ADVANCES IN FOOD RESEARCH VOLUME I
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ADVANCES IN FOOD RESEARCH VOLUME I
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ADVANCES IN FOOD RESEARCH VOLUME I
Edited by E. M. MRAK GEORGEF. STEWART Iowa State College, Ames, Iowa
University of California, Berkeley, California
Editorial Board E. C. BATE-SKITH R. M. CONRAD W.H. COOK W. F. G E D D ~ M. A. JOBLYN
A. J. GIJYVEB
S. LEPKOVBKY B. E. PROCXOR
0. B. WILLIAMS
P. F. SHARP W. M. URBAIN
1948
ACADEMIC PRESS INC., PUBLISHERS NEW YORK, N. Y.
Copyright 1948,by ACADEMIC PR;ESS INC. 125 EAST 2 3 STREET ~ ~ NEW YORK 10, N. 9.
PRINTED
IN
THE UNITED STATES OF AMEBICA
CONTRIBUTOR0 TO VOLUME 1 GEORGE L. BAKER,University of Delaware Agricultural Experiment Station, Newark, Delaware. E. C. BATE-SMITH, Low Temperature Research Station, University o j Cambridge and Department of Scientific and Industrial Research, Cambridge, England. KENNETH C. BEESON,U. S. Plant, Soil and Nutrition Laboratory, Zthaca, New York. L. E. CLIFCORN, Research Department, Continental Can Co., Chicago, Illinois. HARRYL. FEVOLD, Quartermaster Food and Container Znstitute for the Armed Forces, Chicago, Illinois. SAMUEL LEPKOVBKY, University of California, Berkeley, Calijmia. HOWARD D. LIGHTBODY, Quartermaster Food and Container Znstitute for the Armed Force's, Chicago, Illinois. BELLELOWE,Zowa State College, Ames, Zowa. A. FRANKR o ~ BCornell , University, Zthuca, New York. G. FREDSOMERB, U. S. Plant, soil and Nutrition Laboratory, Zthaca, New York. EARLR. STADTMAN, University of Calijornia, Berkeley, Calijornia. ORVILLEWYBS,University of Texas, Austin, Texas.
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Foreword Food research has been accelerating and expanding at a very rapid rate during the past few years. This has been accompanied by a realization of the importance of, and the necessity for, undertaking the work at a fundamental as well as a t an applied level. Research workers trained in the basic sciences are entering the field, and empirical methods of investigation are rapidly being replaced by those baaed on the scientific method. As a result of this trend toward specialization, new professional groups, new societies, and technical publications are continuaIly making their appearance and workers in the various phases of food research are gradually losing contact with one another. At the present time, research relating to foods and human nutrition is being carried on in agricultural experiment stations, federal laboratories, endowed colleges and universities, industrial laboratories, research institutes, and others. The results of original investigations appear in such varied publications as the Industrial and Engineering Chemistry, Food Research, Journal of Nutrition, Journal of Bacteriology, Chemistry and Industy, Journal of Experimental Psychology, Journal of Dairy Science, British Food Journal and Hygienic Review, Journal of Dairy Research, Bulletins of the U.S . Department of Agriculture and of Experiment Stations, etc. It is obviously impossible for any one person to keep informed in more than a very restricted area of food research. The present state of affairs emphasizes the need for the coordination and integration of food research to promote an orderly and systematic development of scientific knowledge in this important fieId. Advances i n Food Research is offered as a partial answer to fulfill this need. Its raison d’btre is to provide a medium in which every phase of food research may be exhaustively and critically reviewed on a continuing basis. The editors will endeavor to achieve a.nd maintain in Advances i n Food Research a, high level of scientific competence. Contributions will be sought which are exhaustive, critical, integrating, and of fundamental importance to the development of food research as a whole. The cooperation of persons from all segments of food research is needed in order that these objectives may be met. Advice and council leading to better subjectmatter coverage and format, and constructive criticism on other matters of editorial policy are earnestly solicited.
SUBJECT MATTERAREAS Food research may be defined as that field of scientific investigation concerned with foods and their relationship to man. The work embraces a large number of scientific disciplines, including such applied sciences a8 Vii
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FOREWORD
human nutritioa, food acceptance, public health, agriculture, food technology, home economics, aa well as certain segments of the more basic sciences of biochemistry, botany, microbiology, physiology, psychology, and 50010gy. Some of the major objectives in food research are: 1. To establish the qualitative and quantitative nutritional requirements of man. 2. To determine the physiological and psychological factors affecting human appetite and food acceptance. 3. To ascertain the factors affecting the incidence of infectious and toxigenic agents in food and their transmission from food to man. 4. To establish principles and practices for control of the nutritive value, acceptability and safety of foods during production, harvesting or slaughtering, processing, storage, distribution, and utilization. Subject matter areas in food research fall under several headings. A brief discussion of their significance and the plans of the editors for suitable coverage of these fields follow. Human Nutrition. Man’s basic need for food is to provide him with the nutrients required for growth and maintenance of body tissues, reproduction, and energy for his voluntary and involuntary activities. The field of human nutrition is, therefore, one of the basic asgments of food research. Experimental work in this area is still in its infancy, but the rapid progress made during the past few years, using both experimental animals and human beings, suggests that significant advances pertaining more directly to human nutrition itself are in the offing. Reviews will be sought in all phases of human nutrition. Special emphasis will be placed on obtaining contributions pertaining t o criteria and methodology for establishing the requirements for individual nutrients, interrelationships in the utili~ationof food nutrients and the effects of environment, age, sex, and physiological activity on nutritive requirements, and availability of nutrients in foods. Food Acceptance. No matter how nutritious a food may be, it must be eaten to provide nourishment, and food acceptance is, therefore, one of the basic phases of food research. The objective of food acceptance research is to discover the underlying factors which determine why and when various foods are consumed by man. Important food acceptance problems relate to appetite, sensory perception (senses associated with organoleptic qualities of food, such aa sight, taste, odor, touch, temperature, pain) food habits and preferences (including the effects of geography, bias, habit, race, religion), and subjective (palatability) and objective (physical and chemical) tests for acceptability. Food acceptance research has just begun to receive the attention it really deserves. Considerable effort will be made to secure reviews which will
FOREWORD
ix
not only establish the present status, but also those which will stimulate scientific interest in this field. Many disagreements exist with regard to methodology, objectivity of approach, and other phases of work in this area. Ample opportunity will be provided for presenting all sides of controversial topics. An example of a review in this field, which is considered suitable for Advances, is to be found in the contribution by Lepkovsky. Agriculture. The nutritional and organoleptic qualities of the basic foodst&s are dependent on the genetic constitution of the plant or animal from which they are derived as well as on the environmental and cultural practices used to produce them. In the past, agricultural research has emphasized mainly disease resistance and productivity, but more and more attention is now being given to those factors which influence the acceptability and nutritive value of foods. Because of their strategic importance, technical developments in agriculture will profoundly influence those in food research aa a whole. The type of review which the editors fee1 is an appropriate contribution to this topic may be surmised by reading the contribution by Somers and Beeson. Microbiology and Public Health. The successful preservation of foods is primarily a matter of “preventing” microbiological spoilage. Scientific and technological advances in this area have been spectacular, but many problems still remaic unsolved. For instance, it is now apparent that the means which have been developed for destroying or inhibiting microbial agents frequently produce significant changes in nutritive value and (or) acceptability. This emphasises the need for preventing or minimiliing microbiological contamination in the handling of foods, aa well as for finding means to inhibit or destroy microorganisms, while at the same time retaining a mrwrimum of their nutritive value and acceptability. The contribution by Wyss summarims present knowledge concerning some of the fundamentals involved in the chemical inhibition of microorganisms and emphasims the need for additional research work in this area. The transmission of toxigenic agents (chemical and biological) and pathogenic organisms from food to man has been recognised aa a serious problem in the handling of food since the time of Paateur. While much has been accomplished in this area, a great deal of additional work is needed in order that the consumer may be provided always with foods which are free from toxic agents. Reviews in this area will be sought on such topics aa botulism, undulant fever, ententic poisoning, etc. Other poeaibilities asd include reviews on toxic spray and fumigant residues (i.e., D.D.T.) toxic preservatives (i.e., monoohloracetic acid). Biochemietry and Histobgy. The nutritive value and acceptability of
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FOREWORD
an individual food are directly related to its physical and chemical composition. The results of histological and biochemical investigations of foodstuffs, therefore, require correlation with nutritive value and acceptability. In addition, fundamental studies on the composition, reactions, and interactions of the various components of foods (lipids, carbohydrates, proteins, pigments, inorganic compounds, etc.) are important in gaining a better understanding of the properties of individual foods. Such basic information permits a more intelligent development of ways and means for controlling the nutritive value and acceptability of foods during their production, processing, storage, distribution, and utilization. Advances in our knowledge of physical and chemical properties of carbohydrates, fats, proteins (including enzymes), vitamins, pigments, etc., are being made at a rapid rate. The editors hope to secure reviews which correlate these fundamental studies with changes in nutritive value and acceptability of various foods. The contribution by Baker on pectin is an example of a review in this category. Food Technology and Engineering. The modern mass production and distribution of fresh and processed foods are tributes to the efforts of those engaged in research and development in food technology and engineering. The establishment of scientific principles and practices for the processing, packaging, storage, distribution, and utilization of foods and their byproducts is very important to the advancement of food research as a whole. A large number of important problems remain to be solved if all of our important foods are to be economically handled without serious loss in nutritive value, acceptability, and safety. Specific reviews in food technology and engineering are planned which relate to the pasteurization, sterilization, dehydration, freezing, packaging, cooking, and baking of foods as well aa those that relate to by-product utilization and waste disposal. The contribution of Clifcorn is an example of a suitable review in this category. Entomology and Zoology. Losses and wastes occasioned by infestation of food commodities with insects, rodents and other pests, and their excrement reach enormous proportions each year. An important segment of food research concerns itself with developing ways and means for eliminating or minimizing losses brought, about by these various food-consuming posts. Reviews in this area will be sought for purposes of ascertaining the present state of knowledge as well as for stimulating further research. Commodity Areas. A large amount of industrial food research is concerned with problems relating to the nutritive value, acceptability, utility, storage life, health hazards, or sanitary quality of individual foods. The objective of such studies is to improve an existing food product or to create a new one. Reviews on such important commodities as cereal
xi
FOREWORD
products, fats and oils, meat products, vegetable products, fish products, dairy products, fruit products, and egg products will be sought for Advances in Food Research. The reviews in specific commodity fields will not only show progress and indicate the need for further investigation, but will also suggest possible applications t o other foods. This volume contains the following reviews in this category: “Factors Affecting the Palatability of Poultry with Emphasis on Histological Post-mortem Changes,” Lowe; “Physiology and Chemistry of Rigor Mortis, With Special Reference to the Aging of Beef,” Bate-Smith; “Deterioration Problems in Processed Potatoes,” Ross; “Nonenzymatic Darkening of Fruit Products,” Stadtman; “Biochemical Factors Influencing the Shelf Life of Dried Whole Egg and Means for Their Control,” Lightbody and Fevold. GEORGE F. STEWART E. M. MRAK Food Technology Diviaion University of California Berkeley, California
Poultry Husbandry Department Iowa State College Amea, Iowa
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CONTENTS
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Contributora to Volume I Foreword
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The Physiology and Chemistry of Rigor Mortis. with Special Reference to the Aging of Beef
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BY E. C BATI-SMITH. Lour Temperature Re-seurch Station. University of Cambridge and Department of Scientijic and Industrial Rcseurch. Cambridge. England
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I Introduction . . I1 RigorMortis . . I11 StorageandAging I V. Conclusions . . References
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3 21 33 34
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39 40 52 89 98 100
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Factors Influencing the Vitamin Content of Canned Foods
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BY L. E CLIFCOBN. Raseurch Department. Continenla1 Can Co., Chicago. IUircois
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I Introduction . . . . . . . . . . . . . . . I1 Vitamin Content of Canned Foods . . . . . . . . . I11 Effect of Canning Operations on Vitamins . . . . . . I V The Effect of Storage on the Vitamin Content of Canned Foods V. Relationship of Type of Container to Vitamin Content . . .
The Physiological Basis of Voluntary Food Intake (Appetite?) BY SAMUELLEPKOVBKY. Univermly of Calijmiu. Berkeley. California
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I Introduction . . . . . . . . . . . . . . . . . . 106 I1 Can Human Beings Choose Wisely in Accordance with Their Nutritional
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111. Can Animals Choose Food Correctly? . . . . . . . . I V Factors Influencing Food Intake . . . . . . . . . V The EfTect of Proteins on Food Intake . . . . . . . . VI The Effect of Amino Acids on Food Intake . . . . . . VII. The Effect of Water-Soluble Vitamins on Food Intake . . . VIII The Effect of Fa& and Fat-Soluble Vitamins on Food Intake . I X The Role of Essential Minerala on Food Intake . . . . . X . EndocrinesandFoodInteke . . . . . . . . . . X I. The Role of Deleterious Compounds on Voluntary Food Intake XI1 Protection Against Deleterious Compounds . . . . . . XI11 Physiological Mechanisms Determining Food Intake XIV Integrative Summary Acknowledgment . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . xiii
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107 111 113 113 115 116 118 119 122 124 126 129 139 141 141
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CONTENTS
Biochemical Factors Influencing the Shelf Life of Dried Whole Eggs and Means for Their Control BY HOWARD D . LIQETBODY AND HARRY L. FIDVOLD. Bureau of Agricultural and Industrial Chemktrq. Agricultural Research Administratim. U . S Department of Agriculture
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I. Introduction . . . . . . . . . . . . . . . . . . I1. Chemical Composition of Whole Egg . . . . . . . . . . . I11. Variation in the Compositibn of Liquid Egg . . . . . . . . . IV. Variations of the Raw Materials Related to Egg Storage Qualities . . V. Physical Properties of Whole Egg Powders . . . . . . . . . VI Criteria of Quality and Deterioration . . . . . . . . . . . VII Chemical and Physical Changes Associated with Deterioration . . . . VIII. Quality Retention Measures . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .
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149 149 152 154 156 157 168 187 194
Factors Affecting the Palatability of Poultry with Emphasis on Histological Post-mortem Changes BY BELLBLOWE.Zowa State Cdlege. A m . Iowa
I. Introduction . . . . . . . . . . . . . . . . . . 204 I1 Composition of Edible Portion of Chicken . . . . . . . . . 206 I11. The Proteins of Muscle and the Structure of Muscle as Related to Poultry
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Cookery . . . . . . . . . . . . . . . . . . . IV Poultry Fat and Palatability . . . . . . . . . . . . . V . Factors Influencing Flavor . . . . . . . . . . . . . . VI Factors Influencing Juiciness . . . . . . . . . . . . . VII . Factors Influencing Tenderness . . . . . . . . . . . . . VIII . Postmortem Changes and Rigor in Poultry Muscle . . . . . . . IX. The Relation of Microscopic Appearance of the Muscle Fibers to Palatability . . . . . . . . . . . . . . . . . . . . X Information Lacking in the Literature . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .
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207 210 219 225 230 232
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234 253 253
Deterioration of Processed Potatoes BY A. FRANK Ross. Cornell University. Zthaca. New York
I. Introduction . . . . . . . . . . . . . I1. Common Types of Deteriorative Changes . . . . . I11. Factors Influencing Fhte and Extent of Deterioration . IV. Chemical Changes during Storage of Dehydrated Potatoes V. Control of Browning . . . . . . . . . . . VI.Summwy . . . . . . . . . . . . . . References . . . . . . . . . . . . . .
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257 259 259 273 279 285 286
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The Influence of Climate and Fertilizer Practices upon the Vitamin and Wineral Content of Vegetables
SOMERB AND KENNETH C. BEESON.U . 9. Plant. Soil and BY G. FRED Nutrition Laboratory. Zthaca. N . Y .
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I Introduction . . . . . . . . . I1. Influence of Climate on Vitamin Content . I11 Influence of Fertilizers on Vitamin Contebt
. . . IV. Influence of Fertilizers on Mineral Content . V.Discussion. . . . . . . . . . . M.Summary . . . . . . . . . . . References . . . . . . . . . . .
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291 295 307 310 316 318 319
Nonemymatic Browning in Fruit Products University of California. Berkeley. California BY EARLR . STADTMAN.
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I. Introduction . . . . . . . . . . . . . . . I1. Methods of Color Measurement . . . . . . . . . I11. The Effect of Storage Temperature on Browning . . . . . IV. The Effect of Processing and Drying Temperature on Browning V. The Influence of Moisture on the Rate of Browning . . . . V I. The Influence of Oxygen on Deterioration . . . . . . . VII. Changes in Chemical Composition Which Accompany Browning VIII. The Use of Inhibitors to Delay Browning . . . . . . . IX . Status of the Browning Problem in Fruit . . . . . . . X . Needed Research . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . .
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325 328 329 333 334 336 346 360 366 368 369
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373 374 377 380 385 389 391
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396 396 406 421 422
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Microbial Inhibition by Food Preservatives BY ORVILLE WYBB.University of T e r n . Austin. Texas
I. Introduction . . . . . . . . . I1. Interference with the Genetic Mechanism . I11. Interference with the Cell Membrane . . IV. Interference with Enzyme Activity V. Applications . . . . . . VI . Summary and Conclusions . . References . . . . . . .
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High-Polymer Pectins and Their Deesterification
BY G ~ O R GLE. BAKER.University of Debware Ag7.icultuTai Experiment Station. Newark. Delaware
I. Introduction . . . . I1. Highly Polymerized Pectin I11. Deesterification of Pectins IV. Future Considerations .
. . References . . . . . . Author Index . . . . . Subject Index . . . . .
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The Physiology and Chemistry of Rigor Mortis. with Special Reference to the Aging of Beef
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BY E C BATESMITH
Low Tencperature Reclearch StutiOn. Univmsitg of Cambridge and Department of
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Scientific and InduatriaE Resmrch. Cambdge. Engktnd CONTENTS
pose I Introduction . . . . . . . . . . . . . . . . . . . 1 I1 RigorMortis . . . . . . . . . . . . . . . . . . . 3 1 The Proceasee Concerned with the Formation of Acid in Muscle 3 6 2 Consequencea of the Posbmortmm Formation of Lactic Acid in Muscle 3 Recent Theoriea of Rigor Mortie . . . . . . . . . . . 8 4 Further Consequences of the Formation of Acid . . . . . . . 10 11 5 Effect of Antemortam Treatment on Glycogen Content of Muscle a.Exerciae . . . . . . . . . . . . . . . . . 11 b.Reeting . . . . . . . . . . . . . . . . . 12 c Feeding . . . . . . . . . . . . . . . . . 13 d.Fatigue . . . . . . . . . . . . . . . . . 14 6 Variation in pH of Meat . . . . . . . . . . . . . . 15 7 SignificcmceofpH . . . . . . . . . . . . . . . . 16 8. “Dark-cutting” Beef . . . . . . . . . . . . . . . 17 9 Electrical Resistance and pH . . . . . . . . . . . . 18 10 Growth of Bacteria . . . . . . . . . . . . . . . 18 11. Enzymea . . . . . . . . . . . . . . . . . . 21 I11 Storage and Aging . . . . . . . . . . . . . . . . . 21 1 Storage Above the Freezing Point . . . . . . . . . . . 21 2 Inhibition of Microorganiama . . . . . . . . . . . . 22 . . . . . . 23 3 Growth of Bacteria within the Meat: Bone Taint 4. The Conditioning. Ripening. or Aging of Beef . . . . . . . 24 5 Changes in Tendernets During Ripening . . . . . . . . . 24 a Measurement of Tendemem . . . . . . . . . . . 24 b ExperimentslReaulta . . . . . . . . . . . . . 25 c Mechaniam of Changes in Tendernets . . . . . . . . 27 d. Autoly& . . . . . . . . . . . . . . . . . 29 6 Tendernem and Freezing . . . . . . . . . . . . . . 31 I V Conclusions . . . . . . . . . . . . . . . . . . . 33 RBferencea . . . . . . . . . . . . . . . . . . . 34
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I . INTRODUCTION In the two decades that have elapsed since the preparation of the British Food Investigation Board Report entitled “Post-mortem changes in animal tissues-the conditioning or ripening of beef” (Moran and Smith, 1929), fundamental knowledge of the physiological and biochemical properties and behavior of muscle has increased out of all recognition. Perhaps
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because of the bewildering rate of growth of this fundamental knowledge and the constantly changing conception of muscle which has resulted, there has not been so striking an advance in knowledge of the particular processes involved in the prolonged storage of meat, nor any striking application of the principles of modern biochemistry to the technology of handling of meat animals and meat. It is one of the intentions of this review to endeavor to visualize possibilities of such application. It will be found, however, that there is a necessary preponderance of the space devoted to the discussion of physiological and chemical facts over that devoted to consideration of the opportunities which have been taken or can be suggested for the application of those facts. There are two points to which concentration of interest can be directed. The first is the vital role which the pH of flesh plays in every phase of meat technology. This general principle affords a very useful plane to which the complicated events which take place in muscle post-mortem can be related, and upon which they can be oriented. In fact, in dealing with post-mortem change, the scale of change in pH is a far more useful reference scale than the scale of time. Equipped, as most modern laboratories are, with a glass electrode assembly, the determination of pH presents little more difficulty nowadays than the determination of temperature. The second focal point t o which particular attention is drawn is the technique of evaluation of quality by panel judgments based on sensory tests. Explicitly or implicitly, an evaluation of quality is a necessary concomitant of any statement which contains a reference to a change in quality, such, e.g., as improvement or deterioration resulting from a period of storage. If a product treated in a certain way is said to be better or worse as a result of that treatment, it is better or worse with respect to particular qualities, specified or implied. If change in weight is the property concerned, that is a quality amenable to physical determination; but if change in bloom, or flavor, or tenderness is the subject of study, these are questions which must be decided by reference to the eye or the palate. If objective methods are devised for their determination, the validity of the measurements so made must be calibrated against the “organoleptic” evaluation and the measurements will continue to be only so valid and accurate as that calibration has shown them to be. There is probably no problem of quality more difficult to solve in quantitative terms than that of the improvement effected by ripening of beef, since there is no means of holding over a “control” to be judged alongside the stored sample, and evaluation of quality has, therefore, to be carried out by scoring in terms of a scale of imaginsry, memorized standards. Experience in this field during recent years has led to notable advances in technique, and what is more, to a realization of the potentialities of developing, with
PHYSIOLOGY AND CHEMISTRY OF RIGOR MORTIB
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discipline, a reasonably exact methodology of panel evaluation. Thus an investigation such as that upon which Dr. T. Moran and the author embarked exactly 20 years ago would now be started with a far greater offensive armory not only of biochemical, but also of “tropho1ogical”l techniques. It is not inappropriate, therefore, to develop this review from the point at which the earlier report left the subject. It will not be possible to deal in the detail it deserves with the considerable work published during this period on testing for palatability. It is a subject which demands a separate full-scale review and it is hoped that a reviewer will be encouraged to undertake this task at an early date.
11. RIGORMORTIS 1 . The Processes Concerned with the Formation of Acid in Muscle
A resting muscle has a neutral reaction and normally contains a considerable amount ol glycogen. After death the reaction becomes acid due to the production of lactic acid and the glycogen disappears in amount equivalent to the lactic acid produced. In terms of elementary chemistry this reaction requires no more than the addition of water to glycogen, (CsHlOO6), nHzO + ZnCaHeOa,but between the initial and final states an extremely complicated series of transformations is interposed, and a considerable amount of energy is liberated. When the conditions are appropriate, some of this energy can be used to enable the muscle to do work by contracting. The first step in the elucidation of the mechanism of anaerobic breakdown of glycogen was the discovery by Lundsgaard (1930) that contraction could actually occur without recourse to t,his source of energy, by virtue of the breakdown of creatine phosphate to creatine and inorganic lactic acid reaction is phosphate. The normal function of the glycogen to supply energy for the rehabilitation of creatine phosphate; by poisoning the muscle with sodium iodoacetate, this reaction was stopped. Muscles treated with this substance do not therefore become acid after death. They rapidly die and pass into rigor in a state of contracture (Ronsoni, 1931). The next important step was the demonstration by Lohmann and
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The author feels that the term “psychametrical” (which has been suggested by some American workers) is misapplied to the measurement of qualities by sensory evaluation since it is commonly applied to the measurement of mental and intellectual capacity. A word is badly needed to describe the science of the physical and psychological relationship between the consumer and the food he consumes. The term “trophology” is suggested, from T & J ~ food or alimentation. Testing for palatability, the factors governing the formation of food habits, etc., would then be “trophological” studies. (The author is indebted to Mr. K. C. Guthrie for this suggestion.)
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Meyerhof (1934) that adenylic acid intervenes in the breakdown of creatine phosphate, adenosine triphosphate (commonly abbreviated to ATP) being formed as an intermediate. Actually, it is ATP which, in the process’of contraction, directly yields its energy to the muscle in breaking down again to adenylic acid (Needham, 1938). Several other substances which are formed in the course of the step-by-step breakdown of glycogen to lactic acid can similarly rehabilitate ATP. The most recent view of the interrelationship of these processes in what is known as the “glycolytic cycle” is represented diagrammatically in Fig. 1, which is taken from a paper by D. M. Needham (1942). The cycle is also tiiscussed and represented in a review by Mann and Lutwak-Mann (1944). From our immediate point of view, it is important to note that the 6rst stage in the breakdown of glycogen is its linkage with free phosphate to form hexose monophosphate. Thus free phosphate must be present before any breakdown of glycogen can take place, and b r e a k d m of glycogen will occur to the ezteat that free phosphate becomes available. The second point to note is that the cycle represents the anaerobic circumstances only. When sufficient oxygen is available the energy interchanges follow a completely different course, and it is strongly debated whether lactic acid is then formed at all (Sacks, 1938, 1941). Whether this is so or not, the end product of aerobic metabolism is carbon dioxide, and this escapes from the muscle by the same route as oxygen is brought to it, viz., by way of the circulating blood. Thus the process of acidification of the muscle commences sharply with the cessation of the blood supply to the muscle. We are, therefore, able to fix fairly definitely both the moment at which post-mortem changes are initiated, and the kind of reaction which sets the pace for the production of acid. As a result of Lundsgaard’s and Lohmann’s discoveries, the focus of interest in muscle biochemistry had pmsed from the carbohydrate molecules to the phosphorus compounds. It was slowly realized that, far from being a comparatively unimportant coenzyme in fermentation and glycolysis, ATP was the central cog in the machinery (cf. D. M. Needham, 1938); the breakdown of ATP is “among the processes of intermediary metabolism in the muscle, that nearest in time to the contraction of the fibril” (Needham et al., 1941). Whereas, however, the breakdown of creatine phosphate in the course of contraction can be readily demonstrated, that of ATP can only be inferred, because it is instantly rebuilt by transfer of phosphate from creatine phosphate. Hence, one source of the phosphate which ia needed for the production of lactic acid from glycogen is the breakdown of ATP, and the extent of the latter is to be judged partly from the appearance of free creatine and partly from the appearance of lactic acid.
1
6
E. C. BATE-SMITH
Every step in the cycle of reactions depicted in Fig. 1 is effected by an appropriate enzyme. For the breakdown of ATP, a number of enzymes are provided. A real landmark in muscle physiology was the discovery by Engel’hasdt and Ljubimova (1939) that the contractile protein of muscle, myosin (or an enzyme so closely associated with it that its removal in an active condition was impossible (Bailey, 1942)), was effective in splitting ATP and in some way making available the energy which it contained for the performance of muscular work. It is not necessary, however, for a muscle to be stimulated to contract in order that the ATP be broken down. Other enzymes are present which can release the highenergy phosphate groups of ATP, hence the possibility of glycolysis occurring in the complete absence of physiological activity. Two such enzymes have been described by Sakov (1941)) the first able to transfer phosphate from ATP to hexose monophosphate, the second resulting in its complete degradation to inorganic phosphate. The question of the rate of formation of lactic acid thus becomes a question of the circumstances in which these, and possibly other enzymes whose presence has not yet been demonstrated, are active. Apart from the relationship between pH and activity in muscle extracts, little definite is known about these circumstances, but it can be surmised that the energy arising from the breakdown of ATP in an inactive muscle is needed for the maintenance of life in the muscle, and the level of activity found post-mortem will, therefore, be related to the level of resting metabolism existing in the muscle before the animal is killed. This is in fact a most important factor in the post-mortem history of the muscle.
8. Consequences of the Post-mortem Formation of Lactic Acid in Muscle The first direct consequence is that the pH of the muscle steadily falls from the moment the circulation stops. The precise pH of the muscle immediately prior to cessation of the circulation depends on the recent history of muscular activity. If the muscles have been completely inert and well supplied with oxygen for a good while before death, metabolism will have been completely aerobic and the lactic acid content will be very low. All the evidence points to the pH of the muscle with nil or minimal lactic acid being in the neighborhood of 7.4 (Voegtlii et al., 1934; BateSmith, 1938~). Thereafter the pH decreases pari passu with the production of lactic acid, but for 2 comparatively minor factors which have the effect of decreasing the fall of pH: the liberation of carbon dioxide from bicarbonate and breakdown of creatine phosphate; the net effect is as if the initial pH of the muscle were 7.6 instead of 7.4. From this starting point, the effect of lactic acid on pR now depends entirely on the buffering power of the muscle. This is not constant, but varies with pH according
PHYSIOLOGY AND CHEMIBTRY OF RIGOR MORTIB
7
to the concentration and dissociation constants of the ionized acidic and basic groups present at any particular pH. The substances which can act as buffers over the range in question are quite limited (Bate-Smith, 1938a); they are the proteins, the phosphate compounds, carnosine and anserine (i.e., 8-alanylhistidine and 8-alanylmethylhistidine, respectively) and, at the lower end of the range, lactic acid itself. Roughly speaking, the buffering power is about 50 m. eq./100 g. of muscle per pH, which means that the production of 1% of lactic a>cidwill usually cause a shift of about 1.8 pH units. Since the glycogen content is usually just about 1%, the lactic acid formed is usually about 1.1% and the pH reached in full rigor about 5.6. The variation found in practice and its causes will be discussed in greater detail later on. This change in pH has at various times been regarded as the actual cause of rigor mortis (von Furth, 1919). It is true that a mere change in pH profoundly alters the physical properties of the muscle proteins, especially of myosin, and some of the effects seen in rigor, such as the change in color, the increased ease of expression of juice, and the easier penetration of salts are attributable to the shrinkage of the fibrils as the isoelectric point of myosin, pH 5.3-5.5 (Weber and Meyer, 1933;BateSmith, 1937), is approached. But one fact alone is sufficient to dispel any idea that rigor mortis is due to production of acid; i.e., that rigor sets in at times without any change in acidity whatsoever. Claude Bernard (1877)was the first to mention this in relation to muscles poor in glycogen; Best el al. (1926)made similar observations on animals deprived of glycogen by administration of insulin or by feeding thyroid; and it is a common observation in the case of animals poisoned with iodoacetate, where, aa has been mentioned, breakdown of glycogen to lactic acid is blocked. Nevertheless, it is a puzzling fact that, when sufficient acid is produced, rigor always sets in when the muscle reaches a pH in the neighborhood of 6.3 (Bate-Smith, 1939). An explanation of this paradox has recently been put forward by Bate-Smith and Bendall (1947). The connecting link suggested is one of the two enzymes described by Sakov (1941)which produces inorganic phosphate from ATP, the one which he calls the mineralizing phosphataae. This enzyme is comparatively inactive at the starting pH of muscle, and comes increasingly into action at pH 6.5 and below. It was shown by Erdos (194142) that the onset of rigor was accompanied by the disappearance of ATP from muscle, and Bate-Smith and Bendall, confirming this for alkaline as well as for acid rigor, conclude that it is in fact the removal of ATP which is the immediate cause of the stiffening of muscle in rigor. The fall in pH to 6.5 and below, by bringing into action the mineralieing phosphatase, hastens the destruction of ATP to such an ex-
8
1. C. BATE-SMITH
tent that rigor must immediately follow. The connection between the onset of rigor and the reaching of a given pH is thus reasonably explained, but the equally pueeling question is substituted aa to how the mere removal of ATP can possibly influence the physical consistency of the muscle. 3. Recent Theories of Rigor Mortis Two theories have recently been advanced to account for this phenomenon. Szent-Gyorgyi (1945) has put forward a revolutionary theory of the structure of muscle and the mechanism of contraction and rigor based on his own and his colleagues’ work at Seeged during the war years. The essential components of the system are: 1. Myosin. A protein extracted immediately after death by means of 0.3 M KC1 0.15 M K phosphate at pH 6.5. The low viscosity and double refraction of flow (DRF) of solutions in the presence of ealts indicate a fairly small particle size. This myosin must not be confused with the myosin of earlier workers (Engel’hardt and Ljubimova, 1939;Bailey, 1942;von Furth, 1919;Weber and Meyer, 1933). It will therefore be distinguished by Seent-Gy(Srgyi’s original postscript and termed myosin A. 2. Actin. A protein slowly extracted under the same conditions aa for myosin A. Prepared by treating muscle at about pH 9 with acetone, drying, and extracting with water. The extract has low viscosity and no DRF, but in the presence of 0.1 M KCl these markedly increase owing to the conversion of actin from a globular (G) to a fibrous (F) form. 3. Adenosine triphosphah. 4. K , Ca and Mg ions. When dilute solutions of myosin A and F-actin are mixed, the solution suddenly becomes highly viscous, indicating the formation of a complex, F-actomyosin. The myosin of earlier workers is regarded aa a mixture of myosin A and actomyosin of indefinite composition. In resting muscle myosin A is considered to be present as a stable complex with a particular complement of K, Ca, Mg, and ATP, uncombined with actin, which is in the F form. The effect of a stimulus is to dislodge part of the combined K, and part also of the combined ATP. The loss of K enables F-actin to combine with the myosin A. In such circumstances, actomyosin in vitro contracts violently in the presence of ATP, and the ATP to effect this contraction is provided by the above sequence of events. When the free ATP is removed by the enzyme function associated with contracted actomyosin, the system reverts to that required for the resting condition.
