimally running their houses for color. These procedures could vary from experience of operator to operator on just how hot to get the smokehouse and just how dry the meat felt in order to get optimum smoking of the meat product. This is why an experienced smokehouse operator was an invaluable asset to a company in the meat smoking business. In today’s meat smoking companies, the smokehouse operator is still important, but technology has removed some of the pressure. The pyrolysis of lignin produces phenolic compounds that play a prominent role in flavor development. Three of the primary phenolic compounds are guaiacol, 4-methyl guaiacol, and syringol. Guaiacol is the phenolic primarily associated with smoke flavor, and syringol is the phenolic primarily associated with smoke odor. Along with these three primary phenolic compounds are many minor phenolic compounds and smoke extractives that contribute to the characterization of smoke flavor and odor. These minor compounds number in the hundreds (11,13,18). The carbonyl compounds tend to react with the amino groups on the meat surface, but Dawn (5) found that phenols tended to react with sulfhydryl groups on the meat surface. Sink (24) collected data from trained taste panels that gave significant evidence that a meat product with smoked flavor was more acceptable than the same meat product unsmoked. There were also significant results that smoked meat products retained their acceptability scores for a longer period of time than the same product unsmoked. Bratzler et al. (4) reported that as samples of smoked bologna were taken from the surface to the center of the stick, the phenol level decreased. The phenol level data were compared to taste panel data that indicated a less smoky flavor as samples were taken from the surface to the center of the stick. This made a strong indication that phenolic compounds have a significant effect on smoke flavor. Wasserman (26) utilized taste panels to determine the threshold level of some of the smoke flavor/odor-related phenolic compounds. The majority of the panelists identified the compounds, guaiacol, 4-methylguaiacol, and 2,6 dimethoxyphenol, as having a smoky odor and smoky taste. The threshold levels at which the panelists could detect the presence of the compounds, as odor and taste respectively, was guaiacol, .021 ppm and .013 ppm; 4-methylguaiacol, .090 ppm and .065 ppm; and 2,6 dimethoxyphenol, 1.85 ppm and 1.65 ppm. As with color, the smokehouse operator in the past was an invaluable asset to ensure the appropriate smoke flavor for a particular product. Current technology has removed some of the need for actual smoke experience in an operator and has replaced that with a need for electronics and mechanical background. With today’s technology and the consuming public’s lack of experience in smoke flavor, the process of applying smoke for flavor is becoming much more standardized. This should not belittle the importance of consistency in process and presenting the consumer with high quality smoked products. B. Antioxidant Properties One of the effects of smoke not readily considered is the antioxidant effect on the smoked product. This antioxidant effect is used to counter the pro-oxidant effect of salt, especially in processed smoked meats. The phenolic portion of the smoke is credited with contributing the antioxidant capability. Oxidation in processed meats leads to rancidity development and a negative impact, in most cases, on the sensory response of consumers (7).

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White (28) stored smoked and unsmoked bacon at temperatures ranging from 18°C to 7°C and measured the time stored until an unacceptable rancidity occurred. The bacon smoked for 14 hours lasted an average of 55 days before unacceptable rancidity occurred, and the unsmoked bacon lasted an average of 30 days. This indicated that the effect of smoking contributed antioxidant properties. Watt and Faulkner (27) stated that the degree of antioxidant effect on smoked meat was related to the intensity of the smoke odor. This indicates that the antioxidant component is derived from the phenol portion of the smoke, because the phenol portion has an active role in smoke flavor and odor. When one looks at two of the most commonly used antioxidants, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), it is easy to see that they are both phenolic type compounds. This makes the phenolic portion of smoke a logical choice for antioxidant activity. One product in particular on whose flavor rancidity does not have such a negative impact on is country-cured ham. In this case, the oxidation process adds to the characteristic flavor of the product. This also is a product that is reviewed negatively by the health industry and is very time consuming to produce. Even with the negatives, country-cured ham in the southern United States is still a very popular and very tasty product. C. Antimicrobial Properties Another one of the less considered effects of smoke is its antimicrobial properties. There has been discussion on the true antimicrobial effects of smoke and the techniques that are used to make a determination. In the United States, smoke is generally considered to have a certain amount of antimicrobial properties. Jensen (15) and Gibbons et al. (10) indicated that smoking of pork bellies to produce bacon caused a reduction in the bacterial load of the product. They also pointed out that there was a tremendous amount of information still to be learned concerning the antimicrobial effects of smoke. Fretheim et al. (9) evaluated smoke generated at different temperatures, 350°C and 500°C, and the effects of these smokes on certain bacteria. The condensation from the generated smokes was used to test inhibition and control of E. coli and S. aureus. The study found that concentration of smoke used had more of an effect than temperature at which smoke was generated. Delay of lag phase was shown to occur at a smoke condensate level of 500 ppm for S. aureus and 1250 ppm for E. coli. However, the control of growth did not occur until 1000 ppm for S. aureus and 2500 for E. coli. Growth and toxin production of Clostridium botulinum type A and E spores has been shown to be prevented with a combination of liquid smoke and sodium chloride. The smoke and salt combination was applied to multiple fish species and stored at 25°C for 7 and 14 days. It was found that the normal salt level used could be cut in half when liquid smoke was added, thus showing a synergistic effect of salt and liquid smoke (8). Donnelly et al. (6) evaluated the effect of liquid smoke on lactic acid starter culture with the liquid smoke being added to the meat batter. Levels of 4,6 (recommended levels), and 12 ounces of liquid smoke per 1000 pounds of meat were used. The recommended levels did increase lag phase of lactic acid starter culture, which presented an opportunity for pathogen growth. The conclusion was to not add liquid smoke to the meat batter, but if it is desired it may be added later in the process. Arseculeratine et al. (3) and Alvarez-Barrea et al. (1), in separate studies, evaluated the effects of smoke on Aspergillus flavus. Smoke treatment inhibited aflatoxin production on corpa kernels and fermented sausages, respectively, but had no effect on mycelial growth.

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The question becomes, if smoke has a negative effect on microorganisms, what in the smoke is causing the effect? The answer is still not fully understood because of the many compounds making up smoke, and their interactions. The acids and formaldehydes are the front runners, but phenols can possibly play a part as well. With the current emphasis on food safety in the meat and food industries, these answers will probably be coming much quicker than they have in the past. IV. SMOKE GENERATION Smoke production for the purpose of smoking foods is not just the burning of appropriate wood. As has been discussed earlier, the type of wood and the temperature that the wood is burned has a significant effect on the quality of the finished product. Another variable is the way in which the wood is burned or the way in which the wood releases the smoke components. There are three primary ways that wood is, or has been routinely, burned for smoking meat. A. Friction Smoke generation by friction is a flameless process that has been used more in the past than now. The process is one in which a wood block is pressed against a rotating metal wheel. The wheel rotates fast enough to generate the necessary friction heat to cause the wood to burn. A hole in the center of the wheel allows for variable air movement, and this air is the cooling source for the smoke. The resulting smoke is piped into the smokehouse or smoking chamber. The friction wheel is usually operated on an intermittent basis with the time spent turning much less than the time spent not turning. This method of smoke generation is seldom used today. B. Smoldering The smoldering method of smoke generation is one of the most widely used methods today. This method exposes a metered amount of wood shavings to a dry heat source. The airflow to this contact area is restricted so the shavings will smolder rather than ignite. This smoldering results in the production of smoke, which is piped into the smokehouse. Normally the airflow in the house pulls the smoke in, so that no additional airflow is needed from the smoke source. The temperature at which the smoke is produced has a significant effect on the quality of the smoke. Smoke generator suppliers have specific temperature ranges for their generators so that smoke quality can be optimized. C. Steam The use of steam to generate smoke is becoming a popular method for smoked product producers. In this method the heat source that produces the smoke is a blast of super-heated air blended with steam. The heat source is forced over a thin layer of wood shavings and the result is the production of a wet smoke. The advantage to the smoked product producer is a reduced amount of wood used and a reduced amount of emissions released from the smokehouse into the outside air. This second advantage is becoming important with the heightened awareness of the Environmental Protection Agency on smokehouse emissions. The disadvantage to this method is that the color and flavor are not as distinctive as that of smoke produced by the dry burning or smoldering of wood. As regulations become more

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strict and consumer demands for smoke flavor and color of meats become more generalized, this method could have significant merit. D. Phases Once smoke is produced, it is composed of two phases, a gaseous and a particulate phase, with the particulate giving the cloudy appearance to the smoke. The gaseous phase contains the organic vapors and combustion gases, and the particulate phase contains visible portions such as tars and resins. The two phases can be separated by steam distillation, the gaseous phase being steam soluble and the particulate phase being non–steam volatile. The particulate portion was shown to be the source for the color components and composed about 90% of the total volume of smoke. The gaseous phase was shown to be the source of the flavor and odor components and constituted about 10% of the total volume of smoke (7,14,20). E. Liquid Smoke Smoke is generated and applied to products for the objectives of positive flavor and color attributes along with antioxidant and antimicrobial activity. So the following discussion on liquid smoke is not how a smoke product producer can make or produce liquid smoke, but how a smoke product producer could use liquid smoke to obtain the objectives of smoking. Liquid smoke is produced by the pyrolysis of hardwoods followed by the capturing of beneficial components of the smoke as a liquid. The raw liquid smoke is filtered to give various products. Some products may be more useful for rich color development, others more for full smoke flavor. A producer of smoked meat products would obtain a liquid smoke product that is designed to do the job he or she wants accomplished. The liquid smoke would then be applied to the meat or food product in the form of a drench, dip, atomized mist, or internal addition as an ingredient (2,25). The use of liquid smoke offers advantages to the producer by lowering air emissions as did the steam process, and liquid smoke removes the variability and maintenance of smoke generators. Liquid smoke does have some disadvantages in that precautions need to be taken to prevent undue corrosion of equipment and operators should be careful in handling it due to liquid smoke’s acidic nature. Even so, liquid smoke is gaining in popularity every day, due to its advantages. V. SMOKEHOUSE FUNCTION AND DESIGN A. Size and Types In today’s meat processing industry, the term smokehouse does not mean what it did 25 or more years ago. Then a smokehouse meant a room or building in which meat was put to have smoke applied and possibly undergo a cooking process. Today a smokehouse is referred to as a room or piece of equipment that applies a thermal process to a meat product and in some cases has the option to apply smoke to the product. Technology has given us the ability to process sausages, hams, and other meat products using casings and nets that have been previously treated with liquid smoke and thus do not need further smoke application during the cooking process. Smokehouses can come in a variety of sizes, from those in the back of a restaurant that process a few hundred pounds of product at a time to those used in commercial meat processing that process thousands of pounds per hour. Smokehouses can be of the batch or con-

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tinuous type. In a batch house, the smokehouse is filled with product and the product never changes its position in the house. The product has a number of different cycles applied to it and then the house is emptied of all the product. In a continuous house, the product is constantly being loaded into the front of the house, the product moves through different zones of the house, and there is product continuously coming out of the back of the house. B. Hot versus Cold Smoking In those smokehouses in which traditional smoke is applied, it is usually applied as a cold or hot smoke. A hot smoke refers to the smoke being applied during the thermal processing of the meat product. An example would be in frankfurters where certain stages of the cooking cycle include the application of smoke. Cold smoke is used for products that have had previous thermal processing or that require only low levels of thermal processing. An example of previous thermal processing would be a product that is cooked in an impermeable film, is removed from the film, is placed in the smokehouse, and has the smoke applied to it. An example of a product with low levels of thermal processing would be a dried fermented sausage. C. Airflow Whether a smokehouse is used for thermal processing or actual smoking, there are fundamental functions of the house that apply in both cases. The smokehouse uses heat, moisture, and airflow to accomplish either task, with airflow being the most important for uniform processing. Figure 1 is a very basic illustration of the airflow pattern of a smokehouse. Smokehouses tend to have hot and cold spots when the airflow follows the basic design of Fig. 1. To reduce hot and cold spots, alternating airflow systems are put into smokehouses to ensure that all areas of the house receive equal volumes and velocities or air. Figure 2 is a basic example of alternating airflow.

Figure 1 Smokehouse airflow. (From Ref. 13.)

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Figure 2 Alternating smokehouse airflow. (From Ref. 13.) D. Humidity In the case of hot or cold smoking of a product, the heat and airflow are present to dry the surface of the product, and moisture, in the form of humidity, is added to control this drying process. Once the surface is dry, the smoke is applied with limited airflow and then the heat and airflow are used to dry and heat the surface again to initiate the browning reaction for color development. To finish a product with a thermal process, the heat and airflow are used to uniformly heat the product to the desired temperature. Moisture is also continuously added to control yield loss generated from thermal processing. The initial drying step is very important for the assurances of good smoke color and smoke flavor. If the surface is too dry, the condition called case hardening can occur and the dried proteins on the surface form a shell that does not easily allow the smoke components to penetrate. On the other hand, if the surface is too moist the smoke components cannot fully react during the color setting phase because sufficient drying cannot occur. Also, a surface with too much moisture will cause the flavor components to not be properly absorbed on to the product surface. E. Balance of Smokehouse Functions A smokehouse provides a few basic functions; airflow, heat, moisture, and smoke, any one of which can make or break the acceptability of a smoked product. Even though these basic functions seem simple, there is a complex set of controls and balances within the house system that ensures the accuracy and consistency of each of these areas. Engineers and food scientists spend careers developing the methods and systems to properly control these areas of airflow, heat, moisture, and smoke. VI. SUMMARY Even with the current technology available to meat and food processors, smoking is still as much an art as an understood science. This can be partially attributed to the extensive variety of food products that are smoked and the wide range of variables that can affect smoke color and flavor. Even with this variability, the food-consuming public likes a smoke fla-

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vor and the appearance of a smoke color. This leaves the challenge to the food and meat scientist to develop the appropriate smoke process for a given product. By following the basic concepts of product processing prior to smoking, smoke generation, temperature, air movement, product position, and product processing after the smoking process, the technologist or operator can deliver an acceptable product to the consumer. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

9.

10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Alvarez-Barrea, V., A.M. Pearson, J.F. Price, J.I. Gray, and S.D. Aust. Some factors influencing aflatoxin production in fermented sausage. J Food Sci 47:1773–1775, 1982. Anderson, C. The use of natural smoke flavors in sausage production. Technical Bulletin, Red Arrow Prodcuts Company, Manitowoc, WI, 1994. Arseculeratine, S.N., U. Samurajeewa, and L.V. Welianga. Inhibition of aflatoxin accumulation in smoke substrates. J Appl Bacteriol 41:223–233, 1976. Bratzler, L.J., M.E. Spooner, J.B. Weatherspoon, and J.A. Maxey. Smoke flavor as related to phenol, carbonyl and acid content of bologna. J Food Sci 34:146–148, 1969. Dawn, H. Interaction of wood smoke components and foods. Food Technol 33(5):66–70, 1979. Donnelly, L.S., G.R. Ziegler and J.C. Acton. Effect of liquid smoke on the growth of lactic acid starter cultures used to manufacture fermented sausage. J Food Sci 47:2074–2075, 1982. Draudt H.N. The meat smoking process: a review. Food Technol 17:1557–1561, 1963. Eklund, M.W., G.A. Pelroy, R. Paranjpye, M.E. Peterson and F.M. Teeny. Inhibition of Clostridium Botulinum types A and E toxin production by liquid smoke and NaCl in hot-processed smoke-flavored fish. J Food Prot 45:935–941, 1982. Fretheim K., P.E. Granum and E. Vold. Influence of generation temperature on the chemical composition, antioxidant and antimicrobial effects of wood smoke. J Food Sci 45:999–1002, 1980. Gibbons, N.E., D. Rose and J.W. Hopkins. Bactericidal and drying effects of smoking bacon. Food Technol 8:154–157, 1954. Gilbert, J., and M.E. Knowles. The chemistry of smoked foods: a review. J Food Technol 10(3):245–261, 1975. Ham, H.A., and R.L. Safflle. Isolation and identification of volatile fatty acids present in hickory sawdust smoke. J Food Sci 30:697–701, 1965. Hanson, R.E. Alkar technical manual, Lodi, WI., 1996. Husaini, S.A., and G.E. Cooper. Fractionation of wood smoke and the comparison of chemical composition of sawdust and friction smokes. Food Technol 11:499–502, 1957. Jensen, L.B. Microbiology of meats. Champaign, Ill.: The Garrard Press, 1942. Jensen, L.B. Action of hardwood smoke on bacteria in cured meats. Food Res 8:377–387, 1943. Love, S., and L.J. Bratzler. Tentative identification of carbonyl compounds in wood smoke by gas chromatography. J Food Sci 31:218–222, 1966. Maga, J.A. Smoke in Food Processing. Boca Raton: CRC Press Inc., 1988. Price, J.F., and B.S. Schweigert. The Science of Meat and Meat Products. Westport, Conn.: Food and nutrition Press, Inc., 1987. Porter, R.W., D.J. Bratzler, and A.M. Pearson. Fractionation and study of compounds in wood smoke. J Food Sci 30:615–619, 1965. Randall, C.J., and L.J. Bratzler. Changes in various protein properties of pork muscle during the smoking process. J Food Sci 35(3):248–249, 1970. Romans, J.R., K.W. Jones, W.J. Costello, C.W. Carlson and P.T. Ziegler. The Meat We Eat. 12th ed. Danville, Ill.: The Interstate Printers and Publishers, Inc., 1985. Ruiter, A. Color of smoked foods. Food Technol 33(5):54–63, 1979. Sink, J.D. Effects of smoke processing on muscle food product characteristics. Food Technol 33(5):72–83, 1979.

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Sink, J.D., and L.A. Hsu. Chemical effects of smoke processing on frankfurter quality and palatability characteristics. Meat Sci 3:247–253, 1979. 26. Wasserman, A.E. Organoleptic evaluation of three phenols present in wood smoke. J Food Sci 31:1005–1010, 1966. 27. Watts, B.M., and M. Faulkner. Antioxidant effect of liquid smoke. Food Technol 8:158–161, 1954. 28. White, W.H. Smoke meats II. Development of rancidity in smoked and unsmoked Wiltshire Bacon during storage. Can J Res 22F:97–106, 1944.

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22 Meat Canning Technology ISABEL GUERRERO LEGARRETA Universidad Autónoma Metropolitana–Iztapalapa, Mexico City, Mexico

I. INTRODUCTION II. HEAT-TRANSFER MECHANISMS III. MICROBIAL DESTRUCTION BY HEATING IV. COMMERCIAL HEAT TREATMENTS A. Blanching B. Cooking C. Pasteurization D. Sterilization V. MEAT CANNING PROCESS A. Can Filling B. Exhaustion and Closing C. Sterilization VI. INACTIVATION PARAMETERS A. D- and Z-Values B. F-Values VII. ALTERATIONS IN CANNED MEAT A. Alterations Before Heat Treatment B. Microbial Alterations C. Chemical Alterations D. Physical Alterations VIII. CONCLUSION ACKNOWLEDGMENTS REFERENCES

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I. INTRODUCTION Among the various methods of extending meat shelf life, canning has the advantage of keeping as much as possible of the original chemical, physical, and sensory characteristics of meat. In addition, canning allows storing or transporting meat, a highly perishable material, in environments where no other preservation method is successful. In 1810, Peter Duran and Thomas Saddinton in England first commercially applied the canning technology to extend meat shelf life by placing it into sealed containers and heating them thoroughly. However, earlier efforts to preserve food materials by this method were made by Nicholas Appert in France in 1790. This resulted from the need to supply more varied and microbiologically safer food to Napoleon’s troops. This method, deserving of the prize granted by that French emperor, was called “I’art de l’appertization.” Canning is basically a heat processing operation where heat flows from a hot body (heating medium) to a cold body (food inside the can). The aim of canning is to destroy microbial populations (vegetative cells and spores) and/or enzymes responsible for meat deterioration (MacMeekin, 1982). Depending on the microbial species, vegetative cells of bacteria and fungi can be destroyed when heated at 60° to 90°C. Inactivation of meat enzymes occurs in most cases at 60° to 75°C (Greer, 1989). Bacterial spores require temperatures between 115 and 121°C. This process also ensures sanitary conditions, destroying spoilage vegetative cells and spores, as well as pathogens present in the food material. In this respect the two bacteriological problems to be solved by canning are (a) elimination of vegetative cells and spores that can grow and produce toxins and (b) elimination or inhibition of the development of spoilage microorganisms. Meat canning has a number of advantages when compared to other preservation methods such as smoking, curing, and drying. These are simpler storage conditions, considerably longer shelf life, and quality and nutritional characteristics closest to those of the unprocessed material. Canning is especially appropriate for meats marketed to tropical and subtropical conditions, where temperature and humidity are high. II. HEAT-TRANSFER MECHANISMS In every heat-transfer operation, it is necessary to know the total amount of heat to be transferred. This allows estimation of transport parameters in the system. As in any dynamic process, heat flow is proportional to the driving force and inverse to the resistance to the flow. The mechanisms involved in heat transfer are conduction, convection, and radiation (Karlekar and Desmond, 1985). Conduction is transmitted within a body due to vibrations of adjacent molecules. This mechanism takes place in solids. Therefore, in meat canning, conduction occurs in meat chunks or canned pastes that undergo gelation inside the can, such as luncheon meat. Conduction is based on Fourier’s law, q/A
k dT/ dz indicating that heat transference rate through a uniform material depends on the area (A) and temperature drop (T) but is inverse to the thickness of the material (L). However, conduction heating also depends on another parameter: thermal conductivity of the material (k). In food materials, k is very low. For instance, in meat, (average) k
1.464 kg cal/hr m2 °C (Mittal and Usborne, 1985), whereas in stainless steel, k
45.872 kg cal/hr m2 °C (Perry, 1963). Convection heating occurs in fluids and is the result of a movement of differential Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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densities when the fluid is heated or cooled. This is typical of liquids such as soups, brine, sauces. This type of heat propagation is faster when an external force, such as stirring, is applied, reducing the temperature difference to a minimum. In static containers slow convection takes place. Convection is based on Newton’s law: q
h A T meaning that heating depends on the area of transference (A) and temperature difference (T). As in conduction, a constant, h, is also included in convection equations, depending on the flow properties, the type of surface, and the flow rate. For instance, if boiling water is used as a heating medium, h
1464 to 19520 kg cal/hr m2 °C, whereas if air is applied, h
2.44 to 24.4 kg cal/hr m2 °C (Karel et al., 1975). In most thermal processing (canning) operations, heat is applied from a heating medium (water or steam) through a barrier (the container), to a cold fluid inside the container. Radiation occurs when heat is transmitted by electromagnetic waves, emitted by one body, and absorbed by other. Radiation in the infrared range (wavelength, , from 0.8 to 400 m) is applied as heating medium because this wavelength can be easily absorbed and transformed into heat. Radiation is occasionally used as a heating process together with the other two mechanisms in the heating of foods. However, radiation has no practical applications in meat canning. III. MICROBIAL DESTRUCTION BY HEATING Inactivation of pathogens or spoilage microorganisms by heat is calculated from the point of view of shelf-life extension as well as by alteration of sensory characteristics (Bacus, 1988). During heat treatment, the main problems related to microbial populations are as follow: 1. All microorganisms (cells and spores) feasible to grow and produce toxins must be eliminated. Canned meat, to be a safe food from the public health point of view, must be free of Clostridium botulinum, the most dangerous agent, which produces a fairly heat-stable toxin. 2. Spoilage microorganisms must be reduced to a safe limit. From the commercial point of view, any canned food is sterile if it is free of spoilage microorganism such as Bacillus stearothermophilus or Clostridium perfringens (commercially sterile). Sporeforming thermophiles such as Cl. sporogenes must be considered only when storage temperatures are high, because 40°C is the maximum growing temperature of these spores. This is the case with tropical preserves. Heat treatment eliminating Cl. botulinum and Cl. sporogenes renders heat-stable, long lasting foods, without the need for applying further preservation methods (Boyle, 1990). Table 1 shows the main microorganisms associated with meat spoilage and their growth intervals. Heat treatment severe enough to destroy Cl. botulinum or Cl. prefringens gives as a result a stable food without the need for applying special storage conditions. However, because severe heating can alter sensory characteristics, it is necessary to achieve a compromise between preservation and alteration of sensory attributes. It is important to note that heat treatment can also improve sensory characteristics of meat, such as texture, due to alteration of the muscle fibers, and flavor, due to generation of compounds responsible for aroma. As the rate and efficiency of microbial destruction depends on their resistance and, in turn, it depends on food composition, intrinsic and extrinsic parameters of the Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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Table 1 Microorganisms Associated with Meat Spoilage

Psychrophiles

Growth interval

Microorganism

5° to 35°C

Pseudomonas sp. Achromobacter E. coli Bacilus subtilis Streptococcus thermophilus Clostridium perfringens Clostridium thermosaccharolyticum Bacillus stearothermophilus

Mesophiles

15° to 45°C

Facultative thermophiles

24° to 54°C

Thermophiles

45° to 75°C

Source: Bem and Hechelmann, 1995.

food to be thermally treated must be taken in consideration. Thermal resistance of microorganisms increases because of the following factors: pH and water, fat, carbohydrate, protein, and salt content as well as the presence of other microbial inhibitors (Hanson, 1990). Meat, in general, has low acidity (4.5), with the exception of fermented meats. In this case, preservation is achieved by acid production of lactic acid bacteria. Acids of chemical origin also act as antimicrobial agents. In this case, meats require a mild heat treatment in order to have an extra preservation method (Guerrero and Taylor, 1994). In low acid (pH 4.5) or pH-neutral foods, such as most canned meats, stronger heat treatments are needed because potential pathogens or spoilage microorganism can grow. In most circumstances, Cl. botulinum does not grow and produce toxins under acid conditions (pH 4.5), but this behavior is very active at pH close to neutrality (Ray, 1996). Water activity (aw) is directly related to microbial growth. Dry meat products do not require further heat processing. However, yeast and fungi are more tolerant of low water activity values. The limiting aw values for Cl. botulinum are 0.97 for psychrotropic species and 0.95 for mesophiles (Leistner, 1985). There is a correlation between aw and redox potential. Genus Staphylococcus can develop in low oxygen concentrations. Raw meat has a redox potential around 50 mV. After heating, this potential decreases; for example, sausages have redox potentials from 20 to 100 mV, depending on the degree of grinding and ingredients added. Vacuum and addition of reducing agents can further decrease redox potentials (Thumel, 1995). Carbohydrate, protein, and fat protect microorganisms against thermal destruction because of their low heat transfer coefficients (Mittal and Blaisdell, 1984). Conductivity in meats depends on the direction in which heat is transferred. Pérez and Calvelo (1984) reported that thermal conductivity in lean beef at 78.5% humidity and 0°C, applying thermal flow perpendicular to the meat fiber, is 1.351 kg cal/hr m2 °C, whereas under the same conditions, in lean beef at 75% humidity, if the flow is parallel, conductivity is 1.385 kg cal/hr m2 °C. IV. COMMERCIAL HEAT TREATMENTS Vegetative cells are destroyed at temperatures slightly higher than their maximum growth temperature (Table 1), whereas spores can survive at much higher temperatures. Basically, there are four heating processes applied to food materials, based on temperature increase (Manev, 1984; Mathlouthi, 1986; Watson and Harper, 1988):

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A. Blanching Blanching is applied to inactivate enzymes in products receiving further heat treatment. For meats, volume reduction usually occurs. It is also used to eliminate gas from the tissues or simply to provide an initial cleaning of the food material. Blanching temperatures are around 65°C. B. Cooking Cooking is applied to improve sensory characteristics of the food material, although it also destroys a number of microorganisms and inactivates some enzymes. It takes place at around 85°C. C. Pasteurization Pasteurization destroys pathogenic vegetative cells, but certain heat-resistant microorganisms and spores can survive. Pasteurized foods have short shelf life even at refrigeration temperatures. Pasteurization temperatures are 140° to 150°C for 1 to 45 seconds, or 70° to 73°C for 15 to 20 seconds. D. Sterilization Sterilization destroys vegetative cells as well as spores; the shelf life of sterilized foods is considerably extended even without the application of additional preservation methods. Time–temperature relationship of the sterilization process depends on the thermal resistance of a given microorganism, taken as spoilage indicator. Cl. botulinum and Cl. sporgenes are good spoilage indicators for meat products. V. MEAT CANNING PROCESS Meat canning consists of several steps, although the basic principle is heat treatment of a sealed container. Once the meat and other ingredients are prepared, canning basically includes three main operations: can filling, exhaustion and closing, and sterilzation treatment (heating and cooling). A. Can Filling When a solid material is canned together with a liquid, heat penetration is affected by the solid-liquid ratio as well as the solid distribution within the can. In canned sausages, as they are distributed parallel to the can axis, a convection-conduction mechanism occurs. Solid material packed loosely is heated faster than closely packed material. In general, 30% of the can volume must be a liquid (brine or sauce) in order to have good heat transfer. Liquid is always filled after solids, but when pastes are filled, usually by automatic dosification, care must be taken not to leave air packs in the bottom. In order to calculate heat transfer, headspace must be always taken into consideration. In addition, exhausting efficiency depends largely on the headspace volume. Approximately 0.5% of the total can volume must be left for headspace (Watson and Harper, 1988). B. Exhaustion and Closing Foods easily react with oxygen; color, flavor, and, in general, wholesomeness may change because of reactions with the air components. Air evacuation from the headspace, as well

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as from the bulk of the food, is necessary to achieve good heat penetration and the desired sterilization temperature. When large meat pieces are canned, exhaustion during filling and closing is enough for a complete air evacuation. Conversely, in meat batters, air is incorporated during almost all processing operations if these are not carried out in vacuum. In fact, air can be partially eliminated in previous operations by using vacuum cutters, mixers, fillers, or stuffers. If a meat batter was not processed and placed into a can under vacuum, air bubbles are incorporated into the emulsion. As cooking occurs during heat treatment, small amount of air bubbles will be trapped within the coagulated meat emulsion or in the headspace. The latter can create sterilization problems. Air evacuation from the headspace reduces the risk of causing a rise in the internal pressure during heat treatment, resulting in can blowing or deformation. Air exhaustion also reduces the risk of promoting growth of aerobes, particularly if the product is only pasteurized, such as is some luncheon meat, tongue, or ham. Exhaustion can be achieved by heating, by mechanical operations, and by vapor injection. Heating of water promotes an increase in its vapor pressure, and the air in the container is replaced by the water vapor; the cans are then immediately closed. When the cans are cooled down, vacuum is produced due to condensation of the water vapor. However, if the headspace is too large, some air remains and a sufficient vacuum is not formed. Heating at 75° to 95°C is applied just before filling and closing. Alternatively, cans be conveyed on a belt into an exhausting chamber or tunnel in which the cans are heated at 85° to 95°C, removing 90% or more of the air in the headspace, depending on the temperature and time of residence in the exhauster. Canned meats are stored in sealed containers to avoid recontamination risks. Under these circumstances, aerobes cannot grow. In some cured and canned meats, oxygen is not completely eliminated. In this situation it is possible for microorganisms such as Bacillus subtilis and B. mycoides to grow and cause spoilage (Guerrero and Pérez Chabela, 1999). Vacuum sealing of the cans with or without vacuum vapor injection is not that efficient for emulsion-type meat products. C. Sterilization Thermal treatments include two cycles: heating and cooling. Whereas heating is involved in microbial and enzyme inactivation, cooling is applied for several reasons, such as easy handling and reduction of sensory characteristics deterioration. Being the basic operation during a process of canning, thermal or sterilization treatment is discussed in detail in the following sections. VI. INACTIVATION PARAMETERS Conditions of a thermal process are calculated on the basis of several considerations, such as composition of the food material (i.e., heat sensitivity of the microbial population, depending on the food composition); specific microbial population; conditions necessary to achieve a given shelf life depending on transport and storage conditions; initial microbial load and desired final microbial load, among others. Several inactivation parameters have been developed as mathematical tools to obtain a time-temperature relationship necessary to achieve a successful treatment.

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Table 2 Pathogens Associated with Meats Microorganisms

Lethality D65oC
0.1 min D70oC
0.3 min D55oC
0.17 min D60oC
1.9 min D60oC
0.2 min D60oC
4 min D60oC
0.4 min

Clostridium botulinum Vibrio sp. Aeromonas hydrophila Listeria monocytogenes Salmonella sp. E. coli 0157:H7 Staphylococcus aureus

Source: Stumbo, 1973; Stiebing, 1992; Manev, 1983; Thumel, 1995; Hanson 1990.

A. D- and Z-Values If a microbial population is subjected to temperatures slightly above those for its maximum growth, vegetative cells or spores are destroyed due to the inactivation of enzymes present in the microorganisms. The destruction follows an exponential equation: dc / dt
kc That is, cell concentration (dc) decreases with time (dt) in a direct proportion of cell concentration (c). In other words, 90% of the microorganisms are destroyed in a given time interval if constant temperature is applied. The time interval is different for each microorganism and is called decimal reduction time (D). It represents the minutes necessary to destroy 90% of a given microbial population at constant temperature. Table 2 shows the D values for some pathogens possibly associated with meats. Therefore, it is possible to compare thermal destruction of different microbial populations. D values are expressed at a given temperature (e.g., D120°C) (Table 3). For example, when heating at 110°C, 90% of the population of Cl. sporogenes (i.e., from 105 to 104) is reduced if heating is maintained for 10 min (D110°C
10 min). If the same population is heated at 115°C, the time necessary to reduce the population one logarithmic cycle at 115°C is 3 minutes (D115°C
3 min); and at 120°C, it requires only 1 min (D120°C
1 min) (Müller, 1990). On the other hand, at different heating times, the number of microorganisms destroyed increases with temperature, as shown in the following example: Table 3 Thermal Destruction Depending on the Heating Time at Constant Temperature Heating time (min)

Number of microorganisms (per can)

Population decrease (%)

0 3 6 9 12 15 18

103 102 101 1 101 102 103

— 1D 2D 3D 4D 5D 6D

Source: Stiebing, 1992.

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Figure 1 D-values, depending of the time-temperature relationship. (Stiebing, 1992.) In order to decrease the number of survivors for 6 log cycles (6D), it is necessary to heat at 120°C for 6 min, 18 min at 115°C, or 60 min at 110°C. This is shown in Fig. 1. When 120°C are applied, D
1; but at 110°C, D was increased to 10. Heat resistance for a given microorganism is given by z-values, indicating the temperature required decreasing D-values in 1/10. In Table 3, the z-value is 10°C, as temperature increases 10°C, D value is reduced from 10 to 1 for 90% destruction. B. F-Values Calculation, evaluation, and comparison of different heat treatments are achieved by the socalled F-value. This value represents the extent of thermal death of microorganisms and severity of the treatment in order to predict the product’s shelf life. The practical importance of F-values is that the individual effect of each part of the process is additive. As it is impossible to raise the temperature in the container to 120°C in every point, F
1 concept is applied. It is the lethality effect of heating at 120°C for 1 min. F-values increase, depending on the severity of heat treatment required for a given meat. Fs is the sum of all Fvalues in every parts of the container. According to the F-value concept, each temperature above 100°C has a defined lethal effect; it increases with the temperature’s increment. For instance, heating must be applied during a certain time and temperature in order to have similar heat damage: 100 min at 101°C, 10 min at 110°C, 1 min at 120°C or 0.1 min at 130°C (Watson and Harper, 1988). Thermal treatments, therefore, depend on a time-temperature relationship. Increasing the temperature for 10°C, the time necessary to achieve the same thermal effect is 1/10. Heat treatments are also calculated taking into consideration the survival of spores from two of the most damaging bacteria in meat products, Clostridium botulinum and Cl. sporogenes. However, as heating is not homogeneous in the entire can geometry, calculations are always done considering the temperature rise at the cold point (where heating is the slow-

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est). In this point, the sum of all lethal effects is Fc. The position of the cold point is determined by the type of food material, therefore its main heat transfer mechanisms, and to a certain extent by the agitation of the cans in the retort. When convection heating is the main mechanism involved, the cold point is on the vertical axis, close to the bottom end of the can. In conduction heating, the cold point is located in the geometrical center of the container. For viscous meat material, with cans rotating during the heating cycle, the cold point is close to the geometric center. Rotation in this case does not increase the heating rate substantially. In static heating of liquid or semisolid products, such as meat pieces in brine, where the leading heat transference mechanism is convection, the cold point is one-third from the can bottom end (Stumbo, 1973). Fc is always lower than Fs because heat effect in the center is always lower than in the rest of the container. When a thermal process is calculated for the first time, the cold point is located experimentally, using thermocouples (Fig. 2). A simple method of calculating the lethal effects during heating and cooling phases consists of measuring with thermocouples the temperature at the cold point, and calculating the corresponding F-values. The relationship between D and F, taking into account the amount of cells before and after heat processing, is: F
D (log a  log b) where a
initial cell load; b
final cell load. It is assumed that low-acid foods, as most meats are, are heated at a temperature that assures total absence of Cl. botulinum spores, and are microbial safe. In this case, spore counts must be reduced from 1012 to 100 (Tompkin, 1986), that is, reducing the count 12 log cycles or 12D. This means that heating must be enough to find only 1 Cl. botulinum spore in 1012 cans (i.e., one spore per gram of meat or 1/1012). Cl. botulinum types A and B are reference microorganisms for D values at 120°C and 0.21 min, as follows: F
0.21 (log 10  log 1012) F
2.52

Figure 2 Cold points.

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For Cl. botulinum, D120°C
0.21 min and z
10°C. In order to reduce an assumed number of Cl. botulinum cells 12 log cycles, heat must be 12 times higher during 0.21 min, that is, 2.52 min at 120°C. Heat processing of food around F
2.5 is called “botulinum cook.” Table 4 shows the thermal conditions for several preserves. Shelf-stable products are such that in addition to heat treatments, other preservation methods have been applied in combination with heating, such as low pH, water activity, and chemical preservatives such as nitrite (Leistner, 1985). Lethality is calculated by the equation: (log t  log F) / (log 10)
(250  T)/Z where log 10
1; therefore log (t /F)
(250  T)/Z The destruction rate per minute of a given microorganism at a temperature T in the process corresponds to the time, t, needed for the destruction of microorganism at that temperature. For every minute, lethality can be calculated at a given temperature, obtaining a curve. The area under the curve represents the total lethality of the process (Fig. 3). Another way to calculate the lethality necessary in a given process is by adding all Fvalues during the heating and cooling phases. This gives Ftotal, the sum of all F values. Table 5 shows the data published by Manev (1984) for F calculation in canned beef in brine, filled into the cans at 10°C. The can, 76  116 mm, was aimed to be marketed in tropical conditions.

Table 4 Thermal Conditions for Different Storage Temperature and Time Thermal processing, FC

Type

Storage conditions

Semi-preserves

Up to 6 months at 5°C

65°–75°C

Half-preserve

Up to 12 months at 10°C

Fc
0.4

Three-quarters

Up to 12 months at 10°C

Fc
0.6 to 0.8

Full preserves

Up to 4 years at 25°C

Fc
4.4 to 5.5

Tropical preserves

Up to 1 year at 40°C

Fc
12 to 15

Source: Manev, 1984; Stiebing, 1992.

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Inactivated microorganisms Nonsporulated; surviving cells of Streptococcus faecium and Str. fecalis and spores of Bacillus and Clostridium Inactivation of the above indicated microorganisms, plus spore-forming psychrophiles Inactivation of all the above plus spores of mesophilic species of Bacillus; spores of Clostridium are not destroyed Inactivation of all the above, plust (sterilized) mesophilic species of Clostridium Inactivation of all the above, plus sporulated thermophiles, such as Bacillus and Clostridium

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Figure 3 Lethality rate calculation. VII.

ALTERATIONS IN CANNED MEAT

Canned meat can undergo different type of alterations; most of them affect the quality of the food. Besides alterations before heat treatment, in-line alterations could be caused by microbial activity, chemical reactions of the meat with the container, and physical alterations. A. Alteration Before Heat Treatment These alterations occur when there is a delay in can filling and closing, or prior to heat processing, causing microbial growth to take place. Most pastes and cooked meat products such as sausages are placed into the cans when still hot; therefore a significant delay in processing can be dangerous. It is recommended that processing must not be delayed more than 20 minutes after closing the cans (Russel, 1982). Further heat treatment may sterilize the product, but gas or other microbial metabolites remain in the can. B. Microbial Alterations Alterations due to microorganisms are the result of insufficient thermal treatment. As a consequence, certain microorganisms survive, some of them producing gas, resulting in blown cans (Brown, 1982). Other surviving microorganisms, such as lactic acid bacteria, generate acid without gas (Schillinger and Lucke, 1987). When this alteration occurs, the first step to be taken is to identify the responsible microorganism; in most cases they are sporulated bacteria (Waites, 1988). Process conditions (time-temperature relationship) and microbial quality of raw materials must be checked, as well as sanitation of the equipment, water supply, and so forth. When can alteration always occurs in the same area of the re-

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Table 5 Lethality Calculation Time (min)

Tc (°C)

F-value

Heating

30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57

90 100 103 105 106 108 109 111 112 113 114 114 115 116 116 117 117 118 118 118 119 119 119 120 120 120 120 120

— 0.0077 0.0154 0.0245 0.0308 0.0489 0.0615 0.0975 0.1227 0.1545 0.1945 0.1945 0.2449 0.3083 0.3083 0.3880 0.3880 0.4885 0.4885 0.4885 0.6150 0.6150 0.6150 0.7746 0.7746 0.7746 0.7746 0.7746

Cooling

58 59 60 61 62 63 64 65 66 67 68 69 70 71 72

120 120 120 120 119 118 117 115 113 110 108 105 102 100 98

0.7746 0.7746 0.7746 0.7746 0.6150 0.4885 0.3880 0.2449 0.1545 0.0775 0.0489 0.0245 0.0123 0.0077 —

Phase

Fs

9.4589

Sum F-values Source: Manev, 1984.

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5.1602 14.6191

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tort, this can be due to failure in the operation conditions or defects in the retort or surrounding equipment, such as pipes and valves. In this situation, alterations occur due to insufficient heating caused by formation of air packs within the retort. Insufficient thermal treatment can also be a result of can distribution in the retort, causing a delay in convection. If the cans are tightly loaded there is no free access of vapor or water to all parts of the container, resulting in irregular heating. When cooling after heating is insufficient, thermophiles can grow. As a general rule, canned meat products are cooled at 35°C. At this temperature, the can outer surface rapidly cools down. However, if the cans are stored in large blocks, further cooling is slow, allowing thermophiles to grow. Therefore, cans must be stocked in relatively small blocks and in well-ventilated storage rooms (Buchanan, 1986). This is especially important in tropical areas where ambient temperature and humidity can reach very high values. Microbial contamination also occurs through seals if cooling water lacks adequate sanitary quality (Knudtson and Hartman, 1993). Recontamination after heat treatment is one of the most common problems, and a cause of can blowing. It indicates that the seam failure was already sealed by the food itself, or that the failure acts as a valve allowing contamination to flow from outside. Microorganisms responsible for this alteration are of diverse type: cocci, sporulated and nonsporulated bacilli (Ray, 1996). Unsuitable sanitation in the cooling water is the main factor responsible of this contamination. When cans are cooled down during storage, contamination through seams is less frequent. C. Chemical Alterations Blowing can also occur as a consequence of reactions of meat components with the packaging material, producing hydrogen or stannous sulfide (Guerrero, 1993). This happens during corrosion if the can coating presents failures (pinholes) or if the tin layer is not thick enough. This reaction produces blue-black spots in the food. Tin cans react without producing any visible alteration, although flavor can be drastically altered, producing an astringent, metallic taste. Meat compounds can also undergo chemical changes promoted by heat treatment, such as Maillard reactions (Ledward, 1992). This seldom occurs in meats, being more frequent in canned fruits. D. Physical Alterations Physical alterations occur as the result of mishandling of the sterilization equipment, such as a fast pressure increase in retorts, insufficient vacuum, or excessive can fill. These alterations include deformation of the cans during inadequate use of the retort. If pressure reduction is carried out at a high rate, higher pressure builds up inside the, can producing tensions and distortions that appear as blowing. These alterations mainly occur in cans of large formats (more than 1 kg). Cans undergoing abnormal high pressure usually show deformation in the upper and bottom seals. Even though no failure is present, these cans are discarded due to the risk of fissures. This type of alteration can also occur if layers used to roll the can are not thick enough, and are therefore unable to withstand high pressure during processing. When a can blows in the bottom end and the end can be slightly pressed back to its original shape, the cause is insufficient exhaustion. Presence of air in the can promotes an excessive internal pressure during heating as a result of gas expansion. If cans are transported from higher to lower altitudes, blowing can also occur, even though exhaustion was

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adequate at the processing plant. It is assumed that for each 300 meters of decreasing altitude, 2.5 cm of vacuum are lost. Excessive exhaustion produces the opposite effect—the can collapses. Excessive filling can also cause can deformation during heat processing due to expansion of the food. This mainly occurs in products cooked inside the can, such as luncheon meat, or in products containing, in addition to meat, beans, corn, rice, and so forth, which notably increase their size once cooked. VIII. CONCLUSION Meat canning has the advantage of keeping as much as possible of the original chemical, physical and sensory characteristics of meat. In addition, canning allows storing or transporting meat in environments where no other preservation method is successful. It is basically a heat processing operation where heat flows from a heating medium to food inside the can. As the aim of canning is to destroy the vegetative cells and spores and/or enzymes responsible for meat deterioration, thermal processing calculations must consider the destruction of Cl. botulinum, a pathogen and Cl. sporogenes, a spoilage microorganism. Nonetheless, canned meat can undergo different type of alterations; most of them affect the quality of the food. A compromise must be achieved between meat safety and quality in order to have a stable product without considerably altering its wholesomeness. ACKNOWLEDGMENTS The author thanks Dr. Mario Vizcarra, Department of Process Engineering, Universidad Autónoma Metropolitana, for his comments. REFERENCES Bacus, J. Microbial control methods in fresh and processed meats. In: Proc Reciprocal Meat Conf, pp 7–9, 1988. Bem, Z. and H. Hechelmann. Chilling and refrigerated storage of meat: Microbiological processes. Fleisch Int 2:25–33, 1995. Boyle, M.P. Meat associated pathogens of recent concern. In: Proc Reciprocal Meat Conf, pp 7–8, 1990. Brown, M.H. Meat Microbiology. Applied Science Publishers, London, 1982. Buchanan, R.L. Processed meats as a microbial environment. Food Tech 40(4):134–138, 1986. Greer, G. Red meats, poultry and fish. In: R.C. MacKellar (Ed.). Enzymes of Psychrotrophs in Raw Foods. Boca Raton, FL: CRC Press, Inc., pp 268–292, 1989. Guerrero, I. 1993. Capítulo 7: Productos cárnicos. In: M. García Garibay, R. Quintero and A. López Munguía (Eds.). Biotecnología Alimentaria. pp. 225–262. Editorial Limusa, Mexico City. Guerrero, I. and A.J. Taylor. Meat surface decontamination using lactic acid from chemical and microbial sources. Lebens Wiss und Tech 27:201–209, 1994. Guerrero, I., and M.L. Pérez Chabela, M.L. Spoilage of cooked meats and meat products. In: R.K. Robinson, C.A. Batt and P.D. Patel (Eds.). Encyclopedia of Food Microbiology. pp 1266–1272. Academic Press, 1999. Hanson, R.E. Cooking Technology. In: Proc Reciprocal Meat Conf, pp 109–115, 1990. Karel, M., O.R. Fennema and D.B. Lund. Principles of Food Science. Part II: Physical principles of food preservation. New York: Marcel Dekker, 1975. Karlekar, B.V. and R.P. Desmond. 1985. Transferencia de calor. Editorial Interamericana, Mexico City.

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Knudtson, L., and P.A. Hartman. Enterococci in pork processing. J Food Protec 56:6–9, 1993. Ledward, D.A. 1992. Colour of raw and cooked meat. In: D.E. Johnson, M.K. Knight, D.A. Ledward (Eds.). The Chemistry of Muscle-based Foods. pp 128–144. Royal Society of Chemistry, London. Leistner, L. 1985. Hurdle technology applied to meat products of the shelf stable product and intermediate moisture food types. In: D. Simatos and J.L. Multon (Eds.). Properties of water in foods. pp. 309–329. NATO ASI Series. Series E: Applied Sciences- No. 90. Martinus Nijhoff Publishers, Dordrecht. MacMeekin, T.A. 1982. Microbial spoilage of meats. In: R. Davies (Ed.). Developments in Food Microbiology. pp 1–37. Elsevier Applied Science, London. Manev, G. 1983. La carne y su elaboración. Tomo II. pp 308–402. Editorial Científica y Técnica. Havana, Cuba. Mathlouthi, M. 1986. Food Packaging and Preservation: Theory and Practice. Elsevier Applied Science Publishers, London. Mittal, G. S. and J.L. Blaisdell. Heat and mass transfer properties of meat emulsions. Lebens Wiss. und Tech 17(2):94–98, 1984. Mittal, G.S. and W.R. Usborne. Moisture isotherms for uncooked meat emulsions of different compositions. J Food Sci 50:1576–1579, 1985. Müller, W-D. The technology of cooked cured products. Fleisch Int 1:36–41, 1990. Pérez, M.G.R. and A. Calvelo. Modeling the thermal conductivity of cooked meat. J Food Sci 49:152–156, 1984. Perry, J.H. 1963. Chemical Engineers’ Handbook. 4th Ed. McGraw-Hill Co, New York. Ray, B. 1996. Fundamental Food Microbiology. CRC Press, Boca Raton FL. Russel, A.D. 1982. The destruction of bacterial spores. Academic Press, London. Schillinger, U. and F.K. Lucke. Identification of lactobacilli from meat and meat products. Food Microbiol 4:199–208, 1987. Stiebing, A. 1992. Tratamiento por calor: conservabilidad. In: F. Wirth (Ed.) Tecnología de embutidos escaldados. pp. 171–190. Editorial Acribia, Zaragoza, Spain. Stumbo, C.R. 1973. Thermobacteriology in Food Processing. Academic Presss, New York. Thumel, H. Preserving meat and meat products: possible methods. Fleisch Int 3:3–8, 1995. Tompkin, R.B. Microbiological safety of processed meat: new products and processes. Food Technol 40(4):172–176, 1986. Waites, W.M. 1988. Meat microbiology: a reassessment. In: R.A. Lawrie (Ed.). Developments in Meat Science. pp 317–333. Elsevier Applied Science, London. Watson, E.L. and J.C. Harper. 1988. Elements of Food Engineering. AVI Publishing Co, New York.

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23 Meat Fermentation Technology FIDEL TOLDRÁ, YOLANDA SANZ, and MÓNICA FLORES Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Burjassot (Valencia), Spain

I. INTRODUCTION II. PROCESSING OF FERMENTED SAUSAGES A. Ingredients B. Additives C. Technology D. Microbiology of the Indigenous Flora III. METABOLISM A. Sugar Metabolism B. Proteolysis C. Amino Acid Metabolism D. Lipolysis E. Nitrate and Nitrite Reductase Activity F. Catalase Activity IV. MICROBIOLOGY OF STARTER CULTURES A. Starter Cultures Used for Meat Fermentation B. Requirements for Starter Cultures C. Production, Quality Control, and Application of Starter Cultures D. Strain Improvement V. CONTRIBUTION OF FERMENTATION TO SENSORY ATTRIBUTES AND SAFETY A. Sensory Attributes B. Safety VI. TRENDS FOR ACCELERATION REFERENCES

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I. INTRODUCTION Fermentation by microorganisms is one of the oldest food preservation practices of humankind. The development of a strain of microbial flora succeeds, dominating and displacing other undesirable microorganisms, and producing a fermented meat in which the generated metabolites contribute to the product’s appropriate sensory characteristics. There are many varieties of fermented meats, varying according to region/country, climate, heritage, and culture. Thus, different amounts of raw materials, spices, and condiments and processing lengths are used for fermentation. Traditionally, fermented sausages were dried in the Mediterranean countries and dried/smoked in central and northern Europe. Today, most fermented meat products are produced and consumed in Europe, mainly Germany and the Mediterranean area, although in many other countries, such as the United States, demand as well as production are increasing. II. PROCESSING OF FERMENTED SAUSAGES A microenvironment has to be created during sausage fermentation in order to control the growth of pathogenic and/or spoilage bacteria (i.e., Salmonella spp., Escherichia coli, Clostridium perfringens). So, anaerobic conditions in combination with the presence of curing salts (salt and nitrite), lowered pH, reduced water activity, and drying will contribute as a hurdle to bacterial growth (Leistner, 1992). The main ingredients, additives, and stages in the processing of fermented sausages are described below. A. Ingredients The main ingredients in sausages are chilled raw meat from skeletal muscle tissue (usually porcine alone or mixed with bovine, although other species may be used) and frozen fat tissue, preferably firm pork backfat, with low content of polyunsaturated fatty acids. The use of fat with high unsaturated fat content might oxidize the color, give a turbid appearance of melting fats on the cut surface, and contribute to the development of rancid flavors. B. Additives Salt, with levels in the range of 2% to 3%, exerts a partial bacteriostatic action, an initial reduction in water activity to 0.96, an improvement in protein solubilization and imparts a typical salty taste. On the other hand, it may cause some undesirable effects such as promoting oxidation of pigments and fats, contributing to off-colors and rancid taste. Nitrite, and sometimes nitrate, is added to the curing mixture. The main role of nitrite is as a microbial preservative with a specific protective effect against pathogens, especially C. botulinum. It also prevents oxidation and contributes to the cured meat flavor (Gray and Pearson, 1984), although the full chemical mechanisms are not fully understood because of the number of complex compounds in the sausage and the high reactivity of nitrite (Cassens, 1994). Nitrite also plays an important role in the development of the typical cured meat color. Several mechanisms are involved in the formation of the cured pigment nitrosomyoglobin or nitric oxide myoglobin, which gives the pinky color, more reddish with dehydration. The use of older animals with a higher myoglobin content also contributes to a more intense color. Nitrate is used in Mediterranean countries for the processing of long-ripened products typical of that area. Sodium or potassium nitrate is reduced to nitrite by bacteria with nitrate reductase activity (i.e., Micrococcaceae). These bacteria may be naturally present in the meat or added as starter cultures. However, pH must be kept above 5.4 during the first hours because lower values would inhibit the nitrate reductase activity. A cold resting period before increasing the Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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temperature is advisable in these cases. The use of nitrate in curing is being reduced because of the uncertainty in its conversion to nitrite (Cassens, 1995). In addition, because of the potential toxic effects of N-nitroso compounds, the levels of nitrite have been reduced to those strictly necessary for protecting against botulism (Cassens, 1995). In this sense, the European Directive (1995) allows maximum and residual amounts of nitrates and nitrites in meat products. Sodium ascorbate and/or erythorbate are used as curing adjuncts to ensure the reduction of nitrous acid to nitric oxide and low residual levels to avoid nitrosamine generation. Carbohydrates are used as a substrate for microbial growth and fermentation to lactic acid with subsequent pH reduction. The rate and extent of lactic acid formation is strongly dependent on the type and amount of carbohydrate added (Lücke, 1985). Thus, the pH may drop very fast if readily metabolizable sugars (such as glucose or saccharose) are added, inhibiting acid-sensitive bacteria. The use of slowly metabolized carbohydrates (such as dextrins) reduces the rate of lactic acid generation and thus the pH reduction. A combination of both kind of sugars is used for controlling the rate of the pH reduction in specific products where other enzymatic reactions are looked for. The amount of carbohydrates is also very important because they are directly related to the final amount of lactic acid. Therefore, carbohydrate levels of 0.5% to 0.7% are usually added for reducing the pH to values slightly lower than 5.0. An excessive amount of carbohydrates reduces the pH to values near 4.5, which results in products with a noticeable, at most times unpleasant, acid taste. Small amounts of carbohydrates (below 0.3%) do not produce so discernible pH reduction. The description of microbial starter cultures and their role during the processing of fermented sausages is given below. Finally, the use of spices and condiments such as ground pepper, paprika, garlic, red pepper, and mace is a very extensive practice that contributes to the final specific flavor of the product. C. Technology The chilled meat pieces and frozen fat tissues are comminuted in a meat grinder or cutter and then the additives (salt, nitrate/nitrite, carbohydrates, microbial starters, spices, and sodium ascorbate or erythorbate) are incorporated. The ground mass is mixed for homogenization under vacuum for removing as much oxygen as possible. The homogenized mass is stuffed into natural, restructured collagen, by using vacuum-filling devices. The sausages are placed in artificial ripening chambers with control of temperature, relative humidity, and air flow rate or in natural ripening rooms when producing traditional sausages in an artisanal way. The conditions for fermentation vary depending on the kind of microbial starters and type of product. For instance, there is a clear difference in meat fermentation technology between the United States and most of the European countries. In the United States, the goal is rapid acid production through a fast fermentation in order to inhibit spoilage microorganisms. Starters such as Lactobacillus plantarum or Pediococcus acidilactici are used for fermenting up to 40°C. The product reaches high lactic acid accumulation, pH drops below 5.0 to 4.6, and the flavor formation is restricted because of the high percentage of inhibition of exopeptidases and lipolytic enzymes. Milder fermentation temperatures, around 22° to 26°C, are used in European countries, although other differences may be found within Europe. The ripening/drying period, the length of which is variable depending on the kind of product and its diameter, usually takes from 20 days to 3 months. In general, and depending on the total processing time, three main groups of fermented sausages can be established (Flores, 1997): (a) rapid (less than 7 days), (b) regular (around 3 weeks), and (c) slow (up to 3 to 4 months). The length and conditions of the process as well as the optional smoking have Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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a strong and definitive influence on the sensory properties. Some typical Mediterranean sausages are French saucisson, Spanish chorizo, and Italian salami. On the other hand, German- or Hungarian-style salamis represent some of the typical north European products. There are basic differences between both groups of products (Flores, 1997). The Mediterranean sausages, which are not smoked, undergo a slow process with nitrate addition and very mild temperatures. On the other hand, only nitrite is used in north European sausages, with faster processes and final smoking in most cases—up to 95% of German raw sausages are smoked (Leistner, 1995). The growth of a mold layer on the outer surface, typical of some Mediterranean dry-cured sausages, may contribute to flavor and appearance. Based on the moisture content, most fermented meat products may be classified as dry (weight loss higher than 30%) or semi-dry (weight loss lower than 20%) fermented sausages. D. Microbiology of the Indigenous Flora The origin of the microflora of the raw sausage mix is diverse and its composition varies depending on meat manipulation, the microorganisms present in the environment, and the additives used for manufacture. However, there are a number of factors that impose selectivity in favor of the development of the desirable flora (Micrococcaceae and lactic acid bacteria) and preventing growth of pathogenic and spoilage microorganisms (mainly gramnegative aerobic bacteria). These selective factors include low pH, reduction in water activity, temperature, oxygen depletion, accumulation of metabolic products, and presence of additives (salt and nitrite). The succession of microbial changes during ripening of different varieties of sausages has been described by several authors (Lücke, 1985; Roncalés et al., 1991; Samelis et al., 1994; Sanz et al., 1997, 1998a). An example of the evolution of the main bacterial groups during the processing of a fermented sausage is shown in Fig. 1. Total counts of aerobic mesophilic bacteria initially reach values of from 104 to 106 colony forming unit, CFU/g. The levels of lactic acid bacteria and Micrococcaceae are commonly around 103 to 105 CFU/g. Initial counts of gram-negative bacteria (Enterobacteriaceae, Pseudomonas, Achromobacter, etc.) are around 103 to 104 CFU/g. The initial levels of yeast and molds are a bit lower, with values of 102 to 103 CFU/g or cm2 (Roncalés et al., 1991). The fermentation stage is characterized by a general exponential growth of every microbial group parallel to a decrease in pH as a result of carbohydrate fermentation. Lactic acid bacteria dominate the microflora, reaching levels of 107 to 109 CFU/g that remain almost constant during the drying period. The evolution of this group is, in fact, parallel to that showed by total aerobic mesophilic bacteria. Members of the genus Lactobacillus are the most competitive among lactic acid bacteria, followed by Leuconostoc, Pediococcus, and Streptococcus. The species L. sakei and L. curvatus dominate the flora of traditional European products fermented at temperatures around 20° to 25°C, whereas L. plantarum is found in sausages fermented at higher temperatures. Moreover, strains of other species such as L. alimentarius, L. farciminis, and L. pentosus have also been isolated. Leuconostoc (Lc) and heterofermentative lactobacilli usually do not represent more than 10% of lactic acid bacteria. Among those we can mention L. viridescens, L. brevis, Lc. mesenteroides, and Lc. paramesenteroides (recently reclassified as Weissella paramesenteroides; Samelis et al., 1994; Kröckel, 1995). Micrococcaceae also gain importance in the fermentation stage, reaching levels of 106 to 107 CFU/g. Members of the Micrococcaceae family (Staphylococcus and Micrococcus; now divided into different genera, Stackebrandt et al., 1995) are, however, acid-sensitive and tend to decline during the drying period. The development and survival of this microbial group greatly depends on the degree of acidifi-

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Figure 1 Microbiology of the indigenous flora during the processing of a typical fermented sausage. (•) Mesophilic aerobic bacteria, (䊏) Lactic acid bacteria, (䉱) Micrococcaceae, (䉲) Enterobacteriaceae, (䉬) Yeast. (Adapted from Sanz et al., 1997.)

cation reached in the product. The colonization of staphylococci over micrococci is the result of the ability of the former to grow and metabolize in anaerobic conditions. The strains found in natural fermented products belong mainly to the species S. xylosus and S. carnosus; strains of S. saprophyticus, S. simulans, or S. sciuri constitute a minor proportion of the isolates. Levels of yeast and molds also increase to 106 to 107 CFU/g or cm2 in the fermentation stage. Yeasts are anaerobic facultative, and therefore they are able to grow in the inner and superficial part of sausages, whereas growth of molds is restricted to the surface. Debaryomyces hansenii is the yeast most frequently isolated from natural fermented meats, although species of Candida, Cryptococcus, Pichia, Rhodotorula, and Trichosporon have also been detected. The mycoflora of mold-fermented sausages is mainly dominated by Penicillium spp.; species of the genera Eurotium and Aspergillus develop more extensively in dry-cured hams. Enterobacteriaceae can experience a slight increase in the fermentation stage, reaching values above 105 CFU/g that dramatically decrease during the drying period. In general, levels of gram-negative bacteria (enterobacteria and psychrotrophs) become almost negligible at the end of ripening (less than 103 CFU/g). The growth of pathogenic bacteria such as Salmonella spp. is prevented mainly by the presence of nitrite in the initial stages and the further reduction of water activity and pH. Listeria monocytogenes is inhibited by the low pH, competitive flora, and accumulation of antimicrobial compounds. An adequate fermentation process prevents the growth of and toxin production by Staphylococcus aureus. The presence of Clostridium botulinicum and

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C. perfringens is excluded by the effect of nitrite combined with other selective factors such as presence of sodium chloride and low pH. III. METABOLISM A. Sugar Metabolism Lactic acid is the main product of carbohydrate fermentation. The enantiomers L and D lactic acid are usually present in the final product and their ratio depends on the species of lactic acid bacteria present, more specifically on the action of L and D lactate dehydrogenase, respectively, and the presence of lactate racemase. Usually, the ratio is near 1, a racemic mixture. Sugar metabolism starts after glucose is transported into the cell and metabolization occurs via the glycolytic or Embden-Meyerhof pathway. Some of the key enzymes (see Fig. 2) are (a) aldolases, which generate glyceraldehyde-3-phosphate, (b) pyruvate kinase, which forms pyruvate (the central intermediate in fermentation) from phosphoethanol pyruvate, and (c) lactate dehydrogenase, which generates lactic acid from pyruvate. NADH, originated during the hydrolysis of glyceraldehyde 3-phosphate, is oxidized in the latter step. By far, the greatest part of glucose is decomposed in a homofermentative way. However, although sugar metabolism is primarily homofermentative, trace amounts of other end products, such as acetate, formate, ethanol, and acetoin may result from alternative metabolic pathways. The pH drop, a consequence of lactic acid accumulation, is of paramount importance for preservation of sausages. The pH drop has other interesting contributions, such as flavor due to the formation of metabolites and the consistency of the product because of water holding capacity reduction and protein coagulation as pH approaches the isoelectric point of most of the meat proteins. The combination of the muscle and the lactic acid bac-

Figure 2 Simplified scheme of homofermentative metabolism of glucose in lactic acid bacteria.

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teria enzyme systems also contribute to keeping the environment anaerobic by reducing the redox potential during lactic acid fermentation. B. Proteolysis An important hydrolysis of myofibrillar and sarcoplasmic proteins takes place during sausage fermentation and ripening. This hydrolysis is brought about by the combined action of muscle proteinases (cathepsins and calpains) and exopeptidases (dipeptidylpeptidases and alanyl-, arginyl-, leucyl-, and pyroglutamyl-aminopeptidases) and starter proteases. One of the major challenges is the correct establishment of the relative role of endogenous and microbial enzymes to proteolysis. This would help optimize the processing conditions, but it is extremely difficult because of the high variety of microorganisms with different enzymatic activities used as starter cultures. The major steps are shown in Figure 3. This proteolytic process finally contributes to product consistency by the degradation of the myofibrillar structure and to flavor by the accumulation of small peptides and free amino acids directly related to taste or indirectly as precursors of flavor compounds through amino acid degradation reactions that will be later described. The extent of proteolysis varies depending on the processing conditions and type of starters added to the sausage, but it might be so intense that levels of non-protein nitrogen up to 20% of the total nitrogen content may be easily reached. The percentage of contribution of each group of enzymes is not fully clarified, but according to recent reports (Molly et al., 1997), it appears that protein degradation, especially of myosin and actin, is initiated by cathepsin D, an acid muscle proteinase that is favored by pH decrease. The activity of cathepsins B, H, and L would be restricted more to actin and its degradation products. Serine-, trypsin-like, and metallo-proteinases were concluded to be of no importance during dry sausage ripening. As muscle aminopeptidases have optimal activity at neutral or basic pH (Toldrá and Flores, 1998), the latter stages of proteolysis would be dominated by bacterial peptidases (Sanz and Toldrá, 1999; Flores et al., 1998a).

Figure 3 Major steps in proteolysis.

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Figure 4 Main reactions involved in the metabolism of free amino acids.

C. Amino Acid Metabolism Free amino acids may be subject to a number of chemical transformations such as decarboxylation, deamination, and transamination, producing different compounds that will affect the sensory characteristics of the product (Ordoñez et al., 1999). The microorganisms present in the product constitute the main enzyme source for most of these reactions. Free amino acids, generated through the proteolysis of muscle proteins, act as a substrate for these reactions, as shown in Fig. 4. 1. Degradation Reactions There are nonenzymatic pathways for amino acid conversion such as the Strecker degradation of amino acids that is an oxidative deamination-decarboxylation reaction producing branched aldehydes. The generation of 3-methylbutanal, 2-methylbutanal, and phenylacetaldehyde from leucine, isoleucine, and phenylalanine, respectively, has been found in dry-fermented sausages. The Strecker degradation of sulfur-containing amino acids such as methionine, cysteine and cystine that leads to the production of sulfur compounds that are characterized by low threshold values and therefore, exert a high aromatic impact in meat products (Shahidi et al., 1986). The enzymatic degradation of the amino acid side chain is another reaction occurring in fermented products. The side chain degradation of tyrosine and tryptophane by tyrosine-phenol-lyase and tryptophane-indole-lyase leads to phenol and indole formation (Molinard and Spinnler, 1996). These indole-derived compounds such as 3-methylindole (skatole) may be responsible for unpleasant odors in meat products. 2. Decarboxylation Biogenic amines are produced by the microbial decarboxylation of amino acids (Ordoñez et al., 1999). The enzymatic decarboxylation of the amino acids tyrosine, tryptophane, and phenylalanine produce tyramine, tryptamine, and phenylethylamine, respectively. Similarly, lysine, histidine, and ornithine give cadaverine, histamine, and putrescine, respectively, the last one being a precursor of spermine and spermidine. The presence of these substances not only can affect the flavor but also constitutes a risk for consumers’ health. Selection of appropriate raw material, processing temperature, and starter strains without

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potential for amine generation are important factors in controlling the level of biogenic amines (Ordoñez et al., 1999). 3. Deamination The oxidative deamination of amino acids is produced by several bacterias generating ammonia (Ordoñez et al., 1999). Glutamate dehydrogenase and alanine dehydrogenase generate -ketoglutarate and pyruvate, respectively, and ammonia in the presence of NAD or NADP. There is also another pathway that consists of the nonoxidative deamination of amino acids. The enzymes involved in these reactions are deaminases, whose action is facilitated by the presence of substituent in the carbon atom of the amino acid. The keto acid also can be transformed to aldehyde by decarboxylation; then, this aldehyde can be reduced to the corresponding primary alcohol or oxidized to acid. 4. Transamination In this reaction, the -amino group of the first amino acid is transferred to the  carbon atom from an -keto acid, generating a new amino acid. Amino transferases and transaminases present in bacteria catalyze this reaction. amino acid 1  keto acid 2 ⇔ keto acid 1  amino acid 2 D. Lipolysis The major steps in lipolysis of fat tissue are shown in Fig. 5. Lipolysis has an important contribution to flavor development through the generation of free fatty acids, and those that are unsaturated will act as substrates for oxidation to form volatile compounds with aroma properties. Adipose tissue and intermuscular fats are mainly composed of triglycerides; intramuscular fat also contains phospholipids, rich in polyunsaturated fatty acids. Initial breakdown of triglycerides would be the result of endogenous lipases such as lysosomal acid lipase, usually present in muscle and very active at a pH around 5.0, and neutral lipase, naturally occurring in fat tissue (Motilva et al., 1992). The latter is probably most important for the final lipolysis because fat tissue constitutes the major fraction in the sausage

Figure 5 Major steps in lipolysis.

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(Toldrá, 1992). Most of the lactic acid bacteria (LAB) cannot hydrolyze triglycerides but they can act on mono and diglycerides, thus contributing to hydrolysis of fat and generation of free fatty acids. As in the case of proteolysis—and although it is difficult to establish the relative role of endogenous and microbial enzymes in lipolysis—the percentage of the contribution of endogenous lipolytic enzymes to total fat hydrolysis is estimated to be around 60% to 80%, the rest being due to microbial lipases (Molly et al., 1997). In the case of phospholipids, which constitute a minor fraction of the total fat, muscle phospholipases are the only lipases responsible for their hydrolysis. E. Nitrate and Nitrite Reductase Activity Micrococcaceae are endowed with nitrate reductase activity that is essential for preservation, color development, and aroma formation in cured meat products (Flores and Toldrá, 1993). This enzyme reduces nitrate into nitrite and also recycles nitrite that was converted into nitrate in the sequential reactions with myoglobin. Then, nitrite may be reduced by both nitrite reductases from Micrococcaceae or by chemical degradation at pH 5.4 to 5.5. The nitrate and nitrite reductase activities of S. carnosus have been studied in more detail. The nitrate reductase is a membrane-bound enzyme involved in respiratory energy conservation, whereas the nitrite reductase is a cytosolic enzyme involved in NADH reoxidation (Neubauer and Götz, 1996). The expression of these activities is stimulated by anaerobiosis, nitrate, and nitrite. Molybdenum appears to be an essential cofactor for nitrate reduction (Pantel et al., 1998; Neubauer et al., 1999). Aerobic gram-positive bacteria (Enterobacteriaceae and psychrotrophs) also possess nitrate reductase activity but their prevalence, and thus their role in nitrate reduction, is limited in fermented meat products. Lactic acid bacteria are poor contributors to nitrate/nitrite reduction. Lactobacillus plantarum can reduce nitrate in vitro but not under conditions of meat fermentation (Lücke, 1985). Also, two type of nitrite reductase activity has been described in lactic acid bacteria: (a) a heme-dependent activity with ammonia as sole product, which has been found in strains of L. plantarum, L. pentosus, and P. pentosaceus, and (b) a heme-independent activity that generates NO and N2O, which was found in L. plantarum (Lücke, 1985, Wolf et al., 1990). This activity is not present in L. curvatus and rarely in L. sake strains (Wolf and Hammes, 1988). F. Catalase Activity Micrococcaceae possess catalase activity that mediates the degradation of hydrogen peroxide responsible for color and flavor defects. This activity is characteristic of aerobic and most facultative aerobic bacteria and together with the superoxide dismutase is involved in the degradation of metabolically toxic compounds derived from oxygen. Moreover, grampositive bacteria have more catalase activity per cell than gram-negative bacteria. In staphylococci, catalase is maximally expressed at the onset of the stationary phase, in aerobic conditions and at low glucose concentration (Baier et al., 1995). Lactic acid bacteria can also synthesize two different type of enzymes for peroxide degradation: a heme-containing catalase, produced only in the presence of hematine, and a pseudocatalase or manganese-dependent catalase that is found in a few species. In lactic acid bacteria, these enzymes are physiologically involved in resistance to oxidative stress (Hertel et al., 1998). Recently, the genes encoding the manganese catalase of L. plantarum and the heme-dependent catalase of L. sakei have been characterized (Igarashi et al., 1996; Hertel et al., 1998). In L. sakei, catalase activity is induced by aerobic conditions and the presence of hydrogen peroxide in anaerobic conditions (Hertel et al., 1998). Nevertheless, the contribu-

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Table 1 Example of Some of the Most Important Starter Cultures for Meat Fermentation Microorganism

Genera

Species

Lactobacillus Pediococcus Kocuria Staphylococcus Debaryomyces Candida Penicillium

L. sakei, L. curvatus, L. plantarum, L. pentosus P. pentosaceus, P. acidilactici K. varians S. xylosus, S. carnosus D. hansenii C. famata P. nalgiovense, P. chrysogenum

Bacteria

Yeasts Molds

Source: Adapted from Hammes et al. (1990), Geiues et al. (1992), and Hammes and Kuauf (1994).

tion to reduction of peroxides by lactic acid bacteria is of minor technological importance compared with that of Micrococcaceae. IV. MICROBIOLOGY OF STARTER CULTURES Traditional practices rely on the selection of desirable indigenous flora or the inoculation of up to 5% of a previous fermentation mixture (back-slopping). These techniques are still in use but have been progressively replaced by the application of well-defined starter cultures. The first commercialized starter culture (United States, 1957) consisted of a single strain of Pediococcus acidilactici (Niven et al., 1959; Everson et al., 1970). The first starter culture available in Europe also consisted of a single strain of Kocuria, named M53 (Niinivaara et al., 1964). This market evolved with the development of mixed cultures consisting of different strains that could cover wider spectra of metabolic properties. Currently, the application of mixed starter cultures constitutes a common industrial practice, with the objective of accelerating the fermentation process and ensuring the products hygienic and sensory quality. A. Starter Cultures Used for Meat Fermentation The main microorganisms used in meat fermentation and their relevant properties are summarized in Tables 1 and 2, respectively. Lactic acid bacteria (Lactobacillus and Pediococcus) are essential components of the starter cultures usually accompanied by MicrococTable 2 Properties of Starter Cultures Catalase

Nitrite-reductase

Proteolytic activity

Microorganism

Hemecontaining

Pseudocatalase

Nitratereductase

Hemedependent

Hemeindependent

Endo-

Exo-

Lipolytic activity

L. sake L. curvatus L. plantarum P. acidilactici P. pentosaceus Kocuria Staphylococcus Yeast Molds

        

        

        

        

        

        

        

        

Source: Adapted from Hammes et al. (1990) and Hammes and Kuauf (1999).

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caceae (Kocuria and Staphylococcus). The application of yeasts or molds also constitutes an alternative but has been less exploited so far. 1. Lactic Acid Bacteria The lactic acid bacteria used as starter cultures belong to the genera Lactobacillus and Pediococcus. The species commercially available are listed in Table 1. L. sakei and L. curvatus are the most competitive microorganisms in this environment. These species are psychrotrophic with optimal growth (Ta25° to 30°C) closer to traditional European fermentation temperatures (20° to 24°C). L. plantarum and Pediococcus spp. are mesophilic, showing optimal growth at 30° to 35°C (up to 40°C for P. acidilactici) and, therefore, their development is favored in fermentations at higher temperatures, typically used in the United States. The major role evolved by lactic acid bacteria is related to carbohydrate metabolism that results in the acidification of the meat mixture. This process has the following desirable effects: (a) it ensures hygienic stability by the reduction in pH itself and the generation of organic acids; (b) it imparts characteristic acid taste, (c) it causes coagulation of meat proteins (at pH 5.4 to 5.5) reduction in water holding capacity and facilitates the drying process with consequences in texture and firmness, and (d) it contributes to the development of desirable red color by favoring the reaction of nitrogen monoxide with myoglobin (pH 5.4 to 5.5). Lactobacilli used as starters in meat are all facultative heterofermentative organisms that utilize glucose and hexose-phosphate via the Embden-Meyerhof-Parnas pathway (glycolysis), generating lactic acid as the major fermentation product. Besides aldolase, the enzyme involved in glycolysis, these organisms also possess phosphoketolase, which decomposes pentoses into lactate, acetate (or ethanol), and carbon dioxide. The carbohydrates are usually metabolized via glycolysis but the heterofermentative pathway can be activated in certain conditions, resulting in the production of undesirable fermentation products (acid acetic, hydrogen peroxide, carbon dioxide, acetoin, formic acid, etc). For instance, L. plantarum is known to oxidize lactic acid into acetate and carbon dioxide under aerobic conditions. L sakei and L. curvatus use oxygen to generate hydrogen peroxide and pyruvate. The heterofermentative metabolism of these lactobacilli can also be activated in glucose depletion conditions under anaerobiosis, resulting in the generation of formate, acetate, and small amounts of ethanol. Pediococcus are also homofermentative organisms that generate lactic acid from sugars. However, P. pentosaceus also produce acetate and ethanol from hexoses and pentoses (Kröckel, 1995). The presence of catalase and nitrate/nitrite reductase activities is desirable in selected starter cultures in order to avoid color and flavor defects. These activities are not attributed mainly to lactic acid bacteria but to Micrococcaceae. Despite that, lactic acid bacteria can synthesize a heme-containing catalase and a pseudocatalase or manganese-dependent catalase (Table 2). Nitrate and nitrite reductase activity has also been found in lactic acid bacteria, one heme-dependent, the other heme-independent (Table 2). Proteolytic activity of lactic acid bacteria is thought to partially contribute to flavor development by releasing small peptides and free amino acids (Verplaetse, 1994; Molly et al., 1997). Several exopeptidases have been purified and characterized from L. sakei, showing their potential role in peptide degradation in meat fermentation (Montel et al., 1995; Sanz and Toldrá, 1997; Sanz et al., 1998b). The lipolytic activity from lactic acid bacteria is of limited interest, although lipolytic bacterial enzymes potentially active at the temperatures and pH values typical of meat fermentation process have been described (Papon and Talon, 1988; Naes et al., 1991).

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Lactobacilli can also decarboxylate amino acids, generating biogenic amines with adverse biological activity in humans. This activity is strain-dependent. So far, decarboxylase activity has not been detected in strains of L. sakei, whereas some strains of L. curvatus potentially produce up to four different amines (Hammes and Knauf, 1994; Straub et al., 1995). The selection of strains without ability to decarboxylate amino acids that competitively eliminate amine-producing strains is also essential to avoid health risks. Bacteriophage infection of starter cultures may account for failures in fermentation. Sensitivity to bacteriophages and presence of prophages has been described in L. sakei and L. plantarum (Nes et al., 1988; Leuschner et al., 1993) but never in pediococci. Nevertheless, infections by phages do not constitute a practical problem, because the semi-solid matrix of the fermenting meat mixture does not seem to be suitable for phage propagation. The synthesis of bacteriocins against undesirable organisms constitutes a trait of utmost importance in the selection of starter cultures. Bacteriocinogenic strains of L. sakei, L. curvatus, L. plantarum, and Pediococcus have been found but are not in use as starter cultures yet. The introduction of these strains could contribute to a reduction in hygienic risks. 2. Micrococcaceae Kocuria (exMicrococcus) and Staphylococcus strains are commercialized as starter cultures (Table 1). Staphylococci are, however, more competitive, mainly because of their metabolic activity under anaerobic conditions. The major functions evolved by this microbial group comprise color formation and stabilization and aroma development by means of their catalase and nitrate and nitrite reductase activities and implication in lipid metabolism (Table 2). Nitrate reductases are enzymes associated with the cytoplasmic membrane that carries out the dissimilation of nitrate at very low oxygen concentrations or in anaerobiosis. The enzymatic activity has been characterized in S. carnosus and it is demonstrated to be active in conditions typical of meat fermentation (Neubauer and Götz, 1996). This activity also has been studied in strains of K. varians showing optima that vary depending on the strain. The nitrite generated from nitrate can be reduced by nitrite reductases or chemically transformed. The compounds responsible for the cured red color are susceptible to oxidation, resulting in color defects. The catalase activity of this microbial group plays an important role in color stabilization by the degradation of the hydrogen peroxide. In this way lipid oxidation and rancidity are also prevented. Proteolytic activity is not significant in Micrococcaceae, although some endo- and exoproteolytic activity has been detected in K. varians, S. sciuri, S. xylosus, and S. carnosus (Montel et al., 1992; Fransen et al., 1997). Micrococcaceae seem to be more active in lipid metabolism and generation of volatile aroma compounds (Johansson et al., 1993; Stahnke, 1995). Phage infection of S. carnosus has been detected in some cases but, in practice, the industrial impact is of limited significance. 3. Yeast Debaryomyces hansenii is the predominant species in fermented meat, and together with Candida famata, constitutes the only yeast available as starter culture so far. They have an aerobic and weak fermentative metabolism, allowing their growth in both the surface and the inner part of meat products. The application of selected yeast strains mainly contributes to color stabilization and flavor generation by means of their catalase and lipolytic activity, respectively. Yeast also metabolizes organic acids and produces deaminase activity that may result in a pH increase.

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4. Mold Penicillium nalgiovense and P. chrysogenum constitute the fungi available as starter cultures. They mainly contribute to the appearance, flavor, and safety of fermented products. Molds have an aerobic metabolism, which restricts their growth to the surface. Apart from the external appearance, the implantation of molds exerts a protective effect against adverse effects of oxygen and light, such as discoloration and rancidity; also, drying occurs more evenly. The contribution to flavor is mediated by the activity of lipolytic and proteolytic enzymes. Also, the ability to metabolize organic acids resulting from lactic fermentation causes a decrease in the acidification level and tangy taste. This is also the result of deaminase activity that generates ammonium from amino acids. The application of nontoxic strains protects the product from the adverse effects of the implantation of mycotoxigenic molds. B. Requirements for Starter Cultures Strains used as starter cultures must be “generally regarded as safe” (GRAS) because they are considered to be food additives. Laws regulating the market of starter cultures may vary depending on the country but, overall, there are some requirements for the starter cultures; they must: 1. 2. 3.

Be neither pathogenic, toxic, nor allergenic Have phenotypical and genotypical stability Be competitive in the typical conditions of the process (tolerance to salt, nitrite, low pH and water activity, considerable growth at manufacturing temperatures, etc.) 4. Provide some technological benefits; for instance, on acidification, preservation, flavor formation, quality assurance, etc. 5. Resist phage infection 6. Be identifiable by specific methods

C. Production, Quality Control, and Application of Starter Cultures The composition of the media and growth conditions (temperature, pH, aeration, etc.) are critical for the production of starter cultures, and they must be selected taking into account their cost and benefits in terms of biomass, enzymatic activity, resistance to freezedrying, and stability during storage. Cells are usually collected at the end of the exponential growth phase or at the beginning of the stationary phase and cooled before processing. Concentration of the culture is carried out by centrifugation or ultrafiltration. The cultures are supplied frozen or lyophilized with or without previous immobilization of the cells. Mold cultures are supplied as freeze-dried spore suspensions, and yeast cultures as freeze-dried cells. The product then is packaged according to a declared activity or cell number. The quality control division must attend to the declared shelf life of the starter culture. Generally, tests of acidification are introduced for lactic acid bacteria and tests of nitrate reduction for Micrococcaceae. The absence of pathogenic or spoilage microorganisms as well as toxic contaminant compounds also must be controlled. Apart from controls of the metabolic activity, the stability of the genetic characteristics must be established by DNA fingerprinting and plasmid profile analysis.