+
PAYBIOLOGY AND CAEMIBTRY OF RIGOR MORTIB
9
Thus the resting muscle is easily extensible, i.e., has a low modulus of elasticity, because the myosin A is in globular form and dissociated from actin. If, now, both potassium (by diffusion) and ATP (by enzymic breakdown) are removed from myosin A, as occurs when the muscle dies, actin combines with myosin A to form actomyosin, which, in the extended form, in which it must exist in the absence of ATP, is extremely inextensible and confers on dead muscle the rigidity characteristic of rigor mortis. The present author’s theory (Bate-Smith, 1948) is based on the following facts: 1. The form of the extension-time curve on loading a resting muscle is quite different from that of a stimulated muscle (Bate-Smith, 1939). 2. When a muscle passes into rigor it does not ordinarily contract. Although the extension under a given load is very much decreased, the form of the extension-time curve is characteristic of that of resting muscle (Bate-Smith, 1939). 3. The amount of fibrillar protein extracted by certain salt solutions is much decreased when a muscle is in rigor (Bate-Smith, 1937; Deuticke, 1930). When extracted by an efficient saline extractant, however, the solubility of the fibrillar protein is exactly the same whether the muscle is extracted before or after the onset of rigor (Bate-Smith, 1933). 4. The diameter of the particles in a solution of myosin so extracted, aa observed under the electron microscope, is approximately the same aa that of the ultimate filaments of the muscle fiber, Viz., about 10 mp (Hall et al., 1946). This represents a unit containing a number of myosin molecules of the widths calculated by Astbury (1942) packed side by side. 5. ATP in vitro has the effect of decreasing the viscosity and DRF of myosin solutions (Needham et al., 1941); it increases the amount of myosin extracted from muscle in rigor by an inefficient solvent (Erdos, 194142);it increases the extensibility of myosin threads (Engel’hardt el al., 1941); and it causes the shortening of actomyosin threads (Szent-Gyorgyi, 1945). The stiffening of muscle and the decreased extractability of the proteins in rigor run parallel with the disappearance of ATP from the muscle (Bate-Smith and Bendall, 1947;Erdbs, 1941-42). The inferences from these experimental observations are: first, that the particles present in myosin solutions are fragments of the ultimate contractile filaments of the muscle fiber such as are seen in preparations examined under the electron microscope, and that these, however uniform in properties, are not necessarily composed of homogeneous molecules;
10
E.
C. BATE-SMITH
second, that the process of rigor mortis involves the association of these filaments by weak cross linkages, making them less easily separable from one another by the action of salt solutions but, when once separation has been effected, the properties of tmheparticles differ little from those of the particles prepared from muscle, before the onset, of rigor. The cross linkages, which account also for the decreased extensibility of the muscle, must be formed as a result of total removal of ATP, whereas contraction of the filaments, which results in so striking a change in the form of the extensiontime curve, but not in absolute extensibilit2y,is to be attributed to the action on ATP of myosin ATP-ase without dissociation of the nucleotide from combination with the filamentary protein. These 2 theories are complementary rather than competitive. The author stresses the importance of interfilamentary reactions in rigor; the only implication regarding molecular processes being that any which occur during rigor must be sharply distinguished from those occurring during contraction. Szent-Gyorgyi’s theory on the other hand places the emphasis on intimate molecular processes.
4. Further Consequences of the Formation of Acid The breakdown of ATP proceeds in the dying muscle through successive stages to hypoxanthine as the main end product (Ostern, 1930; Pohle, 1929), other fragments of the molecule being phosphate, ribose, and ammonia. Creatine phosphate breaks down at an early stage in the development of rigor to creatine and phosphate. There is a limited accumulation of intermediate components of the glycolytic cycle such as hexose phosphates when rigor is fully established (Bate-Smith and Bendall, 1947)) but these chemical changes are dominated both in gross magnitude and in its consequences by the formation of lactic acid at the expense of glycogen. As we have seen, this reaction proceeds with increased velocity when a particular pH is reached. Apparently synchronizing with this phase (Rowan, 1940), a fall occurs in electrical resistance and reactance. The interpretation of A.C. conductivity measurements in biological systems is notoriously difficult, but it is reasonable in the present instance t o accept the view of Hemingway and Collins (1932) that the changes observed are due to the virtual elimination of membrane resistance and capacitance, and to infer that the membrane concerned is the sarcolemma (Meyer and Bernfeld, 1946). At about this stage the muscle loses its ability to contract when stimulated, and the free diffusion of ions through the previously impermeable membranes results in a rapid equalization of pH throughout the tissue (Bate-Smith, 193813). This, in fact, is the true point of “death” of the muscle.
PEYSIOLOGY AND CHEMIS’IBY OF RIGOR MORTIS
11
From this point onwards glycolysis continues a t a diminishing rate until either glycogen is exhausted or a second fixed point in pH is reached at which the glycolytic enzyme system is completely, but not irreversibly, inactivated. This point is at or a little below pH 5.4. Even though ample glycogen may be left in the muscle when this point is reached, it is not further broken down unless alkali is added, whereon glycolysis is immediately resumed until the added alkali has again been neutralized. Callow’s work, which will be considered in greater detail later on, has contributed greatly to our knowledge of the glycogen reserves that can normally be expected in livestock and it is clear that glycogen sufficient to allow the muscle to reach pH 5.4 is exceptional. As a rule, therefore, the pH reached in full rigor (termed by Callow the “ultimate” pH) is determined by the amount of glycogen present in the muscles at the moment of slaughter, and control of ultimate pH, whether in order to effect an increase or a decrease, can only be exercised through control of the glycogen content of the muscles of the living animal. There are, in principle, means of doing this. For most purposes it is a high glycogen content which is desired, since high pH, associated with low glycogen, is detrimental to the color, texture and keeping qualities of most meat products. There are indications, however, that for certain purposes a high pH may be an advantage, and this could be secured by low glycogen content in the muscles before slaughter. The means suggested by physiological knowledge for securing an increase or decrease in glycogen are suitable feeding and regulated activity. In recent years a certain amount of work has been done in an exploration of the possibility of applying these principles in the handling of meat.
5. Efect of Ante-mortem Treatment on Glycogen Content of Muscle a. Exercise. The immediate effect of exercise on a muscle is to decrease its glycogen content. If, however, the work is light, and nutrients are available in the alimentary canal and liver, the glycogen is replenished as the work proceeds. Continued heavy work leads to continued depletion of glycogen (Bate-Smith, 1936), so that an animal killed, e.g., after an exhausting hunt, a bull killed in the course of a bull-fight, or a trawled fish, may have no glycogen a t all in the muscles. Animals driven to the slaughtering floor, or struggling violently during slaughter, must have lost glycogen from their muscles. In a well-nourished animal, recovery of glycogen even after exhausting work is usually quite rapid, and the level of glycogen reached may exceed the original resting level. If periods of work and rest alternate, the glycogen content can be built up to the high levels found in “trained” animals (Embden and Habs, 1927;Procter and Best, 1932). If, therefore, an in-
12
1. C. BATE-BMITH
creaaed glycogen content is a desideratum, this, physiologically speaking, is the way to get it. Although undertaken with a different objective in mind, experiments carried out by Mitchell and Hamilton (1933) provide evidence that a higher glycogen content is, in fact, obtained by causing steers to perform intermittent work. The animals were made to walk an average of 8.8 miles per day at 3.1 m.p.h. for 131 successive days. Their muscles were found to contain an average of 3.60% glycogen on a fat-free dry weight basis, while those of a group of unexercised control animals contained 2.54% on the same basis. The effect of exercise was, therefore, to increase the glycogen found (which would in all probability be less than that present at the moment of slaughter) by 41%. Procter and Best (1932) showed that 1-2 weeks of training were sufficient to produce a maximal effect, so that this increase of 41% is likely to be less than existed after a much smaller number of repetitions of the exercise routine. It should be noted, too, that the “exercise” did not represent a great deal more than a beast might be expected to perform when grazing freely. It would be interesting to know whether cattle on free range normally show a lower ultimate pH than animals stall-fed for a period of weeks prior to slaughter. The intention of Mitchell and Hamilton’s experiment was to learn whether the amount of exercise performed had an adverse effect on toughness, or otherwise influenced the quality of the carcass meat. As regards collagen content, palatability rating, and mechanical testing (cf. p. 25 below), contrary to what might have been expected, the exercised group was found to be slightly moTe tender than the unexercised. (This result was confirmed by Bull and Rusk, 1942.) As the authors point out, the lower collagen content of the exercised group suggests that the muscle substance proper was preferentially increased by the exercise. The only other noteworthy effects of exercise were: a general reduction in the nitrogen content of the muscles [calculated on a dry fat-free basis and attributable to a decrease in extractive nitrogen (actually demonstrated in the case of creatine)], the mentioned increase in glycogen, and an increase in lipoids not extracted by ether. b. Resting. It has already been mentioned that a period of rest following exercise is necessary for the restoration of the glycogen stores in the muscles. Actual data for laboratory animals appears to be confined to Jokl’s (1933) on rats and for livestock to Callow’s work (1936, 1938, 1939) on pigs; there appear to be none at all for beef, but common experience can be translated into physiological terms which agree completely with the results obtained by Jokl and by Callow. Jokl exercised rats on a treadmill, in some instances for comparatively short periods (moderate work), in others to complete exhaustion. A num-
PHYSIOLOGY AND CHEMISTRY OF RIGOR MORTIS
13
ber of the animals in the latter group were allowed to recover, without being,,fed, for 3 hours. The average glycogen contents in the muscles were &B follows: in the normal resting animals, 0.576%; after moderate work, 0.338y0; after exhausting work, 0.105?0; after recovery, 0.652oJ,. It seems almost unbelievable that in an animal like the rat (with a high rate of metabolism and negligible reserves of food to draw upon in the alimentary tract), the power of recovery should be such that 3 hours dter exhausting work, which had lasted on the average for 10 hours, the glycogen reserves in the muscles were more than restored. Yet this is no more surprising than the author’s experience, that once a rat has recovered from exhausting work on a treadmill it is impossible to exhaust it when the exercise is resumed, so long M proper opportunities for feeding are provided. The resources for the restoration of glycogen in the exercised rats in these experiments could only have been depot fat. In the case of the pig, it would appear that the depot fat can with difficulty be mobilized for the purpose of replenishing muscle glycogen, since Callow (1939) found that feeding was necessary to increase muscle glycogen after exercise. In his experiments the pigs were shipped by truck to the factory a distance of 1 mile, and then had to walk a quarter of a mile before slaughter. The ultimate pH in the psoas muscles of a group which was rested over-night after this treatment, but not fed, averaged 5.80 as against 5.79 for the unrested; whereas a group which was both rested and fed had an average pH of 5.58 aa against 5.87 for the unrested control group. As Callow (1936) further points out “resting” must, in fact, be resting in order to be effective. Pigs tend to fight when strange groups are mixed together in resting pens and in these circumstances no recovery of glycogen reserves takes place. c. Feeding. Judging from the different behavior of rats and pigs during recovery after exercise, there seem to be species differences in respect to the requirements of food for replenishment of glycogen stores. In this respect, ruminants may possess an advantage in the larger store of carbohydrate material they carry in their alimentary tract, but, even in their caae, it is likely that the maintenance ration consumed during the 24 hours prior t o shipment can only provide for the current needs of 24 hours, and for any longer fast, body reserves must be mobilized, just as in the case of nonruminants. A knowledge of the nutrient content of the alimentary tract of oxen after various periods of fasting would assist in calculating how much food should be given to maintain the carbohydrate balance in the tissues, but as Benedict and Ritmnan (1927) point out “almost nothing is known with regard to the amount of fill in cattle” after fasting, and it can be presumed that nothing at all is known about its nutrient
14
E. C. BATE-SMITH
content. In the absence of this knowledge, it would be safest to assume that food is needed, as in the pig, if the glycogen content of the muscles is to be maintained. There can be no question but that this is true in the case of the pig. Callow’s work is supported by observations published recently in Denmark (Madsen, 194344). Bacon pigs fed for 2 days before slaughter, with the addition to the feed of various amounts of sugar, were compared with animals fed normally and others not fed. The average pH of the psoas muscles (Mgrbrad) of the fed animals was 5.50, that of the unfed 5.87. The glycogen contents of the muscle of fed animals was 1.81% without sugar, and 2.14 for the greatest supplement of sugar; while the unfed was 1.26. It should be noted that the pH of the fed group was so low that the additional glycogen could have no further influence on it. Particularly important was the difference in keeping quality of the 2 groups of animals. The Burfaces of the cured sides from the unfed group were in a slimy condition 11 days after removal from the curing tank, whereas those of the fed group took $1 days to reach the same conditirm. Thus the pH of the mass of the muscular tissue affects the microbial spoilage not only in the interior, but also on the surface of the carcass. d. Fatigue. Of all the factors which affect the level of glycogen in the muscles, fatiguing exertion immediately before death is the most potent. It is possible by this means to eliminate glycogen completely from the muscles, as, for instance, in the case of animals dying in convulsions induced by injection of insulin (Best et al., 1926). The effectsof moderate and exhausting work have already been quoted in discussing Jokl’s experiments (p. 12). Similar reductions in glycogen, inferred from the amount of lactic acid formed in the muscles post-mortem, were observed by the present author in rats exercised on a treadmill (Bate-Smith, 1936). The effects of activity incidental to the handling of livestock can be seen in Callow’s data on pigs. In the first case (Callow, 1936) “exercise” consisted in transportation by rail for distances varying from 25-70 miles. The average ultimate pH of the muscles of groups ranged from 5.60 for the shortest distance to 5.87 for the longest. A second example quoted by Callow (1938) is of groups of pigs which, although rested after their journey for periods ranging from 2Yr17 hours, were walked mile to the slaughterhouse and had average ultimate pH values of 6.00-6.18. Similar groups carried the short distance by truck had average pH values of 5.75 and 5.83. These differences are surprisingly large for such apparently trivial differences in handling. The individual values showed, as would be expected, a wide range of scatter, and it is only by careful statistical work on groups of animals that significant differences could be demonstrated.
PHYSIOLOGY
AND CHEMISTRY OF RIGOR MORTIS
15
6. Variation in p H of Meat
Having exhaustively examined the factors responsible for variation in pH, it is appropriate to review what values various investigators actually fmd. The methods in use earlier than about 1930 for the determination of pH resulted in the most extraordinary variation in the recorded values (see, e.g., Ritchie, 1922, who quotes values from the literature up to 10.0 for the pH of muscle). Since the use of the glass electrode has become widespread, the quoted values can be regarded as more reliable. Schoon and Ooms (1933) and Postma (1934) investigated especially the effect of injury and disease on the pH of the muscles as an aid to coming to a decision in condemning carcasses as unfit for food. The normal variation wm, however, studied in some detail. The first-mentioned authors quote pH about 6 as normal for pork and veal, 6.1-6.2 for mutton. Both authors stress the considerable variation from one muscle to another of the same animal, but agree that in cattle this is much less than in pigs. Thus in the pig, Schoon and Ooms quote a range of 5.7-6.7 for different muscles, those of the limbs having the lower, those of the neck the higher value. Callow (1939) suggests that proximity to bone may be one reason f.or this variation, and quotes the following values: Along the back In the hind leg near the loin In the middle of the hind leg Near the knee A t the back of the knee
5.44-5.51 5.47-5.44 5.67 5.88 6.12-6.20
Neutralization of lactic acid by calcium carbonate in the bone may well be one factor in causing a rise in pH, but it is more likely that variation in connective tissue is the determining factor. As the muscle narrows towards its tendinous insertion, the relative amount of tendon to muscle proper increases, the lactic acid produced per g. of tissue must decrease, and the fall in pH will be correspondingly reduced. Callow (1938, 1939) also showed that muscles vary in their sensitivity to fatigue, citing values for the Psoas and Longissirnus dorsi. The average values for 30 pigs were 5.72 and 5.48, respectively, and the difference increased with increasing pH. In the psoas muscles of 29 representative beef carcasses, the author (Bate-Smith, 1942) found a range of 5.36-5.80; in the thigh muscles (topside) of 6 other beef animals (Bate-Smith, 193%) 5.5-6.0. Similar figures to these are recorded in work of the Danish (Dansk) Institute of Refrigeration (1944). The meat had in this case been frozen and thawed, but this should have had little, if any, effect on pH.
E.
16
C. BATE-SMITH
Beef
Left inner thigh 5.32 Right iriner thigh 5.28 Right outer thigh 5.26 Clod 5.51 Pork Right neck 6.35 6.21 Left neck Right fillet 6.092 Left fillet 5.64 Reviewing these data, it is evident that in beef in full rigor a variation in pH between 5.4 and 6.0 may normally be expected, but lower or higher values may occasionally be encountered. In pork the expected variation is still greater, pH 5.4-6.7. The question must now be considered as to what is the significance of these differences in pH in the subsequent storage and handling of the meat. 7. Signi$cance of p H Within these limits of variation, pH has a marked effect on both the physical and the biological properties of meat: at the upper end of the range the cdlor of the meat is darker, it is slimy and yielding to the touch, juice is not readily expressed from it; its electrical resistance is high, and salt will not readily penetrate into it from the curing pickle. All these properties must be considered as defects, and they are all referable to the condition of the substance of the fibrils of which the muscle is composed. These fibrils lie close-packed along the grain of the meat. The isoelectric point of their main protein constituent, myosin, is a little above pH 5.3, and at this point it has minimum swelling. At the pH of living muscle, myosin has the consistency of a weak jelly, and as the pH falls this jelly begins to shrink until a t a point between 6.5 and 6.0 the fibrils shrink apart and scatter light. Muscle contains a red pigment, myoglobin, similar to, but distinct from, the hemoglobin of the blood. When the muscle is at a high pH the light reffected from the muscle passes through a deep layer of pigment and the color appears a deep dark-red. If, instead of penetrating the muscle, light is scattered by the superficial layers, the color of the muscle will appear much paler, although its actual pigment content is unchanged. This effect misled the author in 1936 when he wrote “it is noticeable to the eye that the fatigued muscle contains more residual blood than the resting.’’ The fatigued muscle, having a pH above 6.5, appeared to be more highly pigmented, but was shown to have in fact only a negligibly higher pigment content than the resting muscle. *This value seem doubtful, sinae this fillet after 230 days in frozen storage had pH 6.67.
PHYSIOLOQY AND CHnMISTRY OF RIGOR MORT18
17
8. “Dark-cuttdng” Beef
From 1925 on this question has commanded considerable attention in the United States, where dark-cutting beef represents a considerable anpual financial loss to the processor (Hall et al., 1944). In 1938 the National Live Stock and Meat Board initiated a cooperative program of research, the results of which were reported to the Board (Anon., 1941). Apart from the solution it offered to this particular problem, the investigation disclosed many facts of the greatest importance to the understanding of post-mortem behavior. The depth of color in beef was shown to be strictly correlated with the pH of the flesh. Carcasses grading dark, “shady,” and light, averaged pH 6.53, 5.68, and 5.58, respectively, in one experiment. In another experiment this trend was confirmed, and it was shown that there was no significantdifference between samples of different depths of color in respect to their content of muscle pigment. It was observed that the highest incidence of dark-cutting flesh occurred when the animals were kept before slaughter in cold, exposed conditions and without food. The syndrome WM established when the dark-cutting condition was induced by injection of insulin, the flesh of one of the treated animals having pH 6.8. The contribution to this study by the Kansas Experiment Station (Hall st al., 1944) shows that what Callow (cf. p. 000) calls the relative “openness” or “closeness” of the structure, in affecting the scattering of light by the tissue, is not the only factor to which the dark color is due. A second factor is that “the demand for oxygen in dark beef is evidently greater than can be supplied by normal transfusion through the tissue, resulting in robbing oxyhemoglobin8 of its oxygen.” As workers of other of the cooperating laboratories have shown, the oxygen comumption of da;rk meat is much higher than that of light, and the “close” structure of the tissue probably prevents diffusion of oxygen as well as of salt (cf. p. 16). The conclusion of the Kansas authors, that “the cause of dark-cutting beef definitely seem5 to rest upon a deficiency of glycogen in the tissue at the time of slaughter” can readily be accepted in the light of the cumulative data of these cooperative investigations. That it is the differences in amount of lactic acid, resulting from the enzymic breakdown of this glycogen, that are directly and specifically responsible for the differences in color seems to be implied by the statement on p. 75 of their bulletin, but the effect of lactic acid is of course exercised only indirectly by virtue of its influence on pH. The experiments of Winkler (1939a) show that the depth of color of meat increases as its pH is raised by addition of ammonia. The data he gives for the variation of imbibition of water
* More correotly, oxymyoglobin.
18
E. C. BATE-SMITH
with pH provide the connecting link. In approximate figures, a t pH 7 the fibers are able to absorb their own volume of water, at pH 6 less than 50a/,, qnd at pH 5, near the isoelectric point, where imbibition is at a minimum, only 25%. 9. Electrical Resistance and p H
The work of Banfield (1935) and Callow (1938) has provided both a theoretical explanation of a number of the puzzling variations in properties of meat from different animals and a lead in attacking the practical problems connected with bacon production. The aspect of that work which concerns us here is the correlation between electrical resistance and pH. Shortly after death, the resistance, measured with a Banfield (1935) probe in conjunction with a “Megger” Earth Tester a t 50 cycles, is 1700-2000 ohms across the fibers and about 70% of this along the fibers. Both values fall as rigor develops, and normally reach 220400 ohms, the difference along and across being much less marked. A correlation that was observed in the course of routine observations on bacon between a high resistance in the muscles and a fatigued condition in the animals led to an investigation of the dependence of resistance on pH, and the striking correlation illustrated in Fig. 2 was obtained. It is to be noted that all the values relate t o animals in full rigor, so that the effects of membrane resistance noted earlier (p. 10) do not apply. The increase in resistance with increasing pH is regarded as being due to the swelling of the fibers, with a consequent narrowing of the channels through which ions can freely move. This view is confirmed by the comparative ease with which pickle salt can penetrate into flesh with a low electrical resistance, and vice versa. The steep part of the curve in Fig. 2 corresponds with a particularly marked change in properties from what Callow terms an “open” to a “closed” structure: The former is associated with a moist feel, a firm texture, and a pale color; the latter with a sticky, dry feel, a flabby texture, and a deeper color. The stickiness, particularly at the higher pH’s, is probably due not only to swelling of the fibers, but also to a certain degree of actual dissolution of myosin (Bate-Smith, 1933). These differences relate to the physical properties of uncooked meat. When the meat is cooked the difference in color between light and dark meat disappears (Hall et al., 1944). Some effect of pH on texture, however, remains. 10. Growth of Bacteria
Callow’s work on electrical resistance and pH cleared up a puzzling situation that had arisen in the Wiltshire curing of hams in Northern Ireland. Whereas negligible trouble from taint occurred in the case of
PHYSIOLOGY AND CHEMISTRY OF RIGOR MORT16
19
farm-killed animals, a high incidence, in some cases over 50%, of tainted hams occurred in packing house-killed animals. It was noticed that the former had lower electrical resistance than the latter, and that in the latter, as the electrical resistance increased, so did the incidence and severity of taint. Thus a group of farm-killed had an average resistance of 233.5 ohms, a group of sound packing house-killed 365 ohms, slightly tainted
PH
Fig. 2. Relation between the degree of acidity (pH) of flesb and its electrical re&tame (Callow, 1936). Each point on the curve represents an average value calculatad from a large number of observations.
476 ohms, and badly tainted 552 ohms. Callow suggests that the origin of this correlation is 2-fold; first, the slower penetration of pickle into the tissues with high resistance; and second, the effect of higher pH, with which the higher resistance is so closely correlated, in favoring the growih of bacteria. At the time it was only possible t o support this possibility with general knowledge of the pH relationships of bacterial growth, but Ingram (1939) has since isolated a number of types of organisms from trhted hams, and has studied the effect on their growth of varying pH.
20
10. C. BATE-SMITE
Fig. 8 ehows the behavior of 3 of these organisms over the range pH 4 to pH 10. In all 8 OM the great increase in rate of growth with rise of pH from a little below pH 6 t o pH 8 is most striking. It shouId be noted,
pnoftlwmd*m
Fig. a. Relation between growth at 37°C. (98.8'F.) of anaerobio organism ieolated from BOWhams (Ingram, 1939).
moreover, that this effect is self-promoting, in that the growth of bacteria in meat at this pH produces a marked shift in pH in favor of increased growth. It is easy to understand, therefore, how a comparatively small
PHYSIOLOQY AND CHEMIBTRY OF RIQOR MORT18
21
diflerence in pH, s.g., between b.9 and 6.1, can determine whether or not a ham becomes tainted. It should also be noted that if meat has reached its ultimate pH with an excess of glycogen still remaining, the growth of microorganisms may not cause an alkaline shift, because production of base will only result in further breakdown of glycogen t o lactic acid (cf. p. 11) and it will not be until the glycogen is completely exhausted that the pW can begin to rise. In other words, from the point of view of resistance to bacterial growth there cannot be too much glycogen in the muscles. 11. Enzymes
From what is known of eneymes, in general, it can be surmised that pH has a considerable effect on the ensymee active in meat after the major eventrs of rigor have come to a standstill, but little of a specific nature has been reported in this connection. As previously mentioned (p. 17), the oxygen uptake of dark-cutting beef is higher than that of light-colored, but this may be attributable to other factors, such as the nature of the respirable substrates present, rather than to difference in pH. Considering the extent to which pH can vary, if ripening is due to enayme action, either wholly or in part, it is cleerly of interest to know the extent to which that action depends on pH. 111. STORAQE AND AQINQ
1 . Storage Above the Freezing Point
The chief problem in storage above the freering point is that of avoiding the growth of microorganisms within and upon the meat. The general and particular aspects of this problem have been dealt with so competently in recent publications that only the highlights need be indicated here. The subject is considered by Haines (1937) under the headings of access of bacteria to the tissues, physiological and biochemical aspects, and control of infection and growth. His own experience, and, in the particular application to the preparation of chilled beef, the experience (Anon., 1938, 1930a, 1939b) of the Australian Council for Scientific and Industrial Research is that prevention of contamination by stringent hygienic precautions on the slaughtering floor and in the chilling rooms is of paramount importance in securing a satisfactory storage life. Next, the physical conditions in the chilling rooms, aiming at a satisfactory rate of cooling of the carows along with a relative humidity sufficiently low to restrain growth of microorganisms on the surface are important factors. Finally, and obviously, the conditions of storage must be those least favorable to microorganisms while, a t the same time, most favorable to the ripening of the
22
E. C. BATE-SMITH
meat. A great deal of attention has been paid to securing such conditions on board ship in the transport of chilled beef, which, in the case of the journey from Australia and New Zealand, requires perfection in technique if the cargo is to arrive in prime marketable condition (Empey et al., 1938). Even in the case of the comparatively short journey from South America there are difficulties yet to be overcome (Heiss, 1939). In the latter case, from slaughter to retail sale a “shelf-life” of about 30 days is expected, in the former case as much as 50 days. T o date the longer period has not been achieved in commercial practice without the assistance of inhibitory agents. A further point to note is that the beef is carried as quarters, and that the cut surface is, therefore, minimal. The bulk of the surface consists of a connective tissue skin covering a layer of adipose tissue, a medium that can be kept dry and, therefore, unfavorable to bacterial growth. Meat which is subdivided into smaller portions, with an increase in cut lean surface, cannot be kept for such long periods. With reasonable precautions, however, it should be possible to hold beef in this form for 17 days in a small butcher’s refrigerator (Haines and Smith, 1933) at the temperature (32”-38”F.) at which this is usually held. Considering the nature of the greater part of the surface of the dressed carcass, it would hardly be expected that the pH of the underlying muscles would exercise much influence on the growth of organisms on it; yet the single piece of evidence bearing on this point seems to show that there is such an effect (p. 14), and that a considerable one.
2. Inhibition of Microorganisms Several chemical and physical means of inhibiting the growth of microorganisms, without decreasing the commercial or nutritional value of the meat, are now in use. For securing the extra 20 days’ life that is needed for the voyage from Australia, the carcasses are held in gas-tight chambers, the air in which is enriched with carbon dioxide. The concentration of this gas is adjusted to the lowest value needed to effect the necessary inhibition, since the “bloom” (fresh color) of the meat is progressively affected as the concentration is increased. In practice 10% is sufficient to produce the desired extension of life with little or no adverse effect on bloom, while 20% is detrimental to bloom with relatively little advantage in prolongation of life (Moran et al., 1932; Brooks, 1933; Empey el al., 1938). A second gaseous inhibitor that has been advocated is ozone. At an eflective concentration of 4-10 parts per million this is germicidal (Haines, 1937), and inhibitory at much lower concentrations (Ewell, 1935, 1938a, 1938b) but the gas is so rapidly absorbed by any form of organic matter
PHYSIOLOQY AND CHEMISTRY OF RIQOR MORTIS
23
that far higher concentrations are needed to deal effectively with bacteria growing on meat. Concentrations in the atmosphere over 3 p.p.m. are irritant and injurious to human beings, and moreover, the chemical changes that ozone produces, especially by oxidation of fats, are deleterious to the commodity. Its use is, therefore, likely to be restricted to the removal of odors in storage rooms, for which purpose a concentration of 1-2 p.p.m. is quite adequate (Ewell, 1935, 1938a, 1938b). Nevertheless, a patent has recently been secured for the inhibition of microorganisms on meat at a concentration of 0.1-1 p.p.m. (Brit. Patent, 1943). The discovery of other gaseous and volatile inhibitors, some of which are already in use in other segments of the food industry, may well revolutionize the storage of meat in the near future. A physical aid to the storage of meat has achieved prominence in recent years. The use of irradiation, both above and below the wave length of visible light, was patented in the United Kingdom (Brit. Patent, 1928). The bactericidal effect of such rays had been known for many years. The use of ultraviolet light for the treatment of various foodstuffs was first reported by the Food Investigation Board (Moran, 1936),and suggestions for its commercial application in meat storage chambers were reviewed by Horne (1937). A disadvantage of the method pointed out by Haines (1937) is that only the surfaces actually exposed to the radiation will be benefited, and pockets of infection shielded from the rays will be as heavily infected as they would be in the absence of irradiation. Ewell (1938a, 1938b) has suggested using low concentrations of ozone in conjunction with ultraviolet irradiation so as to overcome this disadvantage.
3. Growth of Bacteria within the Meat: Bone Taint The measures that have been discussed up to this point were concerned mainly with the microorganisms growing on the surface of the meat. As in the case of hams, trouble is at times encountered in the deeper parts of beef carcasses. The British name for this condition, from its association with the hip joint and upper femur, is “bone-taint”; in the United States it is referred to as “sour” beef (Jensen, 1942; Tanner, 1944). The circumstances of its appearance are very complicated. As a first requirement, the appropriate organisms must be present in, or obtain access to, the affected areas, and secondly, the conditions must be appropriate for their multiplication. As regards the first, Haines and Scott (1940) isolated an anaerobic organism from the hip joint in a typical case of bonetaint, which was absent from 40 normal joints, although 2 of these were infected in pure culture with a Streptococcus and a Proteus, respect,ively. Of the necessary conditions for multiplication, an insufficient rate of cooling is recognized as one, and, significantly, according to trade experience,
24
E. C. BATE-SMITH
a fatigued condition is another. From what has been said about hams, this is not unexpected. Furthermore, with the organism they isolated, Haines and Scott report that good growth occurred from pH 5.8-7.6. There was feeble and limited growth at pH 5.5. The optimum zone was approximately 6.5-7.0. Unfortunately, they do not record the pH of the muscles of the animal from which their organism was isolated. They state, however, that the infection was mainly under the periosteum and discoloration extended for half a centimeter or so near the bone. The organism waa also abundant in the synovial fluid. These details are interesting in relation to what has already been said about the tendency for the pH to be higher in the neighborhood of the bone than in the deeper muscles. Before considering the proceases which occur during the ripening of beef in storage, the prerequisites for securing a satisfactory length of life in store may be summari5ed aa follows: 1. To reduce surface growth of microorganisms by hygienic handling, rapid cooling, and proper conditions in store, and/or by the use of efficient inhibitors. 2. To prevent growth of bacteria, especially in the deeper tissue, by proper ante-mortem treatment and rapid cooling of the carcasses.
4. The Conditioning, Ripening, or Aging of Beef The primary intention of aging meat for a period after slaughter is to make it more tender. In the cme of game, which when freshly killed is usually much tougher than the meat of domestic livestock, the aging process is 80 prolonged that sophisticated taate has come to accept quite an advanced degree of putrefaction as a desirable character, and here ripening implies a corresponding alteration in flavor, This is not the case in beef, where hanging can be allowed to proceed only for so long as the meat remains in reasonably fresh, wholesome condition. Apart from the growth of microorganisms, however, reactions proceed in the tissues which cawe changes in taste and flavor in hung meat as well as in tenderness. 6 . Changes in Tenderness During Ripening
a. Measurement of Tendemas. AR ww mentioned in the Introduction, it is a matter of considerable difficulty to measure tenderness and to evaluate the comparative effectiveness of various times and conditions of hanging in improving this quality. Directly or indirectly, the human muscles and Beme organs mu& be relied upon to perfom the act of measurement; even if mechmical devices are employed to measure certain physical properties related to tenderness, the results obtained by their use must be adjusted to the verdictrs of humaa judges on the tenderness of the
PBYSIOLOQY AND CHEMISTRY OF RIGOR MORTIS
25
samples tested. Often this bmic correlation is neglected by the promotera of a particular apparatus for measuring tenderness, or the data on whioh correlation between human judgment and the mechanical measurement is claimed are not reported in any detail. Several principles have been employed in mechanical methods of measuring toughness. Lehmann (1907) used 2 of these, one of which determined the breaking strength and the other the force required to bite through the sample with 2 cutting edges. Warner’s (1928) (Black et aZ., 1931) apparatus, determines the force needed to draw a knife-edge through the sample against a rigid plate, the force being registered in pounds weight by means of a spring balance. This apparatus has been adopted for most of the subsequent work on tenderness in the United States, e.g. (Griswold and Wharton, 1941; Ramsbottom et al., 1945; Hankins and Hiner, 1940; Hiner et al., 1945) , and much evidence is given in these papers of the dose relationship between the “shear” values and judges’ scoring for tenderness. In Germany, an apparatus designed by Volodkewich (1938) has been employed for detailed and systematic studies of tenderness changes. This employs two blunt “teeth” as shearing edges, and records the work done in cutting through the sample in terms of the area under a loaddisplacement curve. An improved version of this instrument has recently been described (Krumbhols and Volodkewich, 1943). In the same genre but of much simpler construction is Winkler’s (1939b) apparatus. A constantly increasing force is applied in the form of a stream of lead shot and the deformation of the meat between a moving and a fixed jaw is recorded on a synchronized drum. A cutting or puncturing gaug;e and a penetrometer were employed by Tressler, Birdseye, and Murray (1932); by the use of both of these instruments, the data obtained indicated that “the various cuts of steak differ in relative tenderness in about the proportion ordinarily assumed.” Later (Treader and Murray, 1932), these instruments were compared with that of Warner (1928) and it was concluded that the penetrometer gave the most uniform results. The various principles employed may be roughly classified as cutting, tearing, and squeezing. Inasmuch as “chewing” involves all these processes, it would seem to be desirable, at least until a valid correlation is established, to use all 3 methods in measuring toughness by mechanical means. b. Experimental Result8. Bince 1929 many studies of the effect of prolonged hanging of meat have been reported, all of which confirm common experience and the results of earlier work that an improvement in tenderness, more or less proportional to the time of aging, is achieved. Detail has aimed at defining the optimal time and oonditiona of storage, and at elqcidating the mechanism of the decrease in toughness. A useful and
26
E. C. BATE-SMITH
constructive summary of the work up t o 1940 is given by Ewe11 (1940). From a comparison of the temperature coefficients of increase in tenderness and growth of microorganisms, he concluded that lower temperatures were to be preferred to higher. Although the same increase in tenderness might be effected in 3 days at 15.6"C. (60°F.) as in 3 weeks at 1.1"C. (34"F.), the very deep trimming that might be necessary at the higher temperature outweighed any advantages that the greater rate provided. A combination of higher temperature of storage, however, with the protection against growth of microorganisms afforded by ultraviolet light, has come forward for serious consideration. This appears to have been suggested first in 1939 by M. D. Coulter (Anon., 193913). Using the Westinghouse "Sterilamp,'' the optimum conditions were found to be 15.6"C. (60°F.) with relative humidity 8590%. The times required to achieve comparable degrees of tenderness were of the order of weeks at 0.6"C. (33"F.), 5 days at 12.8"C. (55"F.), 2 days at 18.3"C. (65"F.), and a few hours at 29.4"C. (85°F.). After processing, the meat is cooled and stored, when necessary, a t 2.2"C. (36°F.). The tenderness judgments were arrived at by physical, palatability, and consumer acceptance tests. The design, control, and operation of the plant concerned with this process are described by Christensen (1940). The claims for this last method have been investigated by Griswold and Wharton (1941). Tenderness measurements were made with the Warner-Bratzler shear apparatus (Warner, 1928) and by palatability panel. The correlation between judges' scores and shear tests was highly significant. The experimental treatments studied, for each of 5 carcasses used, were as follows (code letters used are the present author's; paired carcasses are bracketed) : Storage conditions 1.1%. (34°F.) without ultraviolet l.l°C. (34°F.) without ultraviolet 15.6"C. (60°F.) without ultraviolet 15.0"C. (60°F.) with ultraviolet 2.2"C. (36°F.) without ultraviolet l6.0"C. (60'F.) with ultraviolet 2.2"C. (36°F.) without ultraviolet
Time, days 9 37 2 2 4
Code letter
A l
i/
E F
B, compared with A, showed but little advantage in tenderness. This was thought to be due to a considerable proportion of any change having occurred in the 9 days before the experiment was started. C and D rated the same for tenderness; the ultraviolet irradiation per se does not, therefore, induce a greater rate of tendering. The values for C and D showed improved tenderness compared with A and B, but these were, of
PHYBIOLOQY AND CHEMISTRY OF RIGOR MORTIS
27
course, not paired carcasses. F was slightly tenderer than E. The difference was in fact considered to be rather surprisingly small. McCarthy and King (1942), in what was primarily a chemical study of the process to which further reference will be made later, mention a specially devised penetrometer, but do not quote any values for tenderness made with it. c. Mechanism of Changes in Tenderness. Steiner’s (1939a, 1939b, 1939c) work is important for the interpretation he places on the changes in mechanical properties observed during storage. Measurements of the toughness of meat with the Volodkewich (1938) apparatus are best made with cooked meat, and Steiner adopted cooking for 1hour in 0.5% salt solution as a routine procedure. Thus any effect that ripening may have on the rate of softening of the tissues by cooking (which is an intrinsically likely possibility) was added to the immediate effect which would be detected in the raw state. Samples were in all instances tested both along and across the grain of the fibers. By this means Steiner deduces the fractional contribution to the toughness of meat of the muscle fibers proper and the collagenous fibers of the connective tissue. He concludes that while both contribute to the resistance acros8 the grain, only the latter opposes cutting along the grain. The diagrams in the 2 cases are quite different (Fig. 4). Across the grain, resistance reaches a maximum and then falls somewhat, and the area under the curve, which represents the work done, is very much greater than in the curve referring to along the grain. The maximum reached, in the first case, is a more reproducible value and more truly characteristic of the toughness of the specimen than the work area. Fig. 4 also illustrates (a) the effect of heating on the toughening of the muscle fibers due to heat coagulation, and (b) the softening of collagen due to heating at 100°C.(212°F.) for an hour as compared with 70°C.(158°F.). There may, however, be a fallacy in this argument, since it is not only the muscle fibers that are arranged parallel to the lines of stress in the muscle: the connective tissue, which is confluent with the tendinous attachments of the muscle, is also disposed so as to resist passive stretch when antagonistic muscles come into action and, therefore, has different properties lengthwise than crosswise of the muscle. The difference between curves 4 and 5 across the fiber, point to a considerable effect of boiling in this direction, also, which can reasonably be referred to decrease in a large connective tissue component offering resistance to cutting. Steiner’s interpretation applied to curves obtained as ripening proceeds leads to the conclusion that the increase in tenderness is due exclusively to an effect upon the muscle fibers rather than upon the connective tissue. This is directly opposed to the view put forward by Moran and Smith (1929), which was based on the generally sccepted belief, backed by sub-
28
E. C. BATE-BMITII
Fig. 4, Load-deformation (taighnw) curvea for raw and oooked beef (Steiner, 1939&,1939b).
stantid experimehtd evidence (Mitchell et al., 1926), that the connective tissue is the major element contributing to toughness af meat and, therefore, the component most likely to be the subject of change during the ripening process. In fact, no direct evidence of such a change could be,
PHYSTOLOaY AND CHEMI@TRY OF RIQOR MORTIS
29
nor has since been, produced, and it must be concluded that Steiner has demonstrated an equal, if not a preponderating, effect on the muscle fibers themselves. Steiner showed further that the rate of change of tenderness during ripening varied very much with the individuaI animal, especially with age and sex. These variables also affected the advantages that could be obtained by raising the temperatures. The muscles of older animals ripened more slowly than those of younger; those of steers more slowly than those of cows. He concluded that these differences must be related to the rate of the autolytic processes which follow rigor mortis. d. Autolyais. The author well remembers how, a t the beginning of his investigations into the ripening of beef, he was warned by the late Sir William B. Hardy, tben Director of Food Investigation, to keep out of “the morass of autolysis.” The warning still holds good, and the attempt will be made to keep to the few small areas of firm ground in this territory. When rigor is fully established and the carcass has ccoled throughout to a temperature not very different from the surrounding air temperature, i.e., 24 hours after slaughter, the muscle contains proteins, fat, lactic acid, phosphate (about a fifth of which may be combined as hexose phosphate) , purine breakdown products of ATP, creatine, and a large number of different organic substances and salt ions, most of them in minute amounts, which were present in the muscle before death. Occasionally some glycogen is present. Among the proteins are a considerable number which function as enzymes, but most of these will have been deprived of their natural substrates by the changes occurring during rigor (cf. Fig. 1). Those remaining active and able to act in the new circumstances will be concerned mainly with the breakdown of protein, and the term autolysis is, in fact, usually employed synonymously with the breakdown of protein by the agency of tissue enzymes, in particular by cathepsin (Mitchell et at., 1926). The process can be followed by determining changes in nonprotein nitrogen. The early stages, in which the breakdown products still behave like proteins toward precipitants, should be revealed by an increase in the amino nitrogen of the coagulable fraction. Since the same changes are brought about by the action of bacteria, the 2 agencies cannot be readily distinguished by their effects, but in studying autolysis bacteria are usually prevented from growing by addition of toluene. Hoagland, McBryde, and Powick (1917)in their classical work on the storage of beef, considered that the changes observed, including increased tenderness, were attributable almost entirely to autolysis, and although molds were present, their action played very little part. The resultij, calculated from their tables, are shown in Table 1.
E. C. BATE-SMITH
30
TABLBJ I Increaae in NomoagzLluMe Nitrogen During Storage of Beef at O0-.%?.8OC.(3L?-S6'F.) a
Days Average yo increase in non-coeg. N. 0
14 30 45 67 68 77 180 14 17 18 22 39 43
-1
Hoagland, McBryde, and Powick (1917).
Table I shows that noncoagulable nitrogen rises progressively as storage proceeds. Since noncoagulable nitrogen represents about 12%% of the total nitrogen, a rise of 8% in this quantity means the hydrolysis to soluble end products of only 1% of the protein present. Hence, after 30 days' storage, only 2% of the protein is degraded to this level, and even after 180 days, only 6%. The tenderness changes have, therefore, to be explained in terms of much less drastic proteolysis than the complete breakdown t o noncoagulable end products. The proteose nitrogen values recorded by these authors, although they also show large relative increases, do not reflect protein breakdown of any greater absolute magnitude than the noncoagulable nitrogen figures. McCarthy and King (1942) followed the increase in sulfhydryl groups during ripening for 30 days at 1.7"C. (35°F.) compared with 2 days at 15.6"C. (60°F.) with ultraviolet irradiation, followed by 28 days a t 1.7"C. (35°F.). The SH groups rose from 0.16 units to 0.23 in the first case, and to 0.29 in the second, a sharp rise occurring during the 2 days at 15.6"C. (60°F.). This increase may be interpreted as a sign of denaturation of the proteins. Denaturation certainly occurs with great rapidity at temperatures not far below body temperature. Thus the author (Bate-Smith, 1933) found a reduction of 30% in soluble protein when rabbit muscle was kept under sterile conditions for 24 hours at 30°C. (86°F.). There was evidence of only slight denaturation in a sample of Argentine chilled beef (Smith, 1934a), which had 57% of soluble intracellular protein compared with 65% in a sample of home-killed beef 24 hours after slaughter. This aspect has, however, never been systematically examined. In any case, there is no reason why denaturation as such should cause meat to become more tender, especially when it is borne in mind that the meat will be much more drastically coagulated when it is cooked. It is obvious that the link between the chemical and mechanical events during ripening, the "mechanochemistry" of the process, is still awaiting discovery. Autolytic processes require some further consideration, however, on account of the changes in flavor that they bring about. Results confirmatory of those of Hoagland, McBryde, and Powick as to the magnitude of
PHYSIOLOGY AND CHEMISTRY OF RIQOR MORTIS
31
the increase in noncoagulable nitrogen have been reported by Haines (1937, p. 5) and McCarthy and King (1942). Smorodintsev and Nikolaeva (1936, 1942) have carried oub painstaking investigations of numerous aspects of autolysis in beef, primarily with a view to establishing chemical or physical coefficients by means of which deterioration can be characterized. These and other investigations have not yet reached a stage a t which the precise nature of the products of autolysis and the amounts produced under stated conditions can be specsed. In general, the activity of cathepsin increases with decrease of pH (Bradley, 1938), but Smorodintsev and Nikolaeva (1936,1942) found that in autolyzing muscle, catheptic activity decreased 4045% in the first 24 hours post-mortem, and 20% further during the next 5 days' storage at O"4.4"C. (32"-40"F.). Peptidase activity, on the other hand, increased 2% times in 10 days after slaughter. The increase in amino acids due to the action of these proteolytic enzymes will have some effect on flavor. That this effect is not particularly dramatic, at any rate over a period of 17 days, is indicated by Moran and Smith's (1929) results. The character of the added flavor would tend to be rather that of bouillon cubes than truly that of meat, and therefore, not particularly agreeable to the consumer. Furthermore, any gain in flavoring substances due to autolysis is likely to be offset by a loss of meat flavor, especially of the volatile flavor associated with the fat. The judgments reported by Hoagland, McBryde, and Powick refer to the 45-77 day meat as having an "old" taste or as lacking in flavor. Similar observations were made by the author regarding the flavor of chickens stored in carbon dioxide from the fourth week of storage onwards (Smith, 193413). It seems likely, therefore, that for periods in excess of 3 weeks the changes in flavor are adverse, and for periods up to 3 weeks too small to be of any consequence.
6. Tenderness and Freezing Tressler and Murray (1932) compared the effects of ripening beef for 6-7 days with a shorter period of ripening (4 days) followed by quickfreezing and storage at -18°C. (-4°F.) for a month or longer. The penetrometer values were consistently in favor of the latter treatment. Hankins and Hiner (1940) showed that air-freezing produced a significant increase in tenderness of beef as evaluated by the Warner apparatus; the samples were cooked before measurement of tenderness. The meat was frozen to equilibrium a t -6.7"C. (20"F.), -23.3% (- 10°F.)or -440OC. (-4O"F.), the 2 lower temperatures effecting a greater increase in tenderness than the higher. Hiner, Madsen, and Hankins (1945) found that beef aged 5 days increased in tenderness progressively as freezing tem-
32
E.
C. BATE-SMITH
perature was lowered and rate of freezing increased, the conditions studied being -7.8"C. (+18"F.), -233°C. (-10°F.) and -40°C. (-40'F.) in still air, R40"C. (-40°F.) in an air blast, and -81.1"C. ( - 114°F.). Histological examination supported the view that tenderness increased in proportion to the intrafibrillar ice formation, and was due to a combination of rupture of muscle fibers and rupture and stretching of connective tissue. If, as these studies suggest, freezing can effect an equal or even greater increase in tenderness than is effected by a long period of aging, and if, moreover, the potential wastage due to trimming of mold and the likelihood of deterioration in flavor involved in ripening for long periods are borne in mind, the possibility of freezing as a means of increasing tenderness clearly calls for close consideration. There are 2 objections to freezing, however. The first is a serious one in the British Isles (but may not be so important elsewhere), that is the stigma at present attached to frozen beef as an inferior product due to the fact that before the recent war it was usually the poorer grades of beef that were frozen. The British meat trade and housewife prefer unfrozen beef ,whether home-killed or imported. The second objection is of almost universal application, that frozen beef produces a quantity of drip when it is thawed, making it unpleasant to handle and suggesting a considerable loss of nutrient material which is none the less important for being apparent rather than real. The amount of drip produced is less from quickly frozen than from slowly frozen beef (Koonz and Ramsbottom, 1939;Bate-Smith, 1944),so that quick-freezing to a low temperature may not only effect the desired increase in tenderness, but mlty also overcome the most objectionable defect of frozen meat. No means are a t present available, however, to secure the required rate of freezing without subdividing the carcass into small cuts. The work of Empey (1933) and Bair and Cook (1938)suggests, however, a means of securing quick-frozen churucter in beef without the need for literal quick.freezing. These investigators found that if beef is frozen at pH 6.3 or above, it produced a minimum of drip, irrespective of the rate of freezing. The reason for this is the high capacity of the muscle proteins for imbibing and holding water at this pH and above, associated with the "closed" structure already discussed (p. IS). If, then, it could be so managed that the pH at which the meat is frozen is above this critical point, a "quick-frozen" product would be obtained under slow-freeBing conditions, and-large cuts, or even whole carcasses, might be "quick-frozen." The principles discussed in Section I1 above suggest 2 ways in which this might, in theory, be done: either, first, immediately after death, by freezing before the muscles have had time to reach this pH; or, second, lowering the glycogen content of the muscles before death. The first is prac-
PHYSIOLOOY AND CREMILITRY OF RIGOR MORTIS
33
ticable and is, in fact, incorporated in a recent patent (Brit. Patent, 1942); the second is not impossible but would be attended by great practical difficulties. It is interesting to note that ripening of meat by the traditional process demands the highest content of glycogen that can be secured, while the method just outlined envisages means of lowering the glycogen content.
IV. CONCLUSIONS In this review the author has endeavored t o gather together some of the threads of physiological and chemical theory underlying the practice of handling meat, with special consideration of the problem of ripening. A common factor in all aspects of the subject is the predominating influence of the acidity of the flesh on its immediate properties and its future behavior. This influence is seen: (1) In the dependence of the rate of growth of the bacteria responsible for bone-taint and souring on pH; (2) in the dependence of the colloidal properties of the muscle proteins on pH, this factor in turn determining the color of the flesh, the rate of penetration of salt during curing, and the amount of drip from frosen meat after thawing; (3) in the dependence of the stiffening process of rigor mortis on the attainment of a certain pH; and (4) in the dependence of the activity of the enzymes responsible for autolysis on pH, this factor in turn, it is presumed, affecting the rate of increase in tenderness during the ripening process. Physiology, in the immediate future, can help the meat processor by defining more precisely the optimum conditions of the meat for operating his processes, whether these are ripening, freezing, or canning, and by devising routine treatments of animals before slaughter which will produce meat of the properties required for particular processes; also by determining the statistical variation in these properties existing among the individuals of given populations of animals. The actual cause of the increase in tenderness during the ripening procws has not been elucidated. The most likely theory is that it is due to proteolysis by tissue proteinase, such as cathepsin. Further research can be expected to indioate the conditions, especially of temperature and pH, optimal for the activity of these enzymes in muscle; and also to suggest further practicable means of retarding the growth of the microorganisms responsible for spoilage on and in the meat. Perhaps the greatest opportunity for research is in the field that has been touched upon only incidentally in this review, that of the quantitative evaluation of the reaction of the consumer to the foodstuff that determines “consumer acceptance.” Of the one aspect of this reaction that most concerns us here, there is, fortunately, little doubt, namely that the increased tenderness is a uni-
E.
34
C. BATE-SMITH
versally desirable quality in meat, and equally, none of the investigators whose work we have reviewed is in doubt that the process of conditioning or ripdhing produces that desired effect.
REFERENCES Anon. 1938. Hygienic methods for the preparation of beef in the meatworks. Cauneit for Sn'. and I&. Res., AuatraEia. Section of Food Pres. and Transport Cdrc. No.
a P.
Anon. 1939a. The cooling of export chilled beef. Council for Sci. and Znd. Res., Australia. Section of Food Presr and Transport Circ. No. 3 P. Anon. 193913. Meat tenderization. Ind. Eng. Chem., New8 Ed. 17, 236. Anon. 1940. Meat tenderization: new information on the Tenderay process. Znd. Eng. Chem., News Ed. 18,291. Anon. 1941. Meat board uncovers some factors responsible for dark-cutting beef. Natl. Prm'simr 104, 15. Astbury, W. T. 1942. X-rays and the stoichiometry of the proteins, with special reference to the structure of the keratin-myosin group. J. Chem. SOC.1942, 337. Bailey, K. 1942. Myosin and adenosinetriphosphate. Biochem. J. 36, 121. Banfield, F. H. 1935. The electrical resistance of pork and bacon. J. SOC.Chem. I&. 64,411T. Bate-Smith, E. C. 1933. Physiology of muscle protein. Ann. Rept. Food Invest. Bd., p. 19. Bate-Smith, E. C. 1936. The effect of fatigue on post-mortem changes in muscle. Ann. Rept. Food Invest. Bd., p. 21. Bate-Smith, E. C. 1937. Native and denatured muscle proteins. Proc. Roy. SOC. London Bl24, 136. Bate-Smith, E. C. 1938a. The buffering of muscle in rigor: protein, phosphate and carnosine. J. Phpiol. 92, 336. Bate-Smith, E. C. 1938b. Physiology of rigor mortis. Ann. Rept. Food Invest. Bd., p. 15. Bate-Smith, E. C. 19380. The carbohydrate metabolism of slaughter-house animals. Ann. Rept. Food Invest. Bd., p. 22. Bate-Smith, E. C. 1939. Changes in elasticity of mammalian muscle undergoing rigor mortis. J. Physiol. 96,176. Bate-Smith, E. C. 1942. The chemical composition of mammalian and avian meat. Chemistry & Industry 61, 373. Bate-Smith, E. C. 1944. Quick freezing of meat. Modern Rejrig. 47,267. Bate-Smith, E. C. 1948. In preparation. Bate-Smith, E. C., and Bendall, J. R. 1947. J . Physiol. 108, 177. Benedict, F. G., and Ritaman, E. G. 1927. The fasting of large ruminants. Proc. Natl. Acad. Sci. U.S. 13, 125. Bernard, C. 1877. Lepons sur le diabete et la glycogenese ttnimale. J. B. Bailliere & Fils, Paris, p. 576. Best, C. H., Hoet, J. P., and Marks, H. P. 1926. The fate of the sugar disappearing under the action of insulin. Proc. Roy. Soc. London,BlOO, 32. Black, W. H., Warner, K. F., and Wilson, C. V. 1931. Beef production and quality as affected by grade of steer and feeding grain supplement on grass, U.S. Dept. Agr. Tech. Bull. 417. Bradley, H. C. 1938. Autolysis and atrophy. Physiol. Reus. 18, 173.
PHYSIOLOQY AND CHEMISTRY OF RIGOR MORTIS
35
Brit. Patant. 1928. No. 325,824. Brit. Patent. 1942. No. 559,849. Brit. Patent. 1943. No. 557,007. Brooks, J. 1933. The effect of carbon dioxide on the color changes or bloom of lean meat. J. SOC.Chem. Ind. 62, 17T. Bull, S.,and Rusk, H.P. 1942. Effect of exercise on quality of beef. Illinois Agr. Expt. Sta. Bull. 488, 105. Callow, E. H. 1935-1938. Progress Repts., Ann. Rept. Food Invest. Bd. Callow, E. H.1936. Transport by rail and its after-effects on pigs. Ann. Rept. Food Invest. Bd., p. 81. Callow, E. H.1938. The aftereffects of fasting. Ann. Rept. Food Invest. Bd., p. 54. Callow, E. H. 1939. Ann. Rept. Food Invest. Bd. (in press). Callow, E. H., and Boaz, T. G. 1937. Transport by rail and its after-effects on pigs. Ann. Rept. Food Invest. Bd., p. 53. Christensen, P. B. 1940. Modern beef chilling. Refrig. Eng. 39, 296. Dansk Kqileinstituut, Meddel. No. 4. 1944. Study of freezing and subsequent storage of beef and pork. Akud. Tekn. Vidensk. Deuticke, H. J. 1930. Changes in the colloidal nature of muscle proteins in death and fatigue. Arch. ges. Physiol. (PfEuger’s)224, 1. Embden, G., and Habs, H.1927. Chemical and biological changes of the musculature after frequently repeated faradic stimulation. 2. physiol. Chem. 171, 16. Empey, W.A. 1933. Studies on the refrigeration of meat. Conditions determining the amount of “drip” from frozen and thawed muscles. J. SOC.Chem. I d . 62, 230T. Empey, W. A., Scott, W. J., and Vickery, J. R. 1938. Notes on the preparation, cooling and transport of export chilled beef. Ice and Cold Storage 41, 161. Engel’hardt, V. A. 1946. Adenosinetriphosphataae properties of myosin. Advances in Enzynwl. 6, 147. Engel’hardt, V. A.,and Ljubimova, M. N. 1939. Myosin and adenosinetriphosphatase. Nature 144, 668. Engel’hardt, V. A., Ljubimova, M. N., and Meitina, R. A. 1941. Chemistry and mechanics of the muscle studied on myosin threads. Compt. rend. acad. sci. U.R.S.S. SO, 644. Erdos, T. 1941-1942. The effect of potassium and magnesium on the contraction of myosin. Studies Inst. Med. Chem. Univ. Szeged 1, 59. Ewell, A. W.1935. The cooler storage of beef. Refrig. Eng. 29, 16. Ewell, A. W.1938a. The use of ozone in cold-storage warehouses. Kulte-Ind. 36,61. Ewell, A. W. 193813. Present and future prospects of ozone in food storage. Food Research 3, 101. Ewell, A. W.1940. The tenderizing of beef. Rejrig. Eng. 39, 237. von Furth, 0. 1919. The colloid chemistry of muscle and its relation to contraction and rigor. Ergeb. Physiol. 17,363. Griswold, R. M., and Wharton, M. A. 1941. Effect of storage conditions on palatability of beef. Food Research 6 , 517. Haines, R. B. 1937. Microbiology in the preservation of animal tissues. Food Invest. Special ReZ;t. No. 46, H. M. Stationery Office, London. Haines, R. B., and Scott, W. J. 1940. An anaerobic organism aasociated with “bonetaint” in beef. J . Hyg. 40, 154. Haines, R. B., and Smith, E. C. 1933. The storage of meat in small refrigerators. Food Invest. Special Rept. No. 43, H. M. Stationery O5ce, London.
36
E.
C.
BATE-SMITH
Hall, C. E., Jakua, M. A., and Schmitt, F. 0. 1946. Investigation of crow striatione and myosin filaments in muscle. BWZ. Btcll. DO, 32. Hall, J. L., Latachar, E. E., and Mackintosh, D. L. 1944. CharacteristioS of darkcutting beef. Survey and preliminary investigation. Kanerrs Agr. Ex@. 8b. Tech. Bull. MI, Part N. Hankina, 0.G., and Hiner, R. L. 1940. Freezing makes beef tenderer. Food Zndr. 12, 49.
Heiss, R. 1939. Investigation of the keeping quality of Argentine chilled meet. Beihfte 2. ges. Kdrltslnd., B, 3. Hemingway, A., and Collim, D. A. 1932. High and low frequency electrioal resistance changes in dying voluntary muscle of rabbib. Am. J. Physiol. 89, 338. Henriques, V., and Lundsgaard, E. 1931.’ Lactic Acid-free Muscle Contraction. Biochem. Z . 286-286. Hiner, R. L.,Medsen, L. L., and Wnnkinn, 0. G. 1945. Histological chsrwtarbtice, tenderness and drip 10of beef in relation to tamperature of freezing. Food Research 10, 312. Hoegland, R., McBryde, C. N., and Powick, W. C. 1917, Changes in fresh beef during cold storage above freezing. U.S. Dept. Agr. B d l . 438. Home, G. A. 1937. The aging and tendering of meat. Ice and R e j ~ gB2, . 169. Ingram, M. 1939. Ann. Rept. Food Inusst. Bd. (in presa). Jensen, L. B. 1942. Microbiology of Meat. Garrard PM, Champaign, p. 150. Jokl, E. 1933. The carbohydrate exchange during muscular exercise in the warmblooded organism. Arch. ges. Physiol. (PjEuger’a) 2S2,687. Koonz, C. H., and Ramsbottom, J. M. 1939. Susceptibility of frozen-defrwted poultry meat to drip. Food Research 4,485. Krumbhob, G., and Volodkewich, N. N. 1943. Gartenbazlwiea. 17, 643. Lehmann, K. B. 1907. Studies of the c a w s for the to~ghnesein meats. Arch. Hyg. 68, 134.
Lohmann, K., and Meyerhof, 0.1934. b y m a t i c transformation of phosphoglyceric acid into pyruvic and phosphoric acids. Bwchem. 2.288,60. Lundsgaard, E. 1930a. Further studies Qn muscle contraction without laotic acid formation. Bwchem. 2. 427, 61. Lundsgaard, E. 1930b. Studies on muscle contraction without lactic acid production. Biochem. 2.217, 162. Lundsgaard, E. 1931. The energetica of anaerobic muscle contraction, Biochem. 2.
ass, 322.
McCarthy, J. F.,and King, C. G. 1942. Chemical changes accompanying tenderization of beef. Food Research 7 , 295. Madsen, J. 1943-44. Investigations on the keeping quality of pork from animals which have been fed feed containing sugar. Nord. Jordbrugsforskning 1948, 5-8, 340 (Chem. Centr., lB44, I , 1939). Mann, T., and Lutwak-Mann, C. 1944. Nonoxidative enzymes, Ann. Reu. Biochem. 18, 25.
Meyer, K. H., and Bernfeld, P. 1946. The potentiometric analysis of membrme structure and its application to living animal membranes. J . Gsn. PhyaMl. SB, 363. Mitchell, H. H., and Hamilton, T. S. 1933. The effect of long-continued muscular exercise upon the chemical composition of the muscles and other tisaues of beef cattle. J. Agr. Research 40, 917. Mitchell, H. H., Zimmerman, R. L., and Hamilton, T. S. 1926. The determination of the amount of connective tissue hi meat. J . Bwl. Chem. 71, 379.
PHYSIOLOGY AND CHEMISTRY OF RIGOR MORTIS
37
Moran, T.1936, Use of ultra-violet light in the preservation of foodstuffs. Ann. Rept. Food Invest. Bd., p. 29. Moran, T., and Smith, E. C. 1929. Postrmortem changes in animal tissues, the conditioning or ripening of beef. Food Inveat. Bd. Special Rept. No. 86, H. M. Ststionery Office, London. Moran, T., Smith, E. C., and Tomkins, R. G. 1932. The inhibition of mold growth on meat by carbon dioxide. J . SOC.C h m . I d . 61, 110T. Needham, D. M. 1937. Chemical cycles in muscle contraction. Perspectivea in Biochemistry. Cambridge Univ. Press, London. Needham, D. M. 1938. Energy-yielding reactions in muscle contraction. Enzgmlo& I, 168. Needham, D.M. 1942. The adenosinetriphosphataseactivity of myosin preparations. BiOch6m. J. S6,113. Needham, J., Shen, 8. C., Needham, D. M., and Lawrence, A, S. C. 1941. Myosin birefringence and adenylpyrophosphata. Nature 147, 706. Ostarn, P. 1936. The purine bodies of rabbit muscles. Biochem. 2. 2'21, 04. Pohle, Konrad. 1929. Producta of profound enzymetic change of muscle adenylic acid. 2. phyeiol. Chem. 186, 9. Poatma, C. 1984. Determinations of pH in meet juices. 2. Fleisch.Mi2chhyg. 44, 182,200. Pmcter, B. A,, and Bat, C. H. 1932. Changes in muscle glycogen accompanying phyeical trSining. Am. J. Phyeiol. 100, 606. Ramebottom, J. M., Strandin0, E.J., and Koonz, C. € 1946. I. Comparative tendernew of representative beef muscles. Food Research 10, 497. Ritchie, A. D. 1922. Reaction of resting and active muscle. J. Physiol. 66, 53. R.5md, E.1981. Phosphate cbangea in chloroform rigor with and without production of lactic acid. Proc. SOC.Exptl. Biol. Med. 28, 712. Rowan, A. N. 1938. The electrical resistance of muscle. Ann. Rep:. Food Inuest. Bd., p. 23. Rowan, A. N. 1946. The electrid resietence of nduscle. Diseertstion, Univ. of Cambridge (Absir. Zndaz Lit. Food Invest. 18, 4, 1942). Sacks, J. 1938. Recovery from musoulsr activity and its bearing on the chemistry of contractions. Am. J. Physiol. 148, 216. h h ,J. 1941, Changing concepta of the chemistry of muscular contraction. Physiol. h. 21,217. Sair, L.,and c)ook, W.H. 1938. Relation of pH to drip formation in meat. Can. J. Rsssorch 16D,226. &&ov, N. E. 1941. Esterifbation by and hydrolysis of adenosinetriphosphoric acid in aqueoua muscle extract. Bwhhimiw 6,261. Schoon, J. G., and O O ~ BA., 191. Acidity of muscle in n o d and diseased animals. Tijhchr. Diarqareuk. 60, 898. Smith, E.C. 1934a. The coagulation of muscle plasma 11. The solubility of myosin. Proc. Roy. SOC.London B l U , 494. Smith, E. C. 1934b. Cold storage of poultry I. Gas storage of chickens. J. SOC. C h .I d . 63,346T. Smith, E. 0. 19%. Scheme for the approximate determination of the proteins of muscle. J , Soc. Chum. Ind. 58,361T. E. C. 1935. The proteins of meat. J. Soc. C h . Znd. k,162T. Smorodintaev, I. A,, and Nikohva, N. V. 1936. Modifbation of c a t h e p h during sci. U.R.S.S., N.S. 8, 376. the autolyeis of muscular tissue. C m p t . r e d . d.
38
E. C. BATE-SMITH
Smorodintsev, I. A., and Nikolaeva, N. V. 1942. Change in activity of peptidase on autolysis of muscular timue. Compt. rend. acad. sei. U.R.S.S. 34, 233. Steiner, G. 1939a. The post-mortem changes in beef muscle at various temperatures. Arch. Hyg. 121, 193. Steiner, G. 1939b. Mechanical measurements of toughness in meat. 2. Fleiech-u. Milchhyg. 60, 61 ; 74. Steiner, G. 19390. The estimation of the quality of meat and the non-microbiological changes occurring during storage. Forschungsdiemt 8, 450. Szent-Gyorgyi, A. 1945. Studies on muscle. Acta PhysioZ. Scand. 9, Suppl. W V . See also Chemistry of Muscular Contraction, Academic Press, New York, 1947. Tanner, F. W. 1944. Microbiology of Foods. 2nd ed., Garrard Press, Champaign, p. 859. Tressler, D. K., Birdseye, C., and Murray, W. T. 1932. Tenderness of meat. I. Determination of relative tenderness of chilled and quick-frozen beef. Ind. Eng. Chem. 24, 242. Tressler, D. K., and Murray, W. T. 1932. Tendernem of meat. 11. Determination of period of aging grade A beef required to produce a tender quick-frosen product. I d . Eng. Chem. 24,890. Voegtlin, C., Fitch, R. H., Kahler, H., and Johnson, J. M. 1934. The hydrogen-ion concentration of the mammalian voluntary muscle under various conditions. Am. J. Physiul. 107,539. Volodkewich, N. N. 1938. Apparatus for meamrements of chewing resistance or tenderness of foodstuffs. Food Research 3, 221. Warner, K. F. 1928. Progress report of the mechanical test for tenderness of meat. Proc. Am. Soc. Animal Production, p. 114. Weber, H. H., and Meyer, K. 1933. Colloidal behavior of muscle proteins. V. Quantitative relationship between muscle proteins and its significance for the structure of striated rabbit muscle. Biochem. 2. 266, 137. Winkler, C. A. 1939a. Color of meat. I. Apparatus for its measurement, and relation between pH and color. Can. J . Research 17D, 1. Winkler, C. A. 1939b. Tenderness of meat. I. A recording apparatus for ita estimation and relation between pH and tenderness. Can. J . Rpsearch 17D, 8.