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Lyophilized cultures must be reconstituted in water before their addition to the meat mixture. The cultures already reconstituted can be directly incorporated into the mix but, in advance, they are usually activated by keeping the culture at room temperature for 18 to 24 hours. The inoculum reaches between 106 and 108 CFU/g. The highest levels are inoculated for the production of rapid fermented sausages. Cultures of lactic acid bacteria and Micrococcaceae are usually combined to inoculate the meat mixture, whereas the application of mold and yeast is only on the outer part, normally by immersion of the sausage into a solution containing the starter culture or by spraying the suspension on the surface. The application of yeast is also combined with the use of bacterial starter cultures. D. Strain Improvement There is an increasing demand for strains with improved properties that fully cover the requirements for meat processing. Efforts have been made to develop strategies for selection of new strains from the meat environment. On the other hand, genetic manipulation of starter cultures is an alternative method of strain improvement, and research is currently in progress. The DNA coding for desirable traits can be part of a plasmid or chromosome. The properties that reside on a plasmid can be transfered by conjugation that is generally regarded as safe (GRAS). However, the use of gene cloning strategies is still controversial in the food industry. Most work on genetic modification of meat starter cultures has been made in molds in order to eliminate the production of mycotoxins, regulate the metabolic activity (proteolytic and lipolytic enzymes), or promote biopreservation (Leistner et al., 1991; Geisen, 1993). In staphylococci, the main interest is the antibiotic resistance of S. aureus and the possibility of transferring genes involved in flavor generation (lipases) from other gram-positive bacteria into the nonpathogenic S. carnosus (Goetz, 1990; Al-Masaudi et al., 1991). Within the genus Lactobacillus, one of the major goals is related to the development of strains that overproduce bacteriocins. Special interest has been focused on inhibition of acid-resistant strains of Listeria monocytogenes by bacteriocins produced by L. sakei (Leistner et al., 1991). The expression of the lysostaphin gene of S. simulans into meat lactobacilli has also been investigated as a means to enhance the antimicrobial potential of these strains (Cavadini et al., 1996). V. CONTRIBUTION OF FERMENTATION TO SENSORY ATTRIBUTES AND SAFETY A. Sensory Attributes The sensory characteristics of fermented products are achieved by the interaction of microbial, physical, and biochemical reactions (Verplaetse, 1994). During the fermentation process, acidification produces reactions and changes that ensure the development of color, texture, and flavor specific to the fermented products as described in Figure 6. 1. Color Visual appearance is a key factor that influences consumers when they are assessing the quality and palatability of meat and meat products. Certain colors influence food acceptance, although the color of the meat itself may be influenced by its moisture and fat content and also by the content of hemoprotein, particularly myoglobin and its relationship with the surrounding environment. The development of the characteristic color of fer-

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Figure 6 Contribution of fermentation to sensory attributes in fermented meat products. mented products is the result of the action of nitrite with myoglobin, producing the red color (Pegg and Shahidi, 1997). Some of the main additives in the formulation of fermented sausages are nitrate and nitrite. Nitrate is transformed into nitrite by microbial nitrate reductase activity. A large proportion of nitrite reacts with meat constituents and induces desirable changes. The formation of curing color in fermented sausages is obtained through several steps (Lücke, 1985): (a) oxygenated myoglobin (red) reacts with nitrous acid to give metmyoglobin (brown) and nitrate; (b) indigenous and exogenous reductants (e.g., ascorbate) reduce nitrous acid to nitric oxide, and metmyoglobin to myoglobin; (c) both combine to form nitric oxide myoglobin (red). The rate of its formation increases with falling pH, and therefore it is accelerated by the activity of lactic acid bacteria in fermented sausages. During ripening, the protein moiety of nitric oxide myoglobin is denatured, giving the formation of nitric oxide myochromogen. This process improves color stability because the nitric oxide dissociates less readily from the heme group. Nevertheless, nitric oxide myochromogen can be attacked by oxidants in several conditions, such as at low pH values and low redox potential. In fermented sausages, the oxidants are peroxide groups from the fatty tissue or are formed by lactic acid bacteria in the presence of oxygen. Therefore, it is worth emphasizing the importance of using fresh firm fat and of introducing as little oxygen as possible into the sausage mixture during its processing. The color defects appear when the peroxides oxidize the iron within the porfirin ring; then, the curing color changes to gray or brown. In addition, the presence of other enzymatic activities such as catalase are responsible for the elimination of peroxides, resulting in stabilization of the color and flavor, avoiding the attack on the porfirin ring and green discoloration (Demeyer, 1992). 2. Texture As mentioned above, microbial activity produces a decrease in pH value. When the pH approaches the isoelectrical point of meat proteins, the water-holding capacity is reduced,

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producing an increase in consistency that will be also accelerated during the drying process. Therefore, the acidification process is necessary to achieve the sliceability typical of the product. The salt added in the formula gives a suitable cohesion and texture during drying by solubilizing proteins, which act as a bridge between the constituting meat fragments. 3. Flavor The characteristic flavor of fermented sausages mainly originates from the breakdown of carbohydrates, lipids, and proteins through the action of microbial and endogenous meat enzymes. But other substances added to the sausage, such as salt and spices, should be taken into account because of their important contribution to flavor. Additionally, there are other pathways, such as autoxidation, that form flavor compounds without direct enzymatic participation. The carbohydrate fermentation is responsible for the typical tangy or sour taste (Lücke, 1985). The interactions between carbohydrate and protein metabolism during meat fermentation determine the rate of pH decline and flavor development (Demeyer, 1992). During carbohydrate fermentation, significant amounts of acetic acid, besides lactic acid, are generated. On the other hand, pH is partially neutralized during drying as the result of further ammonia and free amino acids generation. All of these compounds have an impact on flavor. There are internal and external parameters that influence flavor (Verplaetse, 1994). The internal parameters are chemical (added sugars or spices) or microbiological (starter cultures); external parameters are physical, such as the temperature and humidity during the process. a. Nonvolatile Fraction of Dry Sausage Aroma. The fermentation of carbohydrates, proteolysis, and lipolysis generate many nonvolatile compounds that play an important role in the taste impression (Verplaetse, 1994): 1. Glycolysis results in production of organic acids, the major products being lactate and acetate, which will contribute to the acid taste. However, the excessive production of these acids is not desirable, because of the suppression of global aroma by the acid taste. An excessive production of the D-lactic acid isomer is undesirable because of its unpleasant spicy taste (Ramihone et al., 1988). 2. Lipolysis is carried out by either endogenous meat enzymes and/or starter cultures. This process generates free fatty acids and diglycerides. The free fatty acids generated have a small impact on taste because it is necessary to have a high concentration of these compounds to produce a perceptible effect on sausage taste. The further oxidation of the free fatty acids generates many different compounds responsible for the aroma of the product as will be described. Lipolysis is mainly caused by endogenous enzymes, but lipid oxidation is caused by microbial action; therefore, they must be considered different processes. The presence of yeasts and molds in fermented sausages contributes to the fermented flavor. Their lipolytic enzymes contribute to flavor by generating carbonyl compounds. In the presence of oxygen, molds and yeasts do not only form flavor compounds but also oxidize lactic acid. 3. Proteolysis of meat proteins produces polypeptides, peptides, and free amino acids that are important for taste development in dry sausage (Nishimura et al., 1988, Kato et al., 1989). The extent of proteolysis depends on the acidity of the sausages. In low-acid-

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ity sausages, the proteolytic activity is low and no major proteins are broken down. In medium- and high-acidity sausages, myosin and actin are clearly degraded to fragments of 135 and 38 KDa, showing a pattern similar to that produced by endogenous cathepsins (Verplaetse, 1994). During ripening, there is a change in the composition of hydrophilic peptides that is correlated with sausage taste. The generation of high amounts of hydrophobic peptides is responsible for bitter taste and off-flavors. The amino acids released during proteolysis can be decarboxylated, deaminated, or even further metabolized. Therefore, the generated ammonia and amines cause an increase in pH, which is observed during drying of sausages (Lücke, 1985), and enhance sausage taste by neutralizing the final acidity. A rapid decrease in pH during initial steps of sausage production, as occurs when starters are used, positively affects color development, texture, and homogeneity of drying, although taste may be negatively affected (Flores et al., 1997). Therefore, it is necessary to find an equilibrium between acid production and taste and, last, ammonia production must be intensified to neutralize final acidity, enhancing sausage taste. b. Volatile Compounds in Fermented Sausage Aroma. Many volatile compounds have been identified in fermented products (Berdagué et al., 1993; Edwards et al., 1999) belonging to the following classes: alkanes, alkenes, aldehydes, ketones, alcohols, aromatic hydrocarbons, carboxylic acids, esters, terpenes, sulfur compounds, furans, pyrazines, amines, and chloride compounds. Different pathways are responsible for the formation of these volatile compounds. However, the impact of an odor component on the total aroma depends on a number of factors, such as odor threshold, concentration, solubility in water or fat, and temperature as reported for dry-cured ham flavor (Flores et al., 1998b). The different pathways of the volatile compounds and their impact on sausage aroma are as follows: 1. Lipid oxidation accounts for the generation of nonbranched aliphatic compounds such as alkanes, alkenes, methyl ketones, aldehydes, alcohols, and several furanic cycles. The contribution of alkanes to flavor is almost irrelevant because of their high thresholds. The flavor of alcohols was considered unimportant in comparison with other carbonyl compounds. The straight-chain primary alcohols are relatively flavorless but as the carbon chain increases, the flavor becomes stronger (Shahidi et al., 1986), giving greenish, woody, and fatty floral notes. C3 and C4 aldehydes have sharp and irritating flavors; intermediate (C5-C9) have green, oily, and fatty flavors; and the higher (C10-C12) have a citrus flavor (Forss, 1972). 2. The fermentation process release compounds of low molecular weight such as diacetyl, acetoin, butanediol, acetaldehyde, ethanol, and acetic acid. The generation of the specific volatile compounds during the carbohydrate fermentation depends on the starter used. The generation of compounds such as diacetyl, acetoin, or butanediol imparts a butter and yogurt aroma described in fermented sausage. 3. The catabolism of branched amino acids such as valine, leucine, and isoleucine generates 2- and 3-methylbutanal, 2- and 3-methylbutanol, 2- and 3-methylpentanoic acids, respectively, dimethyldisulfide from cysteine, and benzeneacetaldehyde from phenylalanine. These sulfur compounds are important contributors to meat flavor because of their low threshold values. 4. Animal feedstuffs and contaminants constitute another source of volatile compounds. For example, toluene and xylene isomers are currently found in plants, giving

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sweety and fruity notes (Shahidi et al., 1986). The chloride compounds may originate from pesticide residues ingested by animals. 5. The spices and condiments added in the manufacture of fermented products contribute to a particular flavor, depending on local traditions. In fact, there are many specific flavors due to the high number of available aromatic plants such as pepper, paprika, mustard, nutmeg, cloves, oregano, rosemary, thyme, garlic, and onion (Ordoñez et al., 1999). These compounds have a high impact on the aroma of fermented products. For instance, the high content of terpene hydrocarbons or sulfur compounds found in the headspace of fermented sausages comes from pepper or garlic, respectively. However, it is important to note the diverse pathways leading to the same volatile compounds. This is the case with acetic acid and ethanol, which also can be produced in the catabolism of lipids or amino acids, and some methyl compounds also generated in the degradation of branched amino acids by Strecker degradations. Sulfur compounds such as dimethyl disulfide and methyl-propyl disulfide have been found in the headspace of fermented sausages. Although these compounds can come from the Strecker degradation of cysteine and methionine in instances when garlic is added as a spice, many of the sulfur compounds come from their previous degradation in the garlic itself (Viallon et al., 1996). The volatile-compound content depends on the sausage type. Italian and Spanish sausages mainly contain the following dominant compounds: terpenes (from spices), ketones and aldehydes (from lipolysis and lipid oxidation), and esters. Certain low-acid sausages contain aldehydes, ketones, alcohols, and esters and low quantities of N-containing volatiles, indicating a low proteolysis in the product. On the other hand, medium-acid sausages (pH 5.1 to 5.3) contain aldehydes and ketones (constituents of the 60% of total volatile), furans, sulfur compounds, pyrazines, and amines, indicating a high proteolysis in the product (Verplaetse, 1994). B. Safety Lactic acid bacteria play a critical role in safety and preservation by the fermentative conversion of carbohydrates to organic acids (lactic and acetic acids). The production of organic acids has two concomitant effects: (a) the inhibition of the acid-sensitive spoilage microorganisms by the pH drop and (b) the intracellular inhibition of microbial metabolic process by the penetration of the nondissociate acid form across the cell membrane. In this sense, acetic acid has a greater antibacterial activity than lactic acid due to differences in dissociation constants of these two acids (Weber, 1994). The presence of lactobacilli is not the only factor necessary for producing safe and stable sausages. There are many other factors, such as anaerobic conditions, salt, nitrite, and low water activity, interacting with each other and exerting a hurdle effect (Leistner, 1992). The overall inhibitory functions of lactic acid bacteria are, however, due to a more complex antagonist system, which also includes production of other inhibitory substances. These antibacterial metabolic products exert a protective effect against a wide spectrum of microorganisms and are produced in fewer amounts than lactic and acetic acids (De Vuyst and Vandamme 1994b). Antibacterial compounds include formic acid, free fatty acids, ammonia, ethanol, hydrogen peroxide, carbon dioxide, diacetyl, acetoin, 2,3-butanediol, acetaldehyde, benzoate, D-amino acids, bacteriolytic enzymes, bacteriocins, and antibiotics, as well as several unidentified inhibitory substances (Daeschel, 1989; Piard et al., 1992). The competition for essential nutrients also constitutes a factor of selection in favor of lactic acid bacteria. Food-borne pathogens such as Staphylococcus aureus and Listeria

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monocytogenes can be inhibited by depletion of essential amino acids and vitamins (Iandolo et al., 1965; Degnan et al., 1992). The production of bacteriocins by lactic acid bacteria is considered of great importance for future applications in food preservation. Bacteriocins can be defined as biologically active proteins or protein complexes (protein aggregates, lipocarbohydrate proteins, glycoproteins) displaying a bactericidal action, against other, mostly closely related, microorganisms (De Vuyst and Vandamme, 1994b). These compounds are usually small cationic peptides with high isoelectric points and amphiphilic characteristics, which are active at micromolar concentrations (Leroy and Vuyst, 1999). The bacteriocins produced by lactic acid bacteria can be divided in three major groups on the basis of primary structure, molecular mass, and heat stability: 1.

Lantibiotics or lanthionine-containing bacteriocins, which are composed of unusual amino acids (lanthionine and methyllanthionine). These are heat-stable compounds of small size (19–37 amino acid residues) (e.g., nisin and lactocin S). 2. Non-lantibiotic bacteriocins, which are composed of common amino acids, are small (15,000 Da), and heat-stable (e.g., sakacin A, pediocin PA-1, and carnobacteriocins). 3. Non-lantibiotic bacteriocins, which are composed of common amino acids, are large (15,000 Da) and heat sensitive (e.g., helveticin and caseicin). Many strains of lactic acid bacteria associated with meat and meat products, belonging to the genera Lactobacillus, Pediococcus, Leuconostoc, Carnobacterium, and Enterococcus, are important bacteriocin producers (Aymerich et al., 1998). Two group of bacteriocins produced by lactic acid bacteria also have been defined according to their inhibitory spectrum: (a) bacteriocin, with a narrow inhibitory spectrum (Klaenhammer, 1988), active only against bacteria belonging to the same genus, and (b) bacteriocins active against other bacteria genera. In the latter, antibacterial activity has been described against gram-positive bacteria, such as Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens, C. botulinicum, and Brochothrix thermosphacta. In only a few cases, bacteriocins have been reported to inhibit the gram-negative Aeromonas hydrophila and Pseudomonas putida. The inhibition of other gram-negative bacteria such as E. coli and Salmonella by bacteriocins requires the addition of chelating-like agents (Aymerich et al., 1998). The mode of action involves the adsorption of bacteriocins to specific or nonspecific receptors on the cell surface, resulting in cell death. The primary target of bacteriocins is the cytoplasmic membrane, initiating changes in membrane permeability, disturbing membrane transport, or dissipating the proton motive force. Ultimately, energy production and biosynthesis of macromolecules are inhibited, causing cell death (De Vuyst and Vandamme, 1994a). The application of bacteriocins, as naturally occurring antimicrobial compounds, is promising given the trend to avoid additives in food. Many bacteriocins are heat stable; have a bactericidal and irreversible mode of action; and are stable, digestible, biodegradable, safe to health, and active at low concentrations (De Vuyst and Vandamme, 1994b). However, their application has some limitations: for instance, restricted spectrum of antimicrobial activity, susceptibility to tissue proteolytic enzymes, and significant lower activity in meat systems due to limited diffusion in the matrix and unspecific binding to meat components such as fat particles (Daeschel, 1993; Holzapfel, et al., 1995).

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VI. TRENDS FOR ACCELERATION Large-scale production requires the application of techniques to enhance flavor and reduce ripening time. The use of higher fermentation temperatures constitutes the simplest approach to achieving these objectives, but it may imply hygienic risks and flavor defects. Other strategies have consisted of reducing the water content of raw meat by freeze-drying or its binding capacity by including pale, soft, exudative (PSE) meat. Currently, the inoculation of starter cultures constitutes the most successful way to speed up and control the fermentation process. Most attention has been paid to the acidification ability of lactic acid bacteria, although their exclusive use results in a fast pH reduction that may result in unpleasant acid tastes. This may occur because of an unbalanced development of the complex set of reactions that contributes to the overall flavor. Thus, flavor enhancement still constitutes one of the major challenges for which two intense fields of research are initiated: use of enzymes and use of whole cells from new or modified starter cultures. Lipolysis and proteolysis are directly related to flavor development and, therefore, the incorporation of lipolytic and/or proteolytic enzymes has been considered a way for acceleration and flavor improvement. The first attempts to use proteinases in meat had the sole aim of increasing tenderness, but also a bitter taste impression was associated with enzyme-treated meats. Further, proteases of different origins have been assayed in fermented meat products. The incorporation of commercially available proteinases such as pronase E from Streptomyces griseus, an aspartyl proteinase from Aspergillus ozyzae, papain from Carica papaya, and neutrase from Bacillus subtilis have been tested (Diaz et al., 1997; Zapelena et al., 1997). However, the effect of these enzymes on sensory characteristics is not clearly positive. Despite the fact that proteolysis was stimulated, an excessive softening and bitter taste was frequently found. More promising results have been obtained with the use of the cell-envelope proteinase of Lactobacillus paracasei (Blom et al., 1996). The increased production of amino acids and peptides stimulates the metabolism of lactic acid bacteria, causing a rapid pH drop that results in accelerated gel formation and drying. On the other hand, flavor development is also promoted, reaching scores comparable to those of long-ripening sausages. The application of exoproteolytic enzymes in fermented meats is still a neglected area, as well as the application of microencapsulated enzymes that can be progressively released in the sausage mix. Studies on the addition of exogenous lipases have not revealed acceleration so far (Blom et al., 1996). The second alternative is directed to improving the enzymatic properties developed by meat-related bacteria, an area in which little has been done so far. Most advances have been made on cell modification of dairy organisms. Bacterial cells have been attenuated by physical methods (freezing, heating, chemical treatments, and/or drying) or mutagenesis, with the purpose of inactivating undesirable enzymes (e.g., those related to acidification) and promoting the activity of desirable enzymes (e.g., peptidases). These technologies have been proved to enhance flavor in shorter ripening times. The role of autolysin of lactic acid bacteria is also critical for flavor development through the release of intracellular enzymes. The application of autolytic strains is being exploited for cheese-making but is still obscure in meat fermentation. Therefore, substantial research is needed on all these topics as well as further studies on selection of new starter strains and all physiological and biochemical aspects related to flavor generation in fermented meats.

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REFERENCES Al-Masaudi, S. B., Day, M. J. and Russell, A. D. A review. Antimicrobial resistance and gene transfer in Staphylococcus aureus. J Appl Bacteriol 70:279–290, 1991. Aymerich, M. T., Hugas, M. and Monfort, J. M. Review: Bacteriocinogenic lactic acid bacteria associated with meat products. Food Sci Technol Int 4:141–158, 1998. Baier, S., Wolf, G., Knauf, H. J. and Hammes, W. P. Investigation of catalase activity of Staphylococcus carnosus. Fleischwirtsch 75:1351–1353, 1995. Berdagué, JL., Monteil, P., Montel, MC., and Talon, R. Effects of starter cultures on the formation of flavour compounds in dry sausages. Meat Sci 35:275–287, 1993. Blom, H., Hageb, B. F., Pedersen, B. O., Holck, A. L., Axelsson, L., and Naes, H. Accelerated production of dry fermented sausage. Meat Sci 43:S229–S242, 1996. Cassens, R. G. 1994. Meat preservation. Preventing losses and assuring safety. Food & Nutrition Press Inc, Trumbull, CT. Cassens, R. G. Use of sodium nitrite in cured meats today. Food Technol 49:72–81, 1995. Cassens, R. G. Composition and safety of cured meats in the USA. Food Chem 59:561–566, 1997. Cavadini, C., Hertel, C. and Hammes, W. P. Stable expression of the lysostaphin gene in meat lactobacilli by introducing deletions within the prosequence. Syst Appl Microbiol 19:21–27, 1996. Daeschel, M. A. Antimicrobial substances form lactic acid bacteria for use as preservatives. Food Technol 43:164–167, 1989. Daeschel, M. A. 1993. Applications and interactions of bacteriocins from lactic acid bacteria in foods and beverages. In:. D. G. Hoover and L. R. Steenson (eds.). Bacteriocins of lactic acid bacteria. p 63, Academic Press, San Diego, CA. De Vuyst, L. and Vandamme, E. J. 1994a. Lactic acid bacteria and bacteriocins: their practical importance, In:. L. De Vuyst and E. J. Vandamme (eds.). Bacteriocins of lactic acid bacteria. p 1. Chapman & Hall, Oxford, UK. De Vuyst, L. and Vandamme, E. J. 1994b. Antimicrobial potential of lactic acid bacteria. In: L. De Vuyst, and E. J. Vandamme (eds.). Bacteriocins of lactic acid bacteria. p 91. Chapman & Hall, Oxford, UK. Degnan, A. J., Yousef, A. E., and Luchansky, J. B. Use of Pediococcus acidilactici to control Listeria monocytogenes in temperature-abuse vacuum-packaged wieners. J Food Prot 55:98–103, 1992. Demeyer, D. I. 1992. Meat fermentation as an integrated process. In: J. M. Smulders, F. Toldrá, J. Flores, and M. Prieto (eds). New technologies for meat and meat products. p 21. Audet, Nijmegen, The Netherlands. Diaz, O., Fernández, M., García de Fernando, G., de la Hoz, L. and Ordóñez, J. A. Proteolysis in dry fermented sausages: the effect of selected exogenous proteases. Meat Sci 46:115–128, 1997. Edwards, R. A., Ordoñez, J. A., Dainty, R. H., Hierro, E. M., de la Hoz, L. Characterization of the headspace volatile compounds of selected Spanish dry fermented sausages. Food Chem 64:461–465, 1999. European Directive (1995) Ref. 95/2/CE of 20 Feb. regarding additives different from colorants and edulcorants in foods. European Commission. Everson, C. W., Danner, W. E., and Hammes, P. A. Bacterial starter cultures in sausage products. J Agric Food Chem 18:570–571, 1970. Flores, J. and Toldrá, F. 1993. Curing. In: R. Macrae, R. K. Robinson, and M. J. Sadler (eds.) Encyclopaedia of Food Science, Food Technology and Nutrition. p 1277. Academic Press, London, UK. Flores, J. Mediterranean vs northern European meat products. Processing technologies and main differences. Food Chem 59:505–510, 1997. Flores, J., Marcus, J. R., Nieto, P., Navarro, J. L., and Lorenzo, P. Effect of processing conditions on proteolysis and taste of dry-cured sausages. Z Lebensm Unters Forsch A 204:168–172, 1997.

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Flores, M., Sanz, Y., Spanier, A. M., Aristoy, M. C., and Toldrá, F. 1998a. In: E. T. Contis, C. T. Ho, C. J. Mussinan, T. H. Parliament, F. Shahidi, and A. M. Spanier (eds.) Food flavors: Formation, analysis and packaging influences. p 547, Elsevier Science BV, Amsterdam, The Netherlands. Flores, M., Spanier, A. M., and Toldrá, F. 1998b. Flavor analysis of dry-cured ham. In: F Shahidi, (ed.) Flavor of meat, meat products and seafoods. 2nd ed. p 320. Blackie Academic & Professional, an Imprint of Chapman and Hall, London, UK. Forss, DA. Odor and flavor compounds from lipids. Prog Chem Fats Other Lipids 13:181–258, 1972. Fransen, N. G., O’Connell, M. B. and Arendt, E. K. A modified agar medium for the screening of proteolytic activity of starter cultures for meat fermentation purposes. Int J Food Microbiol 36:235–239, 1997. Geisen, R. Fungal starter cultures for fermented foods: molecular aspects. Trends Food Sci Technol 4:251–256, 1993. Goetz, F. Applied genetics in the Gram positive bacterium Staphylococcus carnosus. Food Biotechnol 4:505–513, 1990. Gray, J. I. and Pearson, A. M. 1984. Cured meat flavor. In: C. O. Chichester, E. M. Mrak, and B. S. Schweigert (eds), Advances in Food Research, p 2, Academic Press, Orlando, FL. Hammes, W.P., Bantleou, A., and Min, S. (1990) Lactic acid bacteria in meat fermentation. FEMS Microbiol. Rev. 87, 165–174. Hammes, W. P. and Knauf. Starters in the processing of meat products. Meat Sci 36:155–168, 1994. Hertel, C., Schmidt, G., Fishcher, M., Oellers, K., and Hammes, W. P. Oxygen-dependent regulation of the expression of the catalase gene katA of Lactobacillus sake LTH677. Appl Environ Microbiol 64:1359–1365, 1998. Holzapfel, W. H., Geisen, R., and Schillinger, U. Biological preservation of foods with reference to protective cultures, bacteriocins and food-grade enzymes. Int Food Microbiol 24:343–362, 1995. Iandolo, J. J., Clark, C. W., Bluhm, L., and Ordal, Z. J. Repression of Staphylococcus aureus in associative culture. Appl Microbiol 13:646–649, 1965. Igarashi, T., Kono, Y., and Tanaka, K. Molecular cloning of manganese catalase from Lactobacillus plantarum. J Biol Chem 271:29521–29524, 1996. Johansson, G., Berdagué, J-L., Larsson, M., Tran, N., and Borch, E. Lipolysis, proteolysis and formation of volatile components during ripening of a fermented sausage with Pediococcus pentosaceus and Staphylococcus xylosus. Meat Sci 38:203–218, 1993. Kato, H., Rhue, M. R., Nishimura, T. 1989. Role of free amino acids and peptides in food taste. In: R. Teranishi, R. G. Buttery, F. Shahidi (eds.) Flavor Chemistry. Trends and Development. p 158. ACS Symposium Series, 388, ACS, Washington. Klaenhammer, T. R. Bacteriocins of lactic acid bacteria. Bichemie 70:337–349, 1988. Kröckel, L. 1995. Bacterial fermentation of meats. In: G. Campbell-Platt and P. E. Cook (eds.) Fermented meats, p 69. Chapman & Hall, London, UK. Leistner, L., Geisen, R. and Boeckle, B. Possibilities and limits to genetic change in starter cultures and protective cultures. Fleischwirtschaft 71:682–683, 1991. Leistner, F. 1992. Meat fermentation as an integrated process. In: J. M. Smulders, F. Toldrá, J. Flores, and M. Prieto (eds.) New technologies for meat and meat products. p 1. Audet, Nijmegen, The Netherlands. Leistner, F. 1995. Stable and safe fermented sausages world-wide. In: G. Campbell-Platt and P. E. Cook (eds.), p 160, Blackie Academic & Professional, London, UK. Leroy, F. and De Vuyst, L. Temperature and pH conditions that prevail during fermentation of sausages are optimal for production of the antilisterial bacteriocin sakacin K. Appl Environ Microbiol 65:947–981, 1999. Leuschner, R. G. K, Arendt, E. K. and Hammes, W. P. Characterization of a virulent Lactobacillus sake phage PWH2. Appl Microbiol Biotechnol 39:617–621, 1993. Lücke, F. K. 1985. Fermented sausages. In: B. J. B. Wood (ed.) Microbiology of Fermented Foods. Vol. 2. p 41. Elsevier Applied Science. London, UK.

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Molimard, P and Spinnler, H. E. Review: Compounds involved in the flavor of surface mold-ripened cheeses: origins and properties. J Dairy Sci 79:169–184, 1996. Molly, K., Demeyer, D., Johansson, G., Raemaekers, M., Ghistelinck, M. and Geenen, I. The importance of meat enzymes in ripening and flavour generation in dry fermented sausages. First results of a European project. Food Chem 59:539–545, 1997. Montel, M. C., Seronine, M. P., Talon, R., and Hebraud, M. Purification and characterization of a dipeptidase from Lactobacillus sake. Appl Environ Microbiol 61:837–839, 1995. Montel, M. C., Talon, R., Cantonnet, M., and Berdagué, J. L. 1992 Activités métaboliques des bactéries lactiques des produits cárnes. In: G. Novel and J-F Le Querler (eds.). Les bacteries lactiques. p 67. Centre de Publications de I’Université de Caen, France. Motilva, M. J., Toldrá, F., and Flores, J. Assay of lipase and esterase activities in fresh pork meat and dry-cured ham. Z Lebensm Unters Forsch 195:446–450, 1992. Naes, H., Chrzanowska, J., Nissen-Meyer, J., Pedersen, B. O., and Blom, H. 1991. Fermentation of dry sausage—The importance of proteolytic and lipolytic activities from lactic acid bacteria. Proc 37th Int Congr Meat Sci Technol, Kulmbach, Germany, p 914. Nes, I. F., Brendehang, J., and Husby, K. O. Characterization of the bacteriophage B2 of Lactobacillus plantarum ATCC 8014. Biochimie 70:423–427, 1988. Neubauer, H. and Götz, F. Physiology and interaction of nitrate and nitrite reduction in Staphylococcus carnosus. J Bacteriol 178:2005–2009, 1996. Neubauer, H., Pantel, I., Lindgren, P. E., and Gotees, F. Characterization of the molybdate transport system ModABC of Staphylococcus carnosus. Arch Microbiol 172:109–115, 1999. Niinivaara, F. P., Pohja, M. S., and Komulainen, S. E. Some aspects about using bacterial pure cultures in the manufacture of fermented sausages. Food Technol 18:147–153, 1964. Nishimura, T., Rhue, M. R., Okitani, A., Kato, H. Components contributing to the improvement of meat taste during storage. Agric Biol Chem 52:2323–2330, 1988. Niven, C. F., Deibel, R. H., and Wilson, G. D. 1959. Production of fermented sausage. U.S. Patent. 2, 907, 661. Ordoñez, J.A., Hierro, E.M., Bruna, J.M., and de la Hoz, L. Changes in the components of dry-fermented sausages during ripening. Crit Rev Food Sci Nutr 39:329–367, 1999. Pantel, I., Lindgren, P.E., Neubauer, H., and Goetz, F. Identification and characterization of the Staphylococcus carnosus nitrate reductase operon. Mol Gen Genet 259:105–114, 1998. Papon, M. and Talon, R. Factors affecting growth and lipase production by meat lactobacilli strains and Brochothrix termosphacta. J Appl Bacteriol 64:107–115, 1988. Pegg, R.B., and Shahidi, F. 1997. Chemistry and processing aspects of nitrite-free cured meats. In: Spanier, A. M., Tamura, M., Okai, H., Mills, O. (eds.). Chemistry of novel foods. p 273. Allured Publishing Corporation, Carol Stream, Illinois, USA. Piard, J.C. and Desmazeaud, M. Inhibiting factors produced by lactic acid bacteria. 2. Bacteriocins and other antibacterial substances. Lait 72:113–142, 1992. Ramihone, M., Simari, J., Larpent, J.P. and Girard, J.P. Gout acide des saucissons secs. Viandes Produits Carnées 9:291–298, 1988. Roncalés, P., Aguilera, M., Beltrán, J. A., Jaime, I., and Peiro, J. M. The effect of natural and artificial casing on the ripening and sensory quality of a mould-covered dry sausage. Int J Food Sci Technol 26:83–89, 1991. Samelis, J., Stavropoulos, S., Kakouri, A., and Metaxopoulos, J. Quantification and characterization of microbial populations associated with naturally fermented Greek dry salami. Food Microbiol 11:447–460, 1994. Sanz, Y. and Toldrá, F. Purification and characterization of an aminopeptidase from Lactobacillus sake. J Agric Food Chem 45:1552–1558, 1997. Sanz, Y., Flores, J., Toldrá, F., and Feria, A. Effect of pre-ripening on microbial and chemical changes in dry fermented sausages. Food Microbiol 14:575–582, 1997. Sanz, Y., Vila, R., Toldrá, F., and Flores, J. Effect of nitrate and nitrite curing salts on microbial changes and sensory quality of non-fermented sausages. Int J Food Microbiol 42:213–217, 1998a.

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Sanz, Y., Mulholland, F., and Toldrá, F. Purification and characterization of a tripeptidase from Lactobacillus sake. J Agric Food Chem 46:349–353, 1998b. Sanz, Y. and Toldrá, F. The role of exopeptidases from Lactobacillus sake in dry fermented sausages. Recent Res Devel Agric Food Chem 3:11–21, 1999. Shahidi, F., Rubin, L.J., and D’Souza, L.A. Meat flavor volatiles: A review of the composition, techniques of analysis, and sensory evaluations. CRC Crit Rev Food Sci Nutr 24:141–243, 1986. Stackebrandt, E., Koch, C., Guozdiak, O., and Schuman, P. Taxonomic dissection of the genus micococcus Kocuria gen. nov., Nesterenkonia gen. nov., Kytococcus gen. nov., Dermacoccus gen. nov. and Micrococccus Cohn 1872 gen. emend. Int. J. Syst. Bacteriol. 45:682–692, 1995. Stahnke, L. H. Aroma components from dried sausages fermented with Staphylococcus xylosus. Meat Sci 38:39–53, 1995. Straub, B. W., Kicherer, M., Schilche, S.M., and Hammes, W.P. The formation of biogenic amines by fermentation organisms. Z Lebensm Unters Forsch 201:79–82, 1995. Toldrá, F. 1992. Meat fermentation as an integrated process. In: J.M. Smulders, F. Toldrá, J. Flores, and M. Prieto (eds). New technologies for meat and meat products. p 209. Audet, Nijmegen, The Netherlands. Toldrá, F. and Flores, M. The role of muscle proteases and lipases in flavor development during the processing of dry-cured ham. Crit Rev Food Sci Nutr 38:331–352, 1998. Verplaetse, A. Influence of raw meat properties and processing technology on aroma quality of raw fermented meat products. Proc 40th Int Congr Meat Sci and Technol, The Hague, The Netherlands, p 45, 1994. Viallon, C., Berdague, J.L., Montel, M.C., Talon, R., Martin, J.F., Kondjoyan, N., and Denoyer, C. The effect of stage of ripening and packaging on volatile content and flavor of dry sausage. Food Res Int 29:667–674, 1996. Weber, W. Dry sausage manufacture. The importance of protective cultures and their metabolic products. Fleischwirtsch 74:278–281, 1994. Wolf, G. and Hammes, W.P. Effect of hematin on the activities of nitrite reductase and catalase in lactobacilli. Arch Microbiol 149:220–224, 1988. Wolf, G., Arendt, E.K., Pfaehler, U., and Hammes W.P. Heme-dependent and heme-independent nitrite reduction by lactic acid bacteria results in different N-containing products. Int J Food Microbiol 10:323–330, 1990. Zapelena, M.J., Zalacaín, I., De Peña, M.P., Astiasarán, I., and Bello, J. Addition of a neutral proteinase from Bacillus subtilis (neutrase) together with a starter to a dry fermented sausage elaboration and its effect on the amino acid profiles and the flavor development. J Agric Food Chem 45:472–475, 1997.

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24 Meat Production YONG-SOO KIM University of Hawaii at Manoa, Honolulu, Hawaii

I. CONTRIBUTION OF MEAT TO HUMAN SOCIETY A. Meat in Human Nutrition B. Meat Industry II. MEAT PRODUCTION SYSTEMS A. General Features of Meat Animal Production Systems B. Types of Production Systems III. ANIMAL SCIENCE AND MEAT PRODUCTION ACKNOWLEDGMENTS REFERENCES

I. CONTRIBUTION OF MEAT TO HUMAN SOCIETY A. Meat in Human Nutrition 1. Source of High-Quality Protein Meat and meat products are an indispensable part of the human diet—they provide easily available protein, minerals, and all the B vitamins. The share of meat and offal in the world dietary protein supply was 17.2% in 1990–1992 (Table 1). The contribution was, however, uneven from developed and developing countries. The contribution of meat to the dietary protein supply in developed countries was almost three times that in developing countries (Table 1), suggesting a close relationship between income and meat consumption. The contribution of meat to the dietary protein supply has increased steadily in both developed and developing countries during the past decade (Table 1). The excellent digestability and well-balanced composition of essential amino acids of meat is even more important than the amount of protein supply to human nutrition. Nine essential amino acids must be supplied from dietary intake to meet the body’s needs because the human body cannot synthesize them. The body’s requirement of each essential amino

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Table 1 Share (Percentage) of Major Food Groups in Total Protein Supply by Economic Group, 1969–71 and 1990–92 World Food group Vegetable products Animal products Meat and offal Milk Fish Eggs

Developed countries

Developing countries

1969–71

1990–92

1969–71

1990–92

1969–71

1990–92

66.4 33.6 15.6 10.4 5.2 2.4

64.5 35.5 17.2 9.6 5.9 2.8

46.6 53.4 24.9 17.4 7.0 4.1

42.4 57.7 28.3 16.6 8.6 4.1

81.0 19.0 8.8 5.2 3.9 1.1

75.8 24.2 11.6 6.0 4.5 2.1

Source: FAO, The Sixth World Food Survey, 1996 (Ref. 16).

acid is different, thus a high-quality protein is the one that provides an appropriate composition of essential amino acids. As was demonstrated in Chapter 1, muscle proteins from various species meet or exceed the estimated ideal profile for all essential amino acids established by the FAO/WHO/UNU (1). One clear advantage of including meat in the diet is that it provides a significant proportion of the daily requirement of protein and essential amino acids in a relatively small serving. For detailed information on the value of meat proteins in relation to human protein nutrition, Pellett and Young (2) provide a good review. Their conclusion is that meat serves as a complete and well-balanced source of amino acids for effectively meeting human physiological requirements. 2. Source of Minerals The mineral content of fresh meat is about 1%. In general, meat is a good source of all minerals except calcium. Meat not only is an excellent source of iron and zinc but also assists the absorption of those minerals from other foods. Although species, age of animal, and type of muscle affect the content of iron and zinc, one serving (85 g) of braised beef can provide up to 19% and 26% of the U.S. recommended dietary allowance (RDA) for iron and zinc, respectively. USDA data indicate that the contribution of meat (red meat plus poultry) to the dietary supply of iron and zinc in the United States is 27.1% and 44.3%, respectively (3). In addition to the total content of those minerals, the bioavailability of iron and zinc in meat is an important contributing factor to the nutritional value of meat. Of particular importance is the contribution of iron in meat to human nutrition. Dietary iron is classified into two groups, heme and nonheme iron. Heme iron is mostly found in hemoglobin and myoglobin, and it is present only in animal organs but not in any plant tissue. About half of the iron present in skeletal muscle is in the heme form. Although the rate of iron absorption is affected by the quantity of iron storage and other nutritional factors, the absorption rate of heme iron is much better than that of nonheme iron probably due to different absorptive pathways (4). An additional advantage of heme iron is that its absorption is not much affected by other dietary factors, such as phytic acid, known to inhibit the absorption of nonheme iron and other minerals, including zinc. Iron deficiency is the most common nutritional problem in most populations throughout the world (5,6). The deficiency is more prevalent in children and women of reproductive age in developing countries (Table 2). USDA data indicate that only 44% of the survey population in the United States had a dietary iron intake that met or exceeded the

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US RDA (7). Iron deficiency is known to cause many adverse health effects, including a reduction in work efficiency, altered immune response, and, in the child, behavioral abnormality and decreased intellectual performance. Zinc is essential for nucleic acid metabolism, appetite control, gustatory function, sexual development, brain development, immune development and function, and membrane function. The presence of phytate, oxalate, lignin, and certain hemicelluloses that are abundant in plant-originated food reduces the uptake of zinc by intestinal cells. Thus, the zinc in meat is more readily available to the body than the zinc from plant sources. Considering the abundance and excellent bioavailability of iron and zinc in meat, there is no question about the contribution of lean meat to iron and zinc nutrition in humans. In particular, the readily available iron in lean meat is important for growing children and women of reproductive age. 3. Source of Vitamins As shown in Fig. 2 in Chapter 1, meat is a valuable dietary source of all the B vitamins, including thiamin, riboflavin, niacin, vitamin B6, pantothenic acid, and vitamin B12. Organ meats, in general, contain higher amounts of vitamins. Liver is an excellent source of vitamins A and B12. According to USDA data (3), red meat, poultry, and fish combined contributed 44.3%, 39.9%, 24.2%, 23.6%, and 51.1% of the dietary intake of niacin, vitamin B6, riboflavin, thiamine, and vitamin B12, respectively, in the United States. B. Meat Industry 1. Meat Production The meat industry is one of the most important in the world economy, providing a large share of wealth and income. The most comprehensive data regarding livestock and meat Table 2 Estimated Prevalence of Anemia (%) and Number Affected, by Geographical Region and Age/Sex Category, Around 1980 Children (0–12 years) Region Africa North America South America Eastern Asiab Southern Asia Europec Oceania Developed regions Developing regions World

Women (15–49 years)

Men (15–59 years)

Pregnant

All women

Percent

No.a

Percent

No.a

Percent

No.a

Percent

No.a

52 11 26 27 53 2 15 9 46 40

95.3 5.2 31.8 8.8 257.9 7.4 0.9 19.4 391.5 410.9

20 4 13 11 32 2 7 3 26 18

23.4 3.1 12.8 6.1 123.6 3.0 0.5 12.0 162.2 174.2

63 — 30 20 65 14 25 14.0 59 51

11.3 — 3.0 0.5 27.1 0.8 0.1 2.0 41.9 43.9

44 8 17 18 58 12 19 11 47 35

46.8 5.1 14.7 8.4 191.0 14.1 1.0 32.7 255.7 288.4

a

Number in millions. Excluding China (no data are available). c Excluding former USSR (no data are available). Source: Adapted from DeMaeyer and Adiels-Tegman, 1985 (Ref. 6). b

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production, consumption, and trade are available from FAO, a UN agency, but it should be realized that the numbers are compiled from data reported by participating nations. Because the reliability of census data from the participating nations varies, the accuracy of the data may be a problem. Nevertheless, it is useful in providing global perspectives. Unless otherwise specified, consumption data are estimated from carcass disappearance, not actual consumption. Meat can be broadly defined as the flesh and edible organs of the mammalian, avian, and aquatic species used for food. Obviously, the flesh is the major part consumed as meat, but other organs and tissues also make a significant contribution to human nutrition as well as being served for prized specialty dishes. By the above definition any species can be used as meat, but in practice the bulk of meat production comes from only a small number of species. Table 3 summarizes world meat production by regions and sources in 1996. Meat from aquatic species is not included in this table. Pig meat (40.0%), poultry meat (26.9%), and beef and veal (24.8%) contributed more than 90% of total world meat production. Other domesticated species, including sheep (3.4%), goat (1.7%), buffalo (1.3%), and horse (0.3%) contribute less than 7% of world meat production. In addition to the popular domesticated species, various other mammalian species are consumed according to availability and local culture (1.6%). These species include seal, polar bear, deer, moose, and caribou in northern latitudes (including Inuit people); kangaroo, walrus, and possum for Australian aborigines; camel in desert areas; giraffe, rhinoceros, hippopotamus, and elephant for some African tribes; dog and cat in some parts of southeast Asia; and whale in Norway and Japan (8). Rabbits, rodents, beaver, bison, and elk, snake, and frog are also consumed in some regions. Game meat usually indicates the meat from nondomesticated animals. From Table 3, it is also clear that the relative contribution of the various species to meat production varies from region to region. The variation may primarily be due to differences in natural resources for the production of livestock. However, culture and the economy also play a role in meat production. Pig meat production is dominant in Asia and Europe, whereas beef and poultry meat production is dominant in North and South America. Buffalo meat is produced only in Asia and Africa. Even within regions, the relative importance of the various sources of meat varies widely from country to country. Table 4 summarizes the five leading meat-producing countries for each type of meat. China (28.1%), the United States (15.8%), Brazil (5.0%), France (3.0%), and Germany (2.7%) lead in total meat production. Almost 50% of total world pork is produced in China. Even though China is far ahead in total meat production, its per capita total meat production is lowest among the five leading countries because of the enormous population. However, its per capita pork production (34.3 kg) is comparable to other leading countries. Islam prohibits the consumption of pork, thus pig production is virtually nonexistent in Islamic countries throughout the world. The United States leads in the total and per capita production of beef. Australia and New Zealand are far behind China in terms of total sheep and goat meat production, but stand out in terms of per capita production. 2. Changes over Time World meat production has been increasing at a higher rate than the population. Changes in world meat production by type between 1970 and 1995 are illustrated in Fig. 1. While the world population increased about 60% between 1970 and 1995, from 3.6 billion to 5.6 billion, total meat production more than doubled during the same period, from 102 to 206 billion metric tons. The increase in pork and poultry production was greater than the increase in beef. The increase in beef, veal, and buffalo production was about 41%, from 40.2 to Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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Table 3 Total World Meat Production by Region in 1996 Meat production (thousands of metric tons)

Area

Population, in millions (percent of total)

World

5,629

Africa N.C. Americab S. Americab Asia Europe Oceania

708 (12.6%) 290 (5.2%) 474 (8.4%) 3,403 (60.4%) 726 (12.9%) 28 (0.5%)

a

Total 217,594 9,387 (4.3%) 42,749 (19.6%) 19,755 (9.1%) 88,666 (40.7%) 52,402 (24.0%) 4,635 (2.1%)

Beef and veal

Buffalo meat

53,938 (24.8%) 3,307 14,436 9,695 10,415 13,674 2,410

2,863 (1.3%) 175 0 0 2,685 3 0

Horse meat

Lamb and mutton

Goat meat

Pig meat

Poultry meat

Other meata

606 (0.3%) 13 154 92 211 112 23

7,397 (3.4%) 959 170 280 3,295 1,580 1,114

3,603 (1.7%) 687 46 67 2,676 115 12

87,104 (40.0%) 785 10,179 2,522 48,255 24,931 432

58,641 (26.9%) 2,367 17,553 6,985 20,191 10,938 607

3,442 (1.6%) 1,094 211 114 938 1,049 37

Other meat includes meat from all domestic or wild animals except cattle, buffalo, sheep, goats, pigs, and poultry. N.C., North and Central; S., South. Source: FAO Production Year Book, 1997 (Ref. 17). b

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Table 4 Meat Production by Types in Five Leading Countries in 1996 Countries production

1,000 MT production

Percent world

Kilogram/capita

28.1 15.8 5.0 3.0 2.7

50.0 128.4 67.8 112.3 71.8

19.7 8.6 8.6 4.8 4.6

42.3 4.1 31.1 3.0 18.0

21.7 6.6 6.3 5.9 5.0

2.0 5.4 39.1 0.7 153.6

48.0 8.6 3.7 2.6 2.5

34.3 28.2 39.4 56.8 37.3

24.9 19.5 7.1 3.7 2.5

54.6 9.4 26.3 37.0 25.2

Total meat production (217,594 thousand MT) China 61,038 United States 34,294 Brazil 10,785 France 6,524 Germany 5,855 Beef and buffalo meat (57,487 thousand MT) United States 11,308 China 4,967 Brazil 4,949 India 2,752 Russian Federation 2,670 Sheep and goat meat (11,069 thousand MT) China 2,404 Pakistan 732 Australia 700 India 653 New Zealand 553 Pork (87,104 thousand MT) China 41,811 United States 7,522 Germany 3,211 Spain 2,248 France 2,170 Poultry (58,641 thousand MT) United States 14,596 China 11,435 Brazil 4,174 France 2,147 United Kingdom 1,469 Source: FAO Production Year Book, 1996 and 1997 (Ref. 17).

56.6 million metric tons. Pork production more than doubled from 37.1 to 84.8 million metric tons, and poultry production more than tripled from 17.7 to 54.7 million metric tons. The increase in beef production has been lagging behind the increase in pork and poultry production probably because beef production requires significant land resources as compared to pork and poultry. Another factor might be that health-conscious consumers in the developed countries have been less enthusiastic about eating beef in favor of eating poultry. The increased demand for meat and meat products was fueled by economic expansion after World War II, in combination with an abundant feed grain supply. Advances in animal breeding, genetics, and disease control have enabled intensive and massive worldwide production of pork and poultry. 3. Meat Consumption Per capita production of meat varies from region to region and from country to country. From Table 3, regional per capita meat production can be estimated by dividing meat production by population. Oceania (165.5 kg), North America (147.6 kg), Europe (72.2 kg),

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Figure 1 World meat production and human population. (From Refs. 17 and 19.)

and South America (41.6 kg) are above the world average (38.6 kg) in per capita production, Asia (26.1 kg) and Africa (13.2 kg) are below the world average. In a broad sense, per capita meat consumption is closely related to per capita meat production. Relatively wealthy regions have higher per capita meat production, indicating that a relationship exists between meat consumption and the state of the economy. High income countries in North America, Europe, and Oceania, such as Australia, Canada, Denmark, Spain, and the United States, consume about 100 kg of meat per capita per year (Table 5). With a few exceptions, most low-income countries consume far less meat than high income countries. Consumption in Japan, one of the highest income countries, is less than in some of the low-income countries, indicating that natural resource endowments, culture, and religion also affect dietary habits. Moslems eat little pork, and Hindus avoid eating beef and pork. Land-rich countries in South America, including Argentina, Brazil, and Uruguay consume larger amounts of beef than their incomes would suggest (Table 5). Eastern European countries such as Poland consume relatively large quantities of pork, probably because of their traditions.

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Table 5 Per Capita Meat Disappearance in Selected Countries, 1996

Countries High-income countriesa Australia Canada Denmark France Germany Japan South Korea Spain United Kingdom United States Low-income countriesa Argentina Brazil China Egypt Ethiopiac Indiac Indonesiac Kenyac Mexico Nigeriac Philippines Poland Uruguay

Per capita GNPa ($)

Beef and veal (kg)

Pork (kg)

Lamb, mutton, and goat (kg)

Poultry (kg)

Totalb (kg)

20,090 19,020 32,100 26,270 28,870 40,940 10,610 14,350 19,600 28,020

37.2 34.5 20.2 25.0 14.5 12.3 10.0 11.0 14.4 31.2

18.4 33.1 67.6 36.8 46.1 16.7 19.2 52.3 23.7 22.3

16.6 0.4c 0.4 5.2 1.1 0.6 0.3c 6.2 6.3 0.5

24.3 30.3 14.5 18.0 11.1 13.4 9.2c 23.4 24.0 40.9

96.5 98.3 102.7 85.0 72.8 43.0 38.7 92.9 68.4 94.9

8,380 4,400 750 1,080 100 380 1,080 320 3,670 240 1,160 3,230 5,760

60.7 29.2 3.6 8.5 4.2 2.9d 1.5 11.3 19.6 2.1 3.0 10.5 61.6

N.A. 9.0 30.0 N.A. N.A. 0.5 3.1 0.2 9.6 1.5 11.0 40.0 N.A.