Factors Influencing the Vitamin Content of Canned Foods BY L. E. CLIFCORN Research Department, C d i & l
Can Co., Chicago, Illinois
CONTENTS
Page 39 11. Vitamin Content of Canned Foods . . . . . . . . . . . . 40 1. The Distribution of Nutrients between the Solid and Liquid Portions 44 2. Effect of Preparation for Serving . . . . . . . . . . 49 111. Effect of Canning Operations on Vitamina . . . . . . . . . . 52 1. Citrus Juices . . . . . . . . . . . . . . . . . 54 2. Tomato Juice . . . . . . . . . . . . . . . . . 56 3. OtherJuiees . . . . . . . . . . . . . . . . . 63 4. Vegetables . . . . . . . . . . . . . . . . . . 64 a. Raw Product Considerations . . . . . . . . . . . 64 b. The Effect of Blanching . . . . . . . . . . . . 66 6.Fruits . . . . . . . . . . . . . . . . . . . 82 6. Effect of Sterilization . . . . . . . . . . . . . . . 84 IV. The Effect of Storage on the Vitamin Content of Canned Foods . . . . 89 V. Relationship of Q p e of Container toVitaminContent . . . . . . 98 References 100
I. Introduction
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . I. INTRODUCTION
The preservation of foods at the times and locations of their abundance, for later consumption throughout the world represents one of the most valuable contributions of science t o mankind during the past two hundred years. Few people realize the importance of processed foods until they are brought to understand how their living would be affected without them. The movement of people away from the soil would be quickly reversed, and large populations would be forced to migrate to more fertile lands and more moderate climates. The success of canning must first be credited to our victory over bacteria, namely, our ability to affect sterilization by heat. Next in importance is the factor of palatability in processed foods. Foods must taste good to be eaten. People eat what they like and if a food is not eaten its nutritive value is of little importance. In addition many social events center around the enjoyment of good food. Food processors know that their products must taste good and have placed major emphasis on this factor. The third important factor for consideration in processed foods is that of nutritive value on which great emphasis is being placed today. Since people eat what they like, it is imperative that the canning industry accepts the responsibility of preserving to the greatest degree the nut,ri39
40
L. E. CLIFCORN
ents with which our natural foods are endowed. Good raw products must be obtained to produce good processed foods. The nutritional qualities cannot be improved by processing. When the first work on vitamins in canned foods was reported, the results were expressed in terms of rat units which in some instances had only a vague correlation to the nutritional requirements of man. As the Rcience of nutrition advanced, more became known about the chemistry of the vitamins, methods for their determination, and their physiological significance to the health of man in terms of curing deficiency diseases and maintainisg buoyant health through proper diet. The National Nutrition Conference for Defense, called by the late President Roosevelt in May 1941, emphatically revealed the need for more information on the nutritive value of processed foods. As a guide to improved health, the Food and Nutrition Board of the National Research Council issued recommended daily allowances for the various nutrients for men, women, and children. In consideration of all of the work done on vitamins in canned foods prior to this time, relatively few of the data could be quantitatively expressed in terms of vitamin potency. More information was needed on vitamin content of canned foods and on the factors affecting vitamin retention from the field through the cannery, through market channels and to the consumer’s table. With this impetus, the canning industry organized a large scale nutrition research program in 1942,which was officiallysponsored by the National Canners Association and the Can Manufacturer’s Institute. Since that time, grants have been allocated to eight universities on the many phases of the problem of the vitamin content of canned foods, and vitamin retention during canning, storage, home preparation for serving, etc. Since the information resulting from the National Canners AssociationCan Manufacturer’s Institute Nutrition Program has been so basic in nature and broad in scope it will be drawn upon very heavily in this review. The author wishes to express his sincere appreciation to all who have taken part in this and other work which has added to our present knowledge on the subject of the nutritive aspects of canned foods. 11. VITAMINCONTENT OF CANNED FOODS Studies conducted during the period 1924-1937 (Kohman, 1937) produced information on the nutritive value of canned foods as determined mainly by animal assay methods. Subsequently, further fractionation of the vitamin B complex into well established entities, together with the development of improved methods for their determination and more suitable methods for the determination of other vitamins, emphasbed the need for further work. In 1941 available critical reviews (Daniel and
FACTORS WFLUENCINO VfTAMIN CONTENT OF CANNED FOODS
41
Munsell, 1937) contained only limited information on the nutritive values of canned foods. In 1942 the National Cannera Association-Can Manufacturer’s Institute arranged for the collection of 823 samples of commercially canned foods representing thirty-two major products canned in the United States (Clifcorn, 1944). It was felt that maximum and minimum vitamin values obtained on a large number of “run of the mill” samples of the same product would serve to fix more closely the ranges of vitamin content for the specificproducts as well &R increase the reliability of the figures for average vittlmin contents. Grants were allocated to various universities where the assays were carried out for the factors ascorbic acid, carotene, rib* davin, niacin, thiamine, and pantothenic acid. Since the methods of analysis employed determine the reliability of the results obtained, the vitamin methods selected for this work were correlated with other existing methods on carefully prepared samples of canned carrots, corn, peas, tomatoes, sdmon, and chopped ham (Guerrant et al., 1945a, b). The results speak well for the vitamin methods which were selected but emphaaize the importance, through the many inconsistencies observed in collaborative work, of further eflort on the part of investigators to make existing methods yield more concordant results when applied to the same food products. The human element seems to be the major contributing factor to the variation in results, together with the fact that methods of vitamin assay which yield satisfactory resulta with one food often yield unsatisfactory results when applied to another food due to the differences in chemical and physical characteristicS. The findings of the ascorbic acid and carotene or vitamin A w a y s of these samples of commercially canned fruits, vegetables, and fish are reported (Pressley et al., 1944) on the bmis of the total contents of the cans for ascorbic acid and carotene, and for the latter also on the basis of the drained solids only. The dichlorophenolindophenol photometric method was used for ascorbic acid. Carotene was absorbed on dicalcium phosphate, eluted, and determined photometrically. The fish products, mackerel, salmon, sardines, shrimp, and tuna, were analyzed for vitamin A content by the Carr-Prioe method. The results of the thiamine and niacin assays for these same canned foods have been reported (Ivea st al.,1944) on the same basis, using the thiochrome method for thiamine and the microbiological method for niacin. This work failed to show any consistent differences between the thiamine content of the larger size wns and that of the smaller consumer size oans as influenced by the longer steriligation times used in the former. The results of the riboflavin and pantothenic both of which were carried out by microbiological techniques, acid -YE, have been reported (Thompson et aZ., 1944). A study of the results
42
L. E. CLIFCORN
from all of the above work showed no correlation with can size, time of harvest during growing period, and pH, with the exception of peas where the thiamine content seemed to be affected slightly by the period of harvest during packing season. In order to increase the reliability of ranges and averages of vitamin content for a large variety of canned foods, many of the same canned foods previously assayed were resampled in 1943 in order to cover as well as possible the year to year variation. I n addition eleven canned products were included which were not among those previously sampled. Not all of the vitamins were determined on all samples, since the assays were scheduled in accordance with the nutritional significance of each particular vitamin factor in each canned food product, and the number of assays for a product thought necessary to see whether the new vitamin values would change the ranges and averages of vitamin content found in the 1942 samples. All of the samples of new products included in the 1943 survey were assayed for all of the vitamins mentioned previously. The carotene, ascorbic acid, and thiamine content of these samples were reported (Hinman et d ,1947) using the same methods as previously used by other workers in this program. In general, the results agreed very well, with a few exceptions, with those obtained on the previous year’s samples. The average value for ascorbic acid in orange juice was significantly lower than that found in the previous year, while significantly higher averages were found for ascorbic acid in both tomato juice and spinach. For thiamine, somewhat higher averages were noted in 1943 for grapefruit products and tomato juice. The riboflavin, niacin, and pantothenic acid content of the 1943 samples was determined (Ives et al., 1945) by essentially the same methods as employed by other workers in this program. The same schedule, previously described, for the vitamin assays of the products was followed. Of the 36 canned food products assayed, most of which had been examined in 1942, there was found an excellent, agreement of the ranges of vitamin content. Since the number of samples analyzed in 1943 was smaller (in most instances) than in the previous year, the ranges of vitamin content were narrower. Niacin in Lima beans is the only cme where both higher and lower values were found than in 1942. As the significance of the newer B-complex factors became more evident, it became increasingly important that more be known about their presence in canned foods. Accordingly, ten samples of each of the following products were assayed for pyridoxine, biotin, and “folic acid” :green asparagus, carrots, green beans, yellow corn, peas, spinach, tomatoes, grapefruit juice, peaches, and salmon. The assays were made on the entire contents of the cans by microbiological methods, “folic acid” being expressed as both
43
FACTORS INFLUENCINQ VITAMIN CONTENT O F CANNED FOODS
I
Tam I
Average Vitamin Valuesfor Smne CmvnerciaUy Canned Fruits and Vegekrbkw
-
mg./100 g.
-
ThiaItem 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
5
Product Apricots, unpeeled halves Aswagus, green Asparagus, white Beans, baked New England style With tomato sauce Beans, green cut Beam, Lima, green Beets Blackberries Blueberries carrots Cherries Corn, white, cream style Corn,yellow, cream style Corn,white, whole kernel Corn, yellow, whole kernel Grapefruit juice Grapefruit segments Kraut Mushroom Orange juice Peaches, halves, clingstone Peaches, halves, freestone Pears, halves Peas, Alaska Peas, sweet, wrinkled Peppers, sweet red Pimentos Pineapple juice Pineapple, sliced Potatoes, sweet Prunes, Italian Spinach Tomatoes Tomato juice Turpip greens
boorbie acid
Carotene
3.9 15.3 15.0
1.29 .31 .029
2.9 2.7 3.3 7.2 3.0 6.2 13.3 2.0 5.9 5.5 5.6 4.7 4.5 32.1 24.8 17.6 1.7 34.3 3.8 2.5 1.6 9.7 9.2 l9.0 .13.0 8.0 5.1 16.2 1.6 13.1 17.0 14.2 19.5
.02 .05 -18 .077 .007 .ll .024 7.34 .52 .012 .08
.017 .086
.007 .007 .031
.006 .097 .26 .20 .006
.29 .26 1.99 1.38 .03 .03 5.94
.72 1.29 -58
.51 Z.64
--
mine 1ydre hlorid Niacin .018 .064 .050
.021 .053 .029 .032 .008
.013 .014 .021 .027 .027 .030 .021 ,026 .028 .029 .034 .016 .073 .007 ,008
.009 .092 .112 .039 .025 .052 .070 .053 .023 .019 .052 .053 .015
Calcium Ribo- pant+ flavin ;henate
-
.35 .82 .71
.022 .094 .057
.094 .19 .12
.24 .91 .32 .54 .14 .21 .19 .35 .19 1.08 .94 .93
.054 .024 .035
.08
.83
.17 .a1 .ll 1.6 .24 .60 .55 .14 .87 1.00 .70
.38 .18 .17 .49 .36 .32 .69
.78 .58
.OM
.0!24
.ole ,014 ,021 .OM .049 .052 .044 .045 .017 .020 .041 .19 .020
.021 .020 .018 .054 .057 .081 ,065 .018 .021 .043 .020 .095 .027 .028
.OW
Baaed on 1942 and 1943 samplea of the NCA-CMI Nutrition Pro~pam.
.lo .062 .ll .079 .078 .068
.13 .12 .21 .22 .2u .21 .13 -13 .093
.94
.12 .043 .050
.023 .15 .15 .10 .18 .lo 10 .43 .65 .053 .23 .26 a
.068 -
44
L. E. CLIFCORN
S.lacti8 activity and L. cuad activity. These results have been reported
(Ives el al., 1946) on the basis of 101 samples analyred. Salmon, yellow corn, and tomatoes were found to be good sources of pyridoxine, while salmon had the highest biotin content. “Folic acid” wm found in the highest quantity in canned green vegetables. In addition to vitamin content, the proximate composition and calcium, phosphorus, and iron content of all of the samples collected in this program were obtained (Kramer, 1946). Although some information of this type was available, the large number of samples of various canned food products collected in proportion to their production over the entire United States offered the opportunity to obtain more reliable information of this type. The two yeam’ survey by five universities has included the analysis of 1,309 samples of 43 canned food products for ascorbic acid, carotene, thiamine, riboflavin, niacin, pantothenic acid, calcium, phosphorus, iron, and proximate composition. In addition, some of the newer vitamin B factors, namely, biotin, “folic acid,” and pyridoxine were determined on ten canned food products in which they might be expected to be present in significant quantities. The average vitamin values of 43 nonformulated canned food products are shown in Tables I, 11, and 111. With regard to the significance of the protein, carbohydrate, fat, and mineral content of various canned food products, a classification system was developed (Kramer, 1946) which provides a means of readily determining the proximate and mineral contributions of specific canned foods to the diet. These are shown in Tables IV and V. During the period of the War, the need was answered for more extensive and aocurate information on the nutritive qualities of canned foods for Army feeding and Lend-lease purposes. This information is now available and may be used in the more intelligent planning of human dietaries. One can now determine which canned foods are excellent, good, fair, and poor sources of the specific nutrients, The contributions of canned foods to the tentative human nutritional requirements can now be evaluated as shown in Table VI. 1. The Distribution of Nutrient8 between the Solid and Liquid P o r t i m
Since canned vegetables and fruits are almost entirely either brine- or sirup-packed, the solid and liquid phase distribution of the nutrients, particularly those which are water soluble, is a very important consideration. With regard to the water-soluble nutrient8 it was thought that a good picture of such distribution could be obtained by the determination of ascorbic acid, thiamine, and riboflavin in the solid and liquid portions of
FACTORS INFLUENCING VITAMIN CONTENT OF CANNED FOODS
45
several types of canned products. The proportions of these three vitamins in the solids and in the brine of eight different canned vegetables was determined (Brush et al., 1944) in both consumer siee and No. 10 cans, and in the solids and in the sirup of seven canned fruits in consumer siee cans only. It was found that “In most canned vegetables the solid weight, TAB- I1 Average Vitomin Vduea for Soma CommcrcicrUy Canned Se4 Foods. rng./100 8. ~
I‘hismine
Item
Vitamin
hydrochloride
,029
,034 .021
A
Product
1 Mackerel
’
Salmon, red Salmon, pink Bardines in Oil sardine8 in tomato muce shrimp1 dry w k Shrimp, wet peck
I
.087 .019 .009
-
.017 .018 .008
C8lCiUm
7.06 7.81
.29 .67
4.71 4.78 2.23 1.64 10.2
.38 .47 .29 21 .17
.20
.l6
-
-
.024 .010
.n
.032 ,031 .14
.009 .008
.037
-
-
.14
Preesley ef d. (1944).
TABWI11 Atwag6 Pyridoxim, Bidin, and “Folic Acid” Content of Soma C o m w W g Canned Foods. rg./100 g.
Product
I
Pyridoxine
I
Biotin
1
“Folic A6id”
L. carsi
8.h t i a Fcrctm
Faetm
’
Berne, green
80 32
Corn, yellow Grapefruit juice Perrcherr
88 14 16
A s P ~ f 4 wgreen l
csrrote PW
Won Spinach
Tomatma IVW ef
d. (1946).
23
45 130 80 71
~
1.7 1.3 1.6 2.2 0.3 0.2 2.1 9.9 2.3 1.8
6.8 2.9 1.3 1.7 0.6 0.6 1.7 2.6 7.4 2.7
-
9.0 7.7 4.1 6.6 1.2 1.s 4.4 6.9 20.7 6.4
L. E. CLIFCORN
46
being 60% to 73% of the total can contents, carried 46% to 68% of the ascorbic acid, 62% to 72% of the thiamine, and 70% to 80% of the riboflavin. Spinach was the outstanding exception to these ranges for with solid weights ranging from 48% to 55% of the total, all vitamin contents of the solid were correspondingly lower. In fruits, the solid weights of the packs showed more variation, being 46% to 67% . . . with the ascorbic acid and thiamine percentages in the solid agreeing closely with the weight percentages, and riboflavin paralleling them a t about 5% to 12% TABLE IV Classification of Commercially Canned Foods According io Content of Proximate Food Componenlsa Protein (approximate)
Products
%
Shrimp, dry pack; tuna fish Salmon;sardines in oil; mackerel Sardines in tomato sauce Shrimp, regular pack Beans, baked Beans, Lima;peas Mushrooms Corn, vacuum pack; corn, cream style; spinach Corn, whole kernel; asparagus; sweet potatoes; turnip peeBeans, green; tomatoes; tomato juice; pimentos; peppers; orange juice; kraut; cherries; beets Blackberrieq carrots; apricote; blueberries; grapefruit; grapefruit 0.7 juice; peachea 0.5 or lese Prunes; pears; pineapple juice; pineapple segments
27 21 18 15 6 4 3 2.5 2 1
Fat (approximate)
% 20 12 7 2 0.7 0.5 0.3 or lese
Kremer (1946).
Products Sardines; tuna fish Mackerel Salmon Beans, baked; shrimp, dry pack Shrimp, regular pack; corn, cream style; corn, yellow, whole kernel Corn, white, whole kernel; peas, sweet ; peppers; pimentos; apinach Apricots; asparagus; beam, green;beans, L m ;blackberries; blueberries; carrots; cherries; grapefruit juice; grapefruit segments; kraut; mushrooms; orange juice; peaches; peas, Alasks; pineapple; pineapple juice; prunes; tomatoes; tomato juice; turnip greens; beets; pears; sweet potatoes
FACTORS INPLUENCINGI VITAMIN CONTENT OF CANNED FOOD8
47
TABLEI IV (Continued) ~~
Carbohydrates (approlrimate)
Producta
% 26 22 19
14 12 9 4
2 1 or less
Sweet potatoes; pineapple alicea; peaches, freestone Apricots; corn, yellow, cream style; perqches, cling; prunes; blackberries Beans, baked; corn, white, cream style; corn, yellow, whole kernel; grapefruit segments; pears Corn, white, whole kernel; pineapple juice Beans, Lima; blueberries; cherries; grapefruit juice; orange juice; peas, Alaska Beets;peas, sweet; peppers; carrots Asparagus, bleached; beans, green; kraut; pimentos; tomatoes; tomato juice Asparagus, green; mushrooms; sardines in tomato sauce; spinach; turnip greens Mackerel; aahon; sardines in oil; shrimp; tuna fish
higher level.” With reference to pyridoxine, biotin, and “folic acid” the distribution between the solids and the liquids has been determined (Ives el al., 1946) for eight canned vegetable products. Their results show that with the solids being about two-thirds of the contents of the cans, approximately two-thirds of the pyridoxine, and 68% to 99% of the biotin were found in the solids. The two green vegetables found to be highest in “folic acid” contained 64% of this factor in the solid portion. Analyses for carotene (Pressley et aZ., 1944) on a number of canned vegetables and fruits, made on the basis of the total contents of the cans and on the drained solids,have shown that the assumption that the carotene is present in the solid portion only is correct. Vitamin A m a y s were made on canned mackerel and tuna from which the oil had been drained in order to conform more closely to the practices of the housewife. Further work will undoubtedly show that such oil contains appreciable quantities of the fat-soluble vitamins, particularly vitamin A and some vitamin D. Upon the suggestion of the Food and Nutrition Board of the National Research Council, work was conducted on the proximate and mineral composition of the solid and liquid portions of canned foods. Analyses were conducted (Kramer, 1945) on canned asparagus, green beans, Lima beans, beets, carrots, whole kernel corn, peas, and spinach. The proportion of the protein content in the liquid portion varied from 6% to S%, respectively, for spinach and corn to almost 33% for beets. Insignificant amounts of fat and fiber were found in the liquid portion. Due to added
L. E. CLIFCORN
48
salt the ash content of the liquid portion was about equal to that of the solid portion. The carbohydrate fraction in the liquid portion varied considerably, from 9% for Lima beam to well over 25% for asparagus, beeta, and carrots. The proportion of calcium in the liquid portion varied more than for any other constituent. For asparagus, Lima beans, and corn, the calcium content of the liquid portion approached that of the solid, TABIJO V Claseification of Comme*cially Canned Food8 According lo Content of Eesential Mineralsa
Calcium (approximate) nag. % 360 200 100 50
Products SrUdh-8
Mackerel, salmon Shrimp, dry pack; spinach: turnip greens Beans, baked; shrimp, regular pack 40 Beans, green; beans, Lima; carrots; kraut; pineapple slim;sweet potatoes hparagus; beets; blackberries; grapefruit segments; peas; pine20 apple juice Apricots; blueberries; cherries; qtqefruit juice; orange juice; to10 mato juice; peppers; p m ~ tuna ; hh 6 Corn,whole kernel; corn, yellow, oream style; mushrooms; peaches, freestone; peam; pimentos; tomatma 4 or lees Corn, white, cream style; pewhe, cling
Phosphorue (approximate)
Products ~~
w7. % 400 300
-
-
sardines in oil Salmon, mackerel Shrimp, dry pack; tuna f%h 226 Sardines in tomato sauce; shrimp, regular paak 176 100 Beans, b W 66 Beans, Lima; corn, cream style; corn, vacuum pack; corn, yellow, whole kernel; mushroom; peae; sweet potatoee 45 Corn,white, whole kernel; asparqua, green 30 Asparagus, bleached; beans,green; beets; carrots; peppers; spinsoh; tomah;t h p 20 Apricota; blaakbemea; grapefruit segments; kraut; orange juice; pimentw; tomato juice 13 Cherries; grapefruit juice; peaches; pineapple juice; prunea 10 or less Blueberries; peare; pineapple slioea
FACTORS INFLUENCINQ V"TAMXN CONTENT OF CANNED FOODB
49
Tasm V (Continued) (approximate) Iron
I
Products
w. 5%
Blueberries; kraut; sardines in tomato sauce; turnip greens Aapmgus, green; beans, baked; beans, Lima; beets; cherries; blackberries; sardines in oil;shrimp; spinach Apricots; asparagus, bleached; beans, green; mackerel; peas; pep 1.6 pem; pimentos; pineapple slices; prunes; tomato juice; tuna fish 0.7 or less Carrots; corn; grapefruit juice; grapefruit segments; mushroom; orange juice; peaches; pears; pineapple juice; sweet p0tatoe.a; salmon; tomatoes 6.0
3.0
while the amount of calcium in the liquid portion of spinach was negligible. In canned spinach and beets the phosphorus content of the liquid portion approached that of the solid portion. Iron was freely distributed in the liquid portions of all but the spinach samples.
S. Efect of Preparation for Semkg The vitamin content of canned foods at the time they are eaten is the most important consideration from the standpoint of the consumer. Work has been reported (Hinman et al., 1944, 1945) on the effects of various methods of preparation of canned foods for serving, on their ascorbic acid, thiamine, and riboflavin contents. Home preparation methods using consumer size cans and institutional preparation using several No. 10 cans were studied. In home preparation, two methods of cooking were employed: (1) heating the total contents of the can to a boil and serving the solids only, and (2) boiling the liquid portion to onehalf to one-fourth the original volume, adding the solids, bringing to a boil and serving the entire contents. In the institutional preparation, the methods were guided mainly by Army and Navy interest. The total contents of several No. 10 cans were boiled for 30 minutes, after which two methods of serving were employed: (1) serving with all the liquid, and (2) serving solids with a slitted spoon. The results of this investigation are shown in Table VII. In addition to the institutional methods, holding on a steam table for one and one-half hours after preparation was also studied. Insignificant losses were observed upon such holding for asparagus and tomatoes, while significant losses were observed for some of the other vegetables. An explanation of why asparagus should retain 92%-99% of its ascorbic acid under such treatment aa compared with 30%-33% retention for green
cn
0
TABLEVI Contribution of Ascorbic Acid, Carotene and Thiamin3 by
I
~QZ.
Ascorbic Acid
Serving of Canned Food to the Daily Ad& Did Carotene
Thiamine
~~
Product Asparaw Beans, green cut h t s carrots Corn, yellow, whole kernel Grapefruit juice Kraut Orange juice Peaches,halves, clingstone Peas,sweet Potatoes,sweet Pineapple juice Spinach Tomatoes a
% Supplied Average content MiniRecornmumb mg./lOOg. mendedn 15.5 3.3 3.0 2.1 4.5 33.2 17.3 35.2 3.8 9.3 16.2 8.5 13.1 17.1
23.5 5.0 4.5 3.2 6.8 52.5 26.1 55.6 5.7 14.1 24.5 13.5 19.8 25.8
% Supplied Average content mg./lOOg.
58.5 12.5 11.3 7.9 17.0 130.0 65.4 138.0 14.4 35.2 61.1 33.3 49.5 64.5
Allowances recommended by the National Research Council.
* Minimum allowances of the Food and Drug Administxation.
.31 .18 .007 7.37 .086 .007 -031 .087 .26 .26 5.94 .03 3.44
.58
Recommended
Minimum
11.7 6.8 0.3 279.0 3.3 0.3 1.2 3.4 9.83 9.83
14.6 8.5 0.3
224.0 1.2 130.0 21.9
346.0 4.0 0.3 1.5 4.3 12.2 12.2 279.0 1.5 162.0 27.2
% supplied Average content mg./lOOg. .061 .028 .008
.021 .026 .028 .ON
.074 .007 .112 .053 .052 .020 ,053
Recommended
Minimum
4.6 2.1 0.6 1.6 2.0 2.1 2.6 5.6 0.5 8.4 4.0 3.9 1.5 4.0
6.9 3.2 0.9 2.4 2.9 3.2 3.8 8.4 0.8 12.7 6.0 5.9 2.3 6.0
?
m d
n
8
TABLEVII
Per Cent Retenlion of V$hrnimaftw Preparation for Servings (originalCanned Product-100%)
I I
Liquid concentrated h r bic
Vegetable Asparagus
Beans, baked Beans, cut green Beans, greenLima Cm0t.a
--
acid
81 62 41
50 51
Thiamine
Rib ilavin
-
-
98 97
100 93 100 98 100
--
96 92 100
-
Corn, yellow, whole kernel PW3 Spinach
-
Tomatoes
Heated-All served 97 100 100
-
-
Liquid discarded
Pscorbic acid I
49 39 I
37 30 38
-
!2
Institutional preparation (boiled 30')
Home preparation
Solids and liquids
1
d
Drained solids
--
Ascorbic acid
Thiamine
Rib* ilavin
Ascorbic acid
mins
Rib tlavin
72
76
Thia-
2
z
-- -- -~ 95
53
45
40 32
77
95 90 92 94 95 78
96
100 8 5 100 90 98 98
96
Boiled 30 mins. 97 83 100
72 30 29 72 7 52
54
-
-
d
60 73 74 57
70 72 80
3M 3
70
67
69
0
g c)
!i
M
U
I
+4
0 0
E
52
L. E. CLIFCORN
Lima beans and 29%-56% for cut green beans was not offered. Spinach retained 87% of its ascorbic acid content. The low pH of tomatoes would explain their 92% retention of ascorbic acid under these conditions. The one and one-half hour holding period on a steam table was not detrimental to the thiamine or riboflavin content. No information is available on the effect of such holding on the carotene or vitamin A content of canned foods. Work was also done on the effects of preparation for serving and holding on the vitamin content of canned peas (Fenton, 1946). Canned peas were prepared for serving by the following methods: (1) Evaporating the liquid to its volume before adding the peas. (2) Heating on top of a stove to 208'F. (3) Heating in an unopened No.10 can in a steamer. (4) Heating the contents of five No. 10 cans in an uncovered dishpan in a steamer. The retention ranges of the four vitamins were: ascorbic acid 33%-46%, thiamine 5670-70%, riboflavin 62770-76770, and niacin 44Y0-87%. The best retention of ascorbic acid was obtained by using cooking method (3) and the poorest by method (I). The best retention of thiamine, riboflavin, and niacin on the other hand was obtained by method (1) and the poorest by method (3). Holding tests were conducted for drained peas heated by method (2) which showed that the ascorbic acid content decreased from 35%-27%. Slight increases were noted in the thiamine, riboflavin, and niacin content which were explained by the author to be due to reabsorption of these B vitamins from the liquor in which the peas were simmered for the 180-minute holding period. It is believed that the above mentioned investigations (Hinman et d., 1944,1945; Fenton, 1946) give a good cross sectional picture of the changes taking place in the vitamin content of canned foods during preparation for serving in home kitchens and in institutional or mess hall operations. 111. EFFECT OF CANNING OPERATIONS ON VITAMINS It is important in considering this subject that the right conception and understanding of the problem be acquired. One must first appreciate the fact that from the standpoint of flavor and nutritive value there is nothing better than t o gather high quality produce direct from the garden and prepare it immediately for the table by the most acceptable cooking methods. As W M shown in the previous section preparatory and cooking practices are detrimental to vitamin content. Riboflavin is affected by light, thiamine by heat, ascorbic acid by oxidation, etc. The chemical and physical properties of the specific vitamins are very important considerations in work on maximum retention of the natural nutrients
FACTORS INFLUENCING VITAMIN CONTENT OF CANNED FOODB
53
in preparation, cooking, canning, freezing, pickling, etc. It is therefore imperative that the properties of the specific vitamins be understood for a most intelligent discussion of the effects of canning operations on vitamin content. A t present no food can be preserved without some sacrifice in T-ABLII VIII
Some Properties Which Influence the Stability of the Better Knoum Vitumins in Fooda hluble in
Atrected bY heate
Mecbd by light
Vitamin
Water
Fata
Mected bY oxygen
Vitamin A (Carotene and Vitamin -4) Thiamine (Vitamin BJ Riboflavin (Vitamin BI or G) Niacin (P-p Factor) Ascorbic Acid (Vitamin C) Vitamin D
No
YeS
Yes
No
Possibly
Ye.¶
No
No
Yes
No
YeS
No
No
No
Yes
Ye.¶
No
No
No
No
Yes
No
Yes
No*
Possibly
No
Ye.¶
Yes
No
No
4
Assuming absence of oxygen or oxidizing substances.
vitamin content. Preparation , processing, and storage losses must all be considered. The canning industry has conscientiously undertaken the task of determining where in the course of operations vitamins are lost, factors affecting these losses, and how through changes in practice and/or equipment these losses may be minimized. Canning operations vary a great deal from plant to plant dependent upon the products being canned and the equipment and practices preferred by individual canners. In general, however, most commercial vegetable canning operations involve: (1) cleaning, washing, cutting, and grading of the raw product; (2) blanching or scalding; (3) filling the containers; (4) the addition of brine; (5) exhausting or expelling air from the product and headspace by heat, vacuumizing, or steaming; (6) sealing the containers; (7) heat sterilization; and (8) cooling. Since vegetables fall into the socalled non-acid class (over pH 4.5), the sterilization of the product in the closed can is usually carried out under steam pressure 112.8"-121.1"C. (235"-250°F.) for timea varying from approximately 15 minutes to one hour, dependent upon the product, the can size, the nature of the pack, etc.
54
L. E. CLIFCOBN
The same general procedure is followed in canning fruits except that sirup is commonly added instead of brine. For the fruit juices and tomato juice, the juice is extracted, screened or “finished,” heated to 87.8°-96.10C. (19Oo-205”F.) , possibly deaerated before or after heating, filled hot and either processed further, or the cans simply inverted after closing and water or air cooled. The use of tubular flash heat exchangers is becoming common in citrus juice and tomato juice canning practices. Tomatoes are filled into cans, tomato juice added and the cans exhausted of air, closed, and usually processed in boiling water. The fruits and tomatoes, and their juices, fall into the classification of acid products (those having a pH less than 4.5) and can be most satisfactorily sterilized in boiling water in relatively short times (10-50 minutes). In consideration of this discussion one can readily appreciate the complexity of factors in the various canning operations which affect the vitamin content of the products being canned. 1. Citrus Juices
The citrus fruits and tomatoes may be classed as supplementary foods because of their high ascorbic acid content. With an average serving of either of these products an adequate supply of ascorbic acid in the diet is assured. With the development of a relatively simple method for the determination of ascorbic acid, it has been possible to survey many problems involved in the production, processing, storage, and consumer practices of canned citrus products. It is of vital concern to the citrus juice canners that their products, which have such a high ascorbic acid content at the time of canning, retain this property to the consumers table. Unfortunately, one of the first surveys on the effect of canning operations on the ascorbic acid content of grapefruit juice (Floyd and Fraps, 1942) lacked the proper control for comparison of the ascorbic acid content of the canned product with that of the raw grapefruit. Samples of canned grapefruit juice averaged 33.7 mg./100 g. as compared with samples of juice from tree ripened, random-picked, raw fruit which averaged 41.2 mg./100 g. The observation was made that freezing the raw fruit decreased the ascorbic acid content of the juice. Juice prepared with machines using corrugated rollers or a rotary grater contained a little more ascorbic acid than that prepared by screen pressing or hand reaming. They reported that freshly extracted juice lost 11.2% of its ascorbic acid content if allowed to stand 5 minutes before pasteurization, and 34.7% in 30 minutes. However, recent work (Lamb, 1946a), in which a number of tests were conducted on holding freshly extracted grapefruit and orange juice, has shown that samples of raw, screened grapefruit juice from five canneries held from 2-5% hours showed no loss of ascorbic acid content.
FACTORS INFLUENCINQ VITAMIN CONTENT OF CANNED FOODS
55
Similarly, only negligible losses were observed during the holding of freshly extracted orange juice up to 6 hours. Other workers (Hamburger and Joslyn, 1941; Scoular and Willard, 1944; Smith et al., 1944) have confirmed the fact that ascorbic acid in citrus juices exhibits remarkable stability under conditions which promote its rapid oxidation in other products. A number of excellent surveys are now available reporting recent work on the overall retentions of ascorbic acid during the canning of citrus juices and the effects of varied practices and specific canning operations. In most of the surveys, samples were taken: (1) at the extractors; (2) after screening; (3) at the holding tanks; (4) at the filler bowls; and (5) from the closed cans. Such a survey in twelve canneries in the Rio Grande Valley (Wagner et al., 1945) showed retentions of ascorbic acid during the canning of grapefruit juice from 92.2%-!39.6y0 with an average retention for all plants of 96.7%. The low retention of 92.2y0 in one plant appeared to be due to the practice of holding the hot juice after pasteurization. A similar survey of twelve canneries in the Florida area (Moore el al., 1944) showed retentions of 88.7~0-101.1%with an overall average of 97%. The low value of 88.7% was attributed partially to traces of copper from copper alloy fittings a t this particular plant. Surveys of operations in the California and Arizona areas have shown (Lamb, 1946a) retentions varying from 91.6%-107.5% for three California and four Arizona grapefruit juice canneries, and 94.4%-101.6% for five California orange juice canneries. The average for both products and all plants was 98%. The low value of 91.6y0 waa explained by the long holding period for the heated juice in this particular run, while the high value of 107.5y0 was explained by the condensation of the juice by flash boiling without return of the condensate. This work together with that of others confirms the fact that there is no particular problem with regard to the retention of ascorbic acid during the canning of citrus juices. Although the extent of the losses of ascorbic acid during the canning of citrus juices has been noted to be small, it has been possible to make a successful evaluation of all of the specific operations and practices. The factors which appear to contribute most to the high retention of ascorbic acid in canned grapefruit juices have been outlined (Wiederhold et al., 1945) as follows: (1) Absence of copper or other metals which might catalyze the oxidation of ascorbic acid in the juice, (2) avoiding the incorporation of air in juice during any step in processing, such as extraction, screening, filling of holding tanks, and storage of juice in these tanks, (3) thorough deaeration of the juice, (4) use of tubular flash pasteurizers rather than kettle type pasteurizers, (5) high vacuum in cans and reduction of headspace to a practical minimum. Because of the relative importance of ascorbic acid in canned citrus
56
L. E. CLIFCORN
juices little emphasis has been placed on the fate of the other vitamins. The low pH and mild heat treatment required for sterilization should be favorable to a high retention of all vitamins. However, in studies of the loss of thiamine in fruit juices during their preparation and processing, it was found (Harris and Proctor, 1940) that thiamine retention ranged from lOOyoin canned orange juice to only 12% in canned grapefruit. 2. Tomato Juice
Tomato juice, like the citrus juices, haa enjoyed an enlarged popularity because of its high vitamin C content. Unfortunately, tomato juice does not possess as good an environment for the stability of vitamin C during preparation and canning as do the citrus juices. Orange and grapefruit juices containing large quantities of air may be allowed to stand for several hours without appreciable losses of vitamin C, while tomato juice under the same conditions loses large amounts of this important vitamin (Lamb, 1946b). Under conditions of heating the differences are even more marked. Work on the extent and causes of the losses in vitamin C content are particularly important, since canners are interested in obtaining an efficient retention of this important vitamin during the preparation and canning of tomato juice. The point has been stressed (Sanborn, 1938) that increased losses may occur when the hot juice is exposed to excessive aeration, e.g., during centrifugal pumping and tank storing of the juice. It was observed in one instance that during a 13-minute period required to heat the juice in a tank prior to filling into the cans, a 19% losaafaambic acid occurred. During the 1943 and 1944 canning seasons a rather extensive survey of tomato juice canning plants in California (Lamb, 194613) revealed that the ascorbic acid retention ranged from 32%-96%, while there appeared to be no loss of thiamine, niacin, and riboflavin in the juice from three canning plants. The large variation in ascorbic acid retentions is explained by the differences in the speeds a t which the canning operations were conducted particularly in reference to the length of time during which the tomatoes or tomato juice were heated in the presence of air. The temperature to which the juice was heated in the presence of air also appeared to influence the extent of destruction of ascorbic acid, the greatest losses being noted at the higher temperatures. This is easily explainable by the acceleration of the oxidative destruction of ascorbic acid by heat. It was reasonable to expect the high retentions of 93 and 94% of the original ascorbic acid in two canneries where efficient deaerators were being operated in the canning line. Eleven samplings of tomato juice from six different canneries were assayed (Guerrant et al., 1946) to determine the retention during the canning
FACTORS INFLUENCINQ VITAMIN CONTENT OF CMTNDD FOODS
57
operations of the following vitamins: ascorbic acid, carotene, niacin, and riboflavin. The ascorbic acid content wm found to range from 5692% of that of the chopped tomatoes. The lowest ascorbic retention (54%) was found in the juice from the chopped tomatoes which had been subjected to prolonged preheating [35 min. 87.8”C.(190”F.)],while the highest ascorbic acid retention was in the juice from chopped tomatoes which had been preheated to 57.2”C.(135OF.)for only 15 seconds. The carotene retention waa found to range from 60-7401,, which was considerably less than the percentage retained by canned tomatoes. This difference may be due to the mechanical loss of carotene during the juicing operation where seeds and fibrous material are removed. Thiamine retention ranged from 76-86%, while riboflavin and niacin ranged from 89406% and 94-107%, respectively. Surveys (Strachan and Atkinson, 1946) on Canadian tomato juice canning operations have indicated that the loss of ascorbic acid in processing under good conditions based upon two tests at one plant waa 11.7-13.5% The loss at another factory was only slightly greater, while at a third plant the losa was lS.1-26.9%. It is very important to note here that these results are baaed upon the juice immediately after hot extraction and not upon the ascorbic acid content of the original tomatoes. Since considerable loss of ascorbic acid takes plaee during the hot extraction of tomatoes, if this operation were taken into consideration in the above surveys, the results for the two surveys at the first plant should be 17-23% loss instead of 11.7-13.6’210loss. There has been considerable debate as to whether the hot “bre&”l or cold “break” of tomato juice is best for the retention of vitamin C. In general, it may be assumed that a cold break produces juice which immediately after extraction has a temperature of less than 48.9’C. (120OF.). Some of the early work (Sanborn, 1938) reported that the best conservation of ascorbic acid was found in those plants employing the “hot break” for the tomatoes, or crushing the tomatoes while hot. The effect of “breaking” temperature on ascorbic acid retention ww further studied (Robinson et al., 1945). Six runs were made in two tomato juice canning plants at the following “breaking” temperatures 93.3”,85”,76.7”,68.9’, 61.1°, and 26.7OC. (200°, 185”,170°, 166”,124O, and SOOF.). An over-all loas of 16-19% of ascorbic acid was reported which was independent of the “breaking” temperature. It is interesting to note in this regard that aa a result of many plant surveys (Clifcorn, 1945) no difference could be shown in the retention of ascorbic acid of the final canned juice whether prepared from the juice of heated tomatoes or from cold extracted juice. 1 The term “breakJ’and extrsction are synonymous and tm used interchangeably in this paper.
58
L. E. CLIFCORN
The over-all retentions were 51-79% for the hot break juice and 56-78% for the cold break juice, with averages of 68% for each. A very important observation was made, however, on the effects of the two methods of extraction upon ascorbic acid. Based upon the raw tomatoes, the retention of ascorbic acid in the hot extracted juice, immediately after extraction, ranged from 68-9970 with an average of 840/,, while for cold extracted juice the retentions ranged from 93-99% with an average of 94%. It was pointed out (Lamb, 1946b) that ascorbic acid is quite stable in chopped raw tomatoes upon standing in air with no loss being noted after 45 minutes. On the basis of the above, it i s difficult to understand the report (Strachan and Atkinson, 1946) that tomatoes pulped and cold extracted through a screen or centrifugal juices had the lowest retention of ascorbic acid as compared to hot extracted juice. The problem of hot extraction vs. cold extraction is directly correlated with the problem of enzyme activity in tomatoes and tomato juice. Although there is little published information on the mechanism of the destruction of ascorbic acid in tomato juice canning operations, some emphasis has been placed on the presence of an enzyme as the oxidative catalyst. It was found (Wokes and Organ, 1943) that the skin of ripe as well as unripe tomatoes contained the highest concentration of the oxidizing enzyme. However, recent work (Lamb, 1946b) shows that there is no significant enzyme problem. A lot of tomato juice was divided into several portions which were treated as follows: a. No treatment-held a t room temperature b. Heated to 82.2"C. (180°F.) in 2 minutes and quickly cooled-held at room temperature c. Same as a-held a t 37.8"C. (100°F.) d. Same as b-held a t 373°C. (100°F.). All treatments were carried out in the presence of air, and air bubbled through the samples during the subsequent holding. Samples for ascorbic acid determinations were taken after 30, 60, 120, and 180 minutes. The small differences between the heated and unheated samples at both temperatures could not be justifiably explained on the basis of enzymatic activity. The rates of ascorbic acid decomposition in raw tomato juice saturated with air a t different temperatures, and in enzyme free juice (heated and cooled), similarly treated, have also been recently reported (Clifcorn and Peterson, 1947). Figs. 1 and 2 clearly demonstrate two very important points: (1) that no significant differences in ascorbic acid loss were noted in enzyme free as compared with raw tomato juice; and (2) the extreme importance of the time-temperature relationship of tomato juice heated in the presence of air, At a given temperature, under the condition of
FACTORS INFLUENCING VITAMIN CONTlINT OF CANNED FOODS
59
(2
tomato juice saturated with air, the losses were typical of those following a first order reaction in which the formula
=
ck) applies. Most of
the inconsistencies in the literature on the merits of hot break vs. cold break methods could be clarified if additional information on the length
Fig. 1. The effect of tempemture on the decomposition rate of ascorbic acid in raw and heatedand then coded tomato juice in the presence of an excesa of air. A-Enzyme free juice, %raw juice (Clifcorn and Peterson,1947).
of time and the temperature of the juice at the various stations of the operations were given. The important thing to realize in this connection is that it is possible to obtain a higher vitamin C content in tomato juice immediately after extraction by the cold extraction process. If the canner wishes to retain this high vitamin C content one or preferably both of the following must be done: deaerate to eliminale the ozygen and hancEEB the juice in &sequent OperatiMlS to prevent further incorporation of air, or
60
L. E. CLIFCORN
heat the juice a8 quickly a8 possz%le to a high temperature for immediate canning. These fundamental principles as applied to hot extracted juice need particular emphasis because the juice coming out of the extractor has a disadvantage from the standpoint of vitamin C retention in that it contains a high air content and is hot. As shown by Fig. 2 the tempera-
Fig. 2. The effect of temperature on the dewmpoaition rate of ascorbic acid in tomato juice in the preeence of an excesll of air (Clifcorn and Peterson, 1947).
tures approach those of the most rapid rate of vitamin C destruction. Many investigators (Robinson et aZ., 1945; Lamb, 1946b; Clifcornand Peterson, 1947) have observed that there is no loss of ascorbic acid upon processing tomato juice in the can in boiling water for approximately 20 minutes. Although there may be small amounts of oxygen entrapped in the c m during closure, the fact that there is no loss may be explained by the preferential absorption of oxygen by the metal surface of a plain can
FACTOM INFLUENCING VITAMIN CONTENT OF
cmm
FOODS
61
in contact with a metallic substance. It has been found, however (Strachan and Atkinson, 1946), that excessive heating of tomato juice beyond that required for sterilimtion did result in significant destruction of ascorbic acid. For example, 115.6"C. (HOOF.) for 10 minutes wrt8 similar to 98.9"C.
Fig. 3. Retention of ascorbic acid in raw tomato juice with and without the addition of 5 p.p.m. copper (Lamb, 1946b). Unheated: 5 p. p. rn. copper added. 0 No copper added. Heated to 180°F.and cooled: A 6 p. p. rn. copper added. A No copper added. Evacuated and held under N1: 5 p. p. m. copper added. 0 No copper added.
(210°F.)for 10minutes, but 115.6"C.(240°F.)for 20 minutes, and 21.1"C. (250°F.) for 5 and 15 minutes resulted in lower ascorbic acid value8 by 1.0-1.4 mg./100 ml., than when heated to 98.9OC. (210'F.) for 10 minutes. The seriousness of contamination of tomato juice with small amounts of copper from copper equipment with a resultant catalytic effect on the
62
L. E. CLIFCORN
rate of ascorbic acid loss has received the attention of several investigators (Sanborn, 1938; Lamb, 1946b; Clifcorn and Peterson, 1947). A few parts per million of copper accelerate the losses of ascorbic acid in tomato juice, particularly in heated juice. Fig. 3 (Lamb, 1946b) shows the effect of copper on ascorbic acid retention by juice treated in various ways. All samples were held at room temperature and the ascorbic acid content determined periodically. Canners are aware of the danger of the use of too much copper equipment in tomato juice canning operations and fortunately are installing, in most instances, stainless steel equipment. One can realize more emphatically the sign5cance of the previous discussion by observing the results of two plant surveys, one in which bad practices were used and the other in which the best principles had been reduced to plant practice. In the plant survey shown in Table IX (ClifTABLEIX Retention of Ascorbic Acid in Tomato Juice Plank Where Poor Practices Were Employed* Samuline:station % Retention 100 A. Fresh whole tomatoes B. Fresh chopped tomatoes 91 74 C. Preheating tank (copper) 70°C. (158°F.) D. Extracted, holding tank (copper) before heating to 96.1"C. (205°F.) 71 49 E. Filled and closed 86.1"C. (187°F.) F. Processed 30' @ 100°C. (212'F.), water cooled 61 a
Clifcorn and Peterson (1947).
corn and Peterson, 1947) the hot break procedure was employed, the extracted juice was pumped over long distances, held in a number of holding tanks (particularly after being heated), and the whole canning cycle was too long. Unfortunately, the exact time cycle was not obtained, but it is estimated to be 30-40 minutes. Copper equipment was used at various places in the cannery operations. As could be expected, because of the equipment and practices employed, a low over-all retention of 51% of the original vitamin C content was found. I n Table X (Lamb, 1946b), canner A had very efficient operations (including deaeration) in which the whole canning cycle was completed in 2-3 minutes. In this case the tomatoes were extracted cold, heated rapidly to 82.2"-87.8"C. (180"-190"F.), deaerated immediately under a vacuum of 12 inches, heated in a tubular sterilizer to temperatures of 108.3"-110"C. (227"-230"F.), filled and closed, and air cooled. While some question might be raised as to the advisability of preheating the juice prior to deaerating, it seems satisfactory in this
FACTORS INFLUENCING VITAMIN CONTENT OF CANNED FOODS
63
case, because the time required for heating the juice to 87.8"C.(190'F.) prior to deaeration was extremely short. In summary of this subject the author would like to present the following principles which have been found to be concordant with practices which can be employed to bring the retention of vitamin C in tomato juice to high and satisfactory levels: (1) steaming of tomatoes prior to extraction; (2) cold extraction of tomatoes; (3) the elimination of as much air as possible during operations subsequent to extraction; (4) heating of the juice to its top temperature as quickly as possible; (5) if storage tanks are necessary to provide full capacity, it is preferable to hold cold juice rather than TABLIO X Retention of Aacorbic Acid in Tomuto Juice Plants Where Good Practices Were Employed" Sampling station Raw, juiced From finisher After closing Mtar cooling
% Retention 100 97 91
90
Lamb (1946b).
heated juice, and in no instance should heated juice which is not freed of air, be held; (6) elimination of copper equipment; (7) the canning line should be as simple as possible and unnecessary pumping or handling of the juice should be avoided; and (8) the entire canning time should be as short as possible. 3. Other Juices Although a number of fruit and vegetable juices such as apple, grape, pineapple, carrot, and blended vegetable juices are being packed, there is little information as to the effect of commercial canning operations on their vitamin contents. The lack of such information was recognized and studies were carried out (Kirk and Tressler, 1941) on a laboratory scale on the loss of ascorbic acid in bottling strawberry, raspberry, peach, and apple juices. According to these authors most of the ascorbic acid was lost during the preparation of the fruit or juice before heating, while heating caused an inactivation of the ascorbic acid oxidase, thus inhibiting further oxidation of the ascorbic acid. Further, they stated, in general, any process necessitating the handling of the fruit before heating, results in lowered ascorbic acid retention, while any treatment after heating has little effect. They also found that the apple juice packed under laboratory
64
L. E. CLIFCORN
conditions was completely devoid of ascorbic acid. As a rule, fresh apples are low in ascorbic acid content. Furthermore, any treatment the juice receives causes a lowering of the ascorbic acid content, so it is generally recognized that most, if not all, apple juice, contains little or no ascorbic acid, unless fortified. The great difficulty in the preparation and canning of this product is that there is a very active ascorbic acid oxidase system, and the juice is generally extracted in the presence of air, filpered, clarified, etc., before this enzyme is destroyed by heating. The author wishes to venture the opinion that a complete deaeration of whole apples and subsequent extraction of the juice in the absence of air followed by quick heating, preferably in a tubular heat exchanger would produce an apple juice with a significant retention of the natural ascorbic acid content. Since vitamin C in raw apples is almost completely oxidized during mastication because of the active enzymes present (Elvehjem, 1947), consumption of apple juice prepared by the method indicated above would yield a greater nutritive value.
4. Vegetables a. Raw Product Considerations. In surveying the effect of canning operations on vitamins in canned foods, one must include certain steps which are not commonly considered. These are the harvesting of the raw product and the method of handling of the harvested product, prior to its actual preparation for canning. The time of harvest or maturity of the product has a significant effect in most instances on its vitamin content. Of equal significance are variety, type of soil, climatic conditions, fertilizers, etc. (Todhunter and Robbins, 1941; Hansen and Waldo, 1944; Heinze et al., 1944; Schroeder et al., 1943; Hamner et al., 1945; Lee and Whitcombe, 1945; and Hibbard and Flynn, 1945). It is seldom possible to place the freshly harvested raw product directly into a can and sterilize it. Varying periods of time for transporting and handling are usually required before the cycle from the field to the can is complete. Attempts are made to confine this period to the same day in most commercial vegetable canning operations. During such storage, certain changes take place, such as the conversion of starches or cellulose to sugars, and partial hydrolysis or denaturation of proteins. Quantitative changes in protein, fat, carbohydrate, or ash contents do not occur. Moisture losses may occur which vary only with concentration but which do not affect the total amount of any nutrient. Most of the vitamins are quite stable during these preliminary storage periods. Ascorbic acid and riboflavin, however, may undergo serious losses in vegetables if improper conditions are maintained. It was observed (Gleim et al., 1944) that losses of 5-22010 of riboflavin
FACTORS INFLUENCING VITAMIN CONTENT OF CANNED FOODS
65
occurred when crates of spinach and asparagus were exposed to the light of a laboratory room for 24 hours. Upon storage of lots of spinach and asparagus for 1 week at Oo4.4"C. (22"40°F'.) ascorbic acid losses of 35 and 57%, thiamine losses of 15 and lS%, riboflavin losses of 17 and 27%, and carotene losses of 5 and 14%, respectively, were found. Although the conditions here are not comparable with the practices employed in the handling of these products for canning, this work emphasizes the importance of proper handling and storage of these products prior to canning. The following statement was made (Adam, 1941) in respect to this problem: ". . . Various workers have emphasized the importance of avoiding delay between picking the crop and delivery to the canner; significant losses of vitamin C occur a t this stage, particularly in leafy vegetables. . When ascorbic acid oxidase is present and vegetable tissues have been bruised, abraded or cut, losses may be considerable if this period is prolonged." Consideration of the problem of holding raw products between harvesting and canning was given (Lamb, 1946b) with regard to the effects of various holding conditions on the vitamin content of asparagus] green beans, and spinach. Asparagus held at room temperature 20.6"-23.3"C. (69"-74'F.) or in an incubator 27.8'C. (82°F.) showed a rapid loss of ascorbic acid during the first few hours of holding, with a lowered rate of loss upon longer holding. A loss of approximately 50% of ascorbic acid occurred in the first 24 hours at room temperature with the product losing 75% of its ascorbic content upon further storage to 70 hours. Refrigerator storage at 4.4"C. (40'F.) showed a loss of about 50% of the original ascorbic acid content after 70 hours, while this same degree of loss took place in 24 hours at room temperature. Under the latter conditions, green beans and spinach lost 50% and 22% of their ascorbic acid content, respectively. Definite advantages have been reported (Platenius and Jones, 1944) in the use of modified atmospheres in the storage of vegetables under room temperature and refrigerator conditions, particularly when the air was replaced with gaaes such as carbon dioxide and nitrogen to the extent of lowering the oxygen content to approximately 2% with a result that considerable additional preservation of ascorbic acid wm obtained. In consideration of the many factors entering into the final vitamin content of canned foods, the above information emphasizes the importance of further work in attempt to attain more information on the most desirable conditions for holding raw products prior to canning. The realization of this has resulted in the allocation of a project on this phase of the problem at Michigan State College. The vitamin content of canned foods is determined to a great extent by
.
66
L. E. CLIFCORN
the mechanical grading and cutting processes which many products undergo. For example, a given lot of peas is commonly passed through a quality grader for the purpose of separating the most tender peas from the tougher an& more mature peas by a gravity salt brine separation. It was shown (Clifcorn and Heberlein, 1944) that the thiamine content of extra standard or standard peas was significantly higher than that of fancy peas from the same lot. For example, for sieve size No. 3 Alaska peas, the fancy (floaters) peas contained 233 pg./lOO g. thiamine, while the standard (sinkers) peas contained 301 pg./lOO g. In reference to asparagus it was shown that with the edible stalk containing 157 pg./lOO g. thiamine, center cuts contained only 99 pg./lOO g. thiamine, These were packed and labelled as asparagus-whole spears, and asparagus-center cuts, respectively. With regard to Lima beans, the raw product representing a standard pack (90% or more white beans) contained 325 pg./lOO g. thiamine as compared with the extra standard pack (50% or less white beans, the remainder green beans) containing 267 pg./lOO g. thiamine. More extensive work on the concentration of nutrients in the aaparagus stalk by (Lamb, 1946b) showed that the tip cut green asparagus contained 47.2 mg./100 g. ascorbic acid, .323 mg./100 g. thiamine, and 2.31 mg./100 g. niacin as compared with the second cut of the stalk containing 24.3, .113, -94 mg./100 g. respectively, of these nutrients. In this same study various sieve sizes of green beans selected for canning were also found to affect the vitamin content to a lesser degree than the grades of peas and the cuts of asparagus previously mentioned. In consideration of the changes occurring in raw products during their mechanical preparation for canning by grading, cutting, cleaning, washing, etc., canners have become well aware of the fact that when vegetables or fruit tissues are cut, crushed, or damaged in any way, the product thereafter must be handled rapidly and under the best influences possible in order to insure quality in the final canned product and high nutritive content. b. The Efect of Blanching. It was Nicholas Appert (Magoon and Culpepper, 1924), who laid the foundation of modern canning methods and first practiced blanching or scalding in the preparation of certain vegetables for bottling. Asparagus, artichokes, cauliflower, spinach, and chicory were given this preliminary treatment, the object being, as expressed in connection with his discussion on asparagus, “to remove the acridity peculiar to this vegetable.” Experience in the canning of certain food products has indicated that the blanching of freshly prepared raw products in hot or boiling water, or exposing them to the action of live steam for a short time has distinct advantages. In some instances these advantages have been listed although the industry as a whole does not appear to be in good
FACTORS INFLUENCING VITAMIN CONTENT OF CANNED FOOD8
67
agreement concerning them. It may be said, however, that, in general, the canning industry employs the process of blanching or scalding for two reasons: (1) as it affects the appearance and flavor of the final product; and (2) to wilt and/or expel the air from the product in order to insure a more uniform container fill, and to reduce internal can pressures during the heat sterilization. Two other factors are often considered, namely, the elimination of certain bacteria and the destruction of enzymes, but in the main these are not regarded as the primary objectives of the operation. The blanching schedules as followed by the industry in general do not seem to be based upon any fundamental principle. The blanching times may vary from three quarters of a minute in the case of tender snap beans to 12 or 15 minutes for standard peas, and in some instances even longer. The temperature, depending upon the product, may also vary from 76.7"-1OO0C. (170"-212°F.) and in isolated cases may be as low as 54.4"C. (130'F.). Even in the preparation of the same product there are great variations among canners, the time and temperature of the blanch being, in most cases, dependent entirely upon the personal opinion of those in charge of the operation. Thus, generally speaking, in the canning industry the blanching procedures have no scientific background upon which the schedule of time and temperature is based. Hot water blanching is almost universally employed in the preparation of vegetables for canning. The water-soluble vitamins, ascorbic acid, thiamine, riboflavin, and niacin, may be easily extracted from vegetables during hot water blanching, the degree of extraction depending upon the nature and maturity of the product, the amount of the sub-surface of the vegetable exposed through cutting and bruising, and the time and temperature of the blanch. Steam blanching does not in most instances extract significant amounts of the water-soluble vitamins from vegetables although some oxidative destruction of ascorbic acid is believed to take place at the beginning of the blanching period. Owing to the relatively low water solubility of carotene only negligible losses are generally believed to occur in the blanching operation. This fact was substantiated by work (Zimmerman et al., 1941) which showed no significant decreases in the carotene content of asparagus, Lima beans, spinach, and broccoli upon blanching. Insignificant losses of carotene during the blanching procedure have been reported also in other work (Zimmerman et al., 1940; Fitzgerald and Fellers, 1938; Richardson et al., 1937; De Felice and Fellers, 1938; and Stimson et al., 1939). Extensive surveys on the retention of carotene in carrots after various steps in canning (Lamb, 194613) have revealed no loss during steaming. Surveys of commercial canning operations for the products peas and spinach were carried out with consideration to carotene retention (Guerrant et al.. 1947)
68
L. Io. CLIFCORN
with the result that, in some instances, when data wem expressed on the basis of the blanched material or on the dry matter basis, there appeared to have been an actual increase in carotene content where water blanching was employed. When peas were subjected to steam blanching there were small but measurable losses of carotene which were apparent only when the vitamin retention values were calculated on the basis of alcoholinsoluble solids or crude fiber. The steam blanching of spinach was found to have no detectable effect on the carotene content. Carotene retention by carrots during canning was reported to be more than 80% of the original carotene content on a dry weight bmis (Guerrant et al., 1946). Similarly, excellent retentions of carotene were found in asparagus, cherries, spinach, and tomatoes, good retentions in carrots, corn, peas, and tomato juice, and fair in snap beans. It can be stated that there is an appreciable amount of information on the effect of blanching operations on ascorbic acid in vegetables. Data reported in the literature have placed the emphasis on the total loss of ascorbic acid occurring during blanching. No effort has been made to ditrerentiate losses caused by oxidation during heating, from solubility losses due to the extraction. In most instances, the solubility loss is so high that the oxidative destruction effect for a given product under certain blanching conditions is de-emphasized. However, work to differentiate between these two types of losses occurring during blanching would be very helpful. The effects of blanching in boiling water for 1, 3, and 6 minutes or in steam for 3 minutes, have been studied (Adam et al., 1942) using three varieties of fresh peas, three varieties of green beans, Lima beans, whole carrots, sliced carrots, diced carrots, quartered parsnips, two varieties of potatoes, diced swedes, Brussels sprouts, soaked beans, and soaked peas. The results of these studies are best summed up in the words of the authors: “The effects of blanching a representative range of English vegetables for one, three, and six minutes in water and for three minutes in steam have been recorded, the factors studied being the retention of the chief nutritive substance and principal physical changes. Small units of large surface area retain 65 to 81 per cent of their sugars, 70 to 83 per cent of their mineral substances, 78 to 86 per cent of their protein, and 50 to 68 per cent of their vitamin C; the large roota and starchy seeds retain 79 to 90 per cent of their sugars, 84 to 92 per cent of their mineral substances, 92 to 98 per cent of their protein, and 07 to 78 per cent of their vitamin C. All desirable effects are produced in the first two minutes of blanching. Blanching is considered t o be necessary but the time should be as short as possible.” Investigations were carried out (Jenkins et al., 1938) on the effect of time and type of blanch on the ascorbic acid content of peas and the de-
FACTORS INFLUENCINQ VITAMIN CONTENT OF CANNED FOODS
69
struction of the enzyme, catalaae. Table XI summarizes their investigation in which it will be observed that a loss of approximately 10-25% of the original vitamin C content occurred in the first minute of the blanch regardless of whether the blanch was in steam or hot water. The rate of loss of vitamin C decreaaed materially after the first minute. In all the different blanching methods studied the enzyme, catalase, waa destroyed TABLO XI
The Effed of Various Blunching Operations on the Asmbic Acid
Content and
Catcr2ase Activity of Peaso
Content of Ascorbic Acid
Basis Treatment Steam blancher
Scott blancher6
Scott blancher*
Scott blancherb
Berlin-Chapman Blancher
a
b
Jenkins st al. (1938). Water blanching.
Time
Temp.
Fresh
Dry
mc.
-
"C.
-
mg./g. .%4
60 80 100 120 180
100 100 100 100 100
.18 .17 .10 .10 .l0
mg./g. 1.13 .78 .74 .77 .74 .76
-
-
60 130 166
82 82 82
.25 .21 .21 .l8
1.10
-
-
60 90 122 164
88 88 88 88
.26 .20 .20 .17 .l6
1.04 .88 .90 .76 .73
-
-
60 128 163
93 93 93
.26 .21 .17 .l0
1.10 .95 .78 .73
.25 .18 .17
1.04 .78 .77
-
-
40 60
1W 100
.m
.90 .79
Preaence of Catalaee
neg. neg.
neg. neg. neg.
trace w3.
neg.
trace neg. neg. mg-
neg. *g.
neg.
neg.
neg.
70
L. E. CLIFCORN
in 40-130 seconds. In the cooking of peas, which is related somewhat to blanching, it was found that approximately 50% of the vitamin C in peas passed into the cooking water (Fenton et al., 1936) and that most of the destruction of the vitamin took place during the first 2 minutes of cooking. This fact was substantiated by later work of the same authors (Fenton et al., 1938) which indicated that after the enzymes had been inactivated, the destruction of vitamin C was reduced to a minimum although its loss to the cooking water continued. Other work on peas (Olliver, 1937-38; Rostovskaya, 1941) has shown losses in the range of 3540% for vitamin C during hot water blanching. A report of 26 surveys representing work at five canneries on the vitamin retention during the canning of peas has been made recently (Lamb, 1946b). With regard t o ascorbic acid, it was found that retentions ranged from 66-88'% and averaged 77% after blanching. Blanching times varied from 2v2-8 minutes and blanching temperatures from 82.2"-97.8"C. (180°-2080F.). The highest retention was observed for ungraded peas, sieve size No. 3 -6 blanched 4 minutes at 873°C. (190"F.), while the lowest retention was observed for sieve size No. 2 fancy (floaters) peas, blanched 8 minutes at 82.2"C. (180°F.). A number of extensive surveys of pea canning operations were made in Wisconsin in seven different canneries (Wagner et al., 1947a). From results based on fifteen surveys, there was a range of retention of ascorbic acid during blanching of from 63-85y0 with an average of 7101,. In this instance the low value was obtained on ungraded peas blanched 6 minutes at 93.3OC. (200"F.),while the high value was obtained on sieve she No. 5 sweet peas blanched 6 minutes at 87.8O-93.3"C. (19O0-20OoF.), The following year, this group of workers focused their attention more specifically on the variables in the commercial blanching operation as affecting the vitamin content of vegetables. Experimental blanches employing 2% and 8 minute times at 76.7"-82.2"C. (170"-180°F.) and at 93.3"C. (200OF.) were studied on m e r e n t sieve sizes of peas (Wagner et al., 1947b). In Table X I , it will be observed that more mature peas tended to retain a greater per cent of the vitamin than more tender peas, and that the differences caused by the temperature of the blanch were a h o s t insignificant as compared to the differences caused by the increased time from 2% to 8 minutes. In this work, studies also were carried out to determine whether peas blanched before quality grading have a different vitamin content than those blanched after quality grading. It was found on the basis of ascorbic acid, thiamine, and niacin that the differences in vitamin content between peas blanched before quality grading and those blanched after quality grading were very small and probably were not significant. Studies on the effects of the variables, time and temperature of blanch,
71
FACTORS INFLUENCING VITAMIN CONTENT OF CANNED FOODS
using both steam and hot water blanches have been completed recently (Guerrant et al., 1947) on a number of products and the results have been expressed on the basis of the original sample, dry matter, alcohol-insoluble solids, and crude fiber. Hot water blanches were carried out on peas for 3, 6, and 9 minutes at temperatures 82.2", 87.8", and 93.3"C. (180", 190", and 200°F.) and with steam for 1, 2, and 3 minutes a t 98.9"C.(210°F.). TABLEXI1 Sieve Size and Blanching Conditions as Afecting the Retention of Vitamins i n PeaBa
Blanch
Raw product
Sieve size
1, 2, 3 4
5, 6
Ascorbic acid rng./100 g. dry wt.
Thiamine mg./100 g. dry wt.
Niacin mg./100 g. dry wt.
136 103 87
1.94 1.85 1.73
10.80 10.80 11.45
% Retained yo Retained 2.5'@ 76.7"-82.2"C. (170"180"F.)
% Retained
86 86 100
93 85 95
91 103 100
89 99 98
86
8'@76.7"-82.2"C. (17O0-l80"F.)
66 68 76
66 80 70
67 63 64
8'@93.3"C. (200'F.)
64 73 76
72 79 79
72 72
2.5'@93.3"C. (200'F.)
5
84
85 98 78 87
64
Wagner et al. (1947b).
In general, it was observed that the increase in blanching time affected the ascorbic acid content more adversely than did the increase in blanching temperature. The retention of ascorbic acid by peas, water blanched for 1 minute at 82.2"C. (180°F.) was approximately 75% as compared to 40% when blanched for 12 minutes at 93.3"C. (200°F.). Under the most severe conditions of steam blanching, peas retained approximately 75% of their original ascorbic acid content.