1.7 N.A. 1.8 1.4 2.5 0.7 0.5 2.3 1.6 2.5 0.4c 0.1 N.A.

19.0 22.1 3.9 5.4 1.3 0.5 4.9 2.2 12.7 1.5 6.7c 6.9 N.A.

81.4 60.3 39.3 15.3 8.0 4.6 10.0 16.0 43.5 7.6 21.1 70.1 61.6

a

From World Bank, 1998 (Ref. 18). Total, sum of columns to the left. c The values were calculated by dividing total production by population without considering imports and exports (from FAO Production Year Book, Ref. 17). d Includes buffalo meat. N.A., not available. Source: American Meat Institute 1997 (Ref. 20); FAO Production Year Book, 1997 (Ref. 17). b

Table 6 summarizes changes in meat consumption in countries that experienced rapid economic growth during the past three decades. Between 1970 and 1980, when per capita GNP increased from $887 to $16,696, Saudi Arabia experienced a more than quadruple increase in meat consumption, from 9.9 kg to 41.4 kg per capita. A similar pattern of expansion was observed in Japan, Singapore, and Korea. These countries lack land resources to expand livestock production, and the increase in consumption was in large part through imports. The increase in meat consumption in China from 12.6 to 39.3 kg per capita between 1990 and 1996 is tremendous considering the size of the population. The increase was mostly through an increase in pork production, and the phenomenal expansion was probably possible because of the economic reform in the late seventies, which allowed farmers’ rights to the land and free trading of the products. It is, therefore, easy to project that as incomes increase in developing countries, the demand for meat will continue to increase. Ac-

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Table 6 Changes in GNP and Per Capita Meat Disappearance in Selected Countries Per capita GNP ($)

Per capita meat disappearance (kg)

Countries

1970

1980

1990

1996

1970

1980

1990

1996

China Japan South Korea Philippines Saudi Arabia Singapore

120 1,969 266 230 887 916

280 8,873 1,528 690 16,696 4,597

410 26,100 5,770 750 5,174 13,497

750 40,940 10,610 1160 7,040 30,550

— 15.5 5.0 13.6 9.9 30.6

8.3 30.4 14.7 15.4 41.4 57.0

12.6 38.3 23.5 17.8 46.3 73.9

39.3 43.0 38.7 21.1 44.0 66.2

Meat includes beef, veal, buffalo meat, pork, lamb, mutton, goat meat, and poultry meat. The per capita meat disappearance in 1970, 1980, and 1990 was calculated using the following formula: per capita meat disappearance = (total meat production + import of fresh, chilled, and frozen meat - export of fresh, chilled, and frozen meat)/total population. Source: Per capita GNP is from UN Statistical Year Book (Ref. 21). The 1996 data is from American Meat Institute, 1997 (Ref. 20). The China data on per capita meat disappearance is from Colby et al., 1992 (Ref. 22), and other country data is from FAO Production Year Book (Ref. 17) and Trade Year Book (Ref. 23).

cording to a task force report by the international Council for Agricultural Science and Technology (9), total meat consumption in developing countries is projected to more than double by 2020. The task force also reported that global demand for meat is projected to increase by more than 60% of the current consumption by 2020 because developing countries are experiencing rapid population growth. In high-income countries, rising income appears to have little relation to meat consumption, but more effect on demand for meat quality as indicated by a USDA study (10). 4. International Meat Trade Meat is an easily perishable product that requires special attention during transportation; it also can be a carrier of zoonotic and livestock diseases. The uniqueness of meat as a commodity in combination with government restrictions on the import of livestock and its products limited an active international trading of meat. As a result, international trading volume is small as compared to the massive production of meat: in 1970 international trade was about 4.5% of total production, and in 1996 it was about 8% (Table 7). As will be discussed later in this section, the increase in international trade appears to be due to rising income in developing countries and the gradual lifting of trade barriers through negotiations of the General Agreements on Tariffs and Trade (GATT). Table 7 summarizes world meat trade by region in 1970 and 1996, and Table 8 shows meat trade in selected countries in 1996. Trade in Africa is small as compared to other regions. In 1970 Africa was a net exporting region (24 thousand metric tons), but in 1996 it became a net importing region (-212 thousand metric tons). Imports of beef and mutton by Egypt and South Africa are major components of trade in Africa. Asia has been a net importer, and the quantity of net imports was nearly 30 times higher in 1996 as compared to 1970. The expansion can be contributed to the increase in imports by Japan and other East Asian countries, including Hong Kong, Korea, Singapore, and Taiwan that experienced a rapid per capita income growth during this period. Japan leads in importing beef and pork. A substantial amount of broilers was also imported in 1996. The Japanese import of beef, pork, lamb, and broiler meat comprised about 43% and 14% of total Asian and world meat imports in 1996, respectively. Large quantities of

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Table 7 Changes in World Meat Tradea Import Countries World Africa N.C. Americab S. Americab Asia Europe Oceania

Export

Net export

1970

1996

1970

1996

1970

1996

4,438 (4.3%) 65 788 44 405 2973 21

16,216 (7.5%) 477 1,848 361 5,285 8,011 143

4,585 (4.5%) 89 432 822 200 1811 1183

17,579 (8.1%) 264 4,402 1,162 1,968 8,102 3,995

24 356 778 205 1258 1162

212 2,554 802 3,317 92 3852

a

Thousand MT. N.C., North and Central; S., South. The trade is in fresh, chilled and frozen meat. Source: FAO Trade Yearbook (1972, 1998, Ref. 23). b

broiler meat are both imported and exported by China and Hong Kong. Saudi Arabia also imports significant amounts of broiler meat and lamb. Taiwan has long been a major player in the international pork market. In 1996 Taiwan was the leading exporter of pork in Asia and ranked as the third largest pork exporter in the world, behind the United States and Denmark. Taiwanese exports have been mostly for the Japanese market, contributing about 44% of Japanese pork imports in 1996 (11). However, an outbreak of foot and mouth disease (FMD) in Taiwan during early 1997 virtually wiped out Taiwanese pork exports, causing a dramatic increase in pork prices in Japan during the initial period of the disease outbreak. It will probably take 4 to 5 years for Taiwan to obtain FMD-free status and to be able to resume exports of unprocessed pork. North and Central America was a net importer (356 thousand metric tons) in 1970, but changed into a large volume exporter in 1996 (2,554 thousand metric tons). The most significant contribution is from the export of poultry by the United States and of pork by Canada (Table 8). The United States is both the major importer and exporter of beef. The exports are mostly high-value grain-finished beef, whereas the imports are low-value forage-finished beef, which is used mostly for hamburgers or other processed products. South America and Oceania have been traditionally net exporting regions. Between 1970 and 1996, net exports in South America remained relatively stable, but net exports by Oceania more than tripled, from 1,162 to 3,852 thousand metric tons. Major beef exporters in South America are Argentina, Brazil, and Uruguay. Recently, exports of poultry by Brazil have significantly increased, making it the second highest poultry exporting country after the United States. Australia is the leader in beef exports. Lamb exports by New Zealand and Australia are more than 95% of world exports. As expected, the largest volume has been traded in Europe, but much of the trade is within the region. In 1970, Europe was a net importing region, but in 1996 turned into a net exporting region even though the volume was small (92 million metric tons). In Europe, Ireland is the leading beef-exporting country, and Denmark dominates pork exports. France and the Netherlands are the leading poultry exporting countries. Federated Russia imports significant amounts of beef, pork, and poultry, and is second after Japan in net imports of meat. As illustrated, international trade in meat has been steadily increasing, and this trend will continue with population expansion, the removal of trade barriers, and per capita inCopyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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Table 8 Meat Trade in Selected Countries 1996 a Beef and veal Countries Africa Egypt South Africa Asia China Hong Kong Japan South Korea Saudi Arabia Taiwan Europe Denmark France Germany Netherlands United Kingdom Russian Federation North America Canada Mexico United States South America Argentina Brazil Uruguay Ocenia Autralia New Zealand

Imports 110 69

Pork

Exports 0 3

Lamb and mutton

Broiler meat

Imports

Exports

Imports

Exports

Imports

Exports

N.A. N.A.

N.A. N.A.

5 30

N.A. N.A.

N.A. 39

N.A. 2b

N.A. 72 957 163b 30 67

105 5 N.A. 0b 0 N.A.

N.A. 175 822 4b N.A. N.A.

250 10 N.A. 4b N.A. 362

3 N.A. 75 12b 49b N.A.

1 N.A. N.A. 4b 1b N.A.

700 714 520 38b 240b N.A.

390 501 5 0b 0b N.A.

1 35 100 25 172 610

30 90 143 60 12 N.A.

1 3 0 N.A. 2 545

380 50 40 30 19 N.A.

N.A. 25 37 N.A. 21 9

N.A. 1 0 N.A. 2 N.A.

0 10 80 10 5 655

82 381 15 195 36 N.A.

235 75 940

260 2 851

50 30 280

340 5 431

N.A. 16 33

N.A. N.A. 3

68 98 0

30 N.A. 2,090

4 100 N.A.

450 315 170

N.A. 5 N.A.

N.A. 65 N.A.

4 N.A. N.A.

1 N.A. N.A.

25 0 N.A.

13 530 N.A.

N.A. 3

1,097 505

4 N.A.

5 N.A.

N.A. N.A.

280 400

N.A. N.A.

N.A. N.A.

a

Thousand MT. Data from FAO Trade Year Book, 1996 (Ref. 23). N.A., not available. Source: American Meat Institute (Ref. 20). b

come increases in developing countries. Countries in the land-rich regions of North and South America and Oceania will probably continue to supply the demand from countries that have high income but are deprived of the natural resources to expand livestock production. In addition, the land-rich areas of Russian Federation and the Ukraine have the potential to greatly expand livestock production to reduce imports and potentially become net exporters. II. MEAT PRODUCTION SYSTEMS The increase in human population and in demand for meat and meat products will not stop in the near future. Particularly, significant increases in the demand for meat and meat products along with population expansion will be experienced in developing regions, where the efficiency of animal production is far below that of industrialized countries. Thus, the chalCopyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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Table 9 Changes in the Performance of Pork Production

Number of pigs/sow Percent carcass yield Percent lean Ratio lean pork per sow (1951
1) Kilogram of feed/kg of pork

1951

1991

6.2 78 48 1 16.1

12.1 80 50 1.83 9.8

Source: Adapted from Surgeoner and Dalrymple, 1993 (Ref. 24).

lenge is to increase meat production without large increase in farm land use and without adverse impacts on the environment. Improvement in production efficiency is likely to be a key answer to this challenge. In fact, without the improved efficiency in livestock production achieved during the plast century, the current level of meat production would not be possible. Table 9 is an example of the improvement in the efficiency of pork production achieved in Ontario, Canada, during the plast 40 years. In 1951 one sow produced an average of 6.2 finished pigs per year, and by 1991 the average had increased to 12.1 finished pigs. Improvements in carcass yield and lean composition had also occurred during the same period. In 1951 the feed required to produce 1 kg of lean pork was 16.1 kg, and in 1991 it was reduced to 9.8 kg of feed. Without these improvements, almost twice as many sows and twice as much feed would be needed to produce the pork needed to fill today’s demand. The savings in the number of sows means that less feed and other capital and management expenses are used for breeding stock. The savings in feed means that less land acreage is used to produce a unit of pork today as compared to 1951. The improvement in production efficiency is also evident in other sectors of animal production, including dairy, beef cattle, poultry, and egg production. In addition, increases in the production efficiency of feed crops such as corn and soybeans also significantly contribute to reduced land requirements per unit of meat production. Meat production is a complex process, including multiple biological, environmental, economic and social factors. The knowledge of various types of production systems is expected to bring an understanding on the interaction of meat animal production and the environment. Understanding the components involved in meat animal production system is crucial in improving the efficiency of meat production. Because a thorough and detailed description of production systems and the components involved in production is far beyond the scope of this book, a brief description of various types of meat production systems and the general features of components involved in production will follow. A. General Features of Meat Animal Production Systems Figure 2 illustrates a simplified diagram of the general features of a meat-producing system. Three essential biological components of a meat production system are the breeding herd, growing herd, and feed production. These three components are depicted in the central block of the illustration. Supporting the operation of these essential components in producing market animals for slaughter are components associated with system management, environmental (waste) management, animal husbandry, and animal health and welfare. The conversion of market animals into meat involves a slaughter process, meat processing, and distribution.

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Figure 2 General features of a meat-producing system.

Meat production operations takes various forms and degrees of complexity depending on livestock species, geographic conditions, level of a nation’s economic development, social and cultural conditions, national policy, and level of effective demand. Prominent differences in operation characteristics are found between industrialized and developing regions. In industrialized countries, the meat production operation has become intensive and highly commercialized, with meat-producing animals being raised for the sole goal of meat production on a large scale with a high technology and management adoption. The production system is often stratified and specialized, meaning that the three essential components—breeding herd, growing herd, and feed production—may be integrated as an individual operational unit or may be separated into different combinations of each components. In addition, most operations are highly automated, with substantial dependence on mechanical and electrical devices. In developing countries, however, meat production is quite often a part of multi-purpose subsistence operation of animal production. In many of the developing countries in Asia and Africa, a large proportion of agricultural land is still plowed, and merchandise is carried to market by draft animals. Animal wastes supply a significant proportion of fuel energy and are an important source of fertilizer. B. Types of Production Systems Livestock production systems can be classified based on integration with crops, relation to land, agro-ecological zone, intensity of production, and type of product (12). Using the first two criteria, livestock production systems have been divided broadly into grassland-based systems (grazing system), mixed crop-livestock systems, and landless industrial systems (9). Classification into the three systems is helpful in understanding the interactions of live-

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stock production with resource utilization and the environment. Because meat animal production systems are a part of livestock production systems whose essential features are almost identical except for the end products, meat production systems will not be separately discussed from livestock production systems. 1. Grazing In grazing systems, animals (mostly ruminants) generally harvest forages in an area not suited for food crop production. Globally it is estimated that about 23% of beef and veal and 30% of sheep and goat is produced by grazing (Table 10). Grazing systems are distributed in a wide range of agro-ecological zones, including temperate and tropical high lands, humid/subhumid tropics and subtropics, and arid/semi-arid tropics and subtropics. Therefore, matching genotypes with the environment is important in production operations, and a wide range of cattle breeds are found in these systems. Nutritional output is quite variable, depending on regions, and so is the level of production and management intensity. Because nutritional output from this system is variable and often not enough to support the full growth potential of animals, reproductive performance is often compromised, leading to poor production efficiency. For growing animals, inadequate nutrition can make them take longer to reach market weights. The fact that the meat is produced from relatively older animals, and that forage is the sole diet for production, affects meat quality characteristics. Consumers accustomed to feedlot-finished beef often find that forage-finished beef is less palatable than grain-finished beef. In the past the low-input grazing system met a large proportion of regional demand for meat and meat products. Recently, however, in developing regions the demand arising from population expansion, income growth, and urbanization has exceeded production capacity. This has often led to overuse of range land or deforestation of rainforest area, thus generating environmental concerns including the impacts on water resources and biodiversity. More detailed discussion of these issues is available from Seré and Steinfeld (12) and the Council for Agricultural Science and Technology (9). 2. Mixed Crop-Livestock Production Systems In a mixed crop–livestock production system, the livestock element is interwoven with crop production. This system is widely distributed in developed and developing economies in a wide range of agro-ecological zones, and it plays a critical role in maintaining ecological and economic stability by providing draft power, fuel, transportation, and fertilizer (manure) in addition to providing human food. Globally, 65% of beef and veal, 100% of buf-

Table 10 Global Meat Products Produced by the Three Production Systems Grazing Products Beef and veal Buffalo Sheep and goat Pig meat Poultry meat

Mixed crop-livestock

Industrial

1,000 MT

Percent

1,000 MT

Percent

1,000 MT

Percent

12,289 0 2,981 685 796

23.4 0.0 30.0 1.0 1.8

32,249 2,652 6,860 42,821 10,469

65.1 100 69.0 59.8 24.2

6,055 0 100 28,163 31,967

11.5 0 1.0 39.3 73.9

Source: Adapted from CAST, 1999 (Ref. 9).

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falo, 69% of sheep and goat, 60% of pig meat and 24% of poultry meat are produced from the mixed crop–livestock production systems (Table 10). Resources to increase animal production and to improve the efficiency of production are widely variable, depending on ecological locations. In an area where crop production is efficient due to ample rainfall and irrigation, livestock are fed on crop residues and food byproducts, adding value to low opportunity-cost inputs as well as providing nutrient recycling. According to Fadel (15), globally every 100 kg of human food produced yields 37 kg of animal feed by-products, emphasizing the importance of incorporating animal production into human food production systems. In developing countries, inadequate feeding, poor management of animals, inappropriate land utilization policy, and poor infrastructure are often constraints on meat production in these systems. In addition, animal loss from diseases and parasites along with poor management is significant, causing low productivity. One of the biological constraints on meat animal production is poor feed conversion efficiency by the animals. Often adoption of genetics and technologies developed for intensive industrial production systems have failed to improve the efficiency of animal production in developing countries. Careful adoption of appropriate technology will play a key role in improving efficiency of production in these systems. In high population areas in temperate locations, these systems are transforming into intensive operations specializing in one type of animal from multi-purpose animal operations. The soaring demand of meat and meat products arising from urbanization and income growth in developing countries may disturb environmental stability. Soil degradation may result from overstocking animals and from expanding crop production to marginal land or nutrient surpluses on the land may result from heavy commercial fertilizer and manure applications and increased numbers of animals. 3. Landless Industrial Systems In landless industrial systems, feed is introduced from outside the farm; thus, decisions on feed production, feed utilization, and manure utilization/disposal are separately made. High-energy rations, including cereal grains, oil seeds, and their by-products, are fed to animals to support maximum growth. Cereal grains and oil seeds are dense in energy content compared to other forms of feed, so cereal grains can be easily transported to support landless industrial production. The abundance of cereal grains, oil seeds, and their by-products after World War II allowed the development of intensive industrial livestock production systems in developed countries where strong demand existed, and more recently in developing countries where demand has been created by urbanization and rapid income growth. The recent rapid increase in global pork and poultry production is contributable to the expansion of these production systems. About 12% of beef and veal, 39% of pig meat, and 74% of poultry meat is produced from these systems (Table 10). Feed is usually the highest-cost item in almost all meat-producing operations, and this is particularly true for the intensive production of chicken and pigs—feed cost can be up to 80% of the total costs of producing market animals. Feedlot finishing of beef cattle shares similarities with the intensive production of chicken and pigs except that cattle need more fibrous rations. Feedlot finishing beef cattle is highly concentrated in developed countries, where strong demand for high-quality beef exists. In North American feedlot operations, beef cattle are finished to market requirements by feeding relatively high grain rations for 90 to 150 days. The cereal grains used for animal feed often compete with human consumption and industrial uses. Economic factors such as price, supply, and the use and value of the endproducts determine these competing demands. In this regard, feed-conversion efficiency Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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becomes an important productivity indicator and management objective in intensive industrial production systems (12). Genetics are selected and improved to maximize the efficiency of feed utilization and to fit with the production practices. Matching genotypes with the environment is not as much of a consideration as long as the market allows for the costs involved in environment control. In order to take advantage of economies of scale, these systems are often large-scale, capital-intensive, and technology-oriented. Because the amount of manure production often exceeds the capacity for recycling through crop production, or because recycling of manure is not economically permissible in an intensive industrial production system, disposal of manure often creates a major environmental impact as well as adding the dimension of pollution by odors. In addition, the increasing use of grain through the expansion of intensive livestock production systems has become a public concern in association with efficient resource allocation and the environmental impact of expanding crop production for livestock feed supply (13). Although the net amount of grain used for feed has continued to increase, the percentage of world cereal grains used for animal production has stabilized at around 40% since the late 1960 (14). The issue of the impact of cereal grain use for animal production on resource allocation and the environment is complex in nature, involving political, economic, and social aspects. A recent report by the Council for Agricultural Science and Technology (9) provides excellent perspectives on this issue. III. ANIMAL SCIENCE AND MEAT PRODUCTION As an applied science, the primary goal of animal science is to improve the production of animal products, including meat, milk, wool, and eggs, as well as to provide wholesome and safe animal products. To achieve these goals, many disciplines are directly involved in the field of animal science, including biochemistry, nutrition, microbiology, genetics and breeding, physiology, and endocrinology. In addition, other programs such as food science, agronomy and soil science, veterinary medicine, engineering, environmental science, and economics are closely associated with the field of animal science. New disciplinary areas or programs will continuously interact with and enrich the field of animal science in response to new emerging challenges and the development of technology in various fields. The application of a wide range of biotechnology strategies is likely to play a key role in improving the efficiency of meat production as well as the quality and safety of meat and meat products. Chapter 6 in this book provides a detailed account of biotechnology options to improve the efficiency of production and the quality and safety of products. ACKNOWLEDGMENTS Dr. Halina Zaleski is sincerely acknowledged for her critical review of the manuscript. REFERENCES 1.

FAO/WHO/UNU. Energy and Protein Requirements, Reports of the Joint FAO/WHO/UNU Expert Consultation. Technical Report Series No. 724. FAO, WHO, and the United Nations University, Geneva, Switzerland, 1985. 2. PL Pellett, VR Young. Role of meat as a source of protein and essential amino acids in human protein nutrition. In: AM Pearson, TR Tutson, ed. Meat and Health. London and New York: Elsevier Applied Science, 1990, pp 329–379.

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8. 9. 10. 11. 12.

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

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National Research Council (NRC). Designing Food: Animal Product Options in the Marketplace. Washington, DC: National Academy Press, 1988. ER Monsen. Iron nutrition and absorption: dietary factors which impact iron bioavailability. J Am Diet Assoc 88:786–90, 1988. CA Finch, JD Cook. Iron deficiency. Am J Clin Nutr 39:471–477, 1984. E DeMaeyer, M Adiels-Tegman. The prevalence of anemia in the world. World Health Statistics Quarterly 38:302–315, 1985. US Department of Agriculture/US Department of Health and Human Services. Nutrition Monitoring in the United States: A Progress Report from the Joint Nutrition Monitoring Evaluation Committee. DDHS Publication No. (PHS) 86–1255. Washington, DC: US Government Printing Office, 1986. RA Lawrie. Meat Science. 6th ed. Lancaster, PA: Technomic Publishing Company, Inc., 1998, pp 1. Council for Agricultural Science and Technology (CAST). Animal Agriculture and Global Food Supply. Ames, IA, 1999. R Rizek. The 1977–1978 Nationwide Food Consumption Survey. Family Ecom Rev USDA, Sci Ed Adm, 1978 Fall. J Fabiosa, R Clemens, D Hayes. CSF in the Netherlands and FMD in Taiwan: Implications for the World Pork Market. U.S. Meat Export Analysis and Trade News 5(8):1–12, 1997. C Seré, H Steinfeld, J Groenwold. World Livestock Production Systems. FAO Animal Production and Health Paper 127, Food and Agricultural Organization of the United Nations, Rome, 1996. AB Durning, HB Brough. Taking Stock: Animal Farming and the Environment. Worldwatch Paper 103, Washington, DC: Worldwatch Institute, 1991. LR Brown, N Lenssen, H Hane. Vital Signs—The Trends That Are Shaping Our Future. Washington, DC: Worldwatch Institute, 1995, pp 35. JG Fadel. Quantitative analysis of selected byproducts feed stuffs: A global perspective. Anim Feed Sci Technol 79:259–268, 1999. Food and Agricultural Organization of the United Nations. The Sixth World Food Survey. Rome, Italy, 1996. Food and Agricultural Organization of the United Nations. Production Year Book, Rome, Italy, various years. World Bank. World Development Indicators. Washington, DC, 1998. United Nations, UN Population and Vital Statistics Report. New York, NY, various years. American Meat Institute. Meat and Poultry Facts. Mt. Morris, IL: Watt Publishing, 1997. United Nations. UN Statistical Year Book, Washington, DC., various years. WH Colby, FW Crook, SHE Webb. Agricultural Statistics of the People’s Republic of China (1949–90). USDA Statistical Bulletin No. 844. Washington, DC, 1992. Food and Agricultural Organization of the United Nations. Trade Year Book, Rome, Italy, various years. GA Surgeoner, JR Dalrymple. Improving efficiencies in Ontario pork production. Agri-Food Research in Ontario 16(3):8–13, 1993.

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25 Meat Co-Products DENG-CHENG LIU National Chung-Hsing University, Taichung, Taiwan HERBERT W. OCKERMAN The Ohio State University, Columbus, Ohio

I. INTRODUCTION II. CLASSIFICATION, PRODUCTION, AND UTILIZATION III. NUTRITIONAL VALUES IV. UTILIZATION OF BLOOD A. Isolation of Blood B. Removal of Heme from Red Blood Cells C. Usage of Blood Plasma in Food D. Medicinal and Pharmaceutical Usage of Blood V. UTILIZATION OF HIDES AND SKINS A. Stacking of Hides and Skins B. Processing of Leather from Hides and Skins C. Gelatin from Hides and Skins D. Uses of Gelatin in the Food and Pharmaceutical Industry E. Hides and Skins for Food and Sausage Casing F. Medicinal and Pharmaceutical Usage of Hides and Skins VI. UTILIZATION OF BONE A. Gelatin from Bone B. Liquid Extraction from Bone C. Mechanically Separated Meat from Bone D. Medicinal and Pharmaceutical Usage of Bone VII. UTILIZATION OF GLANDS AND ORGANS A. Glands and Organs as Food B. Medicinal and Pharmaceutical Usage of Glands and Skins VIII. UTILIZATION OF EDIBLE TALLOW AND LARD IX. THE PROCESSING AND UTILIZATION OF MEAT EXTRACT X. CONCLUSION REFERENCES

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I. INTRODUCTION Meat co-products are produced by slaughterers, processors, wholesalers, retailers and renderers. Traditional markets for edible meat co-products have gradually been disappearing because of concerns about health and economic returns. In response to these problems, meat processors have directed marketing and research efforts toward inedible applications—for example, pet foods, pharmaceuticals, cosmetics, and animal feeds. The literature indicates that co-products (including organs, fat or lard, skin, feet, abdominal and intestinal contents, bone and blood) of cattle, pigs, and lambs represent 66.0%, 52.0%, and 68.0% of the live weight, respectively. Over 50.0% of animal co-products are not suitable for human or animal consumption due to unusual physical and chemical characteristics (1). A valuable source of potential revenue is lost and the cost of disposal of these products incurred by the meat industry is increased if meat co-products are not efficiently utilized. The USDA Economic Research Service announced that the portion of gross farm economic income from animal co-products is 11.4% for beef and 7.5% for pork for 1986 (2). The cost of live animals often exceeds the selling price of their carcasses and the value of the co-products must pay the expense of slaughter and generate the profit for the meat-slaughtering operation. Bengtsson and Holmqvist have suggested that 7% to 12% of the income from slaughter results from the sale of co-products (3). In addition to economic loss, nonutilization of meat products would create serious environmental pollution of water and air. However, with efficient utilization, meat co-products can be important and result in profits for meat processors. The modern livestock industry in the past has been an effective utilizer of co-products and it has often been stated that all of the pig is used except the squeal.

II. CLASSIFICATION, PRODUCTION, AND UTILIZATION The U.S. meat industry considers everything produced by or from the animal, except dressed meat, as a co-product. Therefore, animal co-products in the United States are divided into two divisions, edible and inedible. In U.S. terminology, offal means slaughter co-products and includes all of the animal that is not a part of the carcass. Variety meats are the wholesale edible co-products that are segregated, chilled, and processed under sanitary conditions and that are inspected by the U.S. Meat Inspection Service. In some areas of the world, and to different degrees, blood is also utilized as an edible product for humans. In the United States, meat trimmings from the head are described as edible offal or edible co-product items, and edible fats are fats obtained during slaughter, such as caul fat surrounding the rumen or stomach, and cutting fat, which is back fat or pork leaf fat, or rumen fat. In English commercial slaughterhouse practice the offal is divided into red (heart, liver, lungs, head, tongue, and tail) and white (fat), set of guts and bladder, set of tripe and four feet and trimmings (4). The English Food Standard Committee also separated offal into two categories (5): List A. Items that may be used in cooked or uncooked products from mammalian species; contain tissues such as diaphragm (skirt, cattle only), head meat (ox cheek, cattle only; bath chip, pig only), heart, kidney, liver, pancreas, tail meat, thymus, and tongue and avian parts such as heart and liver.

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List B. Items that may not be used in uncooked products; contain portions of mammalian species such as blood, blood plasma, brain, feet, large intestines, small intestines, lungs, esophagus meat, rectum, spinal cord, stomach (nonruminant), first stomach (tripe, after cooking), second stomach (tripe, after cooking), fourth stomach, testicles, and udder and parts of avian species such as gizzard and neck. The evaluated quantities of co-products from pork, beef, and sheep are shown in Table 1. The yield of edible meat co-product from animals ranges from 6.7% of the carcass weight for pork and the world production of edible co-products from pig in 1996 is shown in Table 2. A total of 5,655.5 thousand metric tons of pork edible co-products were produced in 1996, and data in Table 2 show that the largest part of the production of pork co-products is from Asia (50.4%) and the second from Europe (37.1%). Asia and Europe are also the two major consumers of meat co-products including beef and lamb (1). Usage of meat co-products often require treatment by the following steps: collection, washing, trimming, chilling, packaging, and cooling. Acceptance of these products depends on factors such as acceptability, regulatory requirements, nutrition, economics, and competitive products. Although customs, culture, and religion often act as major factors when a meat co-product is utilized as an ingredient in meat products, regulatory requirements also are important factors because many countries already had some food regulations on the policy of food safety and quality. An example of an USDA requirement is that mechanically separated meat and variety meats must be specifically identified by showing them as ingredient on labels. If frankfurters and bologna are made with heart meat or poultry mechanically separated meat as an ingredient, it must be listed. A detailed list of potential uses and preparations of meat co-products are listed in Table 3.

Table 1 Percentage of Marketing Live Weight of By-products from Various Species (hog, cattle, and sheep) Hog Item Marketing live weight Carcass Bone Blood Fatty tissue Hide or skin Organs Head Viscera (chest and abdomen) Feet Tail Brain

Cattle

Percent

Kilogram

77.5 17.0 3.0 3.0 6.0 7.0 5.9 10.0 2.0 0.1 0.1

100.0 77.5 17.0 3.0 3.0 6.0 7.0 5.9 10.0 2.0 0.1 0.1

Sheep

Percent

Kilogram

Percent

63.0 16.0 3.0 4.0 6.0 16.0

600.0 378.0 96.0 18.0 24.0 36.0 96.0

62.5 18.0 4.0 3.0 15.0 10.0

60.0 37.5 10.8 2.4 1.8 9.0 6.0

16.0 2.0 0.1 0.1

96.0 12.0 6.0 6.0

11.0 2.0

6.6 1.2

Source: From Refs 1, 3, 80, 81, 82, 83, and 84.

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0.26

Kilogram

0.156

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Table 2 World Production of Edible Co-products from Pigs, 1996 (thousand metric tons) Country

Carcass wt.

Productiona

Percentage

77,985 42,534 40,000 1,264 1,270 9,005 7,765 1,240 1,560 1,560 16,269 3,085 2,193 2,180 1,679 1,619 1,600 1,528 1,355 1,030 15,043

5225.0 2849.8 2680.0 84.7 85.1 603.3 520.2 83.1 104.5 104.5 1090.0 206.7 146.9 146.1 112.5 108.5 107.2 102.4 90.8 69.0 1007.9

100.0 50.4 47.4 1.5 1.5 10.7 9.2 1.5 1.8 1.8 19.3 3.7 2.6 2.6 2.0 1.9 1.9 1.8 1.6 1.2 17.8

World Asia China Japan Taiwan North America U.S.A. Canada South America Brazil Europe Germany France Spain Russia Netherlands Poland Denmark Italy Belgium-Luxemburg European Union a

Based on 6.7% of carcass weight. Source: National Pork Producers Council (NPPC), pork facts, 1997/1998, USA (85).

III. NUTRITIONAL VALUES Edible meat co-products contain many essential nutrients. Some edible meat co-products often are used as medical cures because they contain special nutrients such as amino acids, hormones, minerals, vitamins, or fatty acids. Except for blood, many meat co-products have higher levels of moisture than meat. Examples would be lung, kidney, brain, spleen, and tripe. Some organ meats such as liver and kidney contain a higher level of carbohydrates than other meat material; pork tail has the highest fat and the lowest moisture level of all the meat co-products. Liver, beef tail, ears, and feet have the closest protein level when compared with lean meat tissue but a large amount of collagen is found in the ears and feet (6,7). The lowest protein level of meat co-products is found in the brain, in chitterlings, and in the fatty tissue. USDA (1983, 1986) states that mechanically deboned beef and pork are required to contain at least 14% protein and a maximum of 30% fat (2,8). The amino acid composition of meat co-products is different from that of lean tissue due to the high amounts of connective tissue; this results in a larger amount of proline, hydroxyproline, and glycine and a lower level of tryptophan and tyrosine for co-products such as ears, feet, lungs, stomach, and tripe (9). The vitamin content of organ meats is usually greater than that of lean meat tissue. Kidney and liver contain the highest amounts of riboflavin (1.697 to 3.630 mg/100g) and have 5 to 10 times more than lean meat. Liver is the best source of niacin, vitamin B12, B6, folacin, ascorbic acid and vitamin A. Kidney also is a good source of vitamin B6, B12, and folacin. A 100 g serving of liver from pork and beef contributes 450% to 1100% of the RDA for vitamin A, 65% of the RDA for vitamin B6,

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Table 3 The Potential Uses and Preparation of Edible Meat Co-products Kind Beef and veal Liver Kidney Heart Brains Tongue Tripe

Sweetbread Oxtail Intestine (small and large)

Cheek and head trimmings Udder

Storage and preparation Frozen, fresh, or refrigerate Whole, sliced, or grind Fresh or refrigerate Whole or sliced Frozen, fresh, or refrigerate Whole or sliced Frozen, fresh, or refrigerate Whole Fresh, refrigerate, smoked, or pickled Fresh, refrigerate, precooked, pickled, or soak before use Frozen, fresh, or refrigerate Whole Frozen, fresh, or refrigerate Fresh or refrigerate Remove manure, soaking, washing, and salting before use Frozen, fresh, or refrigerate Frozen, fresh, or refrigerate

Skin Feet Fat

Fresh or refrigerate Frozen, fresh, or refrigerate Frozen, fresh, or refrigerate

Blood

Frozen or refrigerate

Bone

Frozen, fresh, or refrigerate

Pork Liver Kidney Heart

Brains Tongue Stomach

Frozen, fresh, or refrigerate Whole, sliced, or grind Fresh or refrigerate Whole or sliced Frozen, fresh, or refrigerate Whole, sliced Frozen, fresh, or refrigerate Whole Fresh, refrigerate, smoked, or pickled Fresh, refrigerate, or precooked

Methods of usage Braised, broiled, fry, loaf, patty, and sausage Broiled, cooked in liquid, and braised Braised, cooked in liquid Broiled, braised, and cooked in liquid Cooked in liquid Fry, broiled, and cooked in liquid Fry, broiled, braised, and cooked in liquid Cooked in liquid Sausage casing

Cooked sausage, stew, soup, and bouillon Boiled, fried, smoked, and salted Gelatin Jelly Shortening, drippings and chewing gum Black pudding, sausage, blood, and barley loaf Gelatin, soup, jellied products, and refining sugar Braised, broiled, fry, loaf, patty, and sausage Broiled, cooked in liquid, braised, soup, grill, and stew Braised, cooked in liquid, luncheon meat, patty, loaf, and sausage ingredient Broiled, braised and cooked in liquid, poach, and scramble Cooked in liquid, cured, sausage ingredient, salad, and jelly Broiled and cooked in liquid, sausage container, and sausage ingredient

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Table 3 Continued Kind Spleen Tail Intestine (small and large)

Cheek and head trimmings Ear

Storage and preparation Frozen, fresh, or refrigerate Whole Frozen, fresh, or refrigerate Fresh or refrigerate Remove manure, soaking, washing, and salting before use Frozen, fresh, or refrigerate

Fry, pie, melt, and blood sausage

Frozen, fresh, or refrigerate

Smoked and salted, stew with feet Gelatin Jelly, pickled, cook in liquid, boiled, fried Shortening, lard Black pudding, sausage, blood and barley loaf Gelatin, soup, jellied products, and rendered shortening, mechanically deboned tissue Blood preparation and pet food

Skin Feet

Fresh or refrigerate Frozen, fresh, or refrigerate

Fat Blood

Frozen, fresh, or refrigerate Frozen or refrigerate

Bone

Frozen, fresh, or refrigerate

Lung Lamb Liver

Frozen, fresh, or refrigerate

Kidney Heart

Frozen, fresh, or refrigerate Whole, sliced, or grind Fresh or refrigerate Whole or sliced Frozen, fresh, or refrigerate Whole or sliced

Tongue

Frozen, fresh, or refrigerate Whole Fresh or refrigerate

Stomach

Fresh or refrigerate

Sweetbread

Frozen, fresh, or refrigerate Whole

Spleen

Frozen, fresh, or refrigerate

Intestine (small and large)

Fresh or refrigerate Remove manure, soaking, washing, and salting before use Frozen, fresh, or refrigerate

Brains

Cheek and head trimmings Testicles Lungs Feet

Methods of usage

Frozen, fresh, or refrigerate Fresh or refrigerate Frozen, fresh, or refrigerate

Cooked in salt liquid Sausage casing

Cooked sausage

Braised, broiled, fry, loaf, patty, and sausage Broiled, cooked in liquid, braised, fried, stew, and soup Braised, cooked in liquid, roasted, stuff, luncheon meat, patty, loaf, and sausage ingredient Broiled, braised and cooked in liquid, poach, and fried Boiled, stew, jelly, grilled, and cooked in liquid Honeycomb tripe and container for haggis Fry, broiled, braised, poach with sauce, cream and cooked in liquid Pie, melt, blood sausage ingredient, and variety meat Sausage casing

Cooked sausage, stew, and soup Fried Haggis, pet food Jelly

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Table 3 Continued Kind

Storage and preparation

Fat

Frozen, fresh, or refrigerate

Blood

Frozen or refrigerate

Bone

Frozen, fresh, or refrigerate

Methods of usage Shortening, drippings, sweets, oleomargarine, and chewing gum Black pudding, sausage, blood and barley loaf Gelatin, soup, jelly, and mechanically deboned tissue

Source: From Refs. 1, 82, and 86.