72
L. 1. CLIFCORN
It was felt desirable to conduct some tests on the effect of blanching successive lots of peas in the same blanching water so that comparisons could be made between laboratory tests and commercial surveys. The above workers (Guerrant el al., 1947) conducted such a test, blanching successive lots of peas for 6 minutes in the same blanching water at 87.8"C. (190'F.) and attempting to account for ascorbic acid losses. Five batches of peas were blanched and ascorbic acid retentions ranged from 57-72%, the slightly higher retentions occurring in the fourth and fifth batches. Although the ascorbic acid content of the blanching water increased with successive batches of the peas, the percentage of the extracted vitamin present in the blanchidg water decreased. The authors concluded that the data obtained did not indicate any advantage for the practice of blanching successive batches of peas in the same water insofar as increasing ascorbic acid retention was concerned. With regard to the effect of commercial blanching operations upon the ascorbic acid content of green beans, the resu€ts of surveys (Wagner et al., 1947a)in four Wisconsin canneries showed retentions of ascorbic acid during blanching of 4443% with an average of 64%. The lowest retention during blanching was observed on No. 4 sieve size green cut beans blanched in 434 minu- at 81.1'C. (178"F.),while the highest retention was observed on No. 4 sieve size green cut beans blanched 4% minutes at 82.2O-87.8"C. (180'-190°F.). For purposes of later correlation, it is interesting to note that all of the blanches in the cannery studies were conducted at 81.1'87.8'C. (178"-190"F.). Similar surveys on the effect of commercialblanching operations on the ascorbic acid content of green beans were carried out in the Pacific Coast area (Lamb, 1946b) including 26 surveys in five canning plants with results showinga range of 48-96% retention of ascorbic acid and an average of 74%. The range of blanching times varied from 1%3% minutes, while the temperature of the blanching water varied from 71.lo-98.9"C. (16Oo-21O0F.). The low value of 48% retention of ascorbic acid was obtained on uncut No. 2 and No. 3 sieve size green beans blanched 1% minutes at 873°C. (190°F.), while the highest retention, 96%, was obtained on sieve size No. 5 cut green beans blanched 1% minutes at 87.2'C. (189°F.). It is interesting to note that there were 4 blanches carried out on No. 3 and No. 4 sieve size cut green beans of 2% minuteg at 98.9"C. (210°F.) with retentions of 85, 86, 81, and 84%. In varying the time and temperature of commercial blanching of green beans, it waa found (Wagner et d.,1947b) that a 2-minute blanch at 98.9"C. (210'F.) for both whole and cut green beans of sieve size No. 3 and 4, respectively, gave ascorbic acid retentions of 77 and 69%, respectively. This waa greatly superior to retentione obtained by blanching 2 minutes at 71.1'C. (160°F.), 6 minutes at 71.1"C. (16O"F.), or 989°C. (21OOF.).
FACIYIRS INFLUENCINa VITAMIN CONTENT OF CANNED FOODB
73
These authors have commented rm follows with regwd to these findings: "Since the more drastic blanching conditions resulted in a better retention of ascorbic acid than waa obtained in any sample blanched at 160°F., it appears that the lower temperature favors oxidation losses. Losses due to extraction would be greater at the higher temperature. It may be that enqmatic activity is not completely destroyed at 160°F." Variations in time and temperature of blanching for whole No. 2 sieve size green beans and cut No. 5 sieve size green beans were studied (Guerrant et d.,1947) using hot water and steam blanches. Retention of ascorbic acid during hot water blanching of this product ranged from 38-9201,. The lowest ascorbic acid value was obtained for whole green beans which were water blanched for 1 minute at 71.1"C. (160°F.), while the highest value was found after blanching 1minute at 93.3"C. (200°F.). A similar trend in favor of the high temperature-short time blanch was found for cut green beans. The unexpected findings of several investigators (Wagner el al., 1947b; Guerrant el al., 1947; Lamb, 194613) clearly show the advantages of the high temperature-short time blanch for green beans. A comparative study of steam and hot water blanching of green beans has been made (Melnick et al., 1944), and it was found that hot water blanching was more effective in destroying the natural enzymes in this product than steam blanching. A 3-minute hot water blanch at 95°C. (203°F.) was completely effective in the destroying of natural enzymes as compared with a 5-minute steam blanch necessary for a complete inactivation. A 3-minute hot water blanch retained 92% of the ascorbic acid content, while a 3-minute steam blanch retained all of the ascorbic acid in this product. Longer periods of blanching showed rather insignificant further losses for the steam blanch and an additional approximate 10% lose for the hot water blanch. The authors state that the steam blanching method for green beans is to be preferred. Steam blanching experiments (Wagner et al., 1947b) using 4- and 10-minute timea showed only 57-61010 retention of ascorbic acid. A comment waa added that the canned samples reaulting from these blanches were too soft, dark, and poor in flavor to qualify for a high quality pack of green beans. It was suggested that shorter steam blanching times should be tried. Retentions of ascorbic acid in steam blanched beans (Guerrant et al., 1947) ranged from 76-930/, for steam blanches of from 1-5 minutes. With particular reference to studies on the steam blanches for the products, peas and green beans, the effects of such operations upon the flavor and texture of the h a 1 canned product must be considered to be as important or perhaps more so than the effect upon vitamin retention. The most comprehensive surveys available to date on commercial spinach canning operations have been reported (Lamb 1946a; 1946b). Surveys
TABLEXI11 Retartions of Aswrbie Acid, Thiamine, Rz%o$m.n, and Niacin after Blanching (Calculated on the dry solids)a Ascorbicacid Cannery 1
(1)
Type of blancher Steam
Time,
Temp.,
min.
OF.
2%
205
I
Thiamine
I
Niacin
Riboflavin
Average, Actual, Averago,
%
%
%
97
103
102
72
104
95
% % ~-
93
(2)
Draper
2%
188 73 205
96
4%
-166-185
98
3%
186-204
2A
Steam
2
2B
Draper
3
Draper
-I
64
lo7>
57
91
105
109
91
4
(1) (2)
Drum washer Rotary
2% 2%
104
140
109 71
82
206 54
NOTE; The small letters
g31 I 86
~~~
73
(a) and (b) represent individual duplicate runs made on the same blancher.
69
TABLE XI11 (Continued)
1 1%1% Amor:
Type of blancher
Cannery
ROW Immersion Immersion Draper
Time, min.,
Temp.,
1 12 45
160 160 160
OF.
Actual, Averagt
%
89
93
91
200
85
Steam Water wash
991 71
1
- I
I I-98
___
-.
120-130
0
100
*1
99
(2) (3)
42
Rotary Immersion
46
68
67
Draper
59
99
76 55
63
9 -54
-I 10
82
85
93
%
"1 84
~-~
175
~
%
-88
170 192
3 4%
Drum washer
Pverage
%
--
5
I
Niacin
acid
80
")
83
80
TABLEXIII (Continued) Ascorbic acid Cannery
Type of blancher
Time, min.
Temp.,
OF.
I
Niacin
Thir
Actual,
%
Actual, Average.,
%
% 9i -
%
-1-
11
(1)
Draper
3%
185-186
74
70
(2)
Immersion spray
5
160-163 78 73
-
95
"1
98
71} 78
89
97
81
78
~ 69
14
I I
-
=)i 72
I)"
73
173
so/g71
69
FACTORS INFLUENCINQ VITAMM CONTENT OF CANNED FOODS
77
carried out in fifteen canning plants revealed the effects of steam blanching, steam blanching followed by passage through a Draper-type water blancher (in which the spinach moves in such a thick mass that blanch water cannot circulate freely), passage through a rotary-type hot water blancher, immersion in hot water, basket blancher, and blanching in a drum washertype blancher. The results of this survey are shown in Table XIII. The operation at cannery No. 1 consisted of a steam blancher followed by a water blancher. It may be observed that this method of steam blanching showed a much better retention of the vitamins studied than the accompanying water blancher. The next best retention was obtained by the Draper-type blancher when the total blanching period did not exceed 5 minutes. Rotary blanchers proved to be definitely inferior to the Draper blanchers even though comparable times and temperatures of blanch were used. Blanches in excess of 5 minutes were observed to be destructive to vitamins, particularly to ascorbic acid, although here again, the type of blancher appears to have an important role. The author states, in this regard, that considerably more ascorbic acid was lost in the blancher in cannery No. 11 with a total blanching time of 8X minutes than was lost during 13% minutes in the blancher in cannery No. 13 or even the first 12 minutes of blanching in the blancher in cannery No. 5. In a series of laboratory scale tests on the blanching of spinach, it waa observed (Cuerrant et al., 1947) that on the dry weight basis, spinach blanched 7 minutes at 93.3"C. (200OF.) retained only 14% ascorbic acid, while the best retention was 64% for a l-minute blanch at 76.7"C. (170°F.). Steam blanching tests were also conducted, and it was found that the best ascorbic acid retention was 76% for 1% minute blanch at 98.9"C. (210°F.). The results observed in this test correlate very well with those previously discussed (Lamb, 1946b). The effects of water and steam blanching of a number of products, kale, beets, potatoes, cabbage, and carrots, were investigated (von Loesecke, 1942) with regard to their ascorbic acid retention. The results are shown in Table XIV. In studies on the effect of commercial blanching on other vegetables, it was observed (Wagner et d.,1947a) that cut green asparagus retained 89% of its original ascorbic acid after either a 6-minute blanch at 87.8"C. (NOOF.), or a 4-lhinute blanch at 65.6"C. (15OOF.). Following a 1?4minute blanch at 75°C. (167"F.), the retention was increased to 94% for the same batch of asparagus. In a study involving variation of the time of water"blanching and comparing this with steam blanching, ascorbic wid retentions of green whole spear asparagus were observed (Lamb, 1946b) and the results reported on the basis of dry solids and on insoluble solids, the latter recommended M being more nearly correct. On this baais asparagus blanched in steam 2 minutea at 98.9°-100"C.
78
L. 1. CLIFCORN
(210°-2120F.) retained 96% ascorbic acid, while the water-blanched asparagus 2%, 5, 10, and 20 minutes a t 87.8"C. (190°F.) retained 95, 91, 83, and 72%, respectively, of the original ascorbic acid. In studies on the ascorbic acid retention of blanched Lima beans, it was reported (Wagner et al., 1947a) that ungraded, small, and medium Lima beans showed 72 and 62% retention, respectively, after blanching 10 minutes at 87.8"C. (190'F.). In a later report of two surveys on the effect of varying the time and temperature of the blanch (Wagner et al., 1947b), it was observed that the poorest retention for a blend of green and white Lima beans was 77% for a water blanch of about 8 minutes at 76.7"C. T a m XIV
ESed of Tgpe of Bbnch OR Asmbic Acid Retention0 Water blanch
I Steam blanch
% Retention Kale Beeta Potato08 Cabbage carrots 0
63 62 48 55
85 77 82 72
von h e c k e (1942).
(170"F.),while the best retention waB 98% for a 3yrminute water blanch at 873°C. (190°F.). The variations of time and temperature were 2%, 334, 4%, and approximately 8 minutes a t 76.7", 82.2', and 87.8"C. (170°, 180°, and 190°F.). A 5- and 8-minute blanch in steam resulted in 87 and 98% ascorbic acid retentions for the previously mentioned type of Lima bean pack. When only green Lima beans were used with the same time and temperature conditions (omitting the steam blanch) the poorest ascorbic acid retention was 63% when blanched for approximately 8 minutes at either 76.7" or 87.8"C. (170" or 190°F.). The best retention on the other hand was 83% when blanched for 3% minutes at 76.7"C. (170'F.). Other studies on the variations in time and temperature of blanching Lima beans and the resultant effect on ascorbic acid retention have been conducted (Guerrant et al., 1947). The blanching times employed were 2, 4, 6, and 8 minutes at hot water temperatures of 71.1", 82.2", and 93.3"C. (160°, 180", and 200°F.). Steam blanches (98.9"-1OO0C.) (210"212°F.) of 1, 3, and 5 minutes were also used. Ascorbic acid retentions reported on the dry solids basis for the water blanches indicate that the best retention was 94% for 2 minutes at 71.1"C. (160°F.), while the poorest
FACTORS INFLUENCING VITAMIN CONTENT OF CANNED FOOD8
79
retention of ascorbic acid wa;s 6875, obtained in 6 or 8 minutes at 82.2OC. (180°F.).The most severe condition, which was steam blanching for a 3-minute period, resulted in 88% retention although 1 minute indicated a 106% retention of the original ascorbic acid. Although the greatest amount of work has been carried out on the effect of blanching operations upon the ascorbic acid content of vegetables and the results so obtained used generally as an index of solubility losses during this operation, the retention of the various members of the B-complex TABLB XV The Eflect of Blanching on the Thiamine Content of Vegetables0
Item Peas (Sweet) Peas (Almka) Peas (Almka) Pea8 (Alaska) Peas (Alaska) Peas (Alaska) Pea8 (Alaska) Peas (Alaska) Pea8 (Alaska)
- Ungraded
Grade
#1-Fancy #l-Standard #~-FRUCY #2Standard #3-Fancy #3-Standard #4
Blenching schedule
Blanching 10% %
8 min. 206°F. 0 min. 170"-190"F. 6 min. 170"-190"F. 0 min. 170°-190"F.
24 30.4 16.9 21.4 18.1 10.4
8 min. 17O0-190"F. 6 min. 170"-190"F. 0 min. 170"-190"F. 7 min. 170"-190"F. 7 min. 170°-190"F.
None None
11.1
Green beans (whole) Green beans (cut)
#3
R4
0 min. 170"-180"F. 6 min. 180°F.
8.1 16.9
Lima beans Lima bema
Extra standard Standard
12 mh. 208°F. 16 min. 208°F.
38
1% min. 170°F. 1%min. 170°F.
None None
Asparagus (cut) Asparagus (whole spear)
31.9
group of vitamins during blanching has been studied in parallel with many of the investigations previously discussed. From the early literature it was apparent that thiamine was the member of the group that waa most adversely affected by blanching operatlons. It has been observed (Fincke, 1939) that, in general, the trend of the thiamine content of blanched vegetables is toward lower values with increased temperature and longer blanching times. Additional information on the effects of blanching on the thiamine content of peas, asparagus, Lima beans, and green beans have been reported (Clifcorn and Heberlein, 1944). These data are briefly summarised in Table XV.
80
L. E. CLIFCORN
In a series of surveys at seven different Wisconsin pea canneries (Wagner 1947a), thiamine retentions ranged from 79-96% and averaged 88%. The best retention was reported for ungraded sweet peas blanched 6 minutes at 93.3"C. (200"F.), while the poorest retention was found in No. 4 sieve sine sweet peas blanched 6-7 minutes at 87.8O-93.3"C. (190"-200"F.). Similarly, riboflavin retentions ranged from 6349% with an average of 75%. The best riboflavin retentions were obtained with No. 4 sieve size Alaska peas blanched 44/25 minutes at 87.8"-90.6"C. (190'-195'F.) , whereas, the poorest retention was obtained with No. 5 sieve si5e sweet peas blanched 6 or 7 minutes at 87.8"-93.3"C. (190"-200°F.). It is interesting to note that the poorest B-complex retentions were obtained in the same cannery although they did not occur in the same sieve size of peas. In a report of 24 surveys obtained in five different Pacific Coast canneries (Lamb, 1946b),the retention of thiamine for the blanched peas calculated on the dry solids basis ranged from 70-108% and averaged 90%. In 26 surveys in the same canneries, riboflavin retentions for blanched peas were observed to be 61-89% with an average of 76%. Similarly, niacin retentions were 50-94% with an,average of 71%. In connection with the studies of the effect on vitamin retention when the time and temperature of the blanching operation were varied (Wagner et al., 1947a) the retentions for thiamine and niacin which were obtained are found in Table XII. It will be observed that thiamine and niacin retentions in the blanched peas were somewhat higher in the mature peas than in the immature peas, particularly, for the 234-1ninute blanch at 76.7'432.2" or 93.3"C. (170°-180QF.or 200°F.). When the blanching time was increased to 8 minutes, however, the retentions of both thiamine and niacin were somewhat decreaaed with the difference between mature and immature peas also decreasing. In some instances, the dserences in retention between mature and immature peas were actually reversed during the longer periods of blanch with the mature peas retaining slightly lese thiamine and niacin in many instances than was retained in the immature peas. The reasons for this phenomena were not apparent from the data at hand. With further consideration to the work on the variations of times and temperatures of blanching for green beans (Wagner et d.,1947b), it waa observed that with the exception of ascorbic acid, the vitamin retentiom were exceptionally satisfactory, ranging from 9l-l00% for thiamine and 8!3-100% for niacin. The loss of ascorbic acid taking place during similar blanching with results of the order of 41-77% retention indicated, in comparison with the results for thiamine and niacin, that there was either a greater extraction loss of ascorbic acid during blanching or a signifioant oxidation loss, or both. In this particular instance, it would be serioue et al.,
FACTORS INFLUENCING VITAMIN CONTENT OF CANNED FOODS
81
to use ascorbic acid as the index of solubility losses. Work on Lima beans by the same investigators showed retentions of 5547% thiamine and 76-100% niacin retention with variations of time and temperature such as are commercially employed. In surveys of commercial canning operations, the results of many blanching tests have been reported (Wagner et al., 1947a) for asparagus, green beans, and Lima beans, with findings directly parallel to those previously discussed. Retentions of riboflavin also were observed of about the same order as for niacin. With specific reference to asparagus, excellent retention of all the water-soluble vitamins (86-100%) was obtained, which may be explained by the favorable surface area to mass ratio for this product. Others have reported (Lamb, 194613) upon the favorable retention of the water-soluble vitamins during the blanching of asparagus and the lack of necessity for the further improvement of this operation for this product. Under conditions of variations of time and temperature of blanching of many vegetables, it was found (Guerrant et al., 1947) that the most severe conditions of blanching spinach and Lima beans resulted in only a 43 and 27% retention, respectively, of their riboflavin content, whereas green beans showed good retention of this vitamin following water blanching. The loss of riboflavin by vegetables, in general, was insignificant as a result of steam blanching. Spinach and Lima beans were found to retain considerabIy less thiamine following water blanching as the temperature and the duration of the blanch were increased. Under the most adverse conditions, thiamine retentions of only 55 and 25%, respectively, of their original thiamine content were observed. Relatively small losses of thiamine resulted from steam blanching. Only 60% of the original niacin content of Lima beans was retained when water blanched for 8 minutes at 93.3OC. (200°F.). In a study of the effect of blanching on green beans (Lamb, 1946b), excellent retentions of niacin and riboflavin (97%) were found as well as a 94% retention of thiamine. In Table XIII, thiamine, riboflavin, and niacin retentions during the blanching of spinach are included with those for ascorbic acid. The results show a 56100% retention of thiamine, 63-100y0 of riboflavin, and 59-100% of niacin for the commercial blanching studies. In all instances, by comparison, the ascorbic acid losses were more severe than were those for the B vitamins. Their work seems to indicate that the riboflavin and niacin are bound in a somewhat more insoluble form than is thiamine, with the ascorbic acid being the most readily affected of all of the vitamins. It has been reported (Moyer and Stotz, 1945) that negligible losses of ascorbic acid from cabbage occurred when it was electronibally blanched in contrast to losses of 30-40% during steam or water blanching. Electronic blanching of vegetables for canning has been investigated further
82
L. E. CLIFCORN
by these workers, and results will be published soon. In general, for products such as peas and green beans, bitter flavor principles affected the final quality of the canned product. These are normally removed during hot water blanching. 6. Fruits
With regard to the preparatory methods used by canners for fruits, it has been observed that it is very difficult to retain ascorbic acid during the canning of apples, apple sauce, or apple juice. This appears to be due to the high ascorbic acid oxidase activity of apples. It was observed (Kohman et al., 1924) that soaking apples in a 2y0 salt solution for several hours signifkantly preserved the vitamin C content during subsequent canning of apple sauce. It was stated that the protection was due to: (1) the depletion of oxygen content of apples during the soaking period; and (2) the inhibiting action of the salt solution on the ascorbic acid oxidase. Work on the holding of peeled, clingstone peaches (Lamb, 1946b) has shown that exposure of the peeled halves to air for 30, 60, and 120 minutes resulted in losses of ascorbic acid of 29, 34, and 45y0, respectively; the halves held in water for these periods of time lost 30-31%. Peach slices exposed to air and held for similar periods of time lost 42, 52, and 58% of their ascorbic acid, respectively. Recent information on the vitamin content of fruits, as affected by canning, is codned mainly to ascorbic acid studies. This is justified since, generalIy, the fruits are considered to be valuable sources of ascorbic acid, and low in carotene and the vitamin B factors. There are exceptions to this, of course, such as carotene in peaches and thiamine in oranges and pineapple. In a report covering four surveys of red, sour, pitted cherry canning operations (Guerrant et al., 1946) a 96y0 retention of ascorbic acid was found on the dry weight basis together with complete retention of carotene. In a study on canned pineapple (Haagen Smit et al., 1946), the carotene and vitaminA,a thiamine (vitamin BJ, riboflavin (vitamin Ba), vitamin C, pantothenic acid, and p-aminobenzoic acid contents of five types of commercially canned pineapple are given for winter and summer packs. Other than stating that the packs were made by different canning methods, which were not described, the authors do not attempt to account for any differences due to the canning procedures. The only conclusions one can draw from this work are that the vitamin A (and carotene), ascorbic acid, and pantothenic acid contents of the winter-packed pineapple are higher than that of the summer pack, while with thiamine and riboflavin, there are no significant seasonal differences. The ascorbic acid retentions of canned apricots (Lamb, 1946b) varied
* Carotene determined by chemical atmy; vitamin A activity by animal m y .
FACTORS INFLUENCING
83
VITAMIN CONTENT OF CANNED FOODS
from 76-97% (dry weight basis) with an average of 85% for four stages of maturity in two varieties of apricots, and carotene retentions on the same product varied from 78--98% with an average of 89% retention of the original carotene content. The vitamin content of the riper fruit was consistently higher. The same worker (Lamb, 1946b) reported ascorbic acid, thiamine, niacin, and carotene retentions in canned clingstone and Elberta peaches. The results of a number of surveys in several different canneries are shown in Table XVI. In the same plants the lye peeling of clingstone peaches and TABLE XVI Vitamin Retention in Peaches as Affected bg Canning. Clingstone peaches
No. surveys
% Retention
1
Elberta peaches No. surveys
% Retention
~~
Ascorbic acid Range Average Thiamin2 Range Average
Niacin Range Average Carotene Range Average
14
3 63-90 71
59-70 65 3
6
71-93 85
60-79 69 3
5
82-86
87-92 89
84
3
4
59-88 77
88-118 102
Lamb (1946b).
steam peeling of freestone peaches and the effect of these operations on the ascorbic acid in the fruit was determined. Using four varieties of clingstone peaches, losses of ascorbic acid, solids, and weight of peaches during lye peeling were determined with the result that, as the peeling time was increased for a given strength of lye, the losses of ascorbic acid increased. Considering the variation in strength of lye solution, the authors indicated that (I.. . the loss was more nearly proportional to the degree of peeling than to the actual time in the lye bath.” Surveys on freestone peaches, peeled by steam, indicated an unexpected loss of ascorbic acid, retentions on the dry basis ranging from 72-86y0 after steam peeling with an average retention of 77%.
84
L. E. CLIFCORN
6. Effectof Sterilization The heat-labile properties of thiamine have presented a problem with regard to the maximum retention of this vitamin during the sterilization of canned foods by heat. Earlier work has been confirmed (Beadle et al., 19431 showing thiamine to be more stable in solutions of low pH than in neutral or alkaline solutions and also emphasizing the importance of the specific effect of certain buffer salts. It was demonstrated (Greenwood et al., 1943) that cocarboxylase is only slightly more' resistant to moist heat than thiamine itself. It has been concluded (Rice and Beuk, 1945) that the natural occurrence of thiamine as a part of the cocarboxylase molecule does not seem sufficient to explain the superior retention of this vitamin in foods as compared with aqueous solutions. With reference to the variables, time and temperature, as affecting the thiamine content of foods during cooking or sterilization in cans, the thermal destruction of thiamine in pure solutions apparently proceeds according to a.first-order reaction (Watanabe, 1939), but it was not determined whether this holds for food materials. The effect of processing temperatures of from 98.9"-121.loC. (210°-2500F.), upon the thiamine content of pork luncheon meat has been reported (Greenwood et al.,1944) and it was possible to interpret their data upon the basis of a first-order reaction. Other workers (Rice and Beuk, 1945) concluded from their data that the loss of thiamine from pork a t temperatures above 77°C. (171°F.) proceeded at a constant rate throughout the entire heating interval, and that a t these temperatures the reaction seems to be monomolecular and is probably hydrolytic in nature. At temperatures below 77°C. (170.6"F.) the reaction rate (loss) proceeded faster during the first 16-24 hours, perhaps due to some catalytic effect, than it did at later periods when it fell into the constant rate monomolecular class. In studies on the effects of commercial sterilization processes upon the thiamine content of vegetables (Clifcorn and Heberlein, 1944), it was shown that thermal processing destroyed significant amounts of this vitamin. In Table XVII these results may be observed, from which it is readily apparent that a process of 45 minutes at 118.3OC. (245°F.) for peas is more destructive to thiamine than one of 30 minutes a t 115.6"C. (240°F.). The more mature (standard) Lima beans lost less thiamine (29%) upon processing 30 minutes a t 115.6"C. (240°F.) than did the less mature (extra standard) beans (42%). The high degree of retention of thiamine observed in tomatoes and tomato juice was explained by the low pH (which is favorable to the stability of thiamine toward heat) and to the comparatively low sterilization temperature. The low retention of thiamine in whole kernel corn can be explained by the relatively severe process necessary for the
FACTORS INFLUENCINQ
VITAMIN CONTENT OF CANNED FOODS
85
sterilization of this product. In recent surveys of canning operations (Wagner et al., 1947a) attention was given to the effect of processing on the thiamine content of canned asparagus, peas, green beans, Lima beans, and whole kernel corn. It was found that the specific heat sterilization TABLEXVII Stability of Thiamine During Thermal Processinga of Several Vegetables Process
% Retention during
-
Product
Asparagus Whole spears Center cuts Fancy cut Corn, whole kernel White Yellow Green beans Whole, No. 3 sieve size Cut, No. 4 sieve size Lima beans Standard Extra standard Peas, sweet Unsieved, fancy Unsieved, fancy Unsieved, standard Unsieved, standard Peas, Alaska Sieve lq fancy Sieve 1, extra standard Sieve 2,fancy Sieve 2, extra standard Sieve 3,fancy Sieve 3,standard Sieve 4 Sieve 5 Tomatoes Tomato juice 4
Can Size
sterilization process
Min.
OF.
14 14 14
248 248 248
300 300 300
66 63 64
30 30
250 250
2 2
31 47
20 20
240 240
2 2
79 73
30 30
240 240
2 2
71 58
35 45 35 45
240 245 240 245
2 2 2 2
67 60 67 67
35 35 35 35 35 35 35 35 35 20
240 240 240 240 240 240 240 240 212 212
2 2 2 2 2 2 2 2 2 2
64 63 61 64 59 67 64 69 89 74
Clifcorn and Heberlein (1944).
process lowered the thiamine content of asparagus 14-36%, peas 1249%, Lima beans 14-52%, green beans 8-29%, and whole kernel corn 64-66%. These results were almost entirely for the No. 2 can size; but in the cases of green beans and peas, it is interesting to note that the low values in
86
L. E. CLIFCORN
each instance were for No. 10 cans where a more prolonged thermal process is necessary. However, quite extensive data for such comparisons has been obtained (Ives el al., 1944), with the result that for a number of products and a number of samples for each, there was no consistent difference between the thiamine contents of the same products in the two can sizes. Ranges of sterilization losses of about the same order as given above were found by others (Guerrant et al., 1946)) and further, it was stated that blanching accounted for the greatest loss of ascorbic acid in peas, while heat processing was responsible for the greatest thiamine losses. Experiments were conducted (Lamb, 194613) on processing peas in No. 2 and No. 10 cans for various lengths of time a t 122.2"C. (252°F.) with results such as shown in Table XVIII. The effect of increased time of processTABLEXVIII Effect of Processing and Can Size on the Retention of Thinmine i n Canned Peas0 Thiamine Process Wetbasis Code
Can siae
Time, min.
Temp.,
18
252 252 252 252 252 252
"F.
I
Drybasis
mg./100 g.
Retention during processing (dry b-i~), %
_c_
1 2
3 4 5 6
No. 2 No. 2 No. 2 No. 10 No. 10 No. 10
23
30 18 23 30
0.086 0.078
0.071 0.086 0.084
0.078
0.55 0.50 0.45 0.55 0.54 0.50
66 60 54
66 64 60
Raw peas:
Thiamine 0.244 mg./100 g. (wet baais) 0.211 mg./lOO g. (wet basis) Blanched peaa: Thiamine 0.84 mg./100 g. (dry basis)
0
Lamb (1946b).
ing and slower rate of heat penetration in the No. 10 size can is readily apparent. In reviewing this data, it is important to bear in mind that processes of equal lethality for the No. 2 and No. 10 cans, respectively, are 15 and 23 minutes a t 121.1"C. (250°F.). The advantages of higher temperature-shorter time processes for the greater retention of thiamine in pork luncheon meat have been presented (Greenwood et al., 1944). The times a t which 10, 20, and 50Oj, thiamine destruction takes place a t the temperatures 98.9", 110", 118.3",and 126.7"C. (210", 230°, 245", and 260°F.) are shown in Fig. 4 together with a thermal
FACTORS INFLUENCING VITAMIN CONTENT OF CANNED FOODS
87
death time curve of spores of typical spoilage organisms in neutral phosphate medium. It is clearly demonstrated here that with a 10°C. (18°F.) increase in temperature the rate of thiamine destruction is approximately doubled, while the rate of destruction of organisms is increased tenfold. Therefore, to obtain the highest thiamine content in canned foods, it is most desirable to employ the process of the same bacteria killing power (F,) having the highest temperature. This has been confirmed by recent
TEMPERATURE - O F
Fig. 4. Comparative effects of time and temperature on destruction of thiamine and certain bacterial spores (Greenwood et al., 1944).
work (Feaster et al., 1947) comparing the thiamine content of high temperature-short time tubular sterilized cream style corn with that sterilized by the conventiona1 process. The results showed 95% retention of thiamine for the former process and 40% for the latter. With further reference to canned pork luncheon meat (Greenwood et al., 1944) the relationship of the processes as employed for the different can sizes with the percentage retention of thiamine, riboflavin, niacin, and pantothenic acid are shown in Table XIX. It is readily observed that the
88
L. E. CLIFCORN
TABLIIXIX Efeet
of Container Size on Vitamin Retention in Pork Luncheon
Can size
Thiamine
Riboflavin
Xiin
12-02.
73-76 46-60 38-49
86-95 71-81 67
89-97 71-81 73-76
Pantothenic Acid _ .
2X-lb. 61b.
b
62-73 62-68 70-73
% Retention of vitamins after processing. Greenwood et al. (1944).
more severe processes required for the larger can sizes affect the retention of thiamine greatly, and to a lesser degree that of riboflavin and niacin. Fig. 5 shows the distribution of these nutrients in a cross section of a 2s-pound can processed 148 minutes a t 112.8"C. (235°F.). Since the center of the can is the slowest to heat up to approximately retort tem-
Fig. 5. Influence of position in 2Yrlb. can on % B vitamin retantion during heat processing (Greenwoodel al., 1944). Cross-sectional sampling of a Z i c h cylinder of meat mid-distant from ends of a 2X-lb. can. Processed 148 minutea at 235°F.
FACTORS INFLUENCING VITAMIN CONTENT OF CANNED FOODS
89
perature, one would expect the destruction of vitamins here to be less than on the outside of the can. Losses of ascorbic acid taking place during the thermal processing of fruits and vegetables are usually directly proportional to the amount of oxygen entrapped in the can. In surveys of canning operations on the products, peas, asparagus, green beans, Lima beans, and whole kernel corn (Wagner et aZ., 1947a), the most serious losses of ascorbic acid occurred with green beans, i.e., 20 and 26% loss in two surveys specifically due to the sterilization operation. The other products exhibited little or no loss of ascorbic acid, thus warranting the general conclusion of the authors that ascorbic acid, riboflavin, and niacin are not seriously affected by the sterilization operation. Some of the various plant surveys conducted on the effect of the canning operations on the vitamin content of the product (Guerrant et al., 1946) were of the step-wise type. In the few surveys of this type green beans again suffered a significant loss (35%) of ascorbic acid as a result of sterilization, while this nutrilite was relatively unaffected in Lima beans, carrots, whole kernel corn, spinach, tomatoes, and tomato juice. Retentions of ascorbic acid as affected by the sterilization process with asparagus have been shown (Lamb, 194613) to be excellent with an average of 102% retention for green asparagus and an average of 96% ascorbic acid retention for white asparagus. These workers, reporting on the effect of sterilization on ascorbic acid in green beans, did not observe any significant Ioss of the vitamin during that step and concluded that ascorbic acid, riboflavin, and niacin were nearly completely retained in green beans during the sterilization, while thiamine was seriously affected. Studies of the canning operations for clingstone and freestone peaches have indicated (Lamb, 1946b) that the sterilization step caused little or no loss of ascorbic acid, niacin, and carotene, but again, thiamine retentions were poor. It should be noted that this fruit is a poor source of thiamine, and since the thiamine level was rather low, small changes in thiamine content affected the retentions significantly. Other steps in the canning operations, such as lye peeling and holding the peeled peaches before canning had a greater effect on the retentions of ascorbic acid and thiamine than did the sterilization operation.
IV. THEEFFECT OF STORAGE ON THE VITAMIN CONTENTOF CANNEDFOODB Since canned foods are generally packed during a relatively short season and the major consumption of such packs takes place during the period of a year, the effect of storage on the vitamin content of canned foods has been recognized to be an extremely important problem. The tem-
90
t. E. CLIFCORN
perature and length of time of storage are the variables affecting storage losses of vitamins. Although canned foods are generally less affected by adverse storage conditions than most other preserved food products, it is unreasonable t o expect that canning as a means of food preservation can stabilize quality and nutritive value against all conditions of storage. Canned foods can tolerate freezing temperatures, but under high storage temperature conditions the quality (acceptability as food) and the nutrient content are directly proportional to the length of storage. Acceptability is perhaps of greater importance than nutritive content, since as we previously stated, food must taste good to be eaten, and if not eaten, its nutritive value has no benefit. The National Canners Association-Can Manufacturers Institute Nutrition Program has sponsored projects at various universities t o obtain more accurate and extensive information on the effects of maintained constant temperatures on the vitamin content of canned foods, together with a survey of the temperatures existing in canned food warehouses throughout the United States. The following discussion emphasizes the recommendations within the canning industry today that canned foods be stored under "cool" warehousing conditions, and if under elevated storage temperature conditions, for as short a time as possible. There are, of course, specific storage effects as related to the different canned food products which also must be taken into consideration. The problem of the effect of times and temperatures of storage has received a great deal of consideration with respect to citrus and tomato juices. The effects of three storage temperatures, namely, 9.4", 24.4", and 37.2"C. (49", 76", and 99°F.) on the ascorbic acid content of orange juice stored for a 12-month period was reported (Ross, 1944). More recent work (Moschette et al., 1947) has shown results which correlate very well with these data. The results of these two workers are shown in Fig. 6. One can observe readily the seriousness of high temperature storage conditions and conversely the nondetrimental effect of the time element when low temperatures of storage are employed. Although 8% of the original ascorbic acid in this canned product is sacrificed upon 1 year's storage at 18.3"C. (65°F.) the product may still be considered to be an excellent source of ascorbic acid. It must be emphasized that an average storage temperature of 37.2"C. (99°F.) is an abnormal average temperature so far as the commercial warehousing of canned foods is concerned, but is comparable to conditions which were encountered by the Army in their feeding problems in the South Pacific and other tropical areas. In general, warehouses for storing canned foods are maintained at an average yearly temperature from 1Oo-23.9"C. (50"-75°F.). Results were obtained (Moore et al., 1945) which showed that the average retention of ascorbic acid in
FACTORS INFLUENCING VITAMIN CONTENT OF CANNED FOODS
91
canned unsweetened grapefruit juice from twelve central Florida canning plants waa 95% a t the end of 2 months, 90% at the end of 4 months, and 83% at the end of 6 months. Storage experiments upon twelve samples of grapefruit juice from five different packers stored under ordinary temperatures prevailing over a year in the Florida area (Roberts, 1937) indicated 49.F: 5bE 8S.E
8bF: 76T
140-
z
520W
2
k!
10-
Fig. 6. Ascorbic acid retention in canned orange juice at different storage temperatures. (- - - Moschette el d.,1947; -Ross, 1944.)
gradual losses of vitamin C to the extent of 25% a t the end of from 9-15 months. Results of the effect of 18-months’storage at 70°F. on the ascorbic acid content of grapefruit juice and the effect of similar conditions upon the ascorbic acid content of canned orange juice for 12 months have been reported (Lamb, 1946a). These results showed that at the end of 11months both California and Arizona canned grapefruit juice retained 89% of their original ascorbic acid content, while a t the end of 18 months the retention was 81%. Similarly, canned California orange juice showed 92% reten-
92
L. E. CLIFCORN
tion of the ascorbic acid content a t the end of 6% months and 87% a t the end of 12 months. It is important to note that all of these results correlate very well and are generally summarized both for grapefruit juice and orange juice by Fig. 6. With regard to the other vitamins in citrus juices, consideration has been given to the stability of thiamine in grapefruit juice and orange juice upon stbrage (Moschette et al., 1947). In general, it may be said that upon 12-months' storage a t temperatures lo", 18.3", and 26.7"C. (50", 65", and 80°F.) complete retentions, within the degree of accuracy of vitamin methods employed, were found, with the exception of 89Y0 retention in orange juice stored for 12 months a t 26.7"C. (80°F.). The effect of storage on the vitamin content of canned peaches, pineapple, and pineapple juice also has been studied (Moschette et al., 1947) at temperatures of lo", 18.3", and 26.7"C. (50", 65", and 80°F.) for a period of storage of 12 months. It was found that peaches retained 98, 85, and 7270 of the ascorbic acid content, respectively, while pineapple and pineapple juice retained essentially all the ascorbic acid content at the temperatures of 10" and 18.3"C. (50" and 65°F.). Carotene retentions in canned peaches were found to be 95, 90, and 8675, respectively, for simiIar storage conditions, while the niacin retention was found to be complete over a l-year storage period for all temperatures studied. Since an acid condition is undoubtedly favorable for the retention of thiamine, it is not surprising that exceptionally good retentions of this vitamin were obtained on all acid canned food products studied. In general, for the three products under consideration the results were very similar, the average retentions for the group being 94,93, and 86% for the temperatures lo", 18.3", and 26.7"C. (50", 65", and 80"F.), respectively, for a 12-month storage period. Work also has been carried out on the storage effect of products falling generally into the nonacid class which include, ,in the main, vegetables other than tomatoes and products such as canned meats, milk, etc. The acid condition does not exist in these products to the extent that it does in those previously discussed. The variation in stability of the vitamins at various pH levels leads one to believe that the losses occurring during storage should vary with pH. This is particularly true for thiamine. The retentions of vitamin C and carotene in glass-packed peas, spinach, and carrots over a one year period have been reported (Fellers and Buck, 1941). The retentions of ascorbic acid in glass-packed peas after 12-months' storage in both the light and dark a t temperatures of 2.2"-32.2"C. (36"-90°F.) ranged from 71-98701 the lowest and highest retentions correlating inversely with the lowest and highest temperatures of storage. Spinach retained 76% of its original ascorbic acid content when glass packed and
IFACTORB INFLUENCING VITAMIN CONTENT OF CANNED FOODS
93
held in the dark at 21.1"C. (70°F.) for a year. The authors commented that very little loss occurred after the first 2 or 3 months of storage. Storage in the light hastened slightly the destruction of vitamin C in glass-packed foods. Glass-packed spinach, carrots, and peas retained approximately 85% of their original carotene (vitamin A) content after storage in subdued light for a year. Canned green Lima beans and whole kernel yellow corn were stored for a year at temperatures of -1.lo, 5.6", 29.4", and 43.3"C. (30", 42", 85", and 110°F.) and analyzed at various periods for ascorbic acid, carotene, thiamine, riboflavin, and pantothenic acid (Guerrant et al., 1945b). With regard to ascorbic acid, green Lima beans retained 8301, when stored for 270 days at 30°F. and 65y0 after storage at 37.8"C. (110°F.) for the same period. Yellow corn retained 92% of its ascorbic acid content when stored at -1.1"C. (30°F.) for 270 days, but only 47% was retained when stored at 43.3"C. (110°F.). Lima beans and yellow corn retained 78 and 74y0 of their original carotene content at -1.1"C. (30"F.), while Lima beans retained only 54% when stored at 43.3"C. (110°F.) for a year, and corn retained 58y0 after 180 days at 43.3"C. (110°F.). At the end of a year's storage at - 1.1"C. (30"F.), these products had retained essentially 100% of their original thiamine content, while at 43.3"C. (110°F.) for a year, Lima beans retained 25% and yellow corn 20% of their original thiamine content. Relatively satisfactory retentions of riboflavin were observed at all temperatures, the lowest being for tomato juice stored 365 days at 43.3"C. (110°F.) with a 60% retention, and next lowest being an 80% retention by green Lima beans. Almost complete retention of riboflavin was obtained at temperatures of -1.1" and 5.6"C. (30 and 42°F.) and approximately 85-95% a t 29.4"C. (85°F.) for the one-year storage period for all three products. Pantothenjc acid in these products wm not seriously affected by the time of storage, but the temperature of storage again exerted its effect, about 60% retention of pantothenic acid in green Lima beans being noted after a year a t 43.3"C. (IlO'F.). These workers have expanded their investigations to include a wide variety of nonacid products stored a t temperatures of lo", 18.3", and 26.7"C. (50", 65", and 80°F.) for periods up to 18 months. This work has been completed and should appear in the literature in the near future. It has been shown (Clifcorn, 1945) that a tomato juice retaining 68% of the original ascorbic acid content after canning, retained 56y0 when stored a t room temperature for one year, while upon storage at 36.7"C. (98"F.), the retention was 30%. The most extensive work conducted on this subject was reported recently (Guerrant, et al., 1945a and Moschette et al., 1947). Canned tomato juice waa stored for one year at temperatures of -1.1", 5.6', lo", 18.3", 26.7", and 43.3"C. (30°, 42", 50", 65",
94
-I
L. E. CLIFCORN
FACTORS INFLUENCING VITAMIN CONTENT OF CANNED FOODS
95
80°, 85", and 110OF.). In this work ascorbic acid, carotene, thiamine, riboflavin, niacin, and pantothenic acid were considered, As one will observe from Table XX the retentions of all of the vitamins were satisfactory at all of the temperatures except 43.3"C. (110°F.). Under such abnormally high temperature storage conditions all of the vitamins were seriously affected, especially ascorbic acid and thiamine which showed only 20 and 31% retention, respectively. It is interesting to note that tomatoes retained more riboflavin after 12 months of storage under similar conditions than did tomato juice. This leads one to wonder whether some of the materials removed from tomatoes during the preparation of tomato juice may be favorable to riboflavin stability. An excellent correlation has been obtained between the effect of maintained constant storage temperatures on the vitamin content of canned foods and the storage losses that occur in the commercial warehousing of canned foods. Lots of some of the same canned foods stored a t constant temperatures were stored in nine commercial warehouses throughout the United States. The products which were correlated in this manner were canned orange juice, tomatoes, and peas. At the end of various storage periods, samples were removed from the nine warehouses and sent to the universities conducting the storage investigations for vitamin analyses and correlations with results obtained under maintained constant temperatures. Daily temperature readings were made in each of the warehouses in which the correlation samples were stored, and from these daily maximum and minimum temperature readings, average temperature values were calculated. It was of particular importance to determine whether average storage temperatures over a long period of time correlated with maintained constant temperature conditions. The ascorbic acid and thiamine retentions have been reported (Moschette et al., 1947) for one lot of orange juice and two lots of tomatoes stored in the nine warehouses throughout the country. The excellent correlations obtained between the results of storage tests under constant temperature conditions with the results obtained on the warehouse samples on the basis of yearly average temperatures for the two vitamins mentioned are shown in Table XXI. The adverse effect of elevated temperature st orage conditions over relatively long periods of time is readily apparent from the results of this work. Results on samples of peas from the warehouses will be correlated with storage results obtained under constant temperature conditions by the Pennsylvania State College workers. In the planning of storage studies in the National Canners AssociationCan Manufacturers Institute Nutrition Program, it was decided to conduct an investigation with canned tomato juice to determine whether storage for equal lengths of time, 4 months each at 4.4", 21.1°, and 36.7"C.
(0
Q,
TAB^ XXI
Comparison of Warehowre and Constant Temperature Stmags on Ascorbic Acid and Thiamine Retention in Orange Juice and Tomdoes4 Temperature of storage Storage location Constant temperature c h b e r s , University of Chicago No. I No. I1 No. I11
Yearly av., O F .
Daily low av., O F .
Daily high av., O F .
1
Thiamine retention Yearly range, O F .
Orange juice,
%'
Tomatoes,
%"
Orange juice, % b
Tomatoes,
%"
I
80 65 50
79.6 64.9 48.8
80.4 65.2 50.0
75-83 61-68 443-52
76 92 97
82 93 95
a2 80
50-98 54-91 50-92 54-104 51-87 42-79 36-87 28-92 30-78
73
83
81 81 86 92 91
92 100 105 102 101 101 98 106
89
98 100
82 93 94
I
New Orleans, La. Tampa, Fla. Tempe, Ark. Yuba City, Calif. Oakland, calif. Washington, D. C. St. Louis, Mo. Rochelle, Ill. New York. N. Y. 0
b
77 77 72 70 66 63 61 59
58
73 74 68 65 62 63 59 56 57
F
P d
r
Y
q
75 75 66
64 63 63 59
Moschette et d.(1947). Average of 2 lots in constant temperature storage; only 1lot in warehouse storage. Average of 3 lots in constant temperature and for 2 lots in warehouse storage.
91
90 96
96 95 99 99 96 103 95 99 98
83 79 89
88 86
90 89 89 96
8
Ei
FACTORS INFLUENCING VITAMIN CONTENT OF CANNED FOODS
97
(40", 70", and 98"F.), had an effect on ascorbic acid, thiamine, and carotene similar to continiious storage for 12 months a t a constant temperature of 21.1"C. (70°F.). The results as shown in Table XXII suggest that storage of commercially canned tomato juice at temperatures of 21.1"C. (70°F.) or lower for one year should not seriously affect the ascorbic acid, thiamine, or carotene contents. However, any combination of 4-months' storage periods a t 4.4", 21.1", and 36.7"C. (40", 70", and 98"F.), which is essentially an average monthly storage temperature of 21.1"C. (70"F.), results in definite losses of ascorbic acid and thiamine. Carotene was relatively stable during the one-year storage period at temperatures up to 36.7"C. (98°F.). Storage of tomato juice for even short periods of time a t temperatures of 36.7"C. (98°F.) should be avoided. TABLEXXII Efect of Varied Storage Temperaturea on the Retention of Certain Vitamima
Storage temperature during 12-month period, OF. 0-4
4 4
8-12
Ascorbic acid
Thiamine
40" 70"
40"
101 99 51
40"
40" 70" 98" 98" 70" 98"
83 80 54 58 62 55
98"
40"
98"
40" 40" 70" 70" 98" 98" 0
% Retention
70" 98" 70" 98"
40" 70"
70"
40"
83 83 84 83 83 83
00 63
60
Carotene
91 87
90 94 97 100 93 84
Heberlein and Feaster (unpublished).
A study of the effect of storage on the B vitamins in canned pork has been reported (Rice and Robinson, 1944) in which canned pork was stored for a period up to 293 days at various temperatures including -28.9", 3.3", 26.7", 36.7", 48.9", and 6223°C. (-%lo, 38", 80", 98", 120°, and 145°F.) and analyzed a t various lengths of time. The vitamins included were thiamine, niacin, riboflavin, and pantothenic acid. The results of this work are clearly shown in Table XXIII, which again emphasises the importance of time and temperature considerations in stored canned food products. Storage of canned cured pork luncheon meat for one year has indicated (Feaster et al. , 1946) practically complete retention of riboflavin, niacin, and pantothenic acid at storage temperatures of 7.2", 21.1", and 36.7"C.
98
L. E. CLIFCORN
(45", 70") and 98°F.). Thiamine retentions of the processed meat at the end of that time were 89-100% at 7.2"C. (45"F.), 59-76% at 21.1"C. (70°F.)) and 1220% at 36.7"C. (98°F.). Unprocessed cans of the same cured product gave thiamine retentions of 5576% at 7.2"C. (45"F.), 4260% at 21.1"C. (70°F.)) and 7-16y0 at 36.7"C. (98°F.). The authors concluded that neither the can s i ~ nor e the thiamine content of the product immediately after processing had a significant effect on the rate of change of thiamine during storage. Tmm XXIII
Retention of Vitamins in Canned Pork During Storage4.b
I :k'4 I Storage
Vitamin
I
Thiamine
Niacin Riboflavin Pantothenic acid a
Storage temperature, O F . 20"
-I
28 81 235 293 293 293 293
I
38"
-1-1
4.9 5.2 4.9 5.2 34.5 1.8 3.6
4.7 4.9 4.6 4.8 35.5 1.Q 3.7
I
80'
99"
4.6 4.1 3.7 2.7 35.3 1.9 3.8
4.1 3.2 1.0 1.4 35.5 1.9 3.4
120'
145'
2.9 0.9 -
0.8 0.1
-34.0 1.5 2.2
/
33.0 1.2 2.3
Kice and Robinson (1944). PdP.
V. RELATIONSHIP OF TYPEOF CONTAINER TO VITAMIN CONTENT A survey (Newman and Fellers, 1940) of the ascorbic acid content of 21 different foods packed in tin and glass containers as purchased from retail markets showed that the products packed in tin containers contained slightly more ascorbic acid than their glass counterparts. Samples of tomato juice were packed in tin and glass containers (Lueck and Pilcher, 1941) and the ascorbic acid content determined by both animal assays and chemical titrations after various intervals of storage with the result that assays up to 311 days of storage averaged 15.8 mg./100 g. for the plain can and 6.8 mg./100 g. for the glass container. Similarly, other workers (Moore et al., 1943-1944) packed orange juice and grapefruit juice and found the retentions shown in Table XXIV after 6 months of storage at room temperature and approximately 4.4"C. (40°F.). In studies on homecanned tomato juice (Hauck, 1943)) it was concluded that tomato juice canned in tin retains more ascorbic acid than the same when packed in glass for 2%-8% months. Glass containers with tin strips and enamel-
FACTORS INFLUENCING VITAMIN CONTHNT OF CANNED FOODB
99
TABLE Xxrv Cornpariaon of Asmbic Acid Retention in Canned and Bottkd Orawe Juice and Grapefruit Juices
% Retention Orange juice, bottled, room temperature storage Orange juice, canned, room temperature storage Grapefruit juice, bottled, room temperature storage Grapefruit juice, canned, room temperature storage Orange juice, bottled, cold room, 40°F. Orange juice, canned, cold room, 40°F. Grapefruit juice, bottled, cold room, 40°F. Grapefruit juice, canned, cold room, 40°F. 0
after 6 Mo. 76.1 81.7 75.1 82.9 92.1 95.1 89.0 97.1
Moore et al. (1943-44).
lined cans were also included in this comparison with the results as shown in Table XXV. In an experimental pack of orange juice (Boyd and Peterson, 1945), it was found that the removal of air from the headspace of cans showed more beneficial effect from the standpoint of ascorbic acid TABLBX X V Effect of Type of C0nt.ain.e~on tL Redwed Ascorbic Acid Content of Homecanned Tomato Juices Type of container Storage period
Tin
Glass
1-2 days 2-3 days 2% months 3% month 8% months 8% months
mg.jl00 ml. 19.7 (4) 19.9 (4) 19.6 (2) 19.2 (3) 16.4 (3) 16.1 (4)
mg./100 ml. 19.0 (4) 19.2 (4) 15.8 (2) 16.0 (3) 5.7 (3)
.
Glass with “tin insert’’
Tin, enamel-lined
mg./100 ml. 19.1 (4)
mg./100 ml. 17.7 (4)
19.4 (3)
16.4 (3)
15.9 (4)
14.0 (4)
.
* Figures in parentheses represent the number of cam of juice tested. retention in the enameled can than in the plain can, as seen in TabIe XXVI. It seems that the tin can surface in contact with the product produces a desirable reducing atmosphere for the greater retention of ascorbic acid as affected by oxygen in the headspace and perhaps by storage. Further
100
L. E. CLIFCORN
experiments have shown that when orange juice is packed in a plain can most of the oxygen entrapped in the headspace is preferentially used in the metal-acid reaction rather than in the oxidation of the ascorbic acid of the product. This effect may decrease materially as we go from acid products of the citrus family to nonacid products of the vegetable class. TABLE XXVI Relationship of H d p a c e Oxygen and Type of Contcriner to Ascorbic Acid Retentiun in Canned Orange Juices Type of Can Enameled, headapace air removed Enameled, control Plain,headapace air removed Plain, control 4
Ascorbic Acid 26.4 mg./lOO ml. 21.8 28.3 27.0
Boyd and Peterson (1945).
With regard to the other vitamins (McConnell et al., 194445), it has been found that the carotene content of asparagus, green beans, corn, and peas is relatively unaffected by canning and storage in commercial type glass and metal containers. The type of container and exposure to light such as encountered in normal handling had no significant effect. ACKNOW LIDQML6ENT
The author wishes to give sincere thanks for the assistance given by Dr. R. E. Henry, Mr. D. G. Heberlein, and Miss Lois Larson in the preparation of this review.
REFERENCES Adam, W. B. 1941. Factors af'fecting the vitamin C content of canned fruit and vegetables. Progress report. Ann. Rept., Fruit Vegetable Preserv. Research Sia. Campden, Univ. of Briatol 1941, 14-20. Adam, W. B., Homer, G., and Stanworth, J. 1942. Changes occurring during the blanching of vegetables. J . Soc. Chem. Ind. 61,96-99. See also Nutrition Rats. 1, 69 (1943). Beadle, B. W., Greenwood, D. A., and Kraybill, H. R. 1943. Stability of thiamine to heat. I. Effect of pH and buffer salts in aqueous solutions. J . Biol. Chem. 149, 339-347. Boyd, J. M., and Peterson, G. T. 1945. Quality of canned orange juice. Znd. Eng. C h .37, 370-373. Brush, M. K., Hinman, W. F., and Halliday, E. G. 1944. The nutritive value of canned foods. V. Distribution of water soluble vitamins between solid and liquid portions of canned vegetables and fruits. J . Nutrition 28, 131-140. Clifcorn, L. E. 1944. The nutritive value of canned foods. I. Introduction and sampling procedure. J . Nutrition 28, 101-105. Clifcorn, L. E. 1945. Variables influencing vitamin content of processed foods. The Research Department, Continental Can Co., Inc. Bull. No. 6, 3-8. Clifcorn, L. E., and Heberlein, D. G. 1944. Thiamine content of vegetables. Effect of commercial canning. Znd. Eng. C h . 36, 168-171.
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Clifcorn, L. E., and Peterson, G. T. 1947. The retention of vitamin C in tomato juice. "he Research Department, Continental Can Co., Inc., Bull. No. 12, 3-12. Daniel, E. P.,and Munsell, H. E. 1937. Vitamin content of foods. U.S. Dept. A p . Misc. Pub. No. 275. De Felice, D.,and Fellers, C. R. 1938. Carotene content of fresh, frozen, canned, and dehydrated spinach. Proc. Am. Soc. Hart. Sci. 36, 728-733. Elvehjem, C. A. 1947. Univ. of Wisconsin. Private communication. Feaster, J. F., Jackson, J. M., Greenwood, D. A., and Kraybill, H. R. 1946. Vitamin retention in processed meat. Effect of storage. Ind. Eng. Chem. 88, 87-90. Feaster, J. F., Tompkins, M. D., and Ives, M. 1947. Iduence of proceasing technique on vitamin retention. Infomtion Letter, National Canners Assoc. No. 1200, 108-109. Fellers, C. R., and Buck, R. E. 1941. Retention of vitamins C and A in +packed foods. Food Research 6, 135-141. Fenton, F. 1946. Iduence of method of preparation on retention of palatability and vitamins in food. (Cornell Univ.) Private communication. Fenton, F., Tressler, D. K., Camp, S. C., and King, C. G. 1938. Losses of vitamin C during boiling and steaming of carrots. Food Research 3,403-408. Fenton, F., Tressler, D. K., and King, C. G. 1936. Losses of vitamin C during the cooking of peas. J. Nutrition 12, 285-296. Fincke, M. L. 1939. Vitamin values of garden-type peas preserved by frosen-pack method. 111. Thiamine (vitamin B1). Food Research 4, 605-611. Fitegerald, G. A., and Fellers, C. R. 1938. Carotene and ascorbic acid content of fresh market and commercially frozen fruits and vegetables. Food Research 8, 109-120. Floyd, W. W., and Fraps, G. S. 1942. Ascorbic acid content of some grapefruit juices prepared under various processing conditions. Food Research 7, 382-387. Gleim, E. G., Tressler, D. K., and Fenton, F. 1944. Ascorbio acid, thiamine, riboflavin, and carotene contents of asparagus and spinach in the fresh, stored, and frozen states, both before and after cooking. Food R a w ~ c h9,471-490. Greenwood, D. A., Beadle, B. W., and Kraybill, H. R. 1943. Stability of thiamine to heat. 11. Effect of meat curing ingredients in aqueous solutions and in meat. J. BioE. Chem. 149,34!4-354. Greenwood, D. A., Kraybill, H. R., Feaster, J. F., and Jackson, J. M. 1944. Vitamin retention in processed meat. Effect of thermal processing. Ind. Eng. C b m . 86, 922-927. Guerrant, N. B., Vavich, M. G., and Dutcher, R. A. 1945a. Nutritive value of canned foods. Iduence of temperature and time of storage on vitamin contents. I d . Eng. Chem.87, 1240-1243. Guerrant, N. B., Vavich, M. G., and Fardig, 0. B. 1945b. Nutritive value of canned foods. Comparison of vitamin valuea obtained by different methods of assay. Znd. Eng. Chem., A d . Ed. 17,710-713. Guerrant, N. B., Vavich, M. G., Fardig, 0. B., Dutcher, R. A., and Stern, R. M. 1946. The nutritive value of canned foods. Changes in the vitamin content of foods during canning. J. Nutrition 33,436468. Guerrant, N. B., Vavich, M. G., Fardig, 0. B., Ellenberger, H. A., Stern, R. M., and Coonen, N. H. 1947. Nutritive value of canned foods. Effect of duration and temperature of blanch on vitamin retention by certain vegetables. Znd. Eng. Chem. 39,1000-1007. Haagen-Smit, A. J., Strickland, A. G. R., Jeffreys, C. E. P.,and Kirchner, J. G. 1946. Studies on vitamin content of canned pineapple. Food Reseurch 11,142-147.
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Hamburger, J. J., m d Joslyn, M. A. 1941. Auto-oxidation of filtered citrus juices. Food Research 8,599619. Hamner, K. C., Bernstem, L., and Maynard, L. A. 1945. Effects of light intensity, day length, temperature, and other environmental factom on the ascorbic acid content of tomatoes. J. Nutrition 29, 85-97. Hansen, E., and Waldo, G. F. 1944. Ascorbic acid content of small fruits in relation to genetic an&environmental factors. Food Research 9,45-61. Harris, R. S.,and Proctor, B. E. 1940. Effect of processing on the vitamin B1 content of foods. Proc. Zmt. Food Technol. 1940, 109-121, Hauck, H. M. 1943. Ascorbic acid in home canned tomato juice.-Effect of type of container and method of extraction. J. Home E m . 36,295-300. Heberlein, D. G., and Feaster, J. F. Unpublished. Work conducted in the National Canners Association-Can Manufacturers Institute Nutrition Program. Natl. Canners Amoc., Washington, D. C. Heinze, P. H., Kanapaux, M. S., Wade, B. L., Grimball, P. C., and Foster, R. L. 1944. Ascorbic acid content of 39 varieties of snap beans. Food Research 9, 19-28. Hibbard, A. D.,and Flynn, L. M. 1945. Maturity effect on vitamin content of green snap beans. Proc. Am. Soc. Hmt. Sn‘. 46, 350-354. Hinman, W. F., Brush, M. K., and Halliday, E. G. 1944. The nutritive value of canned foods. VI. Effect of large scale preparation for serving on the ascorbic acid, thiamine, and riboflavin content of commercially-cannedvegetables. J. Am. Dietet. Assoc. 20, 752-756. H m n , W. F., Brush, M. K., and Halliday, E. G. 1945. The nutritive value of canned foods. VII. Effect of small-scale preparation on the ascorbic acid, thiamine, and riboflavin content of commercially-canned vegetables. J. Am. Dietet. Assoc. 21, 7-10. Hinman, W. F., Higgins, M. M., and Halliday, E. G. 1947, The nutritive value of canned foods. XVIII. Further studies on carotene, ascorbic acid, and thiamine. J. Am. Dietet. Assoc. 23, 226-231. Ives, M., Pollard, A. E., Elvehjem, C. A., and Strong, F. M. 1946. The nutritive value of canned foods. XVII. Pyridoxine, biotin, and “folic acid.” J . Nutrition ai, 347-353. Ives, M., Wagner, J. R., Elvehjem, C. A., and Strong, F. M. 1944. The nutritive value of canned foods. 111. Thiamine and niacin. J . Nutrition 28, 117-121. Ives, M., Zepplin, M., Ames, S. R., Strong, F. M., and Elvehjem, C. A. 1945. The nutritive value of canned foods. X. Further studies on riboflrtvin, niacin, and pantothenic acid. J. Am. Dietet. Assoc. 21, 357-359, Jenkins, R. R., Tressler, D. K., and Fitzgerald, G. A. 1938. Vitamin C content of vegetables. VIII. Froxen peas. Food Research 3, 133-140. Kirk, M. M.,and Tressler, D. K. 1941. Ascorbic acid content of pigmented fruits, vegetables, and their juices. Food Research 6,395-411. Kohman, E. F. 1937. Vitamins in canned foods. National Canners Association Bull. No. 19-L,4th Revision. Kohman, E. F., Eddy, W. H., and Carlsson, V. 1924. Vitamins in canned foods. 11. Vitamin C destructive factor in apples. Znd. Eng. Chem. 16, 1261-1263. Kramer, A. 1945. The nutritive value of canned foods. VIII. Distribution of proximate and mineral nutrients in the drained and liquid portions of canned vegetables. J . Am. Dietet. Assoc. 21, 354-356. Kramer, A. 1946. Nutritive value of canned foods. XVI. Proximate and mineral compoaition. Food Research 11, 1-8.
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Lamb, F. C. 1945. P r o m report on studies of variability of ascorbic acid content among individual tomatoes and among individual cans of tomatoes. Mimeographed Research Lab. Report No. 11023A, Natl. Canners Assoc., San Francisco, California. Jan. 27, 1945. Lamb, F. C. 194f3a. Nutritive value of canned foods-Factors decting ascorbic acid content of canned grapefruit and orange juices. Ind. Eng. C h m . 88,880-864. Lamb, F. C. 1948b. Studies of factors decting the retention of nutrients during canning operations. National Camera Association, Western Branch Laboratories, Stln Francisco. July, 1946. Summary article in press. Food Research. Lee, F. A., and wbitoombe, J. 1945. The vitamin content of Alderman peas a t BBVeral stagea of msturity. PTOC.Inst. Food Techrwl. 1946, 168-159. von Loesecke, H. W. 1942. Vegetable preparation and processing. Western Cunner and Packer, 34,No. 7, 35-38. Lueck, R. H., and Pilcher, R. W. 1941. Canning fruit juices-technical aapects. Znd. Eng. C h m . 88,292-300. McConnell, J. E. W., Esselen, W. B., Jr., and Guggenberg, N. 1944-45. EtIect of storage conditions and type of container on stability of carotene in canned vegetables. Fruit Products J . 24, 133-135. Magoon, C. A., and Culpepper, C. W. 1924. Scalding, precooking, and chilling as preliminary canning operations. U.S. Dept. Agr. Bull. No. 1266, 1-47. Melnick, D., Hochberg, M., and Oser, B. L. 1944. Comparative study of steam and hot water blanching. Food Research 9, 148-153. Moore, E. L., Wiederhold, E., and Atkins, C. D. 1943-44. Changes occurring in orange and grapefruit juices during commercial processing and subsequent storage of the glass- and tin-packed products. Fruit Products J. 23,270-275. Moore, E. L., Wiederhold, E., and Atkins, C. D. 1945. Ascorbic acid retention in Florida grapefruit juices. 11. During storage of the canned products. The Cunner 100, No. 8, 55-57. Moore, E. L., Wiederhold, E., Atkina, C. D., and MacDowell, L. G. 1944. Ascorbic acid retention in Florida grapefruit juices. I. During commercial canning. The Canner 98, No. 9 , 2 4 2 8 . Moschette, D. S., Hinman, W. F., and Halliday, E. G. 1947. Nutritive value of canned foods--effect of time and temperature of storage on vitamin content of certain commercially canned fruits end fruit juices (stored 12 months). Znd. Eng. Chem. 39,994-999. Moyer, J. C., and Stotz, E. 1945. The electronic blanching of vegetables. Science 102, 68-89. Newman, K. R., and Fellers, C. R. 1940. Vitamin C in packaged foods purchased in retail markets. J. Am. Dietet. Assoc. 16, 895-698. Olliver, M. 1937-38. Vitamin C content of English canned fruits and vegetables. Food 7,48-49. Phtenius, H., and Jones, J. B. 1944. Effect of modified storage on ascorbic acid content of some vegetables. Food Research 9,378-385. Pressley, A., Ridder, C., Smith, M. C., and Caldwell, E. 1944. The nutritive value of canned foods. 11. Ascorbic acid and carotene or vitamin A content. J. Nutrition 28, 107-116. Rice, E. E., and Beuk, J. J. 1945. Reaction rates for decomposition of thiamine in pork a t various cooking temperatures. Food Reaeurch 10,99-107. Rice, E. E., and Robinson, H. E. 1944. Nutritive value of canned and dehydrated meat and meat products. Am. J. Pub. Health 54, 587-592.