3700% of the RDA for vitamin B12, and 37% of the RDA for ascorbic acid. In addition to supplying vitamins, lamb kidney, pork liver, lungs, and spleen are often act an excellent source of iron. The copper content is the greatest in the livers of beef, lamb, and veal. They contribute 90% to 350% of the RDA for copper (2 mg/day). Livers also contain the highest amount of manganese (0.128 to 0.344 mg/100 g). However, the highest level of phosphorus (393 to 558 mg/100 g) and potassium (360 to 433 mg/100 g) are found in thymus and sweetbread when compared with all meat co-products. With the exception of brain, kidney, lungs, spleen, and ears, most other co-products contain sodium at or below the levels found in lean tissue. Among the raw material, mechanically deboned meat has the highest calcium content (315 to 485 mg/100 g). Many organ meats contain more polyunsaturated fatty acid than does lean tissue. Brain, chitterling, heart, kidney, liver, and lung have the lowest level for monounsaturated fatty acids and the highest amount of polyunsaturated fatty acid. In addition to higher levels of cholesterol (260 to 410 mg/100 g), which are three to five times higher than those of lean meat, large quantities of phospholipid also are found in these meat organs. Brain is the highest in cholesterol (1352 to 2195 mg/100 g) and also has the highest amount of phospholipid when compared with other meat co-products (10). Based on these data, USDAUSDHHS recommended limiting the quantity of cholesterol and the amount of these coproducts in the diet because of health-related concerns (11). A high-cholesterol content in many organ meat and the possible accumulation of pesticides, residues of drugs, and toxic heavy metal contribute to the recommendation for limited consumption. IV. UTILIZATION OF BLOOD Animal blood has a high level of protein and heme iron and is an important animal co-product. In Europe, animal blood has been used in making blood sausages, blood pudding, biscuits, and bread for a long time. And in Asia, it also has been used in blood curd, blood cake, and blood yogurt-like blood pudding (12). It is also used in nonfood systems such as in fertilizer, feedstuffs, and binders. The Meat Inspection Act stated that blood is approved for food use when removed by bleeding of an animal that has been inspected and passed for use in meat food products. Basically, blood is usually sterile in a healthy animal and is high (17.0% to 18.0%) in protein that is reasonably well balanced in amino acid composition. Blood is a significant part of the animal’s mass (2.4% to 8.0% of the animal’s live weight) and the average percentage of blood that can be recovered from pigs, cattle, and lambs is 3.0% to 4.0%, 3.0% to 4.0% and 3.5% to 4.0%, respectively. Because use of blood in meat processing results in the final product being dark and often unpalatable, and because

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plasma has a more desirable color and functional properties, plasma is the portion of blood that is of great interest. A. Isolation of Blood Plasma has been widely used in the meat industry because of its excellent functional properties (13). Plasma contains one-third of the total blood protein, and red blood cells contain the other two-thirds (14). On a large-scale processing of edible whole blood, it is separated into two constituents: plasma (60% to 80%) and red cells (20% to 40%). The plasma consisting of 7.0% to 8.0% protein and 91% water, is cooled, frozen or dried. The red cells, consisting of 34% to 38% protein and 62% water, is dried to form a meal or the heme group is removed to obtain globin. Anticoagulants (0.2% sodium citrate or citric acid or 10.0 gram of a mixture of phosphates—22.0% Na2HPO4, 22.0% Na4P2O7, 16.0% Na2H2P2O7 and 40.0% NaCl per liter of blood) are normally used in collecting whole blood and, are injected via a hollow knife if a vacuum transport system is adopted. Continuous blood-separation equipment is used and the separation of the fractions is accomplished with a highspeed centrifuge or separator. After separation, the plasma is frozen or spray dried at low temperature in order to maintain its solubility and functional ability. To freeze blood plasma, it is normally placed on a vertical rotating drum that has a temperature of between 10°C (14°F) and 40°C (40°F) and then the frozen plasma is scraped from the surface in the form of a flake. When blood is dried, great care must be taken to prevent denaturation of the protein because this lowers the quality of the dried fraction. Concentration is the first step for the blood plasma drying process in most plants and generally is accomplished by membrane filtration and evaporation. The drying process of the concentrated plasma is finished by a spray drying system or a fluidized bed drying system. A dried blood plasma can be produced with 96.4% protein and 2.4% moisture by this processing technique. B. Removal of Heme from Red Blood Cells Heme derived from animal blood is a valuable source of organic iron, which may be used as a supplement in foods (15). Heme pigment can be used as a red colorant for food and is used in Chinese semi-dried sausage (16). Several methods have, therefore, been developed to remove the heme group from hemoglobin of red blood cells. These include an acid-acetone method (17), hydrogen peroxide decoloration (18), and decoloration with carboxy methyl cellulose (NaCMC) (19) and sodium alginate (20). With these methods, the globin and heme can be easily separated and recovered. C. Usage of Blood Plasma in Food Blood in food is used as an emulsifier, stabilizer, clarifier, color additive, and nutritional component. Most blood is used in livestock feed in the form of blood meal and used as a protein supplement, milk substitute, lysine supplement, or vitamin stabilizer and is an excellent source of most of the trace minerals. Blood plasma has gel-forming ability because it contains 60.0% albumin and is the best water and fat binder of the blood fraction. Plasma gels appear very similar to cooked egg whites and plasma forms gel at protein concentrations of 4.0% to 5.0% and that its gel strength increases with increasing concentration (21). Cooked ham with the addition of 1.5% and 3.0% frozen blood plasma and hot dog with 2.7% were more satisfactory in color than the control samples (22). In addition to gel forming, blood plasmas also has excellent foaming capacity (22–25). For this reason, blood

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plasma can be used to replace egg whites in the baking industry. Nielsen recently indicated that the application of transglutaminase (TGase) from animal blood and organs or microbes to products in the meat industry is one of the most investigated applications in food processing (26–38). Blood factor XIII is a transglutaminase that occurs as a enzymogen in plasma, placenta, and platelets. The reaction catalyzed by Ca2 dependent factor XIIIa involves the formation of a -( -glutamyl)-lysyl bond between an acyle donor (glutaminyl residue) and an acyl acceptor (lysyl residue) of the proteins fibrin and fibronectin, fibrin and actin, myosin and fibronectin and myosin and actin (39). Therefore, this enzyme catalyzes conversion of soluble proteins to insoluble high-molecular polymers through formation of covalent cross-links (31,40). Transglutaminase extracted from bovine blood at first for improving the binding ability of fresh meat products at chilling temperature and showed how myosin was cross-linked by TGase in 1983 (27). An important property of the TGase reaction was documented when cross-linking between myosin and proteins (soy, casein and gluten) commonly used in meat processing was found (30). Moreover, the restructured meat products without heating, and decreased with salt and phosphates can be made by the addition of TGase from animal blood (28,32,33,41,42). D. Medicinal and Pharmaceutical Usage of Blood Blood can be separated into several fractions that have therapeutic properties. Liquid plasma is the largest fraction (63.0%) and consists of albumin (3.5%), globulin, and fibrinogen (4.0%). In the laboratory, many blood products are used as a nutrient for tissue culture media, as a necessary ingredient in blood agar and peptones for microbial use. Glycerophosphates, albumins, globulins, sphingomyelins, and catalase are also used for biological assay. Many blood components such as fibrinogen, fibrinolysin, serotonin, kalikrenins, immunoglobulins, and plasminogen are isolated and used in the chemical or medical aid. Purified bovine albumin is used to help replenish blood or fluid loss in animals, in testing for the Rh factor in human, as a stabilizer for vaccines, and in antibiotic sensitivity tests. Pork-blood fibrin extract is used as a source of amino acids, which are incorporated into parenteral solutions for nourishing some surgical patients. Superoxide dismutase (SOD) is an enzyme attending a series of reactions of superoxide radical and transfering it into water and oxygen to protect cell membranes of the animal body from serious damage by oxidation (43). SOD can be extracted from cattle blood for curing osteoarthritis, ischemia, and in anti-inflammatory treatment and so forth (44–48). Crude SOD from porcine blood exhibited a higher activity (1570 unit/mg), and the results are similar to the bovine erythrocyte SOD (49). A thin firm can be made from fibrinogen and used to control bleeding in surgery and also can be used as a spray or oral drug for gastric and intestine hemorrhages (50). Otherwise, the citrate-saline treated fibrinogen from porcine blood is an effective hemostat in animal surgery (51). Industrial uses of blood includes uses as an adhesive and film former in paper, plywood, fiber, plastic and the glue industry. Also used as a spray adjuncts with insecticides and fungicides and as a stabilizer in cosmetic base formulations. It also finds use as a foaming agent in fire extinguishers. V. UTILIZATION OF HIDES AND SKINS Animal hides and skins have been utilized for shelter, clothing, and weapons and as food containers by humans since prehistoric times. The hides and skins contain a very notable

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portion, 4.0% to 11.0% (e.g., cattle: 5.1%–8.5%, average: 7.0%; sheep: 11.0%–11.7%; swine: 3.0%–8.0%), of the weight of the live animal and generally are one of the most valuable animal co-products. Examples of finished product from cattle hide co-products, hog skin co-products, and sheep pelts co-products are the following: cattle cured and tanned hides—shoes, bags, belting, rawhide, athletic equipment; cattle corium layer—picking bands, textile shuttle holders and passers, reformed sausage casing, and cosmetic products; calf skin—lightweight leather, gloves, drum heads, and fabric trimmings; pig skin— sausage, edible gelatin, glue, gloves, belts, shoes; sheep slats (skin after wool or fleece is removed)—shoes and slippers, hat sweat bands, fancy shoes, gloves, sporting goods, diplomas; sheep pelts (wool or fleece left on)—heavy coat material, moutons, and shearlings. A. Stacking of Hides and Skins After hides and skins are removed from any animal, they should be quickly cured to stop bacterial and enzymatic decomposition or spoilage. There are four basic treatment for preserving these hides and skins: air drying, salt-pack curing, mixer curing, and raceway curing; salt-curing is commonly used to treat these raw materials. The quality of cured hides and skins are usually evaluated by measuring the moisture and salt content of the hides. The moisture level of the hides are generally maintained from 40% to 48% to result in good condition during storage or shipping. Some chemicals or insecticides such as sodium sulfite, acetic acid, white arsentic (As2O3), sodium silicofluoride (Na2SiF6), 1,2,3,4,5,6-hexachlorocyclohexane (Lindane; C6H6Cl6), 1,4-dichlorobenzene (C6H4Cl2), and pyrethrum, are often used to help protect against insect damage or for short-term preservation before tanning. B. Processing of Leather from Hides and Skins A general description of leather production can be found in the reports of Hague and Ockerman and Hansen and a summary is given of the processing (1,52). The cured hides should be stored in a cooled and well-ventilated tanner’s hide house when they arrive at the tannery. The first step of tanning is grading and sorting the hides into packs of uniform size, weight, and type of hide. The next step is soaking, and this means that enough moisture needs to be added to the cured hides for the succeeding tanning operations. The soaking of hides is finished in half-round cylindrical vats in which the hides are placed with water, wetting agents, and disinfectants; the hides are stirred in this solution by a dip-paddle wheel for 8 to 20 hours in order to let the hides reabsorb the needed water. A washing step after soaking removes the remaining dirt, manure, salt, and blood from the hides. The unhairing procedure is the next step; originally it was carried out by a process known as sweating, in which the hides were placed in the previously described paddle vats or mixer with an unhairing agent in a warm environment. The most common chemical depilatory agents are a saturated solution of calcium hydroxide [Ca (OH)2] and sodium sulfide (NaS) or sodium sulfydrate (NaHS) or milk of lime. Some hides, such as sheepskins and pigskins, contain a large quantity of fat and it is often desirable to reduce this to approximately 3.0% on a dry-weight basis; this process is sometime done by a hydraulic press to remove fat prior to continuation of the tanning process. “Bating” is used to remove the alkaline unhairing chemicals and other nonleather substances in the pelt structure and is performed in a large wooden drum. Bating makes the hide softer, less harsh, and cleaner. The next step is “picking,” which places the pelts in an acid. The hides need to have a low

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pH in order to accept the tanning materials, such as chrome. The major purpose of tanning is to convert the collagen fibers of the skin into a stable non-rotting leather. “Chrome tanning” is the most popular method of tanning today because it can be finished quickly and desirable physical and chemical properties can be produced. After tanning, the next procedure is “setting,” whose purpose is to lower the moisture content, smooth the grain, and remove wrinkles from the hide. Splitting and shaving follow and the purpose of these two steps is to obtain a uniform thickness of the leather desired for its ultimate use. The desired color of leather can be produced by a “dyeing” operation. The aim of coloring is not only to produce the right strength and shade of color but to produce a color that will resist fading and can be dry-cleaned or washed. “Fatliquoring” is used to adjust the firmness or softness of the leather by lubricating the fibers after coloring, and it also can increase the tensile strength of the leather. “Setting out” is accomplished to smooth and stretch the leather and to compress and squeeze out the excess moisture and grease. The purpose of drying is to remove all but equilibrium moisture, and after drying the skin should contain 10% to 12% moisture. The popular drying technique is called “pasting” in which the hides are actually pasted to large stainless steel or glass plates. After drying the leather is hard and fairly unworkable, and the final user always requires varying degrees of softness; additional moisture is applied by shower-like nozzles and this procedure is called “conditioning” or “wetting back.” “Buffing” is necessary to improve the appearance of the leather and to reduce any blemishes by light mechanical sanding. The next process is “finishing,” which is the application of film foaming materials that provide abrasion and stain resistance, enhance the color, and make the leather easy to care for. After the finishing, it must be dried in a long heating tunnel with steam-heated air. If a smooth grain surface or a various grain texture is needed on the leather, this can be accomplished by “platining,” which is obtained by pressing (300 ton/in2) for a few seconds. The final step is “grading” and it is dependent on temper, uniformity of thickness and color, and defects of the leather. The average total time of the processing of leather from raw skins or hides is 4 weeks. Leather is sold by the area; therefore, the hide needs be measured by a planimeter to calculate the total area of the piece of leather, then grouped into batches of four to five hides, rolled into a bundle, covered with paper, and packed in a wooden box for shipping. C. Gelatin from Hides and Skins Gelatin is produced by controlled hydrolysis of a water-insoluble collagen that belongs to a water-soluble, hydrophilic, derived colloidal protein. Gelatin is made from fresh, federally inspected raw materials (hide or bone) that are in an edible condition. The conversion of collagen to gelatin involves the breaking of hydrogen bonds that stabilize the triple-coil helix and transform it into the random coil configuration of gelatin. There are three basic types of new chains: alpha-chain, beta-chain, and gamma-chain in gelatin. A single gelatin has several molecular weights and this determines its characteristics, such as colloidal dispersion in water, viscosity, adhesiveness, and gel strength. The tissues that contain large quantities of collagen that are commercially available as a co-product are usually hides, skins, and bones; therefore, they are a major raw material source for the manufacturing of gelatin. A summary of the general description of gelatin production as described by Hinterwaldner is given as follows (53). The processing of gelatin from hide and skin consists of three major steps: The first step is the elimination of noncollagenous material from the raw material. This is followed

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by controlled hydrolysis of collagen to gelatin, and the final step is recovery and drying of the final extracted product. Generally, a collagen stock will be used to extract gelatin by combinations of an alkaline procedure, an acid procedure, and high-pressure steam extraction. The alkaline procedure is the most widely used commercial system for the processing of collagen to gelatin. A saturated solution of calcium hydroxide made by the addition of lime is used in this procedure and its usage in approximately 10% of the weight of the stock. This procedure causes the noncollagen compounds such as keratin, globulin, mucopolysaccharides, elastin, mucins, and albumins to be changed to more soluble products, and some of the fat is converted into polar compounds that can be removed easily by washing. After the liming of the hides, the collagen fibers are swollen and the internal cohesion of each fiber is decreased. The next step is washing and neutralization; the collagen is washed by cold running water for 1 to 2 days, and the pH of collagen is lowered and the lime is removed. By washing with dilute hydrochloric acid (HCl) or sulfuric acid (H2 SO4) until the collagen is limp, the collagen stock should have a pH between 5.0 and 8.0 and is ready to be extracted. Extraction is normally started at 54° to 60°C for 3 to 5 hours and is continued up to boiling. The highest quality product is obtained at the lower extraction temperature, but yield is increased at higher temperatures. The liquid extract needs be filtered to remove small particles; sometimes, activated carbon is also added to decolorize the gelatin solution. Generally, the extract obtained from higher temperatures needs to be vacuum evaporated in a pan so that a sufficient concentration and gel strength can be obtained when cooled. After concentrating, the gelatin is dried by many drying methods such as a cooled drying tunnel, drum drying, and spray drying. Another extraction procedure for gelatin is acid processing and it is usually applied to pig skin or bone. Pigskins are first washed to remove salt from salted skin and to remove extraneous matter and/or blood. Since pigskin often contains 8.0% to 15.0% fat, pre-extraction of this lipid material is necessary before the acid extraction procedure. This is done by heating in hot water (55°–60°C), two to three times, stirring for 4 to 6 hours, and then washing in 40° to 55°C water. After washing and removal of fat, the skins are soaked in a 5.0% inorganic acid (such as hydrochloric acid, sulfuric acid, or phosphoric acid), which results in a pH of approximately 4.0. This pH causes the collagen to swell and a great deal of solubilization to occur. After 10 to 72 hours of soaking, the acid is then drained and the collagen is washed to raise the pH of the skin to approximately 4.0 to 5.0. At this pH the native collagen is still swollen. After acid treatment, the collagen stock is extracted at a lower starting temperature than cow hides and the procedure is almost the same as the alkaline treatment. However, the gelatin produced from pigskins has a higher gel strength and better clarity and color than alkaline-treated cattle hide products and it also should be recognized that alkaline- and acid-precursor produced gelatin are two different classes of gelatin. Two grades of gelatin may be extracted; Class A, which is a high grade, with relatively undamaged molecular material, and Class B, which is extracted by harsh means and consequently has a range of molecular weights and altered properties. All gelatins are soluble and are able to form gels on cooling from hot solution and thus it is an important food additive. D. Uses of Gelatin in the Food and Pharmaceutical Industry Gelatin is added to a wide range of foods as well as forming the major constituent of confectionery jellies and aspics (54). Its major use is still the production of gel desserts because

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of its melt-in-the-mouth properties, but it is also added to a range of meat products, in particular to meat pies as a jelly component. Gelatin also has been used extensively as a stabilizer for frozen dairy products and other frozen desserts. High-bloom gelatin is added as a protective colloid to ice cream, yoghurt, and cream pies. The gelatin is thought to inhibit ice crystal growth and lactose recrystallization during storage. Approximately 6.5% of the total production of gelatin is used in the pharmaceutical industry, with the largest proportion used for capsule manufacturing (55). Gelatin also can be used as a binding and compounding agent in the manufacture of medicated tablets and pastilles. It is used as an important ingredient in many specialized protective dressings such as zinc gelatin or Unna’s paste for the treatment of ulcerated varicose veins. A hemostatics sponge is formed when a sterile solution of gelatin is whipped into a foam, rendered insoluble by treatment with formaldehyde, and then dried; it can be used as an absorbable sponge in surgery and also to implant a drug or antibiotic directly into a specific area. Gelatin is used as a plasma expander for blood in cases of very severe shock and injury due to its protein character. Gelatin is an excellent emulsifier and stabilizing agent for many emulsions and foam; therefore, it is also used in cosmetic products, flocculation agents, and printing applications such as carbon printing, silk screen printing, and photogravure printing. E. Hides and Skins for Food and Sausage Casing Extraction of gelatin from animal skins and hides can be used for food. The raw material can also be rendered for lard. In the United States and some Asian countries, the pork skin is immersed in a swelling agent, boiled, dried, and then fried to make a snack food (56–58). The collagen of hides and skins also has a role as an emulsifier in meat products because of its hydrophobic nature: it can bind large quantities of fat. This can be an advantage, and it has been suggested that collagen may act as an adhesive between fatty and lean particles and many absorb excess fat. Therefore, collagen is a useful additive or filler for meat products. The collagen also can be extracted from cattle hides to make collagen sausage used in the meat industry (59). Collagen casing products were developed in Germany in the 1920s but only gained popularity in the United States in the 1960s. Regenerated collagen casings are made using extrusion techniques. Collagen casing manufacturing processes do not convert collagen to a soluble product as does the gelatin extraction process; instead, they result in a much more fibrillar product that retains a relatively high degree of the native collagen fiber. The extracted collagen product is suspended in an aqueous solvent and converted to an acidswollen gel or dough that is produced by the alkaline extraction process and is then extruded either by the wet or the dry process. During extrusion the collagen fibers tend to be aligned parallel to the axis of the tube emerging from the extruder. In the wet process, the extruder contains an internal disc that forces the following gel against the sides of the extruder casing. In the dry process the extruder contains twin counter-rotating serrated surfaces. The combination of fiber length and extruder design produces a weave-angle of cross-hatched collagen fibers. The tube of extruded collagen is then passed through a concentrated salt solution and a chamber of ammonia to precipitate the collagen. The swollen gel contracts to produce a film of reasonable strength that can be improved by the addition of plasticisers such as glycerin. The tube is then dried to a 10.0% to 15.0% water content and pleated so that a 15.0 m length of casing is contracted to 18.0 to 20.0 cm.

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F. Medicinal and Pharmaceutical Usage of Hides and Skins An extracted collagen product can assist in stimulating blood clotting during surgery. Pork skin is similar to human skin and can be converted into dressing that can be used for burn or skin-ulcer patients. Pork skin used as a dressing needs to be treated by a series of procedures: cut into strips or into a patch, shaved of hair, split to 0.2 to 0.5 mm thickness, cleansed, sanitized, and packaged. It can be used for skin grafting. When used for skin grafting it is removed from the carcass within 24 hours (60). Gelatin produced from hog skin is also used for coating pills and making capsules in the pharmaceutical industry. VI. UTILIZATION OF BONE Eleven percent of pork carcasses, 15% of beef carcasses, and 16% of lamb carcasses is composed of bone, and these values would be higher if adhering meat is included. In addition to the attached lean, the amount of marrow in a bone can also contribute to the yield of mechanically deboned products (61,62). The marrow can average 4.0% to 6.0% of the carcass weight. For centuries bones have been used to make soup and gelatin. In recent years the meat industry have attempted to get more meat from bone and new separation techniques have been utilized for this purpose. The mechanical deboning or separation technique produces tissue that at times has been called mechanically separated beef, pork, or lamb, mechanically deboned beef, pork, or lamb, and mechanically removed meat. Mechanically deboned or separated meat is now approved for use in meat products (mixed or used alone) in many countries. In 1978, mechanically separated red meat was approved for use in the red meat industry in the United States. A. Gelatin from Bone Ossein is normally produced from bone for gelatin extraction, and at first the bones must be pretreated by cooking at 80° to 95°C to remove adhering meat, gristle, and fat. The bones are then washed several times to get them clean. Next the bones are washed in dilute hydrochloric acid to remove minerals. In general the final ash of the ossein is from 1.0% to 2.0%. The clean ossein is then rapidly dried in hot convection air-drying ovens to an 8.0% to 10.0% moisture level. The products are stored in moisture-proof bags and processed into gelatin within 6 months. The ossein is processed through liming, deliming, washing, and gelatin extraction. The drying of gelatin is similar to the previously described process for gelatin extracted from hide or skin. B. Liquid Extraction from Bone In Asia, for many years chicken bones have been extracted and used as a special flavoring ingredient for meal cooking. In this process, crushed bones are cooked with water for 8 to 12 hours. The product is cooled and the fat is skimmed from the liquid. The liquid contains approximately 5.0% solids. Currently, processing time is reduced to 1.5 to 2.0 hours by using a high-pressure extraction system (4 to 6 kg/cm2 or 57 to 85 lb/in2). This technique yields 66.0% liquid extract, which contains approximately 10% solids, and it can be vacuum concentrated to 60.0% solids. Nine percent salt is added to stabilize the product. These extracted products have been used as a soup base, in noodle products, sauce, stew, and curries as well as in processed hams and sausages.

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C. Mechanically Separated Meat from Bone The technique for muscle separation was developed for the fishery industry in the 1940s and was next applied in the poultry industry because fish and poultry bones have some similarities. The structure of mechanically deboned red meat is a finally ground, paste-like product in which the myofibrils are heavily fragmented. The majority of mechanically separated meat comes from the adhering meat and some of the bone marrow and small quantities of powdered bone is also contained in the final products (63). An average of 30.0% mechanically separated meat based on commercial bone weight is the expected yield for beef, pork, and lamb bone. It is estimated that an average of 6.5 kg of mechanically separated meat could be obtained from a beef carcass and 1.5 kg could be obtained from a pork carcass. The vertebral column, ribs, and sternum would be economically suitable for mechanical separation because of large quantities of high protein matter such as meat tissue and marrow that are contained in these tissues. Mechanically separated meat has at least as good an emulsifying capacity and water-holding capacity, and slightly higher emulsion stability, than hand-deboned products (64). Mechanically separated red meat may be added to ground-beef patties; comminuted fresh, smoked, and cooked sausage-type products; stews, sauces, spreads, and similar products; and even to chunked and formed products. Normally, if mechanically separated red meat is incorporated into products at high levels, the flavor and overall acceptability scores will be reduced, the color becomes darker, and the tenderness and juiciness scores are higher. For these reasons, the practical level of incorporation of mechanically separated meat is usually limited. A 5.0% to 20.0% level in beef patties, hamburger, ground beef, fabricated beef, and a 10.0% to 40.0% level in sausage emulsions have been suggested by the meat industry. Many countries already have regulations on products with mechanically separated red meat. In the United States of America, mechanically separated red meat is not allowed to be use in beef patties, baby food, ground beef, meat pies, and hamburger, and 20.0% is considered to be the maximum level in sausage emulsion (65). In Denmark, if mechanically separated red meat is used at levels of more than 2.0%, this has to be declared on the label. In Australia, a statement of edible mechanically deboned beef or mutton and the maximum calcium and moisture level and minimum protein level must be labeled on the exported products. D. Medicinal and Pharmaceutical Usage of Bone Specially processed xiphoid or xiphisternal cartilage from the breastbone cartilage of young cattle is used by plastic surgeons to replace facial bone. Red bone marrow is used to treat patients who have a low red blood cell count. Bone meal is also a nutritional source of calcium and phosphorus in the diet. VII.

UTILIZATION OF GLANDS AND ORGANS

A. Glands and Organs as Food Animal organs and glands offer various levels of nutritionally attractive contents and have a wide variety of flavor and textures in a range of foods (66). Therefore, they are highly prized and are as valuable as carcass meat in parts of the world, specially in southeast Asia. The glands and organs that are generally recognized as having some use as human foods depends on species; for example, all species: brain, heart, kidney, liver, lung, spleen and

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tongue; bovine: pancreas and udder; porcine: stomach and uterus; ovine and bovine: reumen, reticulum, omasum, and absomasum; ovine and porcine: testes and thymus. Brain, nervous system, and spinal cord are usually prepared for the table rather than for use in manufactured medicine. They are blanched to firm the tissue before cooking because of the soft texture. The pia mater and arachnoid meninges, which is a skin collagenous connective tissue, are peeled off-the brain before cooking. Heart meat is generally regarded as relatively tough (66) and this is possibly due to the nature of the cardiac muscle. Hearts are used as table meats: whole hearts can be roasted or braised, sliced heart meat is grilled or braised (67). Heart meat is also often used as an ingredient in processed meat. Kidneys generally are removed from the adipose capsule, which keeps the kidney between the muscle of the loin and the peritoneum but is still covered by a fibrous capsule. This capsule must be removed and the ureter and blood vessel also need be trimmed before the kidneys are prepared for cooking. Kidneys are used whole or sliced and generally broiled, grilled, or braised. Liver is the most widely used edible organ (68) and is used in many styles of processed meats such as liver sausage and paste. Livers from lamb, veal, and young cattle are prepared for the table because they have a lighter flavor and texture, which is preferred in the United States and Europe. But consumers in southeast Asia prefer livers from pigs. Livers are used sliced and generally fried, braised, or broiled. Pig, calf, and lamb lungs are mainly used to make stuffing and some types of sausages and processed meats (67). The ruminant stomachs from cattle and lamb are composed of four compartments: the rumen, reticulum, abomasum, and omnivore stomach. The rumen and reticulum are the most widely consumed parts of the ruminant stomach. They are generally processed at the place of collection by washing, scalding, and bleaching. They are suitable for poaching or braising, or used in sausage and processed meat, or can be sewn to form casing and stuffed. Pig stomachs are composed mainly of smooth muscle and collagenous connective tissue. They are cleaned and scalded to remove the mucosa lining; they are also suitable for braising and are sometimes used as casing for sausage. Animal intestines are used as food by boiling in some countries. Animal intestines are used for pet food, meat meal, tallow, or fertilizer, but certainly the important economic use of these products is in the production of sausage casing. Animal intestines, when removed from the carcass, are highly microbiologically contaminated. They are fragile and therefore cleaning must be performed immediately after slaughter of the animals. Animal casings come in a wide variety of different shapes and sizes and the preference for a particular type of casing varies tremendously from country to country. When casings are manufactured the following procedures are used: removal of the viscera, separation of the ruffle fat from the intestines, stripping the manure, sometimes (often not used today) fermenting the casings, breaking the inner mucosa membrane and separating it from the casing, removal of all strings, soaking and removing the blood, salting and packaging—described by Ockeman and Hansen (1). The thymus glands are available only from young animals (lamb and calf). The glands are covered by a capsule of fibrous connective tissue that penetrates the gland and divides it into lobules, and the connective tissue and fat will be increased with the age of the animals. The thymus glands from lamb and calf are blanched to firm the tissue and peeled from the capsule before cooking. They are sliced and cooked by frying or stewing.

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The tongues are removed from the carcasses and generally include a small portion of the trachea, larynx, hyoid bones, associated muscle, and salivary glands. An epithelial mucous membrane covering the tongue is removed by a knife before cooking. The tongues are used fresh or salted and are usually boiled or braised. Udders are available only from bovines and the main body is connective tissue and secretory epithelium. Udders are sliced and washed to remove the milk and cooked by frying or boiling. Spleens are minced and used in pie, flavoring, and melt in the United Kingdom but used as variety meat in processed meat in the United States. Uteri are available only from nonpregnant pigs and are collected for human consumption. Fresh pig uteri are generally poached or boiled. B. Medicinal and Pharmaceutical Usage of Glands and Organs Animal glands and organs have been consumed since recorded history. Some have been used in medicine for their curing powers in some countries (such as China, India, and Japan). These glands are called endocrine glands and secrete hormones, or enzymes that regulate body metabolism. These include the liver, lungs, pituitary, thyroid, pancreas, stomach, parathyroid, adrenal, kidney, corpus luteum, ovary and follicle. The glands are collected only from healthy animals and locating the glands requires experience because some of the glands are often small and are often encased in other tissue. Different animals have different glands that are important, and their function is dependent on the species, sex, and age of the animals. The best method of preserving most glands and stoping autolysis and bacterial growth is by quick freezing. Before freezing, the glands must be cleaned and trimmed from surrounding fat and connective tissue. They are then put on waxed paper and kept at 18°C or less. When the glands arrive at the pharmaceutical plant, they are again inspected, then chopped and mixed with different solutions for extraction or placed in a vacuum drier for the drying process. If the dried gland contains too much fat, solutions such as gasoline, light petroleum, ethylene dichloride, benzene, and acetone are used to remove the fat. After drying and defatting, the glands or extracts are milled to a powder form and dispensed as capsules, tablets, or injections or utilized as a dilute liquid. They are tested for safety and potency prior to sale. The adrenal gland consist of two parts, an outer cortex and an inner medulla that secretes at least 20 steroids that are essential for life maintenance. Corticosteroids from the adrenal cortex regulate the body’s utilization of nutrients such as fat, carbohydrate, water, nitrogen, and minerals. Extracted adrenal cortical steroids from cattle, pigs, or sheep are used as anti-neoplastic and anti-inflammatory agents and for treatment of shock and asthma. Epinephrine and norepinephrine can be extracted from the adrenal medullas of cattle, pig, and sheep and are used to arrest hemorrhaging, shrink blood vessels, prolong the effects of local anesthetics, stimulate heart action, and overcome shock. Brains, nervous systems, and spinal cords are a source of cholesterol, which is the raw material for the synthesis of vitamin D3, and steroid pharmaceuticals and is used as an emulsifier in cosmetics. Some materials can be isolated from the hypothalamus of the brain for this purpose. For example, thromboplastin is used as a blood coagulant in surgery, kephalin is prepared to assist in clotting of blood, and lecithin is useful as an emulsifier and antioxidant. The pineal gland is located in the brain cavity behind and above the pituitary. The hormone melatonin extracted from the pineal gland is being evaluated for the treatment of schizophrenia, mental and physical development problems, and mental retardation. The

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pituitary gland is located at the base of the brain and is made up of an anterior and a posterior lobe with separate functions. Hormones such as growth-promoting hormone (GH), thyroid-stimulating hormone (TSH), mammary-stimulating hormone, gonad-stimulating hormones, adrenal-cortex-stimulating hormone (ATCH) (extracted from an anterior of the pituitary), antidiuretic hormone (ADH), and oxytocin hormone (produced from a posterior of pituitary) are used to control growth and metabolism and regulate the activity of other endocrine glands. ACTH is the most commercially extracted hormone from the pituitary and is used as a treatment for rheumatism, arthritis, eye inflammation, and multiple myeloma. Bile consists of bile acid, bile pigment, fatty acids, phospholipids, proteins, cholesterol, and other substances and can be obtained from the gallbladder. It is used for treating digestive disorders, constipation, and bile tract disorders and increasing the secretory activity of the liver, and it is also useful in some fat-digestion disorders. Bile can be purchased as dry or liquid extract preparations from cattle or hogs. Some ingredients of bile, such as prednisone, prednisolone, pregesterone, hydeoxycholic acid, chenodeoxycholic acid and dehydrocholic acid, and cortisone, can be extracted individually and used in the medicinal and pharmaceutical areas. Gallstones are reported to have some mystical aphrodisiac value and are very expensive because they are available in extremely small quantities. They are usually used as ornaments to make necklaces and pendants. Liver is the biggest gland in animals; it will usually average about 5 kg when obtained from market-weight cattle and approximately 1.4 kg from market-weight pigs. Liver extract is produced by extracting raw ground livers with slightly acidified hot water. The stock is concentrated to a paste under vacuum at low temperature and is used as a raw material by the pharmaceutical industry. Liver extract and desiccated liver can be obtained from pork and beef and have long been used as a source of vitamin B12, as a nutritional supplement used in treating various types of anemia, and as an enrichment medium in bacterial numeration (69,70). Heparin can be extracted from the liver as well as the lungs and mucosa (inner) lining of the small intestines. It is used as an anticoagulant to prolong the clotting time of blood; it is used to thin the blood (raise the viscosity) and to dissolve, prevent, or retard blood clotting during surgery and in organ transplants. By using the Scott method 10.2 g of crude heparin can be extracted from 540 g of porcine lung but its activity is not as good as that from commercial products (71). The crude heparin prepared from hog lungs by the alkaline ammonium sulfate method can be combined with a Sephadex G-50 column for chromatography and purification. The purity and activity of the heparin can be improved by this method and was acceptable when compared with commercial products (72,73). Progesterone and estrogens can be extracted from pork ovaries and may be used to treat some reproductive problems, such as functional uterine bleeding, abnormalities of the menstrual cycle, and threatened abortion, and are used in the treatment of breast and prostate cancer. Relaxin, a hormone from pregnant sow ovaries, often is used during childbirth. The pancreas has internal and external sections. The internal section secretes insulin, which regulates sugar metabolism, and the external section secretes chymotrypsin, trypsin, lipase and amylase into the small intestine to assist in digestion of fat, protein, and starch. Insulin is produced by specialized cells in the pancreas called islets of Langerhan and can be extracted from the pancreas by first grinding the hard-frozen gland in acetone and alcohol; then, the crude insulin is salted out and purified. Insulin is used in the treatment of diabetes. Glucagon extracted from the  cells of the pancreas is used to increase blood sugar

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and to treat insulin overdose or low blood sugar caused by alcoholism. Chymotrypsin and trypsin are used to remove dead tissue and improve healing after surgery or injury; chymotrypsin also has been used to facilitate cataract-extraction eye surgery. Animal intestines (sheep and calves) are also used for the manufacture of cat-gut, an internal surgical sutures. It is produced by several steps and twisted into one, two, or three strands and then cut, dried, polished, and sterilized. The pork and beef mucosa lining of the small intestines can be collected during the machining of casings, and it is either preserved in a raw state or processed into a dry powder prior to shipment to heparin manufacturers. VIII. UTILIZATION OF EDIBLE TALLOW AND LARD Animal fats are co-products of the meat packing industry, making supplies of fat available for the preparation of meat for sale or for processed meat products. The major edible animal fats are lard and tallow (74). Lard is defined as the fat rendered from clean, sound edible tissues of hogs in good health at the time of slaughter. Rendered pork fat include bacon skins and fleshed skin cheek meat trimmings, sweet pickle fat, and fats obtained from skimming the rendering tanks. Tallow is hard fat rendered from fatty tissues of cattle or sheep that is removed during processing of beef or sheep. Lard and edible tallow are obtained by dry or wet rendering (75). In the wet rendering process, the fatty tissues are heated in the presence of water, generally at a low temperature, and prime steam lard is obtained; its quality is better than that of the products from dry rendering. Low-quality lard and almost all of the inedible tallow and greases are produced by dry rendering. Rendered lard is used as an edible fat without being subjected to any post-rendering procedure. Due to consumer demand, lard and tallow now are often subjected to hydrogenation, bleaching, and deodorizing treatments before their utilization in food (76). Traditionally, tallow and lard has been used as a deep fat frying medium. However, this use of tallow or lard fried french fries in the fast-food service is changing due to consumer health demands (76). A pouring tallow-oil shortening has been developed for this purpose and resulted in a product where less fat is absorbed. Tallow and lard also have been used in margarine and shortening. Some edible lards are used in sausages or emulsified products. IX. THE PROCESSING AND UTILIZATION OF MEAT EXTRACT Meat extract was first produced in France in the eighteenth and nineteenth centuries by alcoholic extraction (77). The early procedure for manufacturing meat extract included removing the meat from the bones and trimming away the fat; then, the fat-free meat was hashed. The freshly hashed meat was then exposed to clear water and cooked at low temperature (less than 90°C) for extraction. Meat extract is defined as the products obtained by extracting fresh meat with boiling water, removing fat, and concentrating the liquid by evaporation (78). Meat extract should contain more than 75% total solid matter, 8.0% nitrogen, and less than 0.6% fat. The processing procedure for meat extract is summarized as follows (79). Meat stock can be produced from edible meat co-products (e.g., meat trimmings, and mechanically separated meat) that are pressed or soaked in water, or cooked, or obtained from corned beef; then the liquid is skimmed to remove fat and filtered to remove the fines and coagulated protein. The remaining liquid is concentrated under vacuum and later in an open pan to produce meat extract. The concentration of meat stock can be performed by using an evaporator: steam is fed through the line to sterilize the equipment followed by a cold

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water rinse. The stock is reduced in volume until it contains approximately 45.0% to 50.0% solids. The state of the art today accomplishes evaporation in a vacuum (70 cm) evaporator operated at a temperature of 70° to 75°C and producing a product of final concentration of 80% solids. This final product should have a natural color (brown or light brown), odor, and flavor and no bitterness. Meat extract is used for making soups, stews, sauces, casseroles, pot pies, canned meat, bouillon cubes, instant bouillon, and gravies. Meat extract is also an excellent flavoring material in a noodle soup mix, onion soup mix, and chip dip. When meat extract is used as an ingredient in other meat products, it should be listed as “flavorings” in the ingredient statement in the appropriate “order of predominance” position. X. CONCLUSION Meat producers have, for a long time, efficiently used meat co-products in processing into either edible or inedible products. Today, with increasing concerns about health and environmental protection, many new techniques, operating procedures, and research have been developed to permit more efficient processing and utilization of these co-products. The utilization needs become significantly stronger due to competition. This is important because increasing profit and decreasing the cost is required in the future for the meat industry to remain viable. These contributions and efforts are also necessary for the meat industries to change in an innovative manner and to widen the opportunities to utilize meat co-products. However, the saying “the packer uses everything but the squeal” has always existed in the meat industry and will continue to influence the utilization of meat co-products. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

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HW Ockerman, CL Hansen. Animal Co-product Processing. Chichester: Ellis Horwood, 1988, pp 1, 28, 33–37, 89–130. USDA. Livestock and poultry outlook report. USDA, Economic Research Service, Washington, DC, 1986, pp 36–37. O Bengtsson, O Holmqvist. Co-products from slaughtering, a short review. Fleischwirtschaft 64:260–263, & 334–336, 1984. F Gerrard. What is offal? Meat Trades Journal, September 14, 1972. Food Standard Committee of the Ministry of Agriculture, Fisheries and Food. Food standards committee report on offals in meat products. London, HMSO, 1972. MW Vaughn, DP Wallance, BW Forster. Yield and comparison of nutritive and energy value: Pigs’ ears. J Food Sci 44:1440–1442, 1434, 1979. MW Vaughn, DP Wallance, BW Forster. Yield and comparison of nutritive and energy value: Pigs’ feet, pigs’ tails. J Food Sci 48:1320–1322, 1344, 1981. USDA. Agriculture statistics. USDA, Washington, DC, 1983. EE Rice. The nutritional content and value of meat and meat products. In: JF Price, BS Schweighert, ed. The Science of Meat and Meat Products. San Francisco: Freeman, 1971, pp 314–315. JL Weihrauch, YS Son. The phospholipid content of foods. JAOCS 60:1971–1978, 1983. USDA and USDHHS. Nutrition and your health: Dietary guidelines for American. USDA Home and Grarden Bull. No. 232. Washington, DC, 1985. DC Liu, MT Chen, HL Kuo. Studies on the composition and storage quality of blood cake. J Chin Anim Sci Soc 22(4):463–468, 1993. J Wismer-Pederson. Utilization of animal blood in meat products. Food Technol 33:76–80, 1979.

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Liu and Ockerman Y Zhu, A Rinzema, J Tramper, J Bol. Microbial transglutaminase—a review of its production and application in food processing. Appl Microbiol Biotechnol 44:277–282, 1995. DR Kahn, I Cohen. Factor XIIIa catalyzed coupling of structural proteins. Biochem Biophys Acta 6668:490–494, 1981. F Traoe, JC Meunier. Cross-linking activity of placetal FXIIIIa on whey proteins and caseins. J Agric Food Chem 40:399–402, 1992. GS Nielsen, BR Petersen, AJ Moeller. Impact of salt, phosphate and temperature on the effect of FXIIIa on texture in restructured meat. Meat Sci 41:293–299, 1995. TF Tseng. Purification and characteristics of transglutaminase and its application. PhD dissertation, National Chung-Hsing University, Taichung, Taiwan, ROC, 1999. CC Reddy. Antioxdant enzyme. In KJA Davies, ed. Oxidative Damage and Repair. Chemical, Biological and Medical Aspects, Great Britain: Peramon, 1991, pp 591–601. JM McCord, I Fridovich. Superoxide dismutase as enzymic function for erythrocuprein (hemocuprein). J Biol Chem 244(22):6049–6055, 1969a. JM McCord, I Frdovich. The utility of superoxide dismutase in studying free radical reactions. J Biol Chem 244(22):6056–6063, 1969b. L Flohe. Superoxide dismutase for therapeutic use: Clinical experience, dead ends and hopes. Mol Cell Biochem 84:123–131, 1988. JM McCord. Superoxide dismutase: Rational for use in reperfusion injury and inflammation. Free Radic Bio Med 2:307–310, 1986. A Hass, K Brehm. Superoxide dismutases and catalases—biochemistry, molecular biology and some biomedical aspects. The Genetic Engineer Biotechnologist 13(4):243–269, 1993. LC Su. The extraction and properties of superoxide dismutase from porcine blood. Masters thesis, National Taiwan University, Taipei, Taiwan, ROC, 1996. S Divakavan. Animal Blood Processing and Utilization. Central Leather Research Institute, Madras, India, 1982. HJ Huang. Studies on the tested preparation of hemostat from porcine blood and its hemostating effect. Master’s thesis, National Chung-Hsing University, Taichung, Taiwan, ROC, 1987. HM Hague. Leather Tanners’ Council of America. Washington DC, 1949. R Hinterwaldner. Technology of gelatin manufacture. In: AG Ward, A Courts, eds. The Science and Technology of Gelatin. London, New York: Academic Press, 1977, pp 315–360. NR Jones. Uses of gelatin in edible products. In: AG Ward, A Courts, eds. The Science and Technology of Gelatin. London, New York: Academic Press, 1977, pp. 366–392. PD Wood. Technical and pharmaceutical uses of gelatin. In: AG Ward, A Courts, eds. The Science and Technology of Gelatin. London, New York: Academic Press, 1977, pp 415–418. CI Lin, RL Lee. Studies on the manufacture of deep fried pork as a snack food I. Effect of swelling agents and immersing period on the quality of deep fried pork skin. Annual Research Reports of Animal Industry Research Institute of Taiwan Sugar Company. Miaoli, Taiwan, ROC, 1990a, pp 223–232. CI Lin, RL Lee. Studies on the manufacture of deep fried pork as a snack food II. Effect of swelling agents and deep-fat fried temperature on the quality of deep fried pork skin. Annual Research Reports of Animal Industry Research Institute of Taiwan Sugar Company. Miaoli, Taiwan, ROC, 1990b, pp 223–241. CI Lin, RL Lee. Studies on the manufacture of deep fried pork as a snack food III. Effect of packaging methods and storage time on the quality of deep fried pork skin. Annual Research Reports of Animal Industry Research Institute of Taiwan Sugar Company. Miaoli, Taiwan, ROC, 1991, pp 215–222. AJ Bailey, ND Light. Connective Tissue in Meat and Meat Products. London and New York: Elsevier Applied Science, 1989, pp 242–245. HK Lin, PC Yang, RM Chu, JJ Shyu. The application of pigskin xenograft as biological dressings. I. Manufacturing process and methods. J Chin Anim Sci Soc 16(1–2): 51–58, 1987.

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DA Ledward, AJ Taylor, RA Lawrie. Conversion of bone to edible products. In upgrading waste for feeds and food. London, Butterworths, 1983. RA Field. Mechanical deboned red meat. Food Tech 30(9): 38, 40, 42, 44, 46, 48, 1976. RA Field. Mechanically separated meat, poultry and fish. In: AM Pearson, TR Dutson, eds. Edible Meat Co-products. London and New York: Elsevier Applied Science, 1988, pp 83–89. A Pisula, J Rejt. The influence of the addition of mechanically deboned meat (MDM) on the physiochemical properties of meat model blends. Proceedings of 25th European Meeting Meat Research Worker, 1979, pp 865–866. USDA. Standards and labeling requirements for mechanically separated species and products in which it is used. Fed Reg 47:28214, 1982. A Levie. The Meat Handbook. 2nd ed. Westport: AVI, 1970. Anon. The Good Cook—Offal. Time-Life, Amsterdam, 1981. JC Forrest, ED Aberle, HB Hedrick, MD Judge, RA Merkel. Principles of Meat Science. Dubuque: Kendall/Hunt, 1975, pp 335–346. U Ullmann. Die bakteriologische Diagnostik der Vibrio fetus-Infektion des Menschen. Zbl Bakt I Org 1230:480–491, 1975. CS Horng. Manufacture of microbial nutrient enrichment from pork liver and application of pork liver draff on pork liver loaves. Master’s thesis, National Chung-Hsing University, Taichung, Taiwan, ROC, 1999. CL Chen, IK Hwang. Studies on crude heparin preparation. Annual Research Report of Animal Industry Research Institute of Taiwan Sugar Company, Taiwan, 1978, pp 213–219. CW Lin, CT Wang, I Liang. Isolation and purification of heparin from hog lung I. The optimum conditions of alkaline ammonium sulfate method to extract crude heparin and the effect of treatments of raw hog lung on heparin yield and activity. J Chin Agric Chem Soc 25(2):125–130, 1987. CT Wang, CW Lin. Isolation and purification of heparin from hog lung II. Purification of heparin by gel chromatography. J Chin Agric Chem Soc 25(3):233–239, 1987. TJ Weiss. Food Oils and Their Uses. 2nd ed. Westport: AVI, 1983. NO Sonntag. Composition and characteristics of individual fats and oil. In: D Swern, ed. Bailey’s Industrial Oil and Fat Products. 4th ed. John Wiley: New York, 1979, p 289. LRJ Dugan. Meat animal co-products. 3rd ed. Westport: Food and Nutrition Press, 1987, pp 507. J Liebig. Ueber die bestaydtbeile der fliissigbeetin des fleisches. Aundleu der Chemie und Pharmacia 62: 257, 1847. HW Cox, D Pearson. The Chemical Analysis of Food. New York: Chemical Publishing, 1962. W Ockerman, JM Pellegrino. Meat extract. In: AM Pearson, TP Dutson, eds. Edible Meat Coproducts. London and New York: Elsevier Applied Science, 1988, pp 303–337. P Filstrup. Handbook for the meat by products industry. Slaughterhouse Co-products Department, Alfa-Laval, Denmark, 1976. F Gerrard. Meat Technology. 5th ed. London: Northwood, 1977, pp 164–165. MD Judge, ED Aberle, JC Forrest, HB Hedrick, RA Merkel. Principles of Meat Science. Dubuque: Kendall/Hunt, 1989, pp 336–339. JR Romans, W J Costello, CW Carlson, ML Greaser, KW Jones. The Meat We Eat. 13th ed. Danville: Interstate, 1994. KJ Lin, YJ Lin. Investigation on utilization of pork co-products in exported frozen meat packing industry. J Chin Anim Sci Soc 12(1–2):37–49, 1983. National Pork Producer Council (NPPC). Pork facts. Des Moines, IA, 1998. WF Spooncer. Organs and glands as human food. In: AM Pearson, TR Dutson, eds. Edible Meat Co-products. London and New York: Elsevier Applied Science, 1988, pp 198–204.

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26 Occupational Safety TIN SHING CHAO Department of Labor and Industrial Relations, State of Hawaii, Honolulu, Hawaii AHMAD C. K. YU Aloha Hawaii Enterprises, LLC, Keaau, Hawaii

I. INTRODUCTION II. CONGRESSIONAL FINDINGS AND PURPOSE III. THE ACT A. The Act Coverage IV. OCCUPATIONAL SAFETY AND HEALTH LAW V. OSHA INSPECTION A. Inspection Priorities B. Opening Conference C. The Inspection Process D. Closing Conference VI. MOST CITED STANDARDS RELATED TO SAFETY AND HEALTH HAZARDS IN THE WORKPLACE A. Section 5(a)(1). General Duties Clause B. 29 CFR Part 1903. Inspection, Citations, and Proposed Penalties C. 29 CFR Part 1904. Recording and Reporting Occupational Injuries and Illness VII. 29 CFR PART 1910—OCCUPATIONAL SAFETY AND HEALTH STANDARDS A. Section 1910.20—Keeping Medical Records B. Subpart D—Walking-Working Surfaces C. Subpart E—Means of Egress D. Subpart G—Occupational Health and Environmental Control E. Subpart H—Hazardous Materials F. Subpart I—Personal Protective Equipment (PPE) G. Subpart J—General Environmental Controls H. Subpart K—Medical and First Aid I. Subpart L—Fire Protection J. Subpart O—Machinery and Machine Guarding

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VIII. SUGGESTED ELEMENTS OF AN EFFECTIVE SAFETY AND HEALTH PROGRAM A. Management Commitment and Worker Involvement B. Work Site Analysis C. Hazard Prevention and Control D. Safety and Health Training IX. SAFETY RULES AND PRACTICES IN MEAT CUTTING AND MEAT PROCESSING OPERATIONS A. Employee Responsibilities B. Protective Equipment and Clothing C. Handling of Tools X. SOME PHYSICAL HAZARDS THAT A WORKER COULD ENCOUNTER AT THE WORKPLACE A. Electrical Hazards B. Noise C. Pneumatic Stuffers D. Retorts E. Boiler Feedwater F. Conveyors G. Lifts and Hoists XI. SOME CHEMICAL HAZARDS THAT A WORKER COULD ENCOUNTER AT THE WORKPLACE A. Anhydrous Ammonia B. Hydrogen Sulfide C. Liquid Petroleum (LP)-Gas D. Methane E. Carbon Dioxide F. Carbon Monoxide G. Nitrogen H. Plastic Fumes XII. CONCLUSIONS REFERENCES

I. INTRODUCTION More than 90 million Americans spend their days at a job. They are the most important assets of this country. Yet, before 1970, there were no uniform and comprehensive provisions for their safety and protection against workplace hazards. On December 29, 1970, the Senate and House of Representative of the 91st Congress enacted Public Law 91-596. It is also known as the Williams-Steiger Act, or may be cited as the “Occupational Safety and Health Act of 1970.” The Act was later amended by Public Law 101-552, Section 3101, November 5, 1990.

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II. CONGRESSIONAL FINDINGS AND PURPOSE The Congress finds that personal injuries and illnesses arising out of work situations impose a substantial burden upon, and are a hindrance to, interstate commerce in terms of lost production, wage loss, medical expenses, and disability compensation payments. The Congress declares that its purpose and policy is to assure, so far as possible, every working man and woman in the nation safe and healthful working conditions, to preserve our human resources, and to provide for the general welfare of all working people. This is enacted through regulation of commerce among the several States and with foreign nations in several areas (1): 1.

2.

3.

4. 5.

6.

7.

8. 9. 10.

11.

by encouraging employers and employees in their efforts to reduce the number of occupational safety and health hazards at their places of employment, and to stimulate employers and employees to institute new and to perfect existing programs for providing safe and healthful working conditions by providing that employers and employees have separate and independent responsibilities and rights with respect to achieving safe and healthful working conditions by authorizing the Secretary of Labor to set mandatory occupational safety and health standards applicable to businesses affecting interstate commerce and by creating an Occupational Safety and Health Review Commission for carrying out adjudicatory functions under the Act by building upon advances already made through employer and employee initiative for providing safe and healthful working conditions by providing for research in the field of occupational safety and health, including the psychological factors involved, and by developing innovative methods, techniques, and approaches for dealing with occupational safety and health problems by exploring ways to discover latent diseases, establishing causal connections between diseases and work in environmental conditions, and conducting other research relating to health problems often different from those involved in occupational safety by providing medical criteria which will assure insofar as practicable that no employee will suffer diminished health, functional capacity, or life expectancy as a result of his work experience by providing for training programs to increase the number and competence of personnel engaged in the field of occupational safety and health by providing for the development and promulgation of occupational safety and health standards by providing an effective enforcement program which shall include a prohibition against giving advance notice of any inspection and sanctions for any individual violating this prohibition by encouraging the States to assume the fullest responsibility for the administration and enforcement of their occupational safety and health laws by providing grants to the States to assist in identifying their needs and responsibilities in the area of occupational safety and health, to develop plans in accordance with the provision of this Act, to improve the administration and enforcement of

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State occupational safety and health laws, and to conduct experimental and demonstration projects in connection therewith 12. by encouraging joint labor management efforts to reduce injuries and disease arising out of employment (moved to p. 607) III. THE ACT The purpose of the Act is to assure safe and healthful working conditions for working men and women. It is enacted by authorizing enforcement of the standards developed under the Act; by assisting and encouraging the States in their efforts to assure safe and healthful working conditions; and by providing for research, information, education, and training in the field of occupational safety and health (1). A. The Act Coverage The Act extends to all employers and employees in all fifty States, the District of Columbia, Puerto Rico, and all other territories under the Federal Government jurisdiction. Coverage is provided either by Federal Occupational Safety and Health Administration (OSHA) or approved State programs. The Occupational Safety and Health Act of 1970 encourages States to develop and operate their own job safety and health plans. States with approved plans under section 18(b) of the Occupational Safety and Health Act must adopt standards and enforce requirements that are as effective as federal requirements. There are currently 25 state plan states: 23 of these states administer plans covering both private and public (state and local government) employees; the other two states, Connecticut and New York, cover public employees only. Plan states must adopt standards comparable to Federal requirements within 6 months of a Federal standard’s promulgation. Until such time as a state standard is promulgated, Federal OSHA provides interim enforcement assistance, as appropriate, in the states. Criteria for state plans is listed under 29 CFR Part 1902—state plans for the development and enforcement of state standards (1). An employer is any person engaged in a business affecting commerce who has employees, but this does not include the United States or any State or political subdivision of a State. The following are not covered under the act: (a) self-employed persons; (b) farms at which only immediate members of the farm employer’s family are employed; and (1) working conditions regulated by other Federal agencies under other Federal statutes. IV. OCCUPATIONAL SAFETY AND HEALTH LAW The Code of Federal Regulations is a codification of the general and permanent rules published in the Federal Register by the Executive departments and agencies of the Federal Government. The Code is divided into 50 titles, which represent broad areas subject to Federal regulation. Each title is divided into subchapters covering specific regulatory areas. Title 29 refers to Labor laws. It is composed of nine volumes. The Occupational Safety and Health Laws are recorded under 29 Code of Federal Regulations (CFR) Parts 1900–1910.999, Parts 1910.1000 to end, Parts 1911–1925, Part 1926, and Parts 1927 to end (2). V. OSHA INSPECTION Under the Occupational Safety and Health Act of 1970 Act, the Occupational Safety and Health Administration (OSHA) is authorized under the Act to conduct workplace inspec-

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tions to determine whether employers are complying with the OSHA standards issued by the agency for safe and healthful workplaces. OSHA also enforces Section 5(a)(1) of the Act, also known as the “General Duty Clause,” which requires that every working man and woman be provided with a safe and healthful work place. Workplace safety and health inspections are performed by OSHA compliance safety and health officers who are knowledgeable and experienced in the occupational safety and health field, and who are trained in OSHA standards and in the recognition of safety and health hazards. Similarly, states with their own occupational safety and health programs must conduct inspections using qualified state compliance safety and health officers. Inspections are usually conducted without advance notice. In fact, alerting an employer without proper authorization in advance of an OSHA inspection can bring a fine up to $1,000 and/or a 6-month jail term. This is true for federal OSHA compliance officers as well as state inspectors. There are, however, special circumstances under which OSHA may give advance notice to the employer, but such a notice will normally be less than 24 hours. If an employer refuses to admit an OSHA compliance officer or if an employer attempts to interfere with the inspection, the Act permits appropriate legal action, such as obtaining a warrant to inspect (3). A. Inspection Priorities Not all 6.2 million workplaces covered by the Act can be inspected immediately. The worst situations need attention first. OSHA, therefore, has established a system of inspection priorities. Imminent danger situations are given top priority. An imminent danger is any condition where there is reasonable certainty that a danger exists that can be expected to cause death or serious physical harm immediately or before the danger can be eliminated through normal enforcement procedures. Second priority is given to investigation of fatalities and accidents resulting in hospitalization of three or more employees. Such catastrophes must be reported to OSHA by the employer within 8 hours. OSHA will investigate and determine the cause of such accidents and whether existing OSHA standards were violated. Third priority is given to formal employee complaints of alleged violations or standards, or of unsafe or unhealthful working conditions, and to referrals from other government authorities about specific workplace hazards. The Act gives each employee the right to request an OSHA inspection when the employee believes he or she is in imminent danger from a hazard, or when he or she thinks that there is a violation of an OSHA standard that posts physical harm. OSHA will maintain confidentiality if requested, and will inform employee of any action it takes regarding the complaint. Next in priority are programmed inspections aimed at specified high-hazard industries, workplaces, occupations, or health substances, or other industries identified in OSHA’s current inspection procedures. Industries are selected for inspection on the basis of factors such as the injury incident rates, previous citation history, employee exposure to toxic substances, or random selection. Special emphasis programs also may be developed and may be regional or national in scope, depending on the distribution of the workplaces involved. Comprehensive safety inspections in manufacturing will be conducted normally in those establishments with lostworkday injury rates at or above the Bureau of Labor Statistics (BLS) national rate for man-

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ufacturing currently in use by OSHA. States with their own occupational safety and health programs may use somewhat different systems to identify industries for inspection. An establishment can expect a follow-up inspection if an OSHA violation has been issued to the establishment. A follow-up inspection determines if previously cited violations have been corrected. If an employer has failed to abate a violation, the compliance officer informs the employer that he or she is subject to “Failure to Abate” alleged violations and proposed additional daily penalties while such failure to abate or violation continues (3). B. Opening Conference When the OSHA compliance officer arrives at the establishment, he or she will display official credentials and ask to meet with an appropriate employer representative. In the opening conference, the compliance officer will explain how the establishment was selected. The compliance officer also will ascertain whether an OSHA-funded consultation visit is in progress or whether the facility is pursuing or has received an inspection exemption through the consultation program. If so, the inspection may be terminated. Before the inspection, the compliance officer explains the purpose of the visit, the scope of the inspection, and the standards that apply. The employer will be given information on how to obtain a copy of applicable safety and health standards as well as a copy of any complaint that may be involved (with the employee’s name deleted.) The employer will be asked to select an employer representative to accompany the compliance officer during the inspection. An authorized employee representative also is given an opportunity to attend the opening conference and to accompany the compliance officer during the inspection. If a recognized bargaining agent represents the employees, the agent ordinarily will designate the employee representative to accompany the compliance officer. The Act does not require an employee representative for each inspection. Where there is no authorized employee representative, the compliance officer must consult with a reasonable number of employees concerning safety and health matters in the workplace (3). C. The Inspection Process The compliance officer determines the route and duration of the inspection. While talking with employees, the compliance officer makes every effort to minimize any work interruptions. The compliance officer observes safety and health conditions and practices; consults with employees privately, if necessary; take photos and instruments readings, examines records, collects air samples, measures noise levels, and surveys existing engineering controls; and monitors employee exposure to toxic fumes, gas, and dusts. An inspection tour may cover part or all of an establishment, even if the inspection resulted from a specific complaint, fatality, or catastrophe. Trade secrets observed by the compliance officer will be kept confidential. An inspector who releases confidential information without authorization is subject to a $1,000 fine and/or 1 year in jail. The employer may require that the employee representative have confidential clearance for any area in question. Employees are consulted during the inspection tour. The compliance officer may stop and question workers, in private, about safety and health conditions and practices in their workplaces. Each employee is protected under the Act from discrimination by the employer for exercising his or her rights.

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During the course of inspection, the compliance officer will point out to the employer any unsafe or unhealthful working conditions observed. At the same time, the compliance officer will discuss possible corrective action if the employer so desires. Some apparent violations detected by the compliance officer can be corrected immediately. When they are corrected on the spot, the compliance officer records such corrections to help judging good faith in compliance. Although corrected, the apparent violations may still serve as the basis for citation and if appropriate, a notice of proposed penalty (3). D. Closing Conference At the conclusion of the inspection, the compliance officer conducts a closing conference with the employer and the employee representative. It is a time for free discussion of problems and needs, a time for frank questions and answers. The compliance officer also will give the employer a copy of the employer rights and responsibilities following an OSHA inspection and will discuss briefly the information in the booklet and will answer any questions. The compliance officer discusses with the employer all unsafe or unhealthful conditions observed during the inspection and indicates all apparent violations for which a citation and a proposed penalty may be issued or recommended. The employer is informed of appeal rights. During the closing conference, the employer may wish to produce records to show compliance efforts and to provide information that can help OSHA determine how much time may be needed to abate all alleged violation. When appropriate, more than one closing conference may be held. This is usually necessary when health hazards are being evaluated or when laboratory reports are required. If an employee representative does not participate in either the opening or the closing conference held with the employer, a separate discussion is held with the employee representative, if requested, to discuss matters of interest to employees (3). VI. MOST CITED STANDARDS RELATED TO SAFETY AND HEALTH HAZARDS IN THE WORKPLACE For the purpose of this chapter, the Standard Industry Code (SIC) 2011 meat packing plants, and SIC 2013 sausage and other prepared meat products, SIC 2015 poultry slaughtering and processing are covered under 29 CFR Parts 1902–1908, Regulatory Standards and Parts 1910 Occupational Safety and Health Standards. It is also commonly known as the General Industry Standard. Most common violations and standards will be discussed below based on data collected from inspections of 271 establishment between January 1, 1998, to March 23, 1999 (4). A. Section 5(a)(1). General Duties Clause Section 5(a)(1) is commonly known as the general duties clause. It is used when no applicable standards could be found to cite a particular hazard. Section 5(a)(1) of the Act states each employer shall furnish to each of his employees employment and a place of employment which are free from recognized hazards that are causing or are likely to cause death or serious physical harm to his employees.

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B. 29 CFR Part 1903. Inspection, Citations, and Proposed Penalties The purpose of part 1903 is to prescribe rules and to set forth general enforcement policies rather than substantive or procedural rules. Such policies may be modified in specific circumstances where the Secretary of Labor or his designee determines that an alternative course of action would better serve the objective of the Act. 1. Section 1903.2 This standard requires each employer to post and keep posted a notice informing employees of the protections and obligations provided under in the Act, and for assistance and information, including copies of the Act and of specific safety and health standards. If there is any question, employees should contact the employer or the nearest office of the Department of Labor. Such notice shall be posted by the employer in each establishment in a conspicuous place or places where notices to employees are customarily posted. This poster is commonly known as the OSHA poster. This standard also requires the employer, who had obtained copies of the Act and applicable rules and regulations, to make such rules and regulations available upon request from any employee or their authorized representative. Any employer failing to comply with the provision of this section may be subject to citation and penalty. 2. Section 1903.8 This section requires the employer to allow a representative of the employees an opportunity to accompany the compliance safety and health officer during the physical inspection of any workplace for the purpose of aiding such inspection. 3. Section 1903.19 OSHA’s inspections are intended to result in the abatement of violations of the Occupational Safety and Health Act of 1970. Within 10 calendar days after the abatement date, the employer must certify to OSHA (state plans may vary) that each cited violation has been abated. The employer’s certification that abatement is completed must include, for each cited violation, the following information: 1. 2. 3. 4. 5.

the employer’s name and address; the inspection number to which the submission relates; the citation and item numbers to which the submission relates; a statement that the information submitted is accurate; the signature of the employer or authorized representative.

There are times abatement plans may be required as is indicated on the citation and the employer must submit an abatement plan for each cited violation within 25 calendar days (state plans may vary) from the final order of the date. An employer who is required to submit an abatement plan may also be required to submit periodic progress reports for each cited violation as it will be indicated on the citation. The date on which an initial progress report must be submitted may be no sooner than 30 calendar days after submission of abatement plan (states plans may vary). The employer is also required to inform affected employees and their representative(s) about abatement activities by posting a copy of each document submitted to the Agency or a summary of the document near the place where violation occured.

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C. 29 CFR Part 1904. Recording and Reporting Occupational Injuries and Illness The purpose of Part 1904 is to provide guidance on the record keeping of the various incidences. 1. Section 1904.2 Each employer is required to maintain the log of occupational injuries and illnesses and enter each recordable injuries and illnesses on the log and summary as early as practicable but no later than 6 working days after receiving information that a recordable injury or illness has occurred. This form is commonly known as the OSHA 200 Log. 2. Section 1904.4 In additional to the OSHA 200 Log the employer is required to keep the workmen’s compensation, insurance, or other reports or records if they contained the information required by Form OSHA101. 3. Section 1904.5 Each employer is required to post an annual summary of the occupational injuries and illnesses log for each establishment. The summary shall be compiled by February 1 of the next calendar year and shall be posted for the entire month of February of that year. 4. Section 1904.6 The employer is required to retain records provided for in Section 1904.2, 1904.4, and 1904.5 in each establishment for 5 years following the end of the year to which they relate. VII.

29 CFR PART 1910—OCCUPATIONAL SAFETY AND HEALTH STANDARDS (11)

Section 6(a) of the Williams-Steiger Occupational Safety and Health Act of 1970 allows the Secretary of Labor to promulgate occupational safety and health standards. 29 CFR 1910 is commonly known as the General Industry Standards. A. Section 1910.20—Keeping Medical Records Section 1910.20 discusses the requirements on how the employer shall keep medical records. 1. Section 1910.20(d)(1)(i) This standard requires the employer to preserve and maintain medical records for each employee for at least the duration of employment plus 30 years. Exception to this paragraph include health insurance claims records maintained separately from the employer’s medical program and its records, first aid records if made on-site by a non-physician and maintained separately from the employer’s medical program and its records. The medical records of employees who have worked less than one year for the employer are provided to the employee upon termination of employment.

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2. Section 1910.20(d)(1)(ii) This standard requires the employer to preserve and maintain each employee’s exposure record for at least 30 years. Exceptions to this paragraph includes: 1.

background data to environmental (workplace) monitoring or measuring, such as laboratory reports and work sheets 2. material safety data sheets and records concerning the identity of a substance or agent 3. biological monitoring results designed as exposure records by specific occupational safety and health standards, which shall be preserved and maintained as required by the specific standard. 3. Section 1910.20(g)(1)(i) This standard requires the employer to inform current employees upon their first entering into employment and at least annually thereafter, of the existence, location, and availability of any records covered by 29 CFR 1910.20. B. Subpart D—Walking-Working Surfaces Slips, trips, and falls are among of the most reported causes of occupational injuries. Falls are not always from elevation. Many falls are at the same level from slips and trips. 1. Section 1910.22(a)(1) This standard requires all places of employment, passageways, storerooms, and services be kept clean and orderly and in a sanitary condition. Poor housekeeping could be one of the biggest elements contributing to an unsafe working place. 2. Section 1910.22(a)(2) This standard requires the floor of every workroom be maintained in a clean and, so far as possible, a dry condition. 3. Section 1910.22(d)(1) This standard requires every building or structure used for mercantile trade, business, industrial use, or storage to post the load limit signs approved by the building official in a conspicuous place in each space to which they relate. 4. Section 1910.23(a)(1) This standard requires every stairway floor opening be guarded by a standard railing. 5. Section 1910.23(c)(1) This standard requires every open-sided floor or platform that is 4 feet or more above the adjacent floor or ground level be guarded by a standard railing. 6. Section 1910.23(c)(3) This standard requires open-sided floors, walkways, platforms or runways which are above or adjacent to dangerous equipment, pickling or galvanizing tanks, degreasing units, and other similar hazards be guarded with a standard railing and toe board. This is regardless of height.

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7. Section 1910.23(d)(1) This standard requires that every flight of stairs having four or more risers be equipped with standard stair railings or handrails, the requirements as listed under Section 1910.23 (d)(1)(i)–(v). 8. Section 1910.24(b) This standard requires fixed stairs be provided for access from one structure to another where operations necessitate regular travel between levels, and for access to operating platforms at any equipment which requires attention routinely during operations. Fixed stairs shall also be provided: 1.

where access to elevation is daily, or at each shift, for such purposes as gauging, inspection, regular maintenance, etc. 2. where such work may expose employees to acids, caustics, gases, or other harmful substances, or 3. for which purpose the carrying of tools or equipment by hand is normally required. 9. Section 1910.25 This section is intended to prescribe rules and establish minimum requirements for the construction, care and the use of the common types of portable wood ladders. 10. Section 1910.25(b)(2)(xv) This standard requires that a ladder, which is used to gain access to roof, shall extend at least 3 feet above the point of support, at cave, gutter, or roofline. 11. Section 1910.26 This standard requires metal ladders be designed without structure defects, or accident hazards such as sharp edges, burrs, etc. The metal selected shall be of sufficient strength to meet the test requirements, and shall be protected against corrosion unless inherently corrosion-resistant. C. Subpart E—Means of Egress A means of egress is a continuous and unobstructed way of exit travel from any point in a building or structure to a public way. This consists of three separate and distinct parts: the way of exit access, the exit, and the way of exit discharge. A means of egress comprises the vertical and horizontal ways of travel and shall include intervening room spaces, doorways, hallways, corridors, passageways, balconies, ramps, stairs, enclosures, lobbies, escalators, horizontal exits, courts, and yards. 1. Section 1910.37(f)(1) This standard requires all exits shall be so located and exit access be arranged so that exits are readily accessible at all times. The most common problem found was blocked exits which were not readily accessible.

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2. Section 1910.37(f)(6) This standard requires the minimum width of any way of exit access shall in no case be less than 28 inches. 3. Section 1910.37(q) This standard requires exits shall be marked by a readily visible sign. Any door, passage, or stairway which is neither an exit nor a way of exit access, which is so located or arranged as to be likely to be mistaken for an exit, shall be identified by a sign reading “Not an Exit” or similar designation. It can also be identified by a sign indicating its actual character, such as “To basement,” “Storeroom,” “Linen Closet,” or the like. 4. Section 1910.38(a) This standard applies to all emergency action plans required by a particular OSHA standard. The emergency action plan shall be in writing except for those employers with 10 or fewer employees, who may communicate the plan orally to employees. The following elements at a minimum shall be included in the plan: 1. 2. 3. 4. 5. 6.

emergency escape procedures and emergency escape route assignments procedures to be followed by employees who remain to operate critical plant operations before they evacuate procedures to account for all employees after emergency evacuation has been completed rescue and medical duties for those employees who are to perform them the preferred means of reporting fires and other emergencies names or regular job titles of persons or departments who can be contacted for further information or explanation of duties under the plan.

5. Section 1910.38(a)(5) This standard requires that the employer, before implementing the emergency action plan, shall designate and train a sufficient number of persons to assist in the safe and orderly emergency evacuation of employees. 6. Section 1910.38(b) This standard applies to all fire prevention plans required by a particular OSHA standard. The fire prevention plan must be in writing, except for employers with 10 or fewer employees, who may communicate the plan orally to employees. The following elements at a minimum shall be included in the plan: 1.

a list of major workplace fire hazards and their proper handling and storage potential ignition sources and their control procedures, and the type of fire protection equipment or systems which can control a fire involving them 2. names or regular job titles of those personnel responsible for maintenance of equipment and systems installed to prevent or control ignitions or fires; 3. names or regular job titles of these personnel responsible for control of fuel source hazards. 7. Section 1910.38(b)(4) This standard requires the employer to apprise employees of the fire hazards of the materials and processes to which they are exposed. The employer shall also review with each em-

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ployee upon initial assignment those parts of the fire prevention plan for which the employee must know to protect the employee in the event of emergency. D. Subpart G—Occupational Health and Environmental Control 1. Section 1910.95 Protection against noise exposure shall be provided when employees are being exposed to sound level exceeding an 8-hour Time-Weighted Level of 85 decibels (the Action Level) or an 8-hour Time-Weighted Level of 90 decibels (the Permissible Exposure Level). The employers are required to monitor to determine if employees are being exposed to noise over the action level. The employer shall notify each employee exposed at or above the action level. The employers are required to establish and maintain an audiometric testing program and establish a valid baseline audiogram within 6 months of an employee’s first exposure at or above the action level. At least annually after obtaining the baseline audiogram, the employer shall obtain a new audiogram for each affected employee. Hearing protectors shall be available to all employees exposed to an 8-hour Time-Weighted Level of 85 decibels or greater at no cost to employees. Hearing protectors shall be replaced as necessary. The employer shall institute an annually repeated training program for the employees who are exposed to noise at or above the action level and to ensure employee participation in such training program. 2. Section 1910.95(c) This standard requires the employer to administer a continuing, effective hearing conservation program whenever employees’ noise exposures equal or exceed an 8-hour TimeWeighted Level of 85 decibels measured on the A scale or, equivalently, a dose of fifty percent. The requirements are listed in 29 CFR Section 1910.95(c)–(o). E. Subpart H—Hazardous Materials 1. Section 1910.101(a) This standard requires the employer to determine whether compressed gas cylinders under their control are in a safe condition to the extent that can be determined by visual inspection. Visual and other inspections shall be conducted as prescribed in the hazardous regulations of the Department of Transportation (49 CFR parts 171–179 and 14 CFR part 103). Where those regulations are not applicable, visual and other inspections shall be conducted in accordance with Compressed Gas Association Pamphlets C-6-1968 and C8-1962. 2. Section 1910.101(b) This standard requires the employer to follow the Compressed Gas Association Pamphlet P-1-1965 for the in-plant handling, storage, and utilization of all compressed gases in cylinders, portable tanks, rail tankcars, or motor vehicle cargo tanks. 3. Section 1910.106 This section talks about the flammable and combustible liquids separated into different classes, their handling and storage requirements.

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4. Section 1910.119 This standard, commonly known as the Process Safety Management (PSM) standard or PSM, contains the requirements for preventing or minimizing the consequence of catastrophic releases of toxic reactive, flammable, or explosive chemicals. These releases may result in toxic, fire, or explosive hazards. There are 137 chemicals listed in Appendix A to this section that are regulated by this standard and their total quantity; if exceeded, the allowed amount will be subjected to all requirements of the standards. Many companies will try to substitute lesser hazardous chemicals in order to avoid complying with this standard because compliance requires much work and it is often quite expensive to comply. When they have to have one of the 137 regulated chemicals on site they will try to keep their total quantity lower than the regulated level so that the standard will not be applied. Those who have operations covered under this standard are subjected to all the requirements of this standard: 1910.119 (c) Employee participation 1910.119 (d) Process safety information 1910.119 (e) Process hazard analysis 1910.119 (f) Operating procedures 1910.119 (g) Training 1910.119 (h) Contractors 1910.119 (i) Pre-start up safety review 1910.119 (j) Mechanical integrity 1910.119 (k) Hot work permit 1910.119 (l) Management of change 1910.119 (m) Incident investigation 1910.119 (n) Emergency planning and response 1910.119 (o) Compliance audits 1910.119 (p) Trade secrets 5. Section 1910.120 This standard is commonly known as the Hazwooper Standard. This section covers: 1.

2. 3. 4.

5.

clean-up operations required by a governmental body—whether Federal, State, local, or other—involving hazardous substances. These include clean-ups conducted at uncontrolled hazard waste sites, any site on the state priority sites list, sites recommended for the Environmental Protection Agency (EPA) National Priorities List (NPL), and sites which are identified for initial government investigation before substance has been ascertained corrective actions involving clean-up operations at sites covered by the Resources Conservation and Recovery Act of 1976 voluntary clean-up operations at sites recognized by Federal, State, local or other governmental bodies as uncontrolled hazardous waste sites operations involving hazardous waste that are conducted at treatment, storage, disposal (TSD) facilities regulated by 40 CFR parts 264 and 265 pursuant to Resource Conversation and Recovery Act (RCRA); or by agencies under agreement with USEPA to implement RCRA regulations and emergency response operations for release of, or substantial threats of releases of, hazardous substances without regard to the location of the hazard

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Most commonly used chemicals that are used as refrigerants, such as anhydrous ammonia, and chemicals used as cleaning-in-process (CIP) for clean up processes, such as chlorine or ozone, could be subjected to 1910.119 and 1910.120 requirements. F. Subpart I—Personal Protective Equipment (PPE) This standard requires the employer to provide protective equipment including personal protective equipment (PPE) for eye, face, head, and extremities. Protective clothing, respiratory devices and protective shields and barriers shall be used, and maintained in a sanitary and reliable condition wherever it is necessary. Such equipment protects individuals from process or environmental hazards, chemical hazards, radiological hazards, or mechanical irritants encountered in a manner capable of causing injury or impairment in the function of any part of the body through absorption, inhalation, or physical contact. 1. Section 1910.132(d)(1) This standard requires the employer to assess the workplace to determine if hazards are present, or are likely to be present, which would necessitate the use of personal protective equipment. If such hazards are present, or likely to be present, the employer shall: 1.

select and have each affected employee use the type(s) of PPE that will protect the affected employee from the hazard identified in the hazard assessment 2. communicate selection decision to each affected employee 3. select PPE that properly fits each affected employee 2. Section 1910.132(d)(2) This standard requires the employer to verify that the required workplace hazard assessment has been performed through a written certification. This certification should include the name of the person certifying that evaluation has been performed; the date(s) of hazard assessment; and a title that identifies the document as a certification of hazard assessment. 3. Section 1910.132(f) This standard requires the employer to provide training to each employee who is required by 29 CFR 1910.132 to use PPE. These employees shall be trained to know at least the following: 1. 2. 3. 4. 5.

when PPE is necessary what PPE is necessary how to properly don, doff, adjust, and wear PPE the limitation of PPE the proper care, maintenance, useful life, and disposal of the PPE

4. Section 1910.133 This standard requires the employer to ensure that each affected employee uses appropriate eye or face protection when exposed to eye or face hazards from flying particles, molten metal, liquid chemicals, acids or caustic liquids, chemical gases or vapors, or potentially injurious light radiation. Protective devices purchased after July 5, 1994 shall comply with American National Standard Institute (ANSI) Z87.1-1989

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5. Section 1910.134 This section deals with the control of those occupational diseases caused by breathing air contaminated with harmful dusts, fogs, fumes, mists, gases, smokes, sprays, or vapors. The primary objective is to prevent atmospheric contamination. This shall be accomplished as far as feasible by accepted engineering control measures. When effective engineering controls are not feasible or while they are being instituted, appropriate respirators shall be used pursuant to the requirements of this standard. 6. Section 1910.135 This standard requires the employer to ensure that each affected employee wears a protective helmet when working in areas where there is a potential for injuries to the head from falling objects. Protective helmets purchased after July 5, 1994 shall comply with ANSI Z89.1-1969. 7. Section 1910.136 This standard requires the employer to ensure each affected employee uses protective footwear when working in areas where there is a danger of foot injuries due to falling or rolling objects, or objects piercing the sole, and where such employees feet are exposed to electrical hazards. Protective footwear purchased after July 5, 1994 shall comply with ANSI Z41.1-1967. 8. Section 1910.138 This standard requires the employer to select and require employees to use appropriate hand protection when employees’ hands are exposed to hazards such as those from skin absorption of harmful substances; severe cuts or lacerations; severe abrasions; punctures; chemical burns; thermal burns; and harmful temperature extremes. G. Subpart J—General Environmental Controls 1. Section 1910.141 This standard requires permanent places of employment to meet the minimum requirements of sanitation facilities. 2. Section 1910.146 This standard contains the requirements for practice and procedures to protect employees in general industry from any operation of the workplace that contains the hazards of entry into permit-required confined spaces. This section does not apply to agriculture, to construction, or to shipyard employment. A confined space means a space that is (a) large enough and so configured that an employee can bodily enter and perform assigned work; (b) has limited or restricted means for entry or exit; and (c) is not designed for continuous employee occupancy. A permit-required confined space means a confined space that has one or more of the following characteristics: 1. 2.

contains or has a potential to contain a hazardous atmosphere contains a material that has the potential for engulfing an entrant

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3.

has an internal configuration such that an entrant could be trapped or asphyxiated by inwardly converging walls or by a floor which slopes downwards and tapers to a smaller cross-section or 4. contains any other recognized serious safety or health hazards If such a hazard exists in the workplace, the employers are required to comply with this standard. 1910.146(d) requires the employer to establish a permit space program 1910.146(e) requires the employer to document the completion of measurements required by 29 CFR 1910.146(d)(3) by preparing entry permits before entry 1910.146(f) requires the employer to establish entry permit documents compliance of this section 1910.146(g) requires the employer to provide training so that all employees whose work is regulated by this standard acquires the understanding, knowledge, and skills necessary for the safe performance of the duties assigned under this standard 1910.146(h) requires the employer to train authorized entrants to understand their duties 1910.146(i) requires the employer to train the attendant to understand their duties 1910.146(j) requires the employer to train the entry supervisor to understand their duties 1910.146(k) requires the employers to train employees who are entering the permit space to perform rescue services and make training available to have persons other than their employees perform permit space rescue 3. Section 1910.147 This standard is commonly known as the control of hazardous energy Lockout/Tagout (LOTO) Standard. This standard covers the servicing and maintenance of machines and equipment in which the unexpected energization or start up of the machines or equipment, or release of stored energy, could cause injury to employees. This standard establishes the minimum performance requirements for the control of such hazardous energy. Appendix A to 1910.147 states a typical minimal lockout procedure for reference. 4. Section 1910147(c)(1) This standard requires the employer to establish a program consisting of energy control procedures, employee training and periodic inspections. Such a program ensures that all energy sources are isolated before any employee performs servicing or maintenance on a machine or equipment, where the unexpected energizing, start up or release of stored energy could occur and cause injury to employee. 5. Section 1910.147(c)(7) This standard requires the employer to train and to ensure that the purpose and function of the energy control program is understood by employees and that the knowledge and skills required for the safe application, usage, and removal of the equipment are acquired by employees.

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H. Subpart K—Medical and First Aid 1. Section 1910.151 This standard sets requirements for medical and first aid. 2. Section 1910.151(c) This standard requires the employer to provide suitable facilities for quick drenching or flushing of the eyes or body when the eyes or body may be exposed to injuries from corrosive materials. I. Subpart L—Fire Protection 1. Section 1910.155 This standard sets the requirements for fire brigades, and all portable and fixed fire suppression equipment, fire detection system, and fire or employee alarm system installed to meet the fire protection requirements of 29 CFR 1910. 2. Section 1910.156 This standard contains the requirements for the organization, training, and personal protective equipment of fire brigades whenever they are established by an employer. 3. Section 1910.157 This standard applies to the placement, use, maintenance, and testing of portable fire extinguishers provided for employee use. Where the employer has an emergency action plan and a fire prevention plan which meets the requirements of 29 CFR 1910.38 then only the requirements of 1910.157(e) and (f) applies. 4. Section 1910.159 This section sets the requirements for all automatic sprinkler systems installed to meet the particular OSHA standard. 5. Section 1910.164 This standard requires all automatic fire detection systems installed to meet the requirements of the OSHA standard. 6. Section 1910.165 This standard applies to all emergency employee alarms installed to meet a particular OSHA standard. The requirements are that maintenance, testing and inspection shall applied to all local fire alarm signaling systems used to alert employees, regardless of other functions of the system. J. Subpart O—Machinery and Machine Guarding 1. Section 1910.212(a)(1) This standard requires the employer to provide one or more methods of machine-guarding to protect the operator and other employees in the machine area from hazards such as those created by point of operation, in-going nip points, rotating parts, flying chips, and sparks.

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2. Section 1910.212(a)(2) This standard requires the guards for the machine be affixed to the machine where possible, or secured elsewhere if for any reason attachment to the machine is not possible. The guard shall be such that it does not offer an accident hazard in itself. 3. Section 1910.215 This standard sets the minimum requirements for safeguarding of abrasive wheels. Specific requirements for maximum exposure angles, tongue guard, tool rests, and other minimum safety requirements are listed in different tables and figures in this standard. 4. Section 1910.219(d)(1) This standard sets the guarding requirements for pulleys that are 7 feet or less from the floor or working platform. Where the point of contact between belt and pulley is more than 6 inches from the floor or platform, it may be guarded with a disk covering the spokes. 5. Section 1910.219(e)(1) This standard covers and ropes guards on horizontal belts and ropes. On horizontal belts and ropes, where both runs of horizontal belts are 7 feet or less from the floor level, the guard shall extend to at least 15 inches above the belt or to a standard height as listed in Table O-12 (Table of Standard Materials and Dimensions) (6). However, when both runs of a horizontal belt are 42 inches or less from the floor, it shall be fully enclosed. 6. Section 1910.219(i)(1) This standard requires all revolving collars including split collars, be cylindrical, and crews or bolts used in collars shall not project beyond the largest periphery of the collar. 7. Section 1910.219(i)(2) This section requires the couplings shall be so constructed as to present no hazard from bolts, nuts, setscrews, or revolving surfaces. Bolts, nuts, and setscrews will, however, be allowed where they are covered with safety sleeves or where they are used parallel with the shafting and are countersunk or else do not extend beyond the flange of the coupling. K. Subpart P—Hand and Portable Powered Tools 1. Section 1910.242 This standard requires the employer to be responsible to the safe condition of the tools and equipment used by employees, including tools and equipment which may be furnished by employees. 2. Section 1910.243 This standard requires the guarding of all portable, power driven circular saws having a blade diameter greater than 2 inches. It shall be equipped with a guard above and below the base plate or shoe. The upper guard shall cover the saw to the depth of the teeth, except for the minimum arc required to permit the base to be tilted for bevel cuts. The lower guard shall cover the saw to the depth of the teeth, except for the minimum arc required to allow proper retraction and contact with the works. When the tool is withdrawn from the work area, the lower guard must automatically and instantly return to covering position.

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L. Subpart Q—Welding, Cutting, and Brazing 1. Section 1910.252 This standard provides the basic precautions and special precautions of the fire protection and prevention responsibilities of welders and cutters, their supervisors, which includes outside contractors and those in management on whose property cutting and welding is to be performed. The standards for fire prevention in use of cutting and welding processes are listed under National Fire Protection Association (NFPA) Standard 51B, 1962, which is incorporated as reference as specified in 29 CFR 1910.6. 2. Section 1910.253(b)(2)(ii) This standard requires that cylinders kept inside buildings be stored in a well-protected, well-ventilated, dry location at least 20 feet from high combustibles such as oil or excelsior. Assigned storage spaces shall be located where cylinders will not be knocked over or damaged by passing or falling objects, or subject to tampering by unauthorized persons. 3. Section 1910.253(b)(4)(i) This standard requires that oxygen cylinders not be stored near highly combustible material, especially oil and grease; or near reserved stocks of carbide and acetylene or other fuelgas cylinders, or near any other substance likely to cause or accelerate fire; or in an acetylene generator compartment. 4. Section 1910.253(b)(4)(iii) This standard requires the storage of oxygen cylinders to be separated from fuel-gas cylinders or combustible materials by a minimum of 20 feet or by a non-combustible barrier at least 5 feet high, having a fire resistant rating of at least one-half hour. 5. Section 1910.253(b)(5) This standard requires cylinders, cylinder valves, couplings, regulators, hoses, and similar apparatus be kept free from oily or greasy substances. Organic materials such as oil and grease can cause oxygen to self ignite and will be an explosion hazard. M. Subpart S—Electrical Safety This subpart addresses electrical safety requirements that are necessary for the practical safeguarding of employees in their workplaces and is divided into four major divisions as follows: 1. Design safety standard for electrical systems contained in Sections 1910.302 through 1910.330 2. Safety-related work practices contained in Sections 1910.331 through 1910.360 3. Safety-related maintenance requirements contained in Sections 1910.361 through 1910.380 4. Safety requirements for special equipment contained in Sections 1910.381 through 1910.398 Definitions applicable to each division are contained in Section 1910.399.

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1. Section 1910.303(b)(1) This standard requires the employer to perform inspections to identify hazards. It requires electrical equipment to be free from recognized hazards that are likely to cause death or serious physical harm to employees. 2. Section 1910.303(b)(2) This standard requires listed or labeled equipment to be used or installed in accordance with any instructions included in the listing or labeling. 3. Section 1910.303(e) This standard requires that electrical equipment may not be used unless the manufacturer’s name, trademark, or other descriptive marking which may identify the organization responsible for the product is placed on the equipment. Other markings shall be provided giving voltage, current, wattage, or other rating as necessary. 4. Section 1910.303(f) This standard requires each disconnecting means be legibly marked to indicate its purpose, unless located and arranged so the purpose is evident. Each service, feeder, and branch circuit, and its disconnecting means shall be legibly marked to indicate its purpose. 5. Section 1910.303(g) This standard requires that for equipment rated 600m volts, normal, sufficient access and workspace shall be provided and maintained to permit ready and safe operation and maintenance of such equipment. 6. Section 1910.304(a)(2) This standard requires no ground conductor may be attached to any terminal or lead so as to reverse designated polarity. 7. Section 1910.304(f)(4) This standard requires the path to ground from circuits, equipment and enclosures be permanent and continuous. 8. Section 1910.305(a)(2)(iii)(F) This standard requires that lamps used for general illumination be protected from accidental contact or breakage. Protection shall be provided by elevation of at least 7 feet from normal working surface or by a suitable fixture or lamp holder with a guard. 9. Section 1910.305(b)(1) This standard requires conductors entering any box, cabinets, or fittings be protected from abrasion, and any openings through which conductors enter shall be effectively closed. Accumulated dust will cause an arc between conductors and will then cause an electrical fire. 10. Section 1910.305(b)(2) This standard requires all pull boxes, junction boxes, and fittings to be provided with covers approved for the purpose. If metal covers are used, they must be grounded.

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11. Section 1910.305(e)(1) This standard requires that cabinets, cutout boxes, fittings, boxes, and panel board enclosures in damp or wet locations be installed so as to prevent moisture or water from entering and accumulating within the enclosures. In wet locations the enclosures shall be weatherproof. 12. Section 1910.305(g) This standard sets the requirement for the use of flexible cords and cables. The most commonly found violation, which is in Section 1910.305(g)(1)(iii) includes: flexible cords and cables used as a substitute for the fixed wiring of a structure: 1. flexible cords and cables running through holes in walls, ceilings, or floors 2. flexible cords and cables running through doorways, windows, or similar openings 3. flexible cords and cables attached to building surfaces 4. flexible cords and cables concealed behind buildings, walls, ceilings, or floors 13. Section 1910.332(a) This standard requires the employer to train employees who face a risk of electrical shock that is not reduced to a safe level by the electrical installation requirements of Sections 1910.303 through 1910.308. 14. Section 1910.322(b)(1) This standard requires the employer to train employees in and become familiar with the safety-related work practices required by Sections 1910.331 through 1910.335. 15. Section 1910.322(b)(2) This standard sets additional requirements for the employer to train unqualified employees who are covered by Section 1910.322(a), but who are not qualified persons. They shall be trained and be familiar with any electrically related safety practices not specifically addressed by Sections 1910.331 through 1910.335 but which are necessary for their safety. 16. Section 1910.334(a)(2)(ii) This standard requires the employer, during visual inspection, to remove any defective or damaged equipment that might expose an employee to injury. No employees may use it until appropriate repairs and tests are done to render the equipment safe. N. Subpart Z—Toxic and Hazardous Substances 1. Section 1910.1030 This standard is commonly known as the Bloodborne Pathogen Standard. It applies to all occupational exposure to blood or other potentially infectious materials as defined in 1910.1030(b). This standard also covers but is not limited to first responder, and employees who are designated as a first-aid provider in their job description. 2. Section 1910.1030(c) This standard requires employers to establish a written exposure control plan designed to eliminate or minimize employee exposure;

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3. Section 1910.1030(d) This standard discuss the different methods of compliance such as universal compliance, engineering and work practice controls; 4. Section 1910.1030(f) This standard requires the employer to make available the hepatitis B vaccine and vaccination series to all employees who have occupational exposure, and postexposure evaluation and follow-up to all employees who have had an exposure incident. 5. Section 1910.1030(g)(1) This standard requires the employer to affixed warning labels and signs to containers of regulated waste, refrigerators and freezers containing blood or other infectious materials; and other containers used to store, transport, or ship blood or other potentially infectious materials. 6. Section 1910.1030(g)(2) This standard requires employers to ensure all employees with occupational exposure participate in a training program, which must be provided at no cost to the employee and during working hours. Such training shall be repeated annually. The elements of the minimum training program requirements are listed under Sections 1910.1030(g)(2)(vii) A–N. 7. Section 1910.1030(h) This standard requires the employer to establish and maintain accurate records for each employee with occupational exposure, in accordance with 29 CFR Section 1910.20 which is the duration of employment plus 30 years. 8. Section 1910.1200 This standard is commonly known as the hazard communication standard or employee right-to-know standard. The purpose of this standard is to ensure that the hazards of all chemicals produced or imported are evaluated, and that information concerning their hazards is transmitted to employers and employees. This is the most cited standard that tops any other OSHA citations. 9. Section 1910.1200(e)(1) This standard requires the employer to develop and maintain at each workplace, a written hazard communication program which describes the criteria set in 29 CFR Sections 1910.1200 (f), (g), and (h) for labels and other forms of warning, material safety data sheets, and employee information and training. 10. Section 1910.1200(f)(5) This standard requires the employer to ensure that each container of hazardous chemicals in the workplace is labeled, tagged, or marked with the following information: 1. identity of the hazardous chemical(s) contained therein 2. appropriate hazard warnings; or words, pictures, symbols, or combination thereof, which provide at least general information regarding the hazards of the chemicals; and which, in conjunction with other information immediately available to employees under the hazard communication program, provide employees

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with specific information regarding the physical hazards of the hazardous chemical(s) 11. Section 1910.1200(g)(8) This standard requires the employer to maintain in the workplace copies of the required material safety data sheets for each hazardous chemical, and shall ensure that they are readily accessible during each work shift to employees when they are in their work area(s). 12. Section 1910.1200(h)(1) This standard requires the employer to provide employees with effective information and training on hazardous chemicals in their work areas at the time of their initial assignment, and whenever a new physical or health hazard the employees have not previously been trained about is introduced into their work area. The elements of minimum training requirements are listed under 29 CFR Sections 1910.1200(h)(3)(i)–(iv). The above discussion represents some of the most cited violations during an OSHA inspection at the workplace. An effective safety and health program could also contribute to a safer workplace (6). VIII. SUGGESTED ELEMENTS OF AN EFFECTIVE SAFETY AND HEALTH PROGRAM Here are some suggested elements of an effective safety and health program: A. Management Commitment and Worker Involvement 1. 2. 3. 4. 5. 6. 7. 8.

Visible top management leadership Employee involvement in structure and operation of program and in decisions that affect their safety and health Clear work site policy on safe and healthful work and working conditions Goal(s) for safety and health program objectives for meeting goal(s) Assignment and communication of responsibility for all aspects of program Adequate authority and resources for parties to meet assigned responsibilities Managers, supervisors, and employees accountable for meeting responsibilities Annual program review to evaluate success in meeting goal(s) and objectives

B. Work Site Analysis 1. 2.

Regular site safety and health inspection Reliable system for employees to report hazards and receive timely and appropriate response

C. Hazard Prevention and Control 1. 2. 3.

Facility and equipment maintenance to prevent hazardous breakdowns Medical programs to minimize injury and illness Emergency plan and drills so that response of all parties will be “second nature”

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D. Safety and Health Training 1. 2.

Training for managers on their safety and health responsibilities Training for supervisors on their safety and health responsibilities and reasons for them 3. Training for employees on general safety and health rules of work site, specific site hazards, safe work practice to control exposure, and role in emergency situations IX. SAFETY RULES AND PRACTICES IN MEAT CUTTING AND MEAT PROCESSING OPERATIONS A. Employee Responsibilities Under 29 CFR 1960.10, Employee Responsibilities and Rights, the Occupational Safety and Health Act requires each employee to comply with the standards, rules, regulations and orders issued by the agency in accordance with Section 19 of the Act. Executive order 12196 held employees responsible for their own actions and conduct. Employees shall also use safety equipment, personal protective equipment, and other devices and procedures provided or directed by the agency and necessary for their protection. Employee shall have the right to report unsafe and unhealthful working conditions to appropriate officials. They are protected by law from being discriminated against by their employer if they chose to exercise this right. B. Protective Equipment and Clothing Proper protective equipment and clothing can avoid many accidents. Many companies have formulated a program of safety for their employees and mandate the use of safety equipment in job performance. Safety equipment should meet the requirements of the American National Standard Institute (ANSI) requirement for safety equipment. Efficiency of performance can be increased and operating cost reduced by the application of proper training of workers. Safety equipment such as safety helmets (hats), must be worn for all jobs where carcasses or products are conveyed on non-captive overhead rails, where carcasses are lugged, and where employees drive ride-on industrial tractors. Helmets are recommended for all dressing floor and cooler operations. Safety glasses or a face shield should be used for the protection of eyes or face from damage or destruction by physical or chemical agents or by radiant energy. This is an integral part of any good industrial safety program. Hand protection can be achieved by using metal mesh gloves. These gloves are made from stainless steel that conforms to the shape of the hand and the fingers to eliminate cutting injuries of workers’ hand. Various kinds of protective footwear are available on the market but steeltoe safety shoes are the best for impact protection. For work done under wet conditions, rubber boots or rubber shoes are available with steel box toes having similar impact specification set by ANSI for foot protection. C. Handling of Tools There are many good reasons for the proper instruction and training of workmen regarding their handling of both hand and power tools.

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The knife is one of the most commonly used tools in meat cutting. The careful use of the knife and observation of safe practices in meat cutting will prevent accidents. The hand saws usually come in two sizes, small and large. It is important not to use excessive force when using the hand saws. Excessive force may cause the blade to bind, jump, and strike the free hand, causing an injury. Cleavers are mainly used to cut through the backbone of carcasses. It is important to keep the hands and fingers away from the path of the cleavers. Do not attempt to use cleaver as a mallet or hammer. Abuse of cleavers may lead to an injury. Automated and hand-powered equipment play a very important role in the food industry, and the meat industry in particular. Powered circular saws should be positioned on retractable power lines. This will keep the equipment high above the head of the worker when not in use. Accidents are often caused by slipping, which throws the worker into the saw. Grease and meat scraps should be removed from the floor at regular intervals. Salt or other skid prevention materials should also be used to prevent slipping. Band saws generally have two pulley wheels with a saw blade circulating around the two wheels. Operators should know the safety factors as well as the care and maintenance of the saw. Not paying attention to the moving and working parts of the band saw is often the cause of accidents. Safety factors concerning the operation of the band saw should observed. Factors needing attention include adjustment of the saw blade tension, adjustment of blade guides, adjustment of the upper guide assembly and back-up blade support, and uneven wear of the movable carriage or meat table. Uneven wear will cause side play of the carriage or meat table. Manufacturers produce a wide variety of grinders for the food services industry. The most important safety measure for meat grinders is to never operate a grinder without a feed pan or tray. When operating a large grinder, a fork or scoop should be used for feeding or loading. A slicing machine should be used to cut chilled and frozen boneless meats into thin slices. It is important to remember to use the product pusher plate to slice end cuts. Fingers must be kept clear of the path of the blade. Do not disregard safety for speed, either. Before cleaning the slicing machine be sure the power control is shut off to prevent accidental starting by touching the control switch. X. SOME PHYSICAL HAZARDS THAT A WORKER COULD ENCOUNTER AT THE WORKPLACE A. Electrical Hazards All portable hand tools and production tools should be equipped with polarized grounded receptacles. All extension cords should be the three-wire type designed to fit the polarized receptacles. In most operations OSHA laws may require a lock-out tag-out program to be implemented to prevent unexpected energization or startup of the machines or equipment or release of stored energy. B. Noise Noise has been recognized as one of several causes of deafness. Employers should perform noise monitoring as required by law to determine if employees are being exposed to an 8hour Time-Weighted Level of 85 decibels or more. If so, a hearing conservation program should be implemented and personnel protective equipment should be provided.

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C. Pneumatic Stuffers Pneumatic stuffers have enough wall thickness to withstand normal stuffing pressure. Older stuffers are sometimes reamed or honed as interior walls become pitted or irregular. State and federal laws require annual inspection of such devices to prevent explosion of the stuffers. D. Retorts Hot water circulation lines should be periodically checked for hammering problems and to determine the loss of wall thickness. All retort attachments should be periodically inspected for wear or cracking. Accumulation of water in insulated retorts will cause early deterioration of the lower metal portions. E. Boiler Feedwater Boiler care and maintenance procedures will vary with size and type of installation. Because water treatment chemicals are an extremely important consideration in both safety and life of boilers, qualified inspectors should be used to check the boiler regularly. F. Conveyors Conveyors present a special kind of hazard. Many conveyors are installed to transfer products from one place to another or from live to a dead roller section. Guarding of the nip point of the rollers on the conveyors is necessary. It is also required by law to safeguard conveyors. All conveyors should be equipped with covers or electronically interlocking devices to prevent injury to workers’ hands. G. Lifts and Hoists Movement of material by lifts, hoists, and cranes requires careful safety scrutiny. Hoisting apparatus has been used sparingly in the meat industry. Most hoisting equipment found in the market conform with ANSI standards. Some common causes of breakdown of hoists are overloading, improvised or makeshift slinging, using the wrong type cables for the size lifted. It is important to select the correct chain for the job. Most new chains have built-in safety and have a breaking point several times greater than the work load limits. Frequent inspection and replacement of non-function parts could make a difference in preventing accidents. XI. SOME CHEMICAL HAZARDS THAT A WORKER COULD ENCOUNTER AT THE WORKPLACE All chemical hazards are covered under 29 CFR Section 1910.1200 Hazard Communication Standard, and the employer is required by law to educate employees on the health and physical hazards of the chemicals they use at their workplace. A. Anhydrous Ammonia Anhydrous ammonia is used as a refrigerant because of its efficiency in absorbing heat, its economy, and plentiful supply. Anhydrous ammonia has a powerful corrosive action on tissue. Starting at 20 part per million (ppm), one could smell odor. At 40 ppm, a few individ-

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uals could suffer slight eye irritation. At 100 ppm, noticeable eye and upper respiratory tract irritation may occur. At 700 ppm, severe eye irritation may occur. At 5000 ppm serious edema, strangulation, and asphyxia may occur. B. Hydrogen Sulfide Hydrogen sulfide gas frequently accumulates in grease interceptor basins or in places where there are large surface areas of low-grade fats. Locations where this gas may accumulate should have good ventilation. C. Liquid Petroleum (LP)-Gas Liquid petroleum torches are frequently used in the meat industry. One great hazard of LPgases is that they are heavier than air and tend to pocket or cloud. A source of ignition will produce a serious explosion from a leaking container. All LP-gases should be stored in a well-ventilated place. D. Methane Sewer gas or methane gas frequently accumulates in manholes. Therefore, before entering such confined space one should follow proper procedures to check for atmospheric hazard before entering. 29 CFR Section 1910.146 covers the requirements of entering a confined space. E. Carbon Dioxide It is also known as CO2 and tends to accumulate at low levels and at the bottoms of enclosures such as pits, silos, tanks, and the like. It is sometimes used in the packing industry for immobilizing animals, and for quick-freezing or cooling fresh meats. Adequately ventilated areas will disperse the gas and prevent accumulation. F. Carbon Monoxide It is also known as CO. It is a colorless, tasteless, and odorless gas, slightly lighter than air. It is formed by incomplete combustion of organic materials. Exposure of approximately 200 ppm will result in headache and nausea. Overexposure could result in death. G. Nitrogen Nitrogen is a colorless, odorless, and tasteless gas. Nitrogen is frequently used for gasflushing certain packaged products to exclude oxygen. Adequate ventilation should be provided when this gas is used. H. Plastic Fumes Fumes are generated when flexible plastic fumes used in packing are heat-sealed. The fumes tend to accumulate in the immediate vicinity of heat elements. The fumes contains methylethyl ketone (MEK), toluene, propylacetate, or other solvents. Both MEK and toluene are known cancer-causing agents. These fumes are irritating to eyes and mucous membranes, and in high concentration can cause headache and drowsiness. Proper venting should be employed at the sealing area.

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CONCLUSIONS

There are many hazards at the workplace, such as ergonomics and workplace violence, that have not been addressed by a specific standard. Those hazards, however, if found, will be covered under Section (5)(a)(1), the general duties clause. Currently, there are only limited numbers of Federal and State OSHA inspectors and certainly not enough to cover inspection of each and every workplace. In real-life situations, different factors could contribute to an unsafe workplace or to a safer workplace. Safety is not the safety officer’s sole responsibility. Everyone in the company, from the president to each employee, is responsible for participating and contributing in his or her own way to make the workplace safer for everyone. Keeping everyone safe at work will preserve America’s most important asset— its people—and keep America’s workforce in a position of world leadership. REFERENCES 1. 2. 3.

Public Law 91-596, Occupational Safety and Health Act of 1970. Code of Federal Regulation 29 CFR 1910. U.S. Department of Labor Occupational Safety and Health Administration publication—OSHA 2098 1998 (revised). 4. Anonymous. www.osha.gov. 5. U.S. Department of labor Occupational Safety and Health Standard for General Industry (29 CFR Part1910) with amendment as of February 3, 1997. 6. U.S. Federal Register, volume 37 number 202, October 18, 1972. Page 22292.

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27 Waste Management ALBERT J. VAN OOSTROM Albert van Oostrom and Associates, Hamilton, New Zealand

I. INTRODUCTION II. WASTE CHARACTERISTICS III. WASTE SOURCES AND WASTE MINIMIZATION A. Overview and Principles B. Stockyards C. Blood Collection and Processing D. Trimming, Cutting, and Boning E. Viscera Processing F. Rendering G. Hide and Skin Processing IV. WASTEWATER TREATMENT A. Primary Physical Treatment B. Physicochemical Treatment C. Anaerobic Treatment D. Aerobic Treatment and Biological Nitrogen Removal E. Disinfection F. Land Treatment V. SOLID WASTE MANAGEMENT VI. CONCLUDING REMARKS ACKNOWLEDGMENTS REFERENCES

I. INTRODUCTION In meat processing, as in most other industries, the production of some waste is unavoidable. The primary product of processing livestock is edible meat for human consumption. All other materials that leave the meat processing plant are by-products or waste.

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For this chapter, by-products are defined as the non-meat components of the animal (and the products produced from them) that are saleable and generate revenue for the meat processor. By-products can include blood and renderable “waste” tissue, as well as the dried blood, tallow, and meat and bone meal produced from such raw materials. The manufactured by-products are also known as co-products, and the revenues from these products can greatly affect the profitability of a meat processing operation. Waste is defined in this chapter as materials resulting from meat and by-product processing operations that have no current economic value to the processor and that normally have a cost associated with their disposal. By this economic definition, what constitutes a waste or by-product can change with market conditions, and with the development of new economic uses for waste materials. Drawing from an adage, one man’s trash is another’s treasure. Although the meat processing industry has made major advances in waste reduction and by-product recovery, it still produces and discharges a significant amount of solid, liquid, and gaseous waste. This waste must be carefully managed and disposed of to avoid creating a nuisance or pollution hazard, and to minimize disposal costs. With increasingly strict standards and restrictions being imposed on how wastes can be disposed of, and on the levels of pollutants considered acceptable in the receiving environment, many meat processors face the challenge of improving their waste management practices in a cost-effective way. To do this they need to develop waste management strategies based on the following hierarchy: 1. 2. 3.

waste avoidance and reduction at source waste recovery, reuse, and recycling waste treatment and disposal

This chapter summarizes the principles and practices of waste management in the meat processing industry. The reader will be taken on a journey through this hierarchy of waste management. Along the way we will look at the sources and characteristics of the waste, as well as consider the potential effects of waste discharges on the receiving environment. II. WASTE CHARACTERISTICS Meat processing operations use large volumes of hot and cold water to maintain hygienic processing conditions. Various quantities of blood, fat, gut contents, feces, and other organic matter are washed down the drain, producing a wastewater with a not surprising resemblance to uncooked, unclean meat soup. In addition to these organic wastes, the wastewater may also contain small amounts of soil and grit from preslaughter washing of the animals, as well as detergents and other chemicals used during processing and cleaning. The volume of wastewater produced in slaughterhouses can vary greatly (Table 1), depending on the types of animal processed, the cost of the water, a company’s attitude to water conservation, and the extent of by-product processing undertaken on site. Table 1 Typical Slaughterhouse

Wastewater Volumes (liters·animal1) for Processing Different Animals Cattle Pigs Sheep and lambs

1200–5000 250–1000 200–800

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Table 2 Typical Concentration Ranges for Pollutants in the Combined

Screened or Settled Wastewater from Slaughterhouses (all units are g·m3, except for fecal coliforms, which are counts per 100 ml) Pollutant

Concentration range

Chemical oxygen demand (COD) Soluble COD 5-day biochemical oxygen demand (BOD5) Total suspended solids (TSS) Fat, oil, and grease Total Kjeldahl nitrogen (TKN) Ammoniacal nitrogen (NH3-N) Total phosphorus (TP) Fecal coliforms

2000–6000 1200–3600 1000–3000 200–2000 100–1000 100–300 10–80 10–30 107–108

As it leaves the processing operations, the wastewater contains varying amounts of coarse separable material. The first treatment step almost always involves primary treatment by screening or sedimentation to remove such solids. Primary treated wastewater typically contains high levels of 5-day biochemical oxygen demand (BOD5, a measure of organic matter that can be aerobically biodegraded in five days at 20°C), nitrogen, phosphorus, and fecal microorganisms, all of which are pollutants of concern. The characteristics of the primary treated wastewater vary widely from plant to plant, and at different times, depending on the amount of water used, the kinds of livestock slaughtered, and the processing operations undertaken. Table 2 gives the ranges of wastewater characteristics typical for slaughterhouses. By comparison, the concentrations of nitrogen and organic matter in slaughterhouse wastewater are typically 5 to 10 times higher than those in domestic wastewater (sewage). Definitions and measurement techniques for

Table 3 Mean Percentage of Settled Meat Processing Wastewater That is Soluble (or Colloidal), as Determined by Filtration Through a Whatman GF/C Glass Fiber Filter Pollutant

%

COD TKN Fat Total solids TP Carbohydrates

60 79 16 80 76 61

Source: From Cooper, 1982.

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Table 4 Fractions of COD in 1 mm-screened Slaughterhouse Wastewater (total COD of 2870 g·m3), as Determined by Filtration through 7.4 m and 0.45 m Filters Fraction Coarse suspended solids Colloidala Soluble

Nominal size range

Percent total COD

1 mm–7.4 m 7.4 m–0.45 m 0.45 m

45–55 20–30 20–30

a

Some of this colloidal fraction is commonly considered part of soluble COD Source: From Sayed et al., 1988.

the various wastewater pollutants discussed in this chapter can be found in Standard Methods for the Examination of Water and Wastewater (APHA, 1998). A high proportion of many components of screened or settled wastewater is in a soluble or colloidal form, as determined by filtration techniques (Tables 3 and 4). Therefore, these components are difficult to recover from the wastewater by physical means, but are more easily broken down by biological treatment than if they were present primarily as coarse particles. It is also useful to know the mass of each pollutant produced in meat processing operations (by determining waste stream volumes as well as pollutant concentrations) and to relate this to a unit of production, such as dressed carcass weight, live weight, or number and type of animals processed. Although this may seem an obvious calculation, there are very few published data expressing pollutants on a unit production basis. Some data for U.S. meat packinghouses are given in Table 5. The large variation reflects differences in the extent and nature of processing undertaken at different plants. To permit comparison between plants, waste production data should be accompanied by a description of the processing operations, as well as the nature of any treatment upstream of the sampling site. Table 6 indicates the range of pollutant loadings expected in the primary treated wastewater from cattle and sheep/lamb slaughterhouses with cutting and boning operations, but with minimal by-products processing done on-site. Similarly, when measuring waste from a specific by-product processing operation, it is useful to relate waste generation to a measure of by-product producted. Some such data are presented in the next section.

Table 5 Mean Pollutant Loadings in the Wastewater from U.S. Packinghouses (data are combined from four surveys; the ranges in parentheses represent one standard deviation about the mean; units are kg per tonne live weight) BOD5 13.3 (8.6–18.0)

TSS

Grease

Nitrogen

10.3 (5.5–15.1)

5.2 (0.2–10.2)

1.3 (0.9–1.7)

Source: From Witherow et al., 1973.

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Table 6 Typical Pollutant Loadings in the Total Screened or Settled Wastewater from Slaughterhouses with Minimal By-products Processing Done On-Sitea (data are based on unpublished measurements at New Zealand slaughterhouses; units are kg per tonne dressed carcass weight; dressed carcass weight is approximately 50% of live weight)

COD Soluble COD BOD5 TKN TP

Cattle

Sheep/lambs

18–36 11–22 9–18 1.2–2.4 0.12–0.24

22–44 14–28 11–22 1.5–3.0 0.13–0.26

a

For plants with blood collection, dressing, cutting, boning, tripe recovery (cattle), stripping of runners (sheep/lambs), and optionally gut cutting and washing. No on-site rendering, blood processing, or hide/skin processing except cooling with water.

III. WASTE SOURCES AND WASTE MINIMIZATION A. Overview and Principles 1. Waste Minimization and Recovery When organic materials enter the wastewater stream they add to its treatment and disposal costs. Some of the particulate solids can be removed by primary treatment systems, but after such treatment the wastewater still contains large quantities of soluble and fine particulate matter, and is generally unsuitable for discharge to the environment. The cost to discharge primary treated wastewater to a municipal sewer (if allowed), or to further treat the wastewater on-site, is usually very high and is directly related to the wastewater volume and pollutant content. Meat processors therefore have a powerful economic incentive to minimize both water use and the amount of material that enters the wastewater stream. This can be achieved, for example, by using “dry-cleaning” techniques, such as sweeping. Often the material recovered by dry-cleaning can be processed into products with a commercial value. Thus, waste minimization not only reduces wastewater treatment and disposal costs but also can reduce water supply cost and increase revenue. Where wastewater pollutant loadings cannot practicably be reduced at source, the next best option is usually to recover material from the wastewater stream by physical and/or physicochemical treatment methods. Generally, this is best accomplished by segregating different types of solids-laden waste streams and recovering the solids as soon as possible. These actions maximize the quantity and potential value of the materials recovered, as explained below. 2. Segregation of Waste Streams When assessing opportunities for reducing and recovering waste in meat processing, it is useful to distinguish between potentially valuable wastes derived from animal tissues (e.g., blood, fat, meat and bone), and low- or no-value wastes, such as animal gut contents, feces, and urine.

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Animal tissues can be processed into saleable by-products such as tallow, meat and bone meal, or dried blood. These materials contribute to waste only if they are washed down the drain. Waste minimization and recovery applied to gut contents and fecal material is more complex. As with waste animal tissues, the collection of gut contents and feces in a relatively dry form (e.g., dry-dumping paunch contents and dry-collection of stockyard wastes), or their partial recovery from the wastewater by primary treatment, can considerably reduce the wastewater pollutant loading and wastewater treatment costs. However, dry collection can be difficult, and the collected solids pose a significant disposal problem for many slaughterhouses. Nevertheless, managing solid waste is usually more cost-effective than treating and disposing of it as a part of wastewater. Initial segregation of wastewater streams containing animal tissues from those containing fecal matter and gut contents allows animal tissues to be recovered without contamination that can downgrade rendering products. Equally, feces and gut contents can be recovered with minimal contamination by animal tissue. This is important because animal tissue increases the potential for the recovered solids to generate odor and attract vermin, which can restrict the utilization and disposal of these solids. For example, gut contents and feces that are relatively free of animal tissues can be stabilized by simple windrow composting, whereas more costly composting techniques or alternative treatment methods are generally required for similar wastes containing animal tissue. 3. Recover Without Delay and Close to Source As soon as solids enter the wastewater stream they begin to break down and release soluble material. This release is increased by turbulence (e.g., by pumping) and by high temperatures. Therefore, if the solids cannot be recovered dry, they should be removed from the wastewater stream as quickly and as close to source as possible. A schematic diagram of the main sources and flows of wastes, in relation to meat and by-product processing operations, is given in Fig. 1. Some of the main sources of waste in meat processing, and techniques for their minimization, are discussed in the following sections. B. Stockyards The waste from the holding and washing of livestock prior to slaughter is mostly fecal matter and urine, although some soil and grit may be present, depending on animal cleanliness. The quantity and characteristics of the waste voided by the livestock depend on several factors: Animal species, size, and recent diet How long the animals were held off feed prior to arriving at the plant How long the animals were held at the meat plant before slaughter Quantities of waste voided by cows and sheep/lambs during holding in yards for 24 hours are given in Table 7. To reduce the amount of fecal waste, animals are sometimes fasted before transport to the meat plant. However, this practice is difficult for meat packers to control and can potentially result in significant dehydration of the animals and carcass weight loss. A further opportunity to reduce stockyard waste is to slaughter directly off the truck, and thus reduce the time the animals are held in the yards. However, the animals will have a fuller intestinal tract, and the wastewater management benefit of “tailgate” slaughter is realized only if the gut contents are recovered undiluted during later viscera handling. Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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Figure 1 Generalized schematic of waste flows and treatment stages in relation to common meat and by-product processing operations. In accordance with good practice, waste streams containing predominantly animal tissues are segregated and treated separately from those containing predominantly fecal matter and gut contents. (Adapted from Jones, 1974.)

A key characteristic of stockyard waste is that typically 50% to 60% of the TKN is soluble organic nitrogen (i.e., soluble TKN less ammoniacal nitrogen). The urea component of urine is the dominant source of this organic nitrogen. This high proportion of urinederived nitrogen limits the effectiveness of dry-cleaning and primary treatment techniques in reducing the amount of nitrogen in the wastewater from stockyards. Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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Table 7 Quantities of Material Voided by Pasture-Fed Sheep and Cattle during 24 h Holding,

and the Percentage of the Pollutants Recoverable by Various Means (Units are g·animal1 unless otherwise specified) Sheep and lambs Cows

Recoverable (%) by

Parameter

Mean

Range

Recoverable (%) by A

Mean

Range

A

B

COD Soluble COD TKN Soluble TKN NH3-N TP

1265 321 109 84 28 8.3

765–1618 237–474 68–158 55–129 12–43 4.8–13

17–49 — 3.6–9.2 — — 5.0–14

142 — 14 — 5.3 1.4

60–230 — 9–19 — 3.0–8.0 0.7–2.3

36 — 11 — — 31

70 — 24 — — 74

The different groups of animals had been withheld from pasture for 5–22 hours (cows) or 0–32 hours (sheep and lambs) prior to arrival at the meat plant. A
Treatment of wastewater with a 0.5 mm wedge-wire screen. B
Dry-cleaning pens followed by washing the pens and screening of wastewater with a 0.5 mm wedge-wire screen. Source: MIRINZ, unpublished data.

The usual practice for managing stockyard wastes is to flush them into the wastewater stream using large volumes of water, then to recover a proportion of the solids by sedimentation or screening. Typically only 17% to 49% of the organic load (measured as COD) and 4% to 12% of the nitrogen (measured as TKN) will be removed by such screening (Table 7). However, recoveries can be increased to 70% and 24%, respectively, by drycleaning the pens before washing (Table 7). In Australasian slaughterhouses, stockyards for sheep and lambs are covered and typically have a raised floor comprising a metal grating through which the feces and urine fall. Most of the urine drains to wastewater, but the feces are often allowed to accumulate under the grating for several days before being washed to the wastewater stream. The accumulated solids consolidate and dry somewhat, and can be recovered quite effectively if screened from the wastewater close to source before the solids disperse/dissolve significantly. Recovery could be improved by collecting the solids dry, but this is not common, as it requires machine access beneath the grating. The dry recovery of fecal matter from cattleyards is more difficult than for sheep and lambs, as cattleyards usually have a solid floor and need frequent washing. C. Blood Collection and Processing 1. Blood Characteristics Blood contains high concentrations of total solids and nitrogen and has a high oxygen demand (Table 8), and thus only small losses of blood into wastewater can significantly increase pollutant loadings and treatment costs. 2. Blood Collection It is normal practice to collect the blood from animals after slaughter. The blood may be collected by special hygienic techniques for edible purposes, but most commonly it is colCopyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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Table 8 Pollutants of Concern in Undiluted Bovine or Ovine Blood Typical concentration (g·m3) Total solids COD BOD5 Total nitrogen Total phosphorus

200,000 300,000 200,000 30,000 200

lected for inedible processing by allowing it to drain into a collection pit or trough. By the time a carcass has moved past the blood collection area, blood flow has usually slowed to a drip, but blood loss from the carcass can increase periodically due to hide pulling, brisket cutting, and head removal. The quantity of blood that drips on the floor after the designated collection site is usually much greater than it looks, as it is spread thinly over a large area and is regularly washed away. In an unpublished MIRINZ study, the volume of blood lost from carcasses at two beef and two ovine processing plants was measured between the end of bulk blood collection and evisceration. Blood loss in this period averaged 1.2 and 2.4 liters per beef animal, 0.12 and 0.22 liters per lamb, and 0.14 and 0.31 liters per sheep. Detailed data for beef at one plant are shown in Table 9. Blood rapidly coagulates once it has left the carcass. Much of the blood loss that occurs after the collection trough can be recovered by dry-cleaning the floor under the carcasses and heads using a squeegee, then pushing the amassed blood into the blood collection system or scooping the mass into a holding bin for processing with other recovered blood. A low nib wall on the floor around the areas to be dry-cleaned helps to contain the blood and minimize its dilution by wash water used in other operations. Thorough dry cleaning is important for all areas where blood accumulates. At one beef plant, on average 96 liters of coagulated blood lining the collection pit was washed into the wastewater at each break in the working day. This loss of blood, which was equivalent to about 1 liter per animal processed, occurred because the pit design made dry-cleaning difficult, and because staff did not realize how much blood was lost by washing nor its effects in the waste stream. Good management and an understanding of downstream consequences are essential in waste minimization. Table 9 Volume of Blood Drip Lost at One Plant from Beef Carcasses and Detached Heads Between the End of Formal Blood Collection and Evisceration

Site of blood loss Between collection through and hide puller During hide removal From detached head Between hide puller and evisceration Total

Elapsed time after throat cut (min:sec)

Mean blood volume collected (ml.animal1)

6:00–14:00 14:00–16:00 15:45–16:00 16:00–18:00

1540 570 220 100 2430

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In addition to the sources of blood loss already mentioned, significant quantities of blood unavoidably enter the wastewater streams from floor washing in carcass chillers, and from the initial washing and cooling of sheep skins and cattle hides. Blood is too sparse in these situations to warrant dry recovery. It is important that slaughterhouses maximize their blood recovery. An increase in blood recovery of, say, 2 liters per beef animal, which equates to about a 15% increase in blood yield, reduces wastewater COD and nitrogen loadings by 600 and 60 g per animal, respectively. 3. Blood Processing For plants that process blood on site, additional losses to wastewater will occur to a degree that depends on the processing method. Usually blood is dried to produce blood meal, a source of animal protein. The most popular method of producing blood meal involves coagulating the blood proteins by steam injection, centrifuging the coagulum from the aqueous fraction, and then drying the coagulum (Fernando, 1992). The efficiency of the coagulation and separation steps, and thus the quantity of blood solids lost to drain in the aqueous phase, depends on: The amount of water added to the blood during collection (less is better) How long the blood was aged prior to processing (ageing improves coagulation) The coagulation temperature (90° to 95°C is optimal) Potentially the loss of blood solids in the aqueous fraction can be as low as 4%, but in practice a loss of less than 10% can be considered satisfactory (Pilkington, 1975). Additional blood losses to wastewater will occur if a water scrubber is used to remove blood dust from the air stream from the blood dryer, and during the cleaning of blood holding tanks. At one facility an inefficient dust removal system on a blood dryer caused almost 10% of the product to be discharged to wastewater (MIRINZ, unpublished data). Blood processing methods that use advanced techniques such as ultrafiltration to separate the blood proteins from whole blood, or involve the drying of whole blood, produce less waste than the coagulation method, but are much less common due to their current higher cost. As blood can be a major source of wastewater organic and nitrogen loading, plants should monitor their blood collection and processing efficiencies. For plants that process the collected blood on-site, this simply requires relating the amount of dried blood produced to the total carcass weight of source animals. For ruminants, a yield of 12 to 15 g of dried blood per kilogram of dressed carcass weight is an achievable goal. D. Trimming, Cutting, and Boning Some animal tissue waste, typically meat and fat trimmings and fine debris from carcass saws, is unavoidable in meat processing. Dry-cleaning methods should be used to collect the solids close to source to maximize recovery for rendering. Gratings and perforated baskets in floor drains are normally used to prevent large pieces from entering the wastewater. If washed into the wastewater, a proportion of the smaller solids that pass through these gratings cannot be recovered by screening and sedimentation, so producing a significant wastewater load requiring treatment.

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E. Viscera Processing Slaughterhouses may carry out one or more viscera processing operations, all of which contribute gut contents and sometimes gut tissue to the wastewater stream. 1. Paunch Emptying Cattle paunches (rumens) are processed for recovery of edible products (e.g., tripe), for pet food production, or for rendering. Clearly the paunch contents must be emptied if paunches are destined for human or pet food. However, even when paunches are simply sent to rendering, they are normally emptied first. The volume and characteristics of cattle paunch contents vary, depending on the size of the animal, its recent diet, and how long it has been off feed and water prior to slaughter. Paunches of pasture-fed cattle typically contain 30 to 40 kg of material (Table 10), but the paunch of a large animal may contain 80 kg or more. Traditionally, the paunches are manually slashed and their contents flushed out with water. This is called wet dumping (Witherow, 1974). Large volumes of water (100 to 200 liters) are used to both clean the paunch sac (for tripe recovery) and carry the paunch contents out of the plant. When paunch contents are wet-dumped, only about 10% to 30% of the nitrogen and phosphorus and 40% of the total solids in the contents can be readily recovered from the wastewater by screening or sedimentation. The resulting waste stream is therefore high in pollutants. This loading on the wastewater system can be mostly avoided by dry dumping the paunch contents or rendering the full paunches (Fig. 2). Dry dumping involves opening the paunches to release their contents without the aid of water. After dry dumping, the paunch sac, still containing about 10% of its original contents, may be washed for tripe recovery or sent to rendering or pet food processing, as shown in Fig. 2. Compared with wet dumping, dry dumping can reduce the wastewater loading from paunch handling by 90% or more. Dry-dumped paunch contents (8% to 10% total solids) are sometimes dewatered by being passed over a screen or through a screw press to make them more manageable. Dewatering to 15% to 20% total solids removes free-draining liquid and this is all that is required. The recovered liquid is obviously high in nitrogen, phosphorus and BOD5, but its volume is low, and the mass of soluble pollutants lost to wastewater is only about half that of wet dumping systems. Nevertheless, the volume of liquid drained or squeezed from the dry-dumped solids should be minimized to maximize the waste management benefits of dry dumping. Conversion from a wet-dump to a dry-dump system is one of the best single methods of reducing the wastewater pollutant loading at beef plants. For a beef slaughterhouse with Table 10 Typical Composition of Paunch Contents of Pasture-fed Cattle Mass (g·paunch1) Total wet weight Total solids Total nitrogen Total phosphorus Sodium

40,000 4,000 100 35 100

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Figure 2 Paunch handling methods. minimal on-site by-products processing, conversion to dry dumping (with washing of the dry-dumped paunch sac) can reduce total plant wastewater solids, nitrogen and phosphorus loadings by 18% to 36%, 9% to 18% and 20% to 46%, respectively, depending on the extent of solids dewatering (van Oostrom and Muirhead, 1996). 2. Viscera Cutting and Washing for Rendering Condemned paunches and other inedible gut material are sent to rendering. Before rendering, this material is typically macerated in a mechanical gut cutter, followed by separation of tissue from gut contents, usually in a rotating wash screen. This washing increases the value of the gut tissues as a rendering feedstock, but the large volume of wastewater produced contains a high pollutant loading from the gut contents and from dislodged fat. Swan et al. (1986) found that the amount of TKN, fat, and COD in the wastewater from four gut cutting and washing systems ranged from 490 to 720 g, 2.0 to 9.8 kg, and 17 to 33 kg per metric ton of dressed carcass weight, respectively. Fat losses were greater for cutters that employed blunt, fast-moving blades than those with sharp, slow-moving blades. A proportion of the gut solids can be recovered from this wastewater by fine screening, but the collected solids are contaminated with fat, which may restrict how they can be treated or disposed. The waste from gut washing can be avoided altogether by rendering the gut intact. However, this is generally not an economic option, as the rendering of gut contents degrades tallow quality and value (by increasing the tallow free fatty acids and color), reduces the meal protein content, and increases rendering energy costs. However, these disadvantages might be outweighed by reduced fat losses, a reduction in wastewater and solid waste Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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loads, and simplification of materials handling equipment (Cooper, 1975). The economic feasibility of this option has to be considered case by case. 3. Sausage Casings When natural sausage casings are made from animal intestines, the first processing step involves squeezing fecal material out of the intestines. In a series of further steps the mucosa and other tissues are removed from the intestinal wall to produce a clean casing (the submucosal layer). Large volumes of water are used to remove the intestinal contents and animal tissue wastes. To reduce water use, the cleaner wastewater streams are sometimes recycled to the initial processing steps. During the processing of lamb intestines, the animal tissue waste consists of approximately 80% protein and accounts for 68% to 77% of the total COD, nitrogen and phosphorus waste loading from the process (Fig. 3). It is good practice to keep the gut content and waste tissue streams separate, to enable the fecal waste to be treated with other gut-content wastewater, and the animal tissue waste to be rendered. Another pollutant from casings manufacture is the salt (sodium chloride) that is used to preserve the casings. Salt waste should be minimized, especially if the wastewater is applied to land, as excessive application of sodium can damage soil structure. F. Rendering Rendering operations, which usually include blood processing, can account for 30% to 45% of the total wastewater volume, and about 50% to 60% of the wastewater nitrogen loading from beef processing plants (Johns et al., 1995).

Figure 3 Pollutants resulting from the production of sausage casings from lamb small intestines. (MIRINZ, unpublished data.)

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The quantity of raw materials and product lost to wastewater depends on how the raw materials are handled, the rendering technology, and the general standard of housekeeping. Rendering technologies have been reviewed by Swan (1991) and Fernando (1992). The main sources of waste from rendering are discussed below. 1. Raw Material Conveyance and Storage Fluid that drains from materials during conveyance to and storage at a rendering plant contains high concentrations of blood, fat, and other animal tissues. This significant source of wastewater pollutant loading can be minimized by: minimizing the addition of water to the materials using dry conveyance systems (e.g., screws and transport bins), which, unlike waterchutes, pumping, and sometimes blow conveying, do not require the addition of water to aid conveyance avoiding compression of the raw materials delaying size reduction until immediately before rendering 2. Condensate from Cookers and Meal Dryers A large proportion of the volatile organic compounds in the hot gases released during cooking and drying processes ends up in the condensate that forms when these gas streams are cooled for heat recovery and odor control. The volume of condensate from dry rendering systems depends largely on the amount of raw material and water loaded into the cookers. The condensate contributes a significant nitrogen and organic matter load to rendering wastewater. The non-condensable gas stream contains several hundred volatile organic compounds (Luo et al., 1999) and normally requires further treatment to remove odor before discharge to atmosphere. 3. Stickwater In wet rendering and low temperature rendering processes, the meal, tallow, and aqueous phases are separated after the cooking stage by various decanting, pressing, centrifugation, and drying steps. The aqueous phase, often called stickwater, contains solubilized fat, protein, and minerals and can be a major source of wastewater pollutant loading. For example, in the MIRINZ Low Temperature Rendering process, the stickwater separated from the tallow by centrifugation contains high concentrations of pollutants (Table 11). Losses of fat and nonfat solids in this stream typically equate to about 2% and 6% of tallow and meat Table 11 Typical Characteristics of Stickwater from the MIRINZ Low Temperature Rendering Process Concentration (g·m3) COD Total solids Fat TKN

30,000–80,000 20,000–60,000 500–10,000 1,500–4,000

Source: Compiled from Brown et al. 1993, Fernando 1982, and unpublished data.

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meal produced, respectively. pH adjustment of the influent and correct operation of the centrifuge are critical in minimizing this product loss to stickwater. At some rendering plants the solids in stickwater are concentrated by evaporation or recovered by physicochemical treatment, and incorporated into the meal product. Ultrafiltration can recover a high proportion of the stickwater solids (Brown et al., 1993), but this technology has not yet been adopted by industry for this application. 4. Tallow Refining Depending on the process, tallow may be further treated by washing and centrifugation to remove fines and other impurities. The wastewater from this process can be another significant source of organic loading. G. Hide and Skin Processing Soon after removal from a carcass, cattle hides and sheep skins are normally washed and cooled in water to remove blood and loose contaminating material. If they cannot be further processed quickly, they must be temporarily preserved (cured) to prevent deterioration. The most common preservative is salt (sodium chloride), as crystals or as brine. Fungicides and other biocides may also be used in addition to the salt, and curing may also involve “fleshing,” which is the removal of flesh or adipose tissue remaining on the hide. The fleshings are normally rendered. Salt and other chemicals lost to wastewater during curing, and washed from the hides and skins during later processing, can represent a significant disposal problem. In the United States this source of waste is increasingly being avoided by processing the hides onsite so that curing is not necessary (Lollar, 1992). However, if further processing is done on-site, this activity produces its own waste streams that must be managed. For example, in New Zealand, sheepskins have for many years been processed (in departments called fellmongeries) immediately after slaughter. As opposed to simply salting and exporting the whole skin with wool, fellmongering operations remove the wool from the skin with the aid of chemicals, and provide advanced preparation of the pelt for manufacturing leather. After cooling and washing the pelts to remove blood and loose dirt, fellmongery processing steps variously include the use of caustic lime and sulfide depilatory paint, lime-sulfide solution, deliming chemicals, enzymes, sulfuric acid, and salt. Each can add to the wastewater load. The resulting wastewater contains high concentrations of COD and nitrogen (from hydrolyzed wool, epidermal tissue, and adipose tissue), as well as sulfide, calcium, and other chemicals used in the process. Typical wastewater pollutant loadings from the major waste-producing operations in the fellmongering of ovine skins are given in Table 12. For sheep and lamb slaughter plants, an on-site fellmongery can double the wastewater nitrogen and increase the COD by 25%. Because of its high sulfide and total sulfur content, the wastewater is normally treated by chemical oxidation and/or aerobic biological treatment (not anaerobic) to oxidize the sulfides to sulfates, and thus avoid odor and toxicity problems from sulfides, as discussed later. The main opportunity to minimize the wastewater loading from skin processing is to maximize the recovery of wool from the skins in the early stages of fellmongery processing, before the skins are immersed in a lime-sulfide solution that chemically degrades the residual wool, adding to the wastewater loading. As wool is a saleable product, there is an economic incentive to maximize its recovery.

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Table 12 Total Pollutants Discharged from the Liming, Deliming and Enzyme-Wash Steps of the Fellmongering Process (Units are g·animal1 unless indicated otherwise) Pollutant

Lambs

Sheep

COD Soluble COD TKN Soluble TKN Fat Total solids Sulfide-S Thiosulfate-S Water use (L·animal1)

168 143 25 23 6 246 6.2 6.1 25

210 173 32 30 9 291 7.7 6.9 32

Source: From Cooper and Russell, 1982.

IV. WASTEWATER TREATMENT Various treatment methods are commonly applied to meat processing wastewater, as discussed below. A. Primary Physical Treatment The initial treatment of meat processing wastewater (termed primary treatment) almost always involves a simple physical separation process to recover particulate solids. The main physical treatment processes are screening, sedimentation, and flotation. 1. Screening Most meat processing plants use fine screens on one or more of their wastewater streams. Common screen types include brushed screens, rotating drum screens, inclined static screens and vibrating screens. Screen materials include woven wire, perforated plate, and wedge-wire. Static and rotating-drum wedge-wire screens are most popular (Fig. 4). The wedge-shaped bars of such screens act to prevent the screen from blinding. The required screen size depends on the flow rate, and the nature and quantity of the particulate solids in the wastewater. Slots of 0.5 to 1.0 mm are suitable for most applications in meat processing. Narrower slots reduce the water flow rate through the screen whereas wider slots reduce the efficiency of solids removal. 2. Gravity Separation Primary sedimentation tanks (primary clarifiers, save-alls, interceptors, grease traps, manure pits) have a long history in meat processing for the gravity-assisted removal of settleable solids and flotable matter from wastewater. The equipment usually consists of a primary clarifier or save-all with top and bottom scrapers, which continuously remove the floating grease and settled solids. For smaller applications and for stockyard waste streams, grease traps and manure pits are sometimes used, respectively, where solids are removed periodically by various means. The removal efficiency for suspended solids depends on the hydraulic retention time and flow characteristics in the tank, and on how often the solids are removed. The hydraulic

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Figure 4 Common types of wedge-wire screens.

retention time for primary clarifiers used in the industry is typically between 30 and 60 minutes. Gravity separation can generally remove a greater proportion of solids (particularly fat) than screening, but has higher operating costs and a greater potential to generate odor. The trend has therefore been to replace gravity separators with screening or, increasingly, a combination of screening and dissolved air flotation technology. 3. Dissolved Air Flotation In the dissolved air flotation (DAF) process, suspended solids in the wastewater are removed by flotation assisted by micrometer-sized air bubbles. The bubbles are produced by dissolving air in the wastewater or a recycle stream at 3 to 5 atmospheres. When the pressurized air-saturated liquid enters the DAF tank, the decrease in pressure results in the release of tiny air bubbles, in the same way that bubbles are released from solution when champagne is opened. The rising bubbles adhere to suspended solids in the wastewater and assist flotation. The floating solids are recovered with scrapers. DAF systems come in a variety of designs that vary in tank geometry, method of solids removal, and method of introducing the air-charged water into the flotation tank. In some systems a pressurized recycle stream is introduced together with the wastewater into the flotation tank, in others these streams enter the tank separately. A simplified flow scheme of the latter type is given in Figure 5. Although DAF has higher capital and operating costs than passive gravity separation, DAF works faster and produces a drier sludge. DAF systems are popular for recovering fat and protein from meat processing wastewater. The technology is commonly applied instead of, or after, screening or settling, and often incorporates upstream chemical addition to augment its effect, as discussed below.

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Figure 5 Schematic of a dissolved air flotation (DAF) process.

B. Physicochemical Treatment Simple physical processes will not remove soluble proteins, fat emulsions, or colloidal material from wastewater. However, by adjusting pH and dosing the wastewater with specific coagulants and flocculants, some of this dissolved and finely dispersed organic matter can be precipitated and agglomerated into larger particles (flocs) that can be recovered by a physical process such as DAF or settling. Figure 6 shows a generalized scheme of physicochemical unit operations. Without physicochemical treatment, many organic materials in the wastewater will not agglomerate and settle because of their small mass and because they carry a surface charge. The repulsive forces between particles are greater than the forces (e.g., gravity) that cause them to aggregate or settle. The mechanisms involved in chemical coagulation and flocculation involve reducing particle charge or overcoming the effects of the charge. For example, dissolved protein molecules carry a net negative charge at pH values around 7. Acidification of the solution reduces this charge, and at a specific pH for each

Figure 6 Unit operations commonly involved in physicochemical treatment systems.

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protein, known as the isoelectric point, the protein carries no net charge. At this point the electrostatic repulsion between protein molecules is minimal, which allows some proteins to flocculate. On the other hand, acidification to pH values below the isoelectric point will result in the proteins carrying a net positive charge. Adjustment of the pH of meat processing wastewater to between pH 4 and 5 will remove many proteins from solution. Within this range, many of the proteins are at or near their isoelectric points and collectively they have minimal solubility. However, some proteins, including the blood protein hemoglobin, do not flocculate at their isoelectric point and cannot be removed from solution by simple pH adjustment (Cooper et al., 1983). The use of cationic and anionic coagulants with pH adjustment can provide more effective removal of protein and other organic material than pH adjustment alone. The most common cationic coagulants used in wastewater treatment are iron (Fe3) and aluminum (Al3) salts. These ions interact with negatively charged proteins and colloids, and for meat processing wastewater, they have maximum effect in the pH range 5.0 to 5.5 (Russell and Cooper, 1981; Travers and Lovett, 1984). Synthetic polyelectrolytes are often added to the wastewater after addition of the salts to assist in the formation of large, stable, dense flocs that are easily separable from the liquid and to aid subsequent dewatering. An advantage of using Fe3 and Al3 salts is that they also precipitate out much of the phosphorus from wastewater. A disadvantage is that these salts can make the recovered solids unsuitable for use as animal feed. Anionic coagulants interact with positively charged proteins and, in contrast to cationic coagulants, are effective at pH levels on the acid side of the isoelectric point. The most useful anions include sodium hexametaphosphate at pH 3.5 (Cooper et al., 1983), lignosulfonate at pH 3 (Foltz et al., 1974), and sodium alginate at pH 3.5–4.5 (Russell et al., 1984). These anionic coagulants remove hemoglobin, which can make up a large proportion of the soluble organic load in wastewater from meat processing. As well, these coagulants are nontoxic, so the recovered solids can be used in animal feeds. Hemoglobin and other proteins can also be removed with a two-stage pH process (Cooper et al., 1982). The wastewater is acidified to pH 3, which precipitates some proteins and irreversibly splits the hemoglobin molecule into globin subunits. The pH is then raised with calcium hydroxide, and the globin units precipitate at their isoelectric point of pH 6.5. To effect good floc formation, the pH is raised to between 8 and 9 and an anionic polyelectrolyte is added. Raising the pH to 9 with calcium hydroxide can also remove much of the phosphorus. Generally, 40% to 70% of the TKN, 50% to 80% of the COD, and more than 80% of the fat can be removed from meat processing wastewater by the physicochemical treatment methods discussed. Depending on the chemicals used, a large proportion of the phosphorus can also be removed. Comparative performance data are given in Table 13. In the meat industry, physicochemical treatment is generally applied before biological treatment or discharge to a public sewer. It is particularly suitable where land area is limited and in situations where solids can be incorporated into a saleable product like meat and bone meal. A disadvantage of chemical treatment is the high cost of the chemicals. Also, the chemicals have the potential to create downstream problems. For example, the use of sulfur-containing chemicals can increase the risk of odor and corrosion if the wastewater is to be treated anaerobically.

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Table 13 Performance Comparison of Different Methods for Chemically Treating a Sample of Meat Processing Wastewater (Units are g·m3)

Effluent Pollutant COD Soluble COD TKN Soluble TKN NH3-N Fat

Influent 2240 1440 165 130 10 250

Isoelectric pH 4.5

Two-stage pH adjustment

Lignosulfonate process

Hexametaphosphate process

950 880 100 90 10 55

890 750 95 80 10 20

750 670 70 65 10 35

580 500 60 50 10 30

Source: Data are from Cooper et al., 1983.

C. Anaerobic Treatment 1. Overview In anaerobic treatment systems, organic matter in the wastewater is converted to methane and carbon dioxide in the absence of oxygen. Three main groups of bacteria are involved: hydrolytic and fermentative bacteria (also known as acidogenic bacteria), acetogenic bacteria, and methanogenic bacteria (Fig. 7). The hydrolytic/fermentative bacteria hydrolyze fats, proteins and complex carbohydrates into subunit fatty acids, amino acids, and simple sugars, and then ferment these to shorter chain fatty acids, acetic acid, formic acid, alcohols, hydrogen, and carbon dioxide.

Figure 7 The main transformations in the anaerobic microbial conversion of organic wastes to methane and carbon dioxide.

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The acetogenic bacteria convert fatty acids and alcohols to acetic acid, formic acid, and hydrogen gas, whereas methanogenic bacteria use the acetic acid, formic acid, and hydrogen gas as substrates for methane production. Anaerobic treatment depends on complex interactions between bacterial activities. For example, the acetogens produce the acetic acid and hydrogen required by the methanogens and consume various fatty acids that are toxic to the methanogens. In return, the methanogens remove hydrogen, which is toxic to the acetogens. A balance between microbial populations is essential for the stability and performance of an anaerobic treatment system. Anaerobic treatment performs best around pH 7, and when there is a high level of bicarbonate alkalinity to buffer the effects of organic acid production. Temperature is also important. The rate of anaerobic digestion at the normal temperature of meat processing wastewater (20° to 35°C) is usually satisfactory, but digestion is more rapid at higher temperatures. Meat processing wastewater is well suited to biological treatment, as it contains all the nutrients required for microbes to grow. The fat and protein it contains are rapidly degraded. Anaerobic treatment processes commonly achieve removal rates of 70% to 90% for COD and BOD5. The main advantage of anaerobic treatment is its low operating cost, due to low sludge production and low energy requirements. For every unit of COD removed anaerobically, only about 5% to 15% ends up as sludge, contrasting with about 40% to 60% for aerobic biological treatment and 100% for physical and physicochemical treatment. The waste sludge from anaerobic and aerobic treatment consists of bacterial biomass, as well as any refractory and slowly biodegradable particulate material present in the process influent. Anaerobic treatment requires little or no energy input and is a net producer of energy if the biogas is recovered as fuel. Methane yields of up to 0.23 kg per kg COD removed have been reported for the anaerobic treatment of meat processing wastewater (Metzner and Temper, 1990; Borja et al., 1995), 92% of the theoretical maximum. This yield translates to 12.8 MJ of energy per kg of wastewater COD removed. Anaerobic treatment does not remove nitrogen or phosphorus. A further disadvantage is that it rapidly reduces organic forms of nitrogen and sulfur to ammonia and hydrogen sulfide, which can be toxic to fish and other aquatic organisms. The hydrogen sulfide can also cause an odor nuisance and corrosion of equipment. (In anaerobic treatment, sulfides are also produced by bacterial reduction of sulfates in the wastewater.) Therefore, anaerobically treated meat processing wastewater usually requires further treatment before discharge to waterways. Anaerobic treatment of meat processing wastewater is generally applied as a treatment step before discharge to a public sewer, aerobic biological treatment, or land application. 2. Anaerobic Lagoons Anaerobic lagoons are a popular method of treating meat processing wastewater because of their simplicity, reliability, and low cost. They are typically between 3 and 6 m deep, with an operating volume that equates to a loading rate of 0.1 to 0.4 kg BOD5m3day1 (approx. 0.2 to 0.8 kg CODm3day1) or a hydraulic retention time of 5 to 15 days. Anaerobic lagoons are shaped to suit their site, but generally the greater the length:width ratio—where the influent and effluent are at opposite ends—the better the performance because short-circuiting of flow is minimized. Sometimes several anaerobic lagoons are operated in parallel or in series.

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A layer of sludge consisting of denser wastewater solids and anaerobic biomass forms at the bottom of the lagoon; fat and other floating solids form a scum (Fig. 8a). Gas belches from the sludge layer causing localized mixing of sludge with the supernatant, particularly near the inlet end of the lagoon, and this mixing aids in the removal of soluble BOD5 from the wastewater. Typically, sludge needs to be removed every 5 to 10 years. Anaerobic lagoons have the potential to cause an odor nuisance, but this risk can be minimized or eliminated by: minimizing wastewater sulfur loading: changing to a low-sulfate water supply (Chittenden et al., 1977) and reducing the use of sulfur-containing chemicals in processing and wastewater treatment can reduce odor promoting a stable scum layer on the lagoon: scum reduces odor production; the rate of scum development can be enhanced during lagoon commissioning by temporarily discharging an increased quantity of paunch contents and fat to the lagoon covering the lagoon with a membrane: this allows the odorous biogas to be collected and burned as fuel or simply flared (Chittenden et al., 1977; Dague et al., 1990) making the lagoon as deep as possible, because reducing the surface area helps minimize odor release

Figure 8 Examples of anaerobic treatment technologies.

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3. High-rate Anaerobic Systems High-rate systems are characterized by high densities of anaerobic microorganisms (typically 4000 to 8000 gm3 measured as suspended solids), allowing BOD and COD loading rates typically 5 to 20 times greater than those of anaerobic lagoons. The relatively small size of high-rate systems makes them most suitable where land area is limited, and biogas collection and/or strict odor control are objectives. In suspended-growth systems, the biomass in the reactor is maintained in suspension as flocs or granules. Suspended-growth technologies differ in the way that they maintain high concentrations of suspended biomass in the digester. In one such technology, the anaerobic contact process (Fig. 8b), the digester contents are stirred. The biomass washed out with the effluent is recovered by gravity in a clarifier and some is returned to the digester. The solids-laden effluent from the digester must be degassed (e.g., by applying a vacuum) to effect good biomass settling in the clarifier (Steffen and Bedker, 1962; Stebor et al., 1990). In the upflow anaerobic sludge blanket process (Fig. 8c), the wastewater passes upward through the sludge blanket at a rate that prevents washout of the biomass, and thus avoids the need for a separate clarifier tank. In attached-growth systems, the biomass is immobilized on media that have a high surface-to-volume ratio. These systems include anaerobic biofilters (Fig. 8d; Metzner and Temper, 1990) and fluidized bed reactors (Borja et al., 1995). Anaerobic sequencing batch reactors work with repeated cycles of fill, react, settle, and decant. (The aerobic counterpart of this process is illustrated in Figure 10.) This relatively new process shows much promise for the treatment of meat processing wastewater (Morris et al., 1998). Increasing concerns about odor from anaerobic lagoon systems will probably result in an increased use of high-rate enclosed anaerobic systems in the meat industry. However, compared with lagoons, such high-rate systems have higher capital and operating costs, and their performance can be more sensitive to variations in organic loading. D. Aerobic Treatment and Biological Nitrogen Removal 1. Overview Before anaerobically treated wastewater is discharged to waterways, it is treated aerobically to remove most residual BOD5 and suspended solids, and to oxidize ammonia and hydrogen sulfide to less harmful nitrate and sulfate. Increasingly, aerobic treatment is being coupled with specialized anoxic treatment to biologically remove nitrogen as well. Aerobic treatment is also commonly used to treat meat-processing wastewater before land application. If the wastewater is not properly treated, the BOD5 and suspended solids can result in oxygen depletion and produce turbidity and color in the receiving waters. Moreover, ammonia and hydrogen sulfide can deleteriously affect aquatic life. Discharges of nitrogen and phosphorus nutrients are increasingly being controlled because they contribute to algal blooms and other undesirable biological growths in waters. Aerobic biological treatment systems can be designed for carbonaceous BOD5 reduction only, but for meat processing effluent they are usually also used for ammonia oxidation (nitrification), and sometimes nitrogen removal by nitrate reduction (denitrification). Sulfide will be rapidly oxidized in these systems without need for special design.

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2. Main Microbial Processes a. Carbon Removal. During aerobic treatment, heterotrophic bacteria remove organic matter (measured as COD and BOD5) from the wastewater by two main methods: by biological oxidation to carbon dioxide and water in the presence of oxygen, and by incorporation into cell biomass, which is subsequently removed as sludge. About 60% to 70% of the COD taken up by the heterotrophic bacteria is incorporated into biomass, while the balance is respired to provide the energy for cell synthesis (Eq. 1). Organic substrate  nutrients  O2 → CO2  bacterial cells  other end products

(1)

As well as growth, there is biomass decay by respiration using cell biomass as an energy source. Decay of biomass under aerobic conditions is illustrated in Equation 2. Bacterial cells  O2 → CO2  H2O  NH 4  energy

(2)

Taking growth and decay into account, the proportion of wastewater COD and BOD5 converted into cell biomass depends on how long the biomass is retained in the aerobic treatment system. The longer sludge stays in the system, the less sludge is produced due to cell decay processes. Because cell decay consumes oxygen, a cost of reducing sludge production is the price of supplying more oxygen. b. Nitrification. Nitrification is carried out by specialized bacteria that sequentially oxidize ammonium to nitrite, and then to nitrate. Two different groups of nitrifying bacteria are involved: ammonium oxidizers, and nitrite oxidizers (Eq. 3). These slow-growing bacteria are autotrophs: they use the energy derived from the oxidation of inorganic nitrogen compounds to fix inorganic carbon (carbon dioxide).   Ammonium oxidizers Nitrite oxidizers NH 4  → NO2 → NO3

(3)

The stoichiometry for complete nitrification including cell synthesis is (USEPA, 1993): NH 4  1.89O2  0.081CO2  → 0.016 C5H7O2N  0.98NO 3  0.95H2O  1.98H

(4)

where C5H7O2N represents the ratio of these elements in new bacterial cells. On a weight basis, each gram of ammonium nitrogen removed requires 4.3 g of oxygen, produces about 0.13 g of nitrifying organisms, and consumes 7.1 g of alkalinity (measured as CaCO3) through the production of hydrogen ions. Nitrification occurs between 4° and 45°C, with an optimal temperature between 30° and 35°C. The optimal pH is between pH 6.5 and 8. Ammonia oxidation causes the wastewater pH to decline (by consuming alkalinity), which in turn can inhibit nitrification. To ensure all the ammonia in a meat processing wastewater is oxidized, it may be necessary to add alkalinity by dosing the wastewater with hydrated lime. Nitrification in meat processing wastewater is particularly sensitive to pH because of the high nitrogen content of the wastewater. This sensitivity is due to the fact that high pH increases the concentration of the un-ionized form of ammonia and low pH increases the concentration of un-ionized nitrous acid. Both un-ionized ammonia and nitrous acid can inhibit nitrification. c. Denitrification. Biological denitrification reduces nitrate or nitrite primarily to nitrogen gas (N2) but also to nitrous oxide gas (N2O). A broad range of bacteria can accomplish denitrification. Denitrifying bacteria are heterotrophic and therefore obtain their energy and carbon from organic compounds.

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Denitrification of nitrate to N2 is approximately represented by the following equation (McCarty et al., 1969), where methanol is the illustrative carbon source:  NO 3  1.08CH3OH  H → 0.065C5H7O2N  0.47N2  0.76CO2  2.44H2O

(5)

On a weight basis, each gram of nitrate nitrogen removed consumes about 2.9 g of COD (net), and produces about 3.5 g of alkalinity and 0.5 g of denitrifying bacteria. Denitrification is an anoxic process. In wastewater treatment, anoxic conditions are defined as the absence of oxygen and, unlike anaerobic (fermentative) conditions, the presence of an alternative terminal electron acceptor for respiration, such as nitrate or nitrite. However, denitrification is considered under aerobic treatment because it depends on the oxygen-dependent formation of nitrate (or nitrite, not shown) (Eq. 5). 3. Aerobic Treatment Technologies a. Aerobic Lagoons. Naturally aerated lagoons, often called oxidation ponds, are popular in the meat processing industry because of their low cost and good reliability (Fig. 9a). The lagoons are typically 1 to 1.5 m deep and commonly follow an anaerobic treatment stage. To maintain aerobic conditions throughout much of their depth, they must have a relatively low organic loading rate (60–120 kg BOD5ha1day1) and natural oxygenation by algal photosynthesis and wind-aided diffusion from the atmosphere. In practice, oxygen concentrations and pH in these lagoons fluctuate greatly with diurnal and season variations in algal activity. Oxidation ponds can reduce soluble BOD5 to low levels (20 gm3); however, a common problem is that they are able to support a high algal population, the biomass of which can contribute to high levels of suspended solids and associated BOD5 in the discharged effluent. Mechanically aerated lagoons (Fig. 9b) are also popular and can be used in various combinations with oxidation ponds. Mechanical aerators oxygenate the wastewater by agitation or by introducing fine air bubbles into the wastewater. Because these lagoons rely largely on mechanical oxygenation, they can be deeper than naturally aerated lagoons. The energy requirements is typically about 1 kWh per kg of oxygen supplied. In one treatment configuration, high concentrations of suspended solids (TSS) discharged from a mechanically aerated lagoon are removed in an oxidation pond (Table 14). Aerated lagoons, when operated with a long hydraulic retention time to prevent washout of nitrifying bacteria, can oxidize a large proportion of the ammonium in the wastewater. Oxidation ponds can also effect some nitrification. However, conventional aerobic lagoon systems seldom remove more than 40% of the influent total nitrogen, as conditions in such lagoons do not favor denitrification, which requires readily biodegradable carbon compounds (e.g., fatty acids) and the absence of oxygen. b. High-rate Systems. Like high-rate anaerobic processes, high-rate aerobic treatment systems are characterized by high densities of microorganisms and can be divided into suspended growth systems (activated sludge processes) and attached growth systems. Activated sludge treatment: The single-stage activated sludge process (Fig. 9c) is the aerobic counterpart of the anaerobic contact process, and consists of an aerated basin (a tank or lagoon) and a clarifier. Oxygen is supplied by mechanical aerators or by blowing air through diffusers in the base of the basin. The sludge age (also called the mean cell residence time) in the system can be controlled by sludge wasting, to effect BOD5 removal only or to achieve nitrification as well.

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Figure 9 Examples of some aerobic wastewater treatment technologies.

As with aerated lagoons, alkalinity dosing may be required to oxidize all of the ammonia in a meat processing wastewater. Figure 9d shows a continuous-flow activated sludge process incorporating an anoxic basin followed by an aerated basin. The process is designed to enhance nitrogen removal by recycling a proportion of the effluent from the aerated basin to the anoxic basin. In the anoxic basin, which is not aerated but gently mixed, the low-BOD, high-nitrate recycle stream mixes with the return sludge and a high-BOD influent. This creates ideal conditions for denitrification: nitrate, microorganisms, and a biodegradable carbon source are all present, and oxygen is absent. The sequencing batch rector (SBR) is an alternative activated sludge process that is particularly suitable for nitrogen removal (Fig. 10). Unlike continuous-flow activated

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Table 14 Characteristics of the Effluent from an Anaerobic Lagoon, Aerated Lagoon, and an Oxidation Pond Serially Treating Beef Slaughterhouse Wastewater (Data are mean  standard deviation for 32 samples)

BOD5 (g·m3) COD (g·m3) TSS (g·m3) TKN (g·m3) NO3-N (g·m3) NO2-N (g·m3) NH3-N (g·m3) Alkalinity (g·m3 as CaCO3) pH Total nitrogen removal (%)

Anaerobic lagoon ~12 days HRT

Aerated lagoon ~10 days HRT

Oxidation pond ~10 days HRT

n.d. 555  115 175  61 210  22 0 0 190  24 910  63 6.9  1.4 n.d.

n.d. 380  68 230  86 75  30 100  38 6.1  12 60  29 115  225 5.9  1.2 13.6  9.2

15  12 145  54 30  17 50  22 83  39 2.4  2.8 45  21 111  150 6.3  1.8 26.5  14

HRT, hydraulic retention time. n.d. not determined. Averaged temperatures were 17.1C for the anaerobic lagoon, 16.6C for the aerated lagoon and 14.2C for the oxidation pond. Source: Data are from Russell and Cooper 1992.

Figure 10 Operation of a sequencing batch reactor for nitrogen removal.

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sludge plants, which spatially separate the treatment steps, an SBR treats the effluent in batches using one basin for all steps. The conditions within the basin are changed with time, and the sequence of steps is repeated in cycles. An SBR typically operates with 1 to 4 cycles per day. Flow balancing must be provided by an upstream equalization tank or lagoon, or by operating two or more SBRs in parallel. A continuously fed variant of the SBR (Young, 1988) avoids this requirement. An important advantage SBRs have over continuous-flow systems is flexibility. Process changes can be made by simply adjusting the cycle conditions, whereas to make similar changes in a continuous process can require major equipment modifications such as resizing of basins. In addition to nitrogen removal, an important benefit of denitrification in activated sludge systems is the recovery of half the alkalinity lost during nitrification, so reducing or eliminating the need for alkalinity dosing. Denitrification also makes the process more energy efficient: the oxygen in nitrate and nitrite is used to oxidize organic carbon and so is not wasted. Activated sludge systems can be used to treat primary-treated wastewater, but this treatment option produces more sludge and requires more energy than if the wastewater is first treated anaerobically to remove much of the organic carbon. However, if total nitrogen removal is an objective, thorough anaerobic pretreatment will not leave enough biodegradable organic carbon to support denitrification. Minimizing the organic load on the activated sludge plant, while still supplying enough organic carbon, can be achieved by partial anaerobic pretreatment (Subramaniam et al., 1994; Slaney and van Oostrom, 1997). Activated sludge treatment can remove more than 90% of the nitrogen and COD in meat processing wastewater, and can also be operated to remove the phosphorus biologically (Subramaniam et al., 1994). Attached-growth systems: A common form of attached-growth system is the trickling filter. Trickling filters consist of a 4 to 10 m deep bed of porous media such as rocks or plastic packing (Fig. 9e). The wastewater is applied to the surface of the bed and trickles downwards through the media, to which the microorganisms are attached. In meat processing, trickling filters are sometimes used as highly loaded roughing filters for the preliminary removal of BOD5 before activated sludge treatment (e.g., Frose and Kayser, 1985). c. Constructed Wetlands. Over the past two decades, wastewater treatment systems involving wetland plants have become popular worldwide for secondary or tertiary wastewater treatment (Fig. 11). For the treatment of meat processing wastewater, wetlands can be useful as a final effluent “polishing” step before discharge to surface waters. Surface-flow wetlands consist of a shallow pond in which wetland plants grow either rooted in the soil base of the wetland or floating in a raft on the water surface. In subsurface-flow wetlands, the wastewater flows through a bed of porous soil, sand, or gravel, in which the wetland plants are rooted. Wetland systems reduce the pollutants in wastewater by a complex variety of biological, chemical, and physical processes associated with the plants, microorganisms, substrates, and sediments. Generally, both aerobic and anoxic zones occur in wetlands. For the final treatment of meat processing effluent, wetlands can outperform tertiary wastewater ponds, producing effluents low in TSS and BOD5 (20 gm3) (van Oostrom and Cooper, 1990). Gravel bed subsurface-flow wetlands generally produce higher quality effluents than surface-flow wetlands, but the gravel is prone to blocking by solids, eventually forcing the wastewater to flow across the gravel surface.

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Figure 11 Two types of constructed wastewater wetland. Decaying plant matter can provide a carbon source for denitrification in surface-flow wetlands, and nitrogen reductions of up 75% have been be achieved when the influent nitrogen was predominantly nitrate (van Oostrom, 1995). E. Disinfection 1. Infectious Microorganisms An important route for the spread of infectious illness in humans is the fecal-oral route, by which a disease-causing microorganism that is shed in the feces of an infected animal or human is subsequently ingested, causing disease. A wide variety of microorganisms can be isolated from animal feces. Some of these are zoonotic, defined as harmful (pathogenic) microorganisms acquired from animals and that have the potential to cause disease in humans. Zoonoses that can be present in meat industry wastewaters include a number of bacteria, such as species of Salmonella, toxigenic strains of Escherichia coli (including E. coli O157:H7), Campylobacter jejuni and C. coli, Yersinia enterocolitica, and Listeria mono-

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cytogenes. Viral zoonoses have not been linked to meat industry wastes, but enteric parasites such as Giardia and Cryptosporidium can be found from time to time in ruminant feces. Wastes from slaughterhouses contain high concentrations of fecal matter and therefore could contain some of these parasites. It is possible for healthy animals to be symptomless carriers and shed zoonoses (Acha and Szyfres, 1987; Donnison and Ross, 1999). Although large numbers of microorganisms need to be ingested to cause some infectious diseases (for example 104 Salmonella cells), this is not always the case. Low-dose zoonoses include the bacteria C. jejuni and E. coli O157:H7, and cysts of the enteric parasites Giardia and Cryptosporidium. For a detailed account of human wastewater microbiology, which has many parallels with meat processing wastewater microbiology, the reader is referred to Wastewater Microbiology (Bitton, 1999). Many of the infectious microorganisms that are found in meat industry wastes are also found in human wastewater. 2. Fecal Indicator Microorganisms When meat-processing wastewater is discharged to the environment, the processor is often required to measure concentrations of fecal microorganisms in the wastewater and sometimes in the receiving environment to ensure there is no health risk. As zoonoses may or may not be present, indicator microorganisms are usually measured to determine the microbiological risk that might result from the discharge. An indicator microorganism is an organism that is consistently found in fecal wastes, so that its presence in an environmental sample demonstrates that the environment has been fecally polluted. Fecal coliform bacteria are widely used as an indicator. However, a more specific indicator of fecal pollution is E. coli, a component species of the fecal coliform group. In temperate climates, the recovery of E. coli from an environmental sample is almost certain proof that that the environment had received a recent input of fecal matter. For fresh waters used for recreation, the Environmental Protection Agency has set criteria based on concentrations of E. coli (USEPA, 1986). 3. Disinfection Treatments Untreated slaughterhouse wastewater can contain up to a hundred million fecal coliforms per 100 ml, and the majority of these are usually E. coli. Treatment in a series of lagoons with a total residence time of 20 to 30 days typically reduces the fecal coliform concentration to 10,000 per 100 ml. Such lagoons, and other biological treatments, rarely reduce fecal coliforms to below 1,000 per 100 ml. The reduction that does takes place is due to a combination of predation by other organisms and removal in sediments and sludge, but ultraviolet (UV) radiation from sunlight in lagoon systems also plays a role in disinfection (Davies-Colley et al., 1999). In some situations, natural disinfection processes are inadequate or too slow, and in such cases effluents may be subjected to disinfection treatment. For light-colored effluents that are low in suspended solids, UV radiation at 256 nm inactivates fecal coliform bacteria but may not be as effective for all potential zoonoses, particularly enteric parasite cysts. Before UV irradiation, wastewater is often sand-filtered to improve the efficiency of the UV treatment, and with sufficient filtration cysts may be removed. Chlorination is widely used to disinfect human sewage. However, chlorination is not usually recommended for meat processing effluents because even after treatment these effluents still contain organic compounds that can react with chlorine to form toxic organochlorines that present their own environmental risks (Donnison, 1996).

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F. Land Treatment Where enough suitable land is available, irrigation of meat processing wastewater can be an attractive disposal option, because it avoids a point source discharge into waterways and can increase the productivity of the land by providing nutrients and water for plants. Pollutants in the wastewater are removed by plant uptake as well as by a range of physical, chemical, and microbial processes in the soil. In this role the soil is a biological filter. Irrigation of meat-processing effluent is not without risks. Wastewater irrigation systems must be designed and managed to avoid such problems as groundwater contamination, nuisance odors, aerosol drift, surface runoff into waterways, and degradation of soil structure and quality. Successful land treatment relies on balancing the amount of wastewater applied with the nutrient uptake by the plants, and ensuring that any excesses do not detrimentally affect the environment (Russell et al., 1991). For irrigation of meat processing wastewater, the annual application rate, and therefore the area required, is usually governed by the nitrogen loading. Nitrogen has the potential to cause nitrate contamination of groundwater if applied in excess. Unlike ammonium and organic nitrogen, nitrate is highly mobile in soils. It may be either formed in the soil from the oxidation of organic nitrogen and ammonium present in the wastewater, or it may already be present in aerobically treated wastewater. Nitrate that is not taken up by plants eventually leaches to groundwater or is converted to nitrogen gases by denitrification and returned to the atmosphere (Fig. 12). Plant uptake of nitrogen followed by nitrogen removal in plant or animal products is the main sink for the applied nitrogen and depends on the cover crop and how it is managed. For example, meat-processing wastewater can be applied to grazed pasture. Because

Figure 12 Scheme showing the main nitrogen inputs, transformations and outputs that occur at wastewater irrigation sites.

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grazing animals recycle about 90% of their nitrogen intake back onto pasture, only 10% of the nitrogen taken up by plants is eventually removed from the site in the form of animal products. More nitrogen can be removed if the pasture or crop is not grazed but instead harvested and taken off-site (e.g., as hay or silage). Harvesting allows higher wastewater nitrogen application rates. After only primary treatment, meat processing effluent can be irrigated without adverse effects on plant growth, and the high carbon content of such wastewater can promote high rates of denitrification in the soil (Russell et al., 1993). However, lagoon treatment and storage of the wastewater is commonly provided before irrigation to avoid potential odor problems and allow irrigation to be delayed during adverse conditions (e.g., during rainfall). Lagoon pretreatment and storage also reduces the microbial risk associated with irrigation of the wastewater (Donnison and Ross, 1992). Various types of flood and spray irrigation systems have been used for applying meat processing wastewater to land. Factors important in selecting an irrigation system include terrain and soil characteristics, crop type, capital and operating costs, aerosol production, and the precision and control of application. Spray irrigation systems provide more control over the application rate than flood systems but have a greater risk of aerosol production. The volume of wastewater applied during each irrigation event should be less than the water-holding capacity of the topsoil to minimize the quantity of nitrogen washed through the root-zone and out of reach of the plants. The wastewater should be distributed evenly and applied slowly to maximize wastewater renovation and avoid surface runoff into waterways. The environmental effects of wastewater components other than nitrogen also need to be considered. Many soils have the capacity to store large quantities of applied phosphorus, and for such soils phosphorus leaching losses are very low. However, excess phosphorus application will eventually saturate the soil and phosphate breakthrough to groundwater will then occur. This usually takes several decades at normal application rates for meat-processing wastewater. Wastewater with high concentrations of sodium relative to calcium and magnesium can cause an accumulation of sodium in the soil and adversely effect soil structure and plant growth. Meat-processing wastewaters generally have favorable salt ratios. However, some processes (e.g., salt curing) produce effluents high in sodium. Where such effluent is irrigated, the effect on soil structure should be closely monitored. V. SOLID WASTE MANAGEMENT As we have already seen, the main organic solid wastes associated with meat processing are animal tissue wastes, fecal and gut content solids, solids and slurries recovered from primary and physicochemical wastewater treatment systems, and sludges produced from biological wastewater treatment systems. In the past it was common practice to landfill at least some of these wastes, but with increased restrictions and costs associated with this practice, landfilling is now uncommon. Obviously the wastes containing predominantly animal tissues should be rendered if possible, to recover some value from these materials and avoid disposal costs. Tissue wastes collected in the plant and recovered from wastewater screens can be transported directly to rendering. Slurries recovered from physicochemical treatment systems, if suitable for use as animal feed, generally need to be dewatered before they can be further processed (Fig. 6). Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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Fecal wastes, gut contents and biological treatment sludges are unsuitable for rendering, but their high nutrient and organic content makes them useful as a fertilizer and soil conditioner. Such wastes are often applied directly onto land, either as a slurry or as dewatered solids. However, these solids generally have a high oxygen demand and can carry pathogens and weed seeds. Like wastewater application, the land application of such wastes must be managed to avoid odor, leaching to groundwater, runoff, and adverse effects on animal and human health. Where it is not possible to avoid such impacts, or where direct land application is not permitted, the solids are commonly treated by anaerobic digestion or composting. These treatments biologically stabilize the solids, to reduce or eliminate odor generation and the attraction of flies and vermin. Anaerobic digesters range from simple sludge lagoons to high-rate systems using fully enclosed heated tanks. Anaerobic solids digestion has the potential to recover methane for energy (Umstadter, 1986). An important disadvantage is that much of the nutrient content from the solids is released into the liquid phase during digestion, creating a highstrength liquid waste requiring further treatment. Composting is a popular method of stabilizing waste solids from meat processing. In the composting process, bacteria and fungi oxidize biodegradable compounds such as fats and proteins into water and carbon dioxide, leaving a stable organic residue of mature compost. The process is accelerated by high temperatures and moist, aerobic conditions within the composting mass. Aerobic conditions are also important for odor minimization. The high temperatures (50° to 80°C) achievable during composting kill pathogens and help to ensure a safe product. A variety of composting methods can be used to stabilize meat processing wastes, from simple windrow composting techniques, to high-rate forced-aeration methods (Keeley and Skipper, 1988; van Oostrom, 1993). Regardless of the type of composting process, the first step is to ensure that the waste has a porous and open structure to assist aeration. This usually involves draining excess liquid from the waste and mixing the waste with a bulking agent, such as sawdust, chopped straw, crushed pine bark, or recycled compost. In windrow composting, the waste is formed into long piles. Oxygen is supplied by mechanically turning the pile, and by passive and convective aeration. In forced-aeration composting, the compost is placed in a pile or vessel over an air distribution system, and oxygen is supplied by forcing air through the composting mass using a fan. With this method, oxygen supply and compost temperatures can be controlled more effectively than with windrow composting, resulting in faster stabilization and a reduced potential for odor generation. All organic waste solids from meat processing operations can be stabilized by composting. Even high-fat semi-liquid dissolved air flotation (DAF) solids can be successfully composted if they are mixed with enough bulking agent. Simple windrow techniques are more suitable for composting paunch manure and fecal solids, although they can treat most meat processing wastes. Forced-aeration systems are particularly suited to stabilizing wastes containing animal tissues, which have a very high oxygen demand and a high potential for odor generation. Several weeks or months are needed for composting to produce a mature product, depending on the process used. A variety of products can be made from meat-processing waste compost, ranging from a basic soil conditioner to a high-quality potting medium (van Oostrom et al., 1988). Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

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VI. CONCLUDING REMARKS Although the meat processing industry has made major advances in waste reduction and by-product recovery, it remains a large producer of waste. To be economically competitive and to meet increasingly strict environmental standards, meat processors face the ongoing challenge of reducing waste at source, finding new uses for waste materials, and selecting cost-effective methods for waste treatment and disposal. In this chapter we have identified the main sources and characteristics of meat processing wastes, and investigated some of the methods that can be used to reduce, recover, treat, and dispose of these wastes. ACKNOWLEDGMENTS The author is grateful to his former employer, MIRINZ Food Technology and Research, for permission to present unpublished MIRINZ data. He also thanks Drs. Andrea Donnison and Patricia Johnstone for their valuable assistance in preparing the manuscript. REFERENCES Acha, P. N., and B. Szyfres. 1987. Zoonoses and Communicable Diseases Common to Man and Animals, 2nd ed. Pan American Health Organization, WHO, Washington, DC. APHA. 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association, American Water Works Association, and Water Environment Federation, Washington, DC. Bitton, G. 1999. Wastewater Microbiology, 2nd ed. Wiley, New York. Borja, R., C. J. Banks, and Z. Wang. 1995. Effect of organic loading rate on anaerobic treatment of slaughterhouse wastewater in a fluidized-bed reactor. Bioresource Technology 52(2):157–162. Brown, G. I., A. Peacham, and R. N. Cooper. 1993. Stickwater ultrafiltration. MIRINZ Publ. No. 923, Hamilton, New Zealand. Chittenden, J. A., L. E. Orsi, J. L. Witherow, and W. J. Wells. 1977. Control of odors from an anaerobic lagoon treating meat packing wastes. Pp. 38–61. In: Proceedings of the Eighth National Symposium on Food Processing Wastes. EPA-600/2-77-184, U.S. Environmental Protection Agency, Cincinnati, Ohio. Cooper, R. N. 1975. Pollution abatement within departments, a discussion. Pp. 57–62. In: Proceedings of the Seventeenth Meat Industry Research Conference. MIRINZ Publ. No. 458, Hamilton, New Zealand. Cooper, R. N. 1982. Characteristics of slaughterhouse effluents. In: Water and Soil Miscellaneous Publication No. 29. Pp. 43–48. National Water and Soil Conservation Organization, Wellington, New Zealand. Cooper, R. N., and J. M. Russell. 1982. Characterization of New Zealand fellmongery wastes and their treatment by manganese-catalyzed oxidation. Journal of the American Leather Chemists Association 77(9):457–468. Cooper, R. N., and J. M. Russell. 1991. Meat industry processing wastes: characteristics and treatment. In: Encyclopedia of Food Science and Technology. Pp. 1683–1691. John Wiley & Sons, Inc., New York. Cooper, R. N., J. M. Russell, and J. L. Adam. 1982. Recovery of protein from slaughterhouse effluents by double adjustment of pH. Pp. 285–293. In: Proceedings of the 37th Industrial Waste Conference. Ann Arbor Science Publishers, Ann Arbor, Michigan. Cooper, R. N., J. M. Russell, and J. L. Adam. 1983. Recovery and utilization of protein from slaughterhouse effluents by chemical precipitation. Pp. 31–49. In: Ledward, D. A. et al. (Eds.) Upgrading Waste for Feeds and Food. Butterworths, London.

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Dague, R. R., R. F. Urell, and E. R. Krieger. 1990. Treatment of pork processing wastewater in a covered anaerobic lagoon with gas recovery. Pp. 815–824. In: Proceedings of the 44th Purdue Industrial Waste Conference. Lewis Publishers, Chelsea, Michigan. Davies-Colley, R. J., A. M. Donnison, D. J. Speed, C. M. Ross, and J. W. Nagels. 1999. Inactivation of faecal indicator micro-organisms in waste stabilisation ponds: interactions of environmental factors with sunlight. Water Research 33:1220–1230. Donnison, A. M. 1996. Options for reducing faecal microorganisms in meat processing waters. MIRINZ Publ. No. 971, Hamilton, New Zealand. Donnison, A. M., and C. M. Ross. 1992. Irrigation of pasture with meat processing effluent: health aspects. Pp. 182–186. In: Gregg, P. E. H., and L. D. Currie (Eds.). Use of Wastes and By-Products as Fertilizers and Soil Amendments for Pastures and Crops. Occasional Report No. 6. Fertilizer and Lime Research Centre, Massey University, Palmerston North, New Zealand. Donnison, A. M., and C. M. Ross. 1999. Animal and human faecal pollution in New Zealand rivers. New Zealand Journal of Marine and Freshwater Research 33:119–128. Fernando, T. 1982. The MIRINZ low-temperature rendering system. Pp. 79–84. In: Proceedings of the Twenty-Second Meat Industry Research Conference, MIRINZ Publ. No. 816, Hamilton, New Zealand. Fernando, T. 1992. Blood meal, meat and bone meal, and tallow. Pp. 81–112. In: Pearson, A. M., and T. R. Dutson (Eds.). Inedible Meat By-products. Advances in Meat Research, Volume 8, Elsevier Applied Science, London. Foltz, T. R., K. M. Ries, and J. W. Lee. 1974. Removal of protein and fat from meat slaughtering and packing wastes using lignosulfonic acid. Pp. 85–106. In: Proceedings of the Fifth National Symposium on Food Processing Wastes. Environmental Protection Technology Series, EPA-660/274-058. U.S. Environmental Protection Agency, Corvallis, Oregon. Frose, G., and R. Kayser. 1985. Effective treatment of wastewater from rendering plants. Pp. 69–77. In: Proceedings of the 40th Industrial Waste Conference. Ann Arbor Science, Ann Arbor, Michigan. Johns, M. R., M. L. Harrison, P. H. Hutchinson, and P. Beswick. 1995. Sources of nutrients in wastewater from integrated cattle slaughterhouses. Water Science & Technology 32(12): 53–58. Jones, H. R. 1974. Pollution Control in Meat, Poultry and Seafood Processing. Noyes Data Corporation, Park Ridge, New Jersey. Keeley, G. M., and J. L. Skipper. 1988. The use of aerobic thermophilic composting for the stablilisation of primary meat waste solids. Pp. 120–131. In: Bhamidimarri, R. (Ed.). Alternative Waste Treatment Systems. Elsevier Applied Science, London. Lollar, R. M. 1992. The tanning process and the production of finished leather goods. Pp. 35–65. In: Pearson, A. M., and T. R. Dutson (Eds.). Inedible Meat By-Products. Advances in Meat Research, Vol. 8. Elsevier Applied Science, London. Luo, J., C. D. Campbell, A. J. van Oostrom, and M. P. Agnew. 1999. Chemical characteristics of influent and effluent gases of odour-control biofilters treating rendering plant emissions. Pp. 199–208. In: Cao, Z., and L. Pawlowski (Eds.). Proceedings of the 12th International Conference on Chemistry for Protection of the Environment. Nanjing University Press, Nanjing, China. McCarty, P. L., L. Beck, and P. St. Amant. 1969. Biological denitrification of wastewaters by addition of organic materials. Pp. 1271–1285. In: Proceedings of the 24th Purdue Industrial Waste Conference, Purdue University, Lafayette, Indiana. Metzner, G., and U. Temper. 1990. Operation and optimization of a full-scale fixed-bed reactor for anaerobic digestion of animal rendering waste water. Water Science & Technology 22(1/2):373–384. Morris, D., S. Sung, and R. R. Dague. 1998. ASBR treatment of beef slaughterhouse wastewater. Pp. 225–236. In: Proceedings of the 52nd Industrial Waste Conference. Ann Arbor Press, Chelsea, Michigan.

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Pilkington, D. B. 1975. Continuous blood coagulation and dewatering: factors in reduction of product loss. Pp. 65–67. In: Proceedings Seventeenth Meat Industry Research Conference. MIRINZ Publ. No 458, Hamilton, New Zealand. Russell, J. M., and R. N. Cooper. 1981. Flocculation of slaughterhouse effluents with aluminium salts. Environmental Technology Letters 2:537–544. Russell, J. M., and R. N. Cooper. 1992. Nitrogen transformations in three lagoon systems treating meat-processing wastes. MIRINZ Publ. No. 898, Hamilton, New Zealand. Russell, J. M., R. N. Cooper, and B. I. Crocombe. 1984. Physico-chemical treatment of meat-processing effluent with sodium alginate and seaweed. Environmental Technology Letters 5:289–294. Russell, J. M., R. N. Cooper, and S. B. Lindsey. 1991. Reuse of wastewater from meat processing plants for agricultural and forestry irrigation. Water Science and Technology 24(9):277–286. Russell, J. M., R. N. Cooper, and S. B. Lindsey. 1993. Soil denitrification rates at wastewater irrigation sites receiving primary-treated and anaerobically treated meat-processing effluent. Bioresource Technology 43:41–46. Sayed, S., J. van der Zanden, R. Wijffels, and G. Lettinga. 1988. Anaerobic degradation of the various fractions of slaughterhouse wastewater. Biological Wastes 23(2):117–142. Slaney, A., and A. van Oostrom. 1997. Meat processing wastewater treatment in a sequencing batch reactor. Pp. 526–527. In: Proceedings 43rd International Congress of Meat Science and Technology. Auckland, New Zealand. Stebor, T. W., C. L. Berndt, S. Marman, and R. Gabriel. 1990. Operating experience: anaerobic treatment at Packerland Packing. Pp. 825–834. In: Proceedings of the 44th Industrial Waste Conference. Lewis Publishers, Chelsea, Michigan. Steffen, A. J., and M. Bedker. 1962. Operation of full-scale anaerobic contact treatment plant for meat packing wastes. Pp. 423–437. In: Proceedings of the 16th Industrial Waste Conference. Engineering Extension Series No. 109. Purdue University, Lafayette, Indiana. Subramaniam, K., P. F. Greenfield, K. M. Ho, M. R. Johns, and J. Keller. 1994. Efficient biological nutrient removal in high strength wastewater using combined anaerobic-sequencing batch reactor treatment. Water Science & Technology 30(6):315–321. Swan, J. E. 1991. Animal by-product processing. In: Encyclopedia of Food Science and Technology. Pp. 42–49. Wiley, New York, NY. Swan, J. E., G. Fox, and E. J. Pooley. 1986. Assessment of four viscera cutting and washing systems. MIRINZ Publ. No. 843, Hamilton, New Zealand. Travers, S. M., and D. A. Lovett. 1984. Treatment of abattoir wastewater by dissolved air flotation— Part 2: wastewater chemically pretreated. Meat Research Report No. 10/84, CSIRO Meat Research Laboratory, Cannon Hill, Queensland, Australia. Umstadter, L. W. 1986. Digestor system yields more than methane. BioCycle 27(3):36–37. USEPA. 1986. Ambient water quality criteria for bacteria—1986. EPA440/5-84-002. U.S. Environmental Protection Agency, Washington, DC. USEPA. 1993. Process Design Manual—Nitrogen Control. EPA/625/R-93/010. U.S. Environmental Protection Agency, Washington, D.C. van Oostrom, A. J. 1993. Meat . . . a reliable source for composting. Waste Management and Environment 4(5):66–68. van Oostrom, A. J. 1995. Nitrogen removal in constructed wetlands treating nitrified meat processing effluent. Water Science and Technology 32(3):137–147. van Oostrom, A. J., and R. N. Cooper. 1990. Meat processing effluent treatment in surface flow and gravel-bed constructed wastewater wetlands. Pp. 321–332. In: Cooper, P. F., and B. C. Findlater (Eds.). Constructed Wetlands for Water Pollution Control. Pergamon Press, Oxford. van Oostrom, A. J., R. N. Cooper, and J. E. van Rossem. 1988. Temperature-controlled, aerated staticpile composting of slaughterhouse waste solids. Pp. 174–184. In: Bhamidimarri, R. (Ed.), Alternative Waste Treatment Systems. Elsevier Applied Science, London.

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