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Richardson, J. E., Mayfield, H. L., and Davis, R. J. 1937. Effect of home preservation on the quality and vitamin content of Golden Bantam sweet corn. Montana Agr. Espt. Sta. Bull. S47,2-22. See C. A. 32, 5950 (1938). Roberts, J. A. 1937. Vitamin C in citrus juice beverages and canned grapefruit juice. Food Resmrch 2, 331-337. Robinson, W. B., Stotz, E., and Kertesz, Z. I. 1945. The effect of manufacturing methods on the ascorbic acid content and consistency characteristics of tomato juice. J . Nutrition 30,435-442. Ross, E. 1944. Effect of time and temperature of storage on vitamin C retention in canned citrus juices. Food Research 9, 27-33. Rostovskaya, Yu. V. 1941. Experimental data on stability of vitamin C in canning vegetables. Proc. Sci. Inst. Vitamin Research U.S.S.R. 8, 246-252. See C. A . 36,2946 (1942). Sanborn, N. If. 1938. Conservation of vitamin C in tomato juice production. Glass Packw 17, 102-103. Schroeder, G. M., Sattedeld, G. H., and Holmes, A. D. 1943. The iniluence of variety, size, and degree of ripeness upon the ascorbic acid content of peaches. J . Nutrition 26,503-509. Scoular, F. I., and Willard, H. 1944. Effect of refrigeration on ascorbic acid content of canned fruit juices after opening. J . Am. Dietet. Assoc. 20, 223-225. Smith, M. C., ROSS,W., and Caldwell, E. 1944. Comparative vitamin C values of Arizona citrus fruits of different varieties and sizes when prepared for consumption several different ways. U.Ariz. Agr. Expt. Sta. Mimeu. Rept. 60, 1-15. Stimson, C. R., Tressler, D. K., and Maynard, L. A. 1939. Carotene (vitamin A) content of fresh and frosted peas. Food Research 4,475483. Strachan, C. C., and Atkinson, F. E. 1946. Ascorbic acid content of tomato varieties and its retention in processed producta. Sci. Agr. 26, No. 2, 83-94. Thompson, M. L., Cunningham, E., and Snell, E. E. 1944. The nutritive value of canned foods. IV. Riboflavin and pantothenic acid. J . Nutritim 28, 123-129. Todhunter, E. N., and Robbins, R. C. 1941. Ascorbic acid (vitamin C) content of red raspberries preserved by the frozen-pack method. Food Research 6, 435444. Wagner, J. R.,Ives, M., Strong, F. M., and Elvehjem, C. A. 1945. Nutritive value of canned foods. VII. Effect of commercial canning and shoretime storage on ascorbic acid content of grapefruit juice. Food Research 10, 469-475. Wagner, J. R., Strong, F. M., and Elvehjem, C.A. 1947a. Nutritive value of canned foods-Effect of commercial canning operations on the ascorbic acid, thiamine, riboflavin, and niacin contents of vegetables. I d . Eng. Chem. 39, 985-990. Wagner, J. R., Strong, F. M., and Elvehjem, C. A. 1947b. Nutritive value of canned foods-Effects of blanching on the retention of ascorbic acid, thiamine, and niacin in vegetables. Ind. Eng. Chem. 39,990-993. Watanabe, A. J. 1939. Kinetics of the thermal decomposition of vitamin BI-HC1 in aqueous solutions. J . Pharm. SOC.Japan 69, 218. Wiederhold, E.,Atkins, C. D., and Moore, E. L. 1945. Ascorbic acid in Florida grapefruit juice. The Canner 100, No. 23, 12-14. Wokes, G.,and Organ, J. G. 1943. Oxidizing enzymes and vitamin C in tomatoes. Biochem. J . 37,259-265. Zimmerman, W. I., Tressler, D. K., and Maynard, L. A. 1940. Determination of carotene in fresh and frozen vegetables. I. Carotene content of green snap beans and sweet corn. Food Research 6,93401. Zimmerman, W. I., Tressler, D. K., and Maynard, L. A. 1941. Determination of carotene in fresh and frozen vegetables by an improved method. 11. Carotene content of asparagus and green Lima beans. Food Rmearch 6,5748.
The Physiological Basis of Voluntary Food Intake (Appetite?) BY SAMUEL LEPKOVSKY University of California. Berkeley. Califmkz CONTENTS
I. Introduction . . . . . . . . . . . . . . . . . . I1 Can Human Beings Choose Wisely in Accordance with Their Nutritional
. Needs? . . . . . . . . . . . . . . I11. Can Animals Choose Food Correctly? . . . . I V. Factors Influencing Food Intake . . . . .
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V. The Effectof Proteins on Food Intake . . . . . . . . . . . VI . The Effect of Amino Acid on Food Intake . . . . . . . . . VII . The Effect of Water-Soluble Vitamins on Food Intake . . . . . . VIII. The Effect of Fats and Fat-Soluble Vitamins on Food Intake . . . . 1.Fats . . . . . . . . . . . . . . . . . . . 2 FatSoluble Vitamins . . . . . . . . . . . . . . IX. The Role of Essential Minerals on Food Intake . . . . . . . . 1 Phosphorus . . . . . . . . . . . . . . . . . 2. Calcium . . . . . . . . . . . . . . . . . . 3. Magnesium . . . . . . . . . . . . . . . . . 4 Sodium . . . . . . . . . . . . . . . . . . 5. Potaasium . . . . . . . . . . . . . . . . . 6 Chlorine . . . . . . . . . . . . . . . . . . 7.Zinc . . . . . . . . . . . . . . . . . . . 8. Cobalt . . . . . . . . . . . . . . . . . . 9.Manganese . . . . . . . . . . . . . . . . . 10. Iodine . . . . . . . . . . . . . . . . . . 11.Selenium . . . . . . . . . . . . . . . . . . 12. Fluorine . . . . . . . . . . . . . . . . . . . X Endocrines and Food Intake . . . . . . . . . . . . . XI The Role of Deleterious Compounds on Voluntary Food Intake . . . X I1 Protection Against Deleterious Compounds . . . . . . . . . 1. Elimination of Deleterious Compounds from the Diet . . . . . a . Heat . . . . . . . . . . . . . . . . . b . Fermentation . . . . . . . . . . . . . . . c . Germination . . . . . . . . . . . . . . . d . Chemical Treatment . . . . . . . . . . . . . e. Destruction in the Digestive Tract . . . . . . . . . f . Conversion into Nontoxic Compounds . . . . . . . . g. Adsorption . . . . . . . . . . . . . . . . 2 Counteraction of Deleterious Compounds by Specific Nutrients . . 3. Physiological Adaptation t o Deleterious Compounds . . . . . 4. Decrease in Food Intake a~ a Protection against Deleterious Compounds . . . . . . . . . . . . . . . . . . 129
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XIII. Physiological Mechanisms Determining Food Intake . . . . . . . 1. Food Intake and Caloric Output . . . . . . . . . . . 2. Growth and Food Intake . . . . . . . . . . . . . 3. Internal Environment and Food Intake . . . . . . . . . 4. The Possible Role of Enzyme Systems . . . . . . . . . 5. The State of TwueIrritabilityandFoodIntake . . . . . . 6. The Gastrointestinal Tract and Food Intake . . . . . . . 7. Intestinal Bacteria and Food Intake 8. Role of Flavors in Food Intake 9. Appetite Centers and Food Intake . . . . . . . . . . 10. Taste Mechanisms and Food Intake . . . . . . . . . . XIV. Integrative Summary . . . . . . . . . . . . . . . 1. Palatability of Food . . . . . . . . . . . . . . 2. Possible Mechanisms ControllingFoodIntake . . . . . . . 3. Variability in the Reaction to Nutrients . . . . . . . . . 4. Glucose and Energy Exchange . . . . . . . . . . . 5. Differentiation of Psychological from Physiological Factors in the Control of Food Intake . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .
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I. INTRODUCTION The literature on the physiology of food intake is immense and so widely scattered that no attempt will be made to cover it completely. Since the scope of this review is limited, only the most pertinent literature is covered. The term “voluntary food intake” will be used in order to avoid the confusion which might accompany the use of such terms as hunger, appetite, preference, hyperphagia, polyphagia, satiety, anorexia, bulimia, etc. An attempt will be made to explore the basic physiological factors which influence the food intake of an animal, increasing or decreasing it. The question has been well put by Brobeck (1946a) when he says: “Why does the animal eat food? What determines how much it will eat? What changes in the internal environment set the animal to eating and what changes are associated with satiety?” To survive, animals must ingest foods which supply the essential nutrients necessary for growth and reproduction. To survive, they must choose adequate foods, for in any given environment animals can easily choose foods which will not meet their nutritional needs. The existence of a rich animal population is proof that they have chosen adequate foods. The question could well be raised whether or not animals are guided in their choice of foods by physiological responses to foods.
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BEINGSCHOOSEWISELYIN ACCORDANCE WITH 11. CANHUMAN THEIRNUTRITIONAL NEEDS? Sir William Roberts (1897) wrote: “The generalized food customs of mankind are not to be viewed as random practices, adopted to please the palate or gratify an idle or vicious appetite. These customs must be regarded as the outcome of profound instincts which correspond to certain wants of the human economy. They are the fruit of a colossal experience accumulated by countless millions of men through successive generations.” Human populations have shown an amazing ability to choose foods in accordance with their physiological needs, provided their choice was not unduly influenced by education, imitation, social, religious, or other considerations. Mursell (1925), who examined the literature of comparative and historical dietetics, wrote: “When we find widely sundered races, with vast differences in tastes and preferences, in food opportunities, and in habitat, still keeping to pretty much the same general balance of rationing, it is difficult to avoid the impression that we are in the presence of a very powerful auto-regulative mechanism, which, given certain external conditions, strongly favors a certain proportion of intake from among the basic nutrient substances.” There is much support to be found for this contention in studies of food habits of certain populations. For example, the inhabitants of the Island of Nauru in the Pacific Ocean, a t one time, included in their diet a “toddy” or fermented drink made from the flower spathe of the coconut palm. The Island was brought under the Commonwealth of Australia and the making of the toddy was prohibited. Infantile mortality increased; its cause was traced to beriberi which resulted from the prohibition of the fermented drink. Toddy yeast was actually used by the natives to cure the babies of beriberi. The origin of drinking this fermented beverage may have been due to a desire for alcohol or the desire to satisfy some craving of the body for an essential nutrient or nutrients present in the yeast (Anon., 1932). An interesting example of how human beings react to food and make surprisingly wise choices has been described by Adolph (1944). In the rural, cereal-consuming areas of North China, cereals are consumed as mixtures and seldom as single grains. The biological value of the mixed cereals used has been found to be invariably superior to that of single cereals. In each area the combination of cereals had been worked out apparently to provide a protein mixture of high, if not even of maximum, biological value. “This may be another case of blind experimentation, examples of which are widespread throughout Asia. The rural peoples
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are generally prepared with a ready response when questioned regarding the relative nutritive value of the food items used in that area. They may state that this food ‘stays with you’ or that food ‘furnishes muscular vigor.’ One of our pastimes has been that of checking up these rural opinions and finding that they often correspond closely to estimates of nutritive values made in the laboratory.” Where men subsist under conditions of near starvation so that they know that the food consumed ensures their survival, it seems probable that the influence of prejudices and of established food habits is greatly minimized. Men somehow learn to distinguish between foods of low and of high nutritional value (Englander, 1945). Such was the situation during World War I1 with a group of American soldiers in a German prison camp. “NO device or illusion could alter the fact that we were slowly starving. Our medical officer who analyzed the German ration said it was falling 200 calories below the minimum necessary to sustain life in a sedentary position. The internees sloughed off as much as fifty pounds apiece.” The great event of the prison camp was the arrival of the Red Cross food parcels. Trading went on among the men and a point system was set up which reflected fairly well the nutritional value of the food. “A can of powdered milk topped the list a t 150 points, meat followed at 120, and so on down the scale.” No values were reported for jam, cheese, sugar, and the D bar which were all among the items traded. It is possible that the war prisoners were able to rate the food items so closely to their actual nutritional value as a result of their physiological reactions to the food. The reason given for the popularity of powdered milk was that “it satisfied-even more than the chocolate-the prisoners’ craving for something rich to eat.” This is especially significant since powdered milk was so unpopular among the American soldiers. The soy bean has played a prominent role in the nutrition of populations in the Orient. It is well known that heating soy beans a t temperatures of over 100°C. (212°F.) or prolonged boiling a t 100°C. greatly improves their nutritional value (Osborne and Mendel, 1917). Orientals, however, do not prepare soy beans by ordinary cooking methods, but instead, subject them to one of several treatments (Miller, 1933) : (1) germination, (2) fermentation (production of soya sauce), and (3) fractionation (preparation of soy curd). Germination is known t o increase the nutritional value of soy beans, although heating improves the‘ germinated product still further (Everson et al., 1944). Fermentation may also improve the nutritional value of soy beans, perhaps by destroying some deleterious compounds present in them. The fractionation process involving the preparation of soy bean
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curd may possibly eliminate the antitrypsin and perhaps other inhibitory compounds which are responsible for the low nutritional value of the soy bean. The ability of children to choose foods correctly has been studied by Davis (1928). The children were found to be governed by their caloric needs in the selection of foods, but they also showed unpredictable preferences. Although, in general, they preferred the protein foods, fruits, and such vegetables as beets, carrots, lettuce, and potatoes, they also had “food jags.” ’ They maintained good digestion and nutritional health. “A tendency was observed in all infants to eat certain foods in waves, i.e., after eating cereals, eggs, meats, or fruits in small or moderate amounts for a number of days, there would follow a period of a week or longer in which a particular food or class of foods was eaten in larger and larger quantities until astonishingly large amounts were taken; after this, the quantities would decline to previous levels.” Religious beliefs may overcome the physiological forces governing the choice of foods (Anon., 1932). An incident illustrating this point has been told of six Russian priests and a boy attendant. The party arrived at Kharborova, Yugor Straits, in the autumn. The priests were prevented from eating meat by their religious vows; they, therefore, subsisted largely on salt fish as there were no vegetables available. According to the story the boy attendant was the only survivor by the next May, having buried his late masters in the snow. The boy had no religious scruples and lived largely on reindeer meat through the winter. Social prejudices and economic availability play critical roles in the proper choice of food. McCarrison (1921)described a “race unsurpassed in perfection of physique and in freedom from disease in general” in the State of Hunza situated in the extreme northernmost point of India. They subsisted on grains, fruits, and vegetables, some milk and butter, and goat’s meat on feast days. Elsewhere in India, populations choose polished rice as the food staple and suffer nutritional diseases curable by the unpolished rice. An investigation into the reasons for the choice of highly milled rice has indicated that the question is quite complex (Aykroyd et al., 1940). Some of the reasons for the choice of polished rice were found to be: 1. Convenience-it is easier to buy milled rice than to prepare “homepounded” rice. 2. Paddy cannot be bought in small amounts and the people neither can afford to buy large quantities nor do they have the place in which to store it. 3. Polished rice is somewhat cheaper than home-pounded rice.
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4. Of lesser importance is the desire of the poor to imitate the well-to-do. 5. The superior keeping quality of the polished rice is not an important
factor in the situation in India. It was suggested that the problem could be controlled by legislation making it obligatory that rice be milIed to contain not less than 1.5 pg. thiamine per g. In passing, it is worth mentioning that the influence of industrialists engaged in t,he manufacture and sale of foods can be very great in determining the choice of food by populations. With advertising as an extra tool, industrial interests sometimes become a very important factor in determining the choice of food by large populations. Therefore, the food industry has an increasing responsibility for the nutritional welfare of populations, a responsibility which most. industries are not aware of or choose to ignore. One of the best known studies on the ability of man to choose foods correctly is that of Orr and Gilks (1931) on the Masai and Akikuyu, 2 tribes living side by side in East Africa. The Masai live almost exclusively on meat, milk, and fresh blood, and they are healthy and vigorous. “Their neighbors, the Akikuyu, are agriculturalists, and although they maintain herds of goats, these are regarded as a form of currency and are rarely used to provide meat or milk. The diet of the men is almost entirely composed of cereals and potatoes, but, it being considered effeminate to eat green vegetables, these enter the diet only of the womenfolk.” As a result the Akikuyu men are “weedy and unhealthy.” The full grown Masai male is on the average five inches taller and 23 pounds heavier than the Akikuyu male. The Akiiuyu children suffer from various bone deformities, anemia, and enlarged tonsils. There are 3 causes here mitigating against the proper choice of a good diet by the Akikuyu: 1. Economic-The goats, instead of providing milk and meat, are used as a form of currency. 2. Social customs-Eating of vegetables is considered effeminate. As a result, the girls eating green vegetables are in better health than the boys. Even so, however, the diet was still insufficient and the Akikuyu girls were in poorer physical condition than the Masai girls. 3. Superstitions-Men ate much less green vegetables than the women because of the prevailing belief that “such food prevented them from being swift of foot if defeated in battle by the Masai.” Man does not always seem to profit nutritionally from experience. In Newfoundland and Labrador during the winter and spring, the population subsists largely on bread. Formerly “brown” flour was used to make the bread and beriberi was unknown. But with the advance of civilization,
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white bread replaced the “brown” bread and beriberi became a common occurrence. In 1910 a ship ran ashore laden with a cargo of whole wheat flour, and a considerable portion of the whole wheat flour, unloaded to lighten the ship, was consumed by the population of the adjacent districts. Following this event no beriberi was reported in that region for a year. Subsequently the population returned to the use of white flour and beriberi reappeared (Anon., 1932). The incident shows 2 things: (a) the human being is slow to learn and to take advantage of nutritional experience, and (b) the people had no objection to eating whole wheat bread, the white bread being used because of its availability and habit. Apparently man, unprejudiced by social, economic, religious, or other conventions, reacts physiologically to food in such a way as to choose a nutritionally adequate diet if it is available. The physiological reactions are, however, overridden by such factors as availability, cost, social habits, advertising, etc. As a result the life and health of populations often hang on a slender thread. There are some nutritional defects to which man reacts decisively and promptly. The best known of these defects is a low protein diet. when fed to human subjects, such a diet becomes unpalatable and the subjects finally lose interest in any kind of food (Sumner and Murlin, 1938). People on salt-deficient diets have a strong desire for salt; similarly, the desire for calcium during deprivation may become pronounced. In East Africa the search for edible earths rich in calcium resulted in tribal wars. Ashes from water plants with a very high calcium content enjoyed the greatest popularity among pregnant women. The most popular edible earths were those with a high calcium, sodium, or iron content (Om and Gilks,1931).
111. CAN ANIMALSCHOOSEFOODCORRECTLY? Animals seem to have retained the ability to choose a nutritionally correct diet to a much greater degree than man. Drummond (1934) described his observations with pigs on normal diets and on diets deficient in calcium and vitamin A. The pigs permitted to exercise in a yard behaved differently depending on what they had been fed. Those on the normal diet were quiet or strolled about the yard contentedly. “Those suffering from a deficiency of lime spent a large part of their time attempting to lick whitewash from the walls or root out a fragment of cement or mortar from the brickwork, whilst the shortage of vitamin A stimulated the animals of the third group to search for the smallest blade of grass or chance weed which might be growing in the cracks of the floor.” Cattle, fed grass growing on soil deficient in phosphorus, develop a craving for bones (osteophagia) and other phosphorus-containing material
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(Green, 1925). Young cattle raised in an area carefully cleaned of all bone debris showed osteophagia as soon as bones are offered them. There seems little doubt that cattle raised on phosphorus-deficient diets can distinguish between phosphorus and nonphosphorus containing foods. It is also known that cattle can distinguish hays of different nutritive qualities. They prefer hays grown on fertilized soil, and, when these are available, they ignore hay grown on unfertilized soils (Albrecht, 1945). Osborne and Mendel (1918) allowed rats a choice of 2 diets; one was nutritionally adequate while the other was inadequate either in vitamins or in the amount or quality of the protein. The rats chose the diet to suit their nutritional needs. Sometimes they ate the inadequate diet for weeks and failed to grow, but eventually they turned to the good diet. Sometimes, after subsisting on the good diet for a time and growing well, they ate the poor diet for a week or two, but they returned to the good diet before suffering any great loss in weight. These workers state, “It is, therefore, interesting to have this evidence that the desire of a young animal for food is something more than the mere satisfaction of its caloric needs. The demand made by the growth impulse must also be met by a food of the proper chemical constitution.” Normal rats can select a well-balanced diet adequate for growth and reproduction from the following food substances: olive oil, casein, sucrose, cod liver oil, wheat germ oil, yeast, sodium chloride, calcium lactate, sodium phosphate, and potassium chloride. They grow as well as rats on the standard McCollum diet, though they consumed 18.7% less calories (Richter et al., 1938a). Chicks allowed a free choice of foodstuffs consumed less protein, less fat, slightly more carbohydrate, and 10% less calories than did the chicks on a mixed diet of similar ingredients. The chicks in both groups grew and laid eggs at about the same rate. Obviously, the chicks given free choice must have utilized their food more efficiently (Pearl and Fairchild, 1921). Dove (1935) fed chicks, allowing them free choice, and found 2 types of behavior among the birds: those which chose wisely and those which chose unwisely. Chicks fed a mixture of the foods which had been selected by the sZow-growing birds grew faster and utilized their food more ejiciently than those which selected the food, while chicks fed a mixture of the foods selected by the fast-growing individuals grew slower and utilized their food less efficiently than those that selected the food. Apparently the order in which the nutrients of a diet are consumed is of importance in determining the nutritional value of the diet. While most of the literature on free choice feeding indicates that animals can select nutrients in accordance with their needs, there are nevertheless contrary findings (Kon, 1931; Scott, 1946a).
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IV. FACTORS INFLUENCINQ FOODINTAKE Mursell (1925) stated that one of the factors influencing food preferences is the “specific positive chemotropism for certain substances” which is registered most manifestly by way of the chemical senses of taste and smell. “Food preferences are ultimately based on the chemical needs of the organism, but with man social conventions come into the picture.” Young (1933) has discussed the maintenance of a constant internal chemical state in the organism as a factor which influences the voluntary food intake of animals. “The mammal maintains a relatively constant internal chemical state, and to do this he requires water, salt, protein, carbohydrate, fat, vitamins, etc. Any disturbance of the internal equilibrium may have its effect on the food preferences of the animal but the precise relationships between them need yet to be discovered. Free choice feeding experiments and studies of special food cravings and aversions show clearly that food selection has some definite relation to the metabolic state of the organism. Are there specific appetites for a particular food or general appetites for classes of foods, e.g., fats, proteins, carbohydrates, etc., as thirst is p craving for liquid?” Basic metabolic reactions involving the emyme systems may also determine food preferences (Richter el al., 1938b). Thus, on thiaminedeficient diets the enzyme system involving the oxidations of carbohydrate is affected, which results in a “biochemical lesion” (Peters, 1936). Such animals quickly lose their appetite for carbohydrates because the enzyme system is defective for the oxidation of some of the intermediates, especially pyruvic acid. The desire for carbohydrates is sharply reduced, resulting in a greatly decreased carbohydrate intake. A t the m e time, the animal, in an attempt to make up for its caloric needs, increases its fat intake. Whatever may be the mechanisms controlling food intake, the chief site of their action must be the cell. “When an added dietary component leads to an appetite stimulation, the explanation is to be found, we believe. in the influences exerted by the substance on the cells themselves” (Rose. 1928). To understand the continuing acceptability of foods, more exact knowledge must be obtained of the factors which control the voluntary intake of each nutrient. An aversion for just a single nutrient or the presence of a single deleterious component may render the diet containing such compounds entirely unacceptable.
V. TEE EFIPECT OF PROTICINS ON FOOD INTLack of protein in the diet has long been known to cut down the food intake. Rose (1938) states: “Growing animals lose their desire to eat
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when food is not suitable for tissue-synthesis, but regain it when all the components required for anabolism are made available.” Thus the presence of proteins in the diet is essential for the maintenance of appetite for food. However, the recognition of the need for proteins by the protein-depleted animal is a matter of disagreement. Scott (194613) states: “Rats either do or do not like casein; if they like it, they eat an average of 3 grams per day and grow well; if they do not, they eat less than 0.1 gram per day, lose weight, and die within a short period.” The effect of protein on food intake was shown by the observation that the rats which ate little casein consumed less than half the total calories ingested by rats eating an adequate amount. Thirty-four out of eighty-seven rats chose poorly but they may have been confused because the arrangement of the four cups containing casein, fat, sugar and salts was continually changed. Kon (1931) found a marked inability on the part of rats to respond to a protein deficiency. Rats offered sucrose or rice starch, casein, and a salt mixture in separate dishes and allowed a free choice consumed, on an average, only 6.5% protein. The animals were fed vitamins separately. On the other hand, Osborne and Mendel (1918) showed that rats can recognize diets of inadequate protein content, regardless of whether the inadequacy is one of quality or quantity of the protein. The ability of rats to choose correctly is often influenced by the conditions of the experiment (Warkentin et al., 1943). Rats offered a free choice of casein, dextrin, lard, yeast, and salt mixture, selected each constituent in such a manner as to support good growth. When the salts and yeast were mixed together, only 5 out of 13 rats chose a good diet because of a low casein intake. The absence of a protein appetite in this caae was possibly due to a low yeast intake which in turn may have been due to its being mixed with the salts. The low yeast intake probably resulted in a thiamine deficiency which depresses the protein intake (Richter et al., 1938b). Unidentified factors present in the proteins of natural foods may influence food intake. Such a factor has been found associated with pancreas protein (White and Sayers, 1942). Feeding pancreas increased body weight and food consumption beyond that which could reasonably be expected from the protein content itself. One or more such factors have been found associated with liver proteins. One such factor in liver increases growth by increasing the food intake. This is apparently a purely appetite effect since no difference of growth has been found in paired feeding experiments (Johnson and Palmer, 1934). Another liver factor increased both food intake and eaciency of food utilization. This may or may not be due to the factor described above. It is interesting to note
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that, when the liver was extracted with alcohol, it lost its growth-promoting properties (Seegers and Smith, 1932). The factor, or factors, in liver and pancreas may or may not be the same one which has been extensively studied by Cary and HaEtman (1943-1947). Animals deficient in the factor voluntarily decrease their food intake, and when the factor is administered, either parentally or orally, the animals will increase their food intake. Severe deficiency of the factor seems to cause kidney abnormalities. As the amount of dietary protein ingested goes up the need for the factor progressively increases. Inclusion of substantial amounts of lactose (25%) in the diet also increases the requirements for the factor. The factor is largely associated with animal proteins. The following foods contain the factor: liquid milk, cheese, liver or liver extracts, meat, fish, egg yolk, lettuce, alfalfa hay, timothy hay, and Kentucky blue grass. The following foods do not contain the factor: cereals, yeast, soy beans, linseed meal, egg white, carrots, and tomatoes From the distribution of this factor in the commonly used foodstuffs, it is hard to see how the food intake (appetite) of populations can be maintained without a liberal intake of foods of animal origin.
VI. THEEFFECT OF AMINOACIDS ON FOOD INTAKE While animals often seem to be able to select adequate quantities of protein, their ability to do so does not seem to be very great. It is possible that proteins, representing a heterogeneous mixture of nutrients depending on their amino acid composition, affect the food intake by virtue of their amino acids rather than the protein molecule itself. Frazier et al. (1947) have shown that each of the 9 essential amino acids is important in maintaining the rat’s food intake. As little as 9 mg. of the dl-tryptophane per 16 g. of food had a marked effect on the food consumption of the rat. The variations in the intake of food during acute amino acid deficiency may be caused by: (a) a decreased palatability of the food giving it a bad taste; (b) inadequate stimulation of the taste organs, thereby reducing the desire for food, or (c) sickness of the rat leading to loss of interest in food generally. After removal of an indispensable amino acid from the ration, the rats continue to eat well for one day, then the food consumption declines and remains low. If taste were the determining factor, it might be expected that the rats woidd have decreased their food intake at once. There is evidence to show that a general metabolic disturbance is the factor responsible for the decreased food intake. Frazier et al. (1947) quote Rose to the effect that human subjects, who are in negative nitrogen balance, feel miserable and their appetite is diminished. After the nitro-
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gen balance is reestablished they again feel well and their appetites improve. “It is possible, therefore, that a similar mechanism may operate in the rats and that their disinclination to eat may be due largely t o the fact that they actually feel too ‘sick’ to be interested in food, even though they are severely undernourished and need the food to promote effective convalescence.” It is possible that the deficient animal recognizes the essential nutrient in 2 ways: (a) either it feels miserable or “sick” and the ingestion of food relieving this condition may result in a sense of “well-being,” or (b) the deficiency of the nutrient may decrease the sensory threshold for the nutrient or otherwise change the taste of the nutrient and may thus impart to it a more pleasant taste. The essential amino acids must, therefore, be present in diets if animals are to consume them in adequate amounts. Yet, a rat subsisting on a diet deficient in an essential amino acid possmses but limited ability to choose the diet containing the needed amino acid when it is confronted with a choice of 2 diets, one containing the amino acid and one lacking it (Geiger, 1947). The remarkable results obtained with the essential amino acids are not duplicated with other amino acids. For instance, the amino acid cystine does not seem to affect food intake very markedly, although it does influence metabolism since it increases the efficiency of food utilization. “There is no more vexing problem than that of food intakes and their interpretation. The writer is of the opinion that there is no evidence in our records which indicates that cystine affected the palatability of the rations fed.” (Haag, 1931). Beadles et al. (1930) did not find that cystine increased food intake, although it increased the weight of the animals in comparison with animals ingesting the same amount of food but without cystine. Griffith (1941) observed that cystine increased body weight and length of rats while increasing their food consumption little or not at all. The principal effect of the cystine was an increased efficiency of utilization of food. Still, under some conditions cystine apparently may cause an increase in food intake (du Vigneaud et al., 1942). For example, it was found that supplementary cystine increases growth and food consumption on a diet containing 0.2% dl-methionine.
VII. THEEFFECT OF WATER~OLUBLE VITAMINS ON FOOD INTAKE Thiaminedepleted rats when offered a choice of 2 diets, one deficient and one containing just sufficient thiamine in some distinctive form (marmite or wheat germ), will invariably prefer the latter diet (Harris et al., 1933). If the diet has more than the required amount of the B vitamin, the rats will no longer eat only the vitamin-containing diet but the other diets as well. If a large selection of diets is placed before the depleted
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rat it may be unable to “discover” the one containing the vitamin. However, if the rat is first “educated,” by being fed the vitamin-containing diet alone for a few days, this experience enables it to make the correct selection when confronted with a large variety of diets. The rat may also be “deceived” by transferring the vitamin concentrate to a diet flavored with cocoa instead of bovril, but it can be “reeducated” again t o choose whichever diet contains the vitamin. The ability of the rat to choose vitamin B-containing diets correctly depends, therefore, on (tsaociating the distinctive property of the diet with an experience of its prompt beneficial effects. The selection of food is often influenced by the presence of deleterious components in the food. In that case, the selection becomes a contest between the need for vitamins and a defense against a deleterious component. Two diets, one containing vitamin B complex in the form of protein-free milk and the other low in vitamin B complex, were offered to rats (Mitchell and Mendel, 1921). If an adequate amount of the first vitamin containing diet was ingested, it furnished enough protein-free milk to cause diarrhea. The rats ate both diets, but they did not eat enough of the diet containing protein-free milk with the result that they did not get sufftcient vitamin B complex for normal growth. When the rats were fed yeast separately, their intake of the vitamindeficient food increased while the intake of the vitamin-containing food decreased. Thiamine differs from the other members of the vitamin B complex. The outstanding characteristic of the thiamine deficiency is a decreased food intake (Cowgill, 1934). The requirement for thiamine is proportional to the body weight of the animal. Decreased food intake is more pronounced in the case of thiamine deficiency than in a deficiency of riboflavin, pyridoxine, or Factor 2 (Dimick, 1940). Thiamine increases the efficiency of food utilization moderately (Sure and Dichek, 1941a), but riboflavin is much more effective in this respect (Sure and Dichek, 1941b). The claim, that thiamine has an effect on growth unrelated to the actual food intake, is disputed. It has been found that rats receiving thiamine lost weight at the same rate and failed to survive as long as their deficient pair-mates (Voris et aE., 1942). (‘Riboflavin, pyridoxine, and pantothenate have specific growth-promoting effects unrelated to appetite. Thiamine does not seem to have a specific effect on growth separate from its effect on appetite, when sexes are not differentiated.”--“With insufEcient thiamine, the appetite of the rat would drop to sub-maintenance levels and the disinterest in eating would continue until additional thiamine was provided. With insufficient riboflavin, the appetite depression appeared to be the result of a general physiological debility of an unspecific nature. With insufficient pyridoxine, there were sudden and temporary lapses in appetite
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followed by an equally prompt recovery. Pantothenate anorexia seemed to follow the pattern of riboflavin insufficiency.” (Voris et al., 1942). Other reports have been made on the effects of vitamin B-complex deficiencies on food intake. Pyridoxine deficiency causes a decrease in the intake and in the efficiency of food utilization of the dog (Street et al., 1941). Food intake also decreases in the pantothenic acid deficient dog (Silber, 1944). In nicotinic acid deficiency, both the food intake and the efficiencyof food utilization of rats decrease (Spector and Mitchell, 1946; Krehl et al., 1946). In rabbits a nicotinic acid deficiency results only in a decreased food intake, without loss in efficiency of food utilization or any pathological changes (Wooley and Sebrell, 1945). Biotin deficiency decreased the food intake of the monkey (Waisman et al., 1945). Choline deficiency decreased the food intake of the dog (McKibbin et al., 1945). The ability of rats to recognize dietary pantothenic acid is apparently weak and, except under special conditions, has been denied. With the help of added flavors, however, rats were made to recognize pantothenic acid (Scott and Quint, 1946). “Appetites for foods containing thiamine, riboflavin, or pyridoxine are developed in rats whose diets have been deficient respectiveIy in these vitamins. These appetites are not simple preferences because they cannot be found in normal animals. They must, therefore, be either learned appetites or hungers.”-“The lack of appetite for pantothenate under the same conditions may be due either to an insufficient stimulus, i.e., incapacity of the animal to appreciate the beneficial effect of this vitamin, or to an inability to distinguish between the two diets, or to both.’’ Thus, an appetite for pantothenate can be demonstrated with foods so flavored that the animal can associate a diet of a certain flavor with the resulting beneficial effect of its consumption. A more frequent use of self-selection techniques could be profitable in nutritional studies. In this way Richter et aZ. (1938b) were able to show that a deficiency of the vitamin B complex caused an anorexia for carbohydrate and protein and a craving for fat and yeast. The following changes took place in the food intake of rats on the vitamin B complex deficient diet: (a) the intake of protein decreased from 26 to 5 caloric Yo; (b) that of carbohydrate decreased from 51 to 11 caloric %; while ( c ) the intake of fat increased from 23 to 84 caloric %. VIII. THEEFFECT OF FATS AND FAT-SOLUBLE VITAMINSON FOODINTAKE
,
1 . Fats
Rats show no special tendency to eat more of a fat-containing than of a fat-free diet, although they grow better on diets containing fat. This
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effect may be due to a greater efficiency of food utilization or to the greater caloric density of the fat-containing diet or both (Henderson et al., 1945). Bile seems to play a role in the fat intake of rats (Richter and Birmingham, 1942), because ligation of the rat’s bile ducts reduces their voluntary fat intake. No difference in food intake had been found in rats on diets with and without the essential unsaturated fatty acids, although deficient rats weighed one-fifth less than the control rats (Burr and Burr, 1930). 2. Fat-Soluble Vitamins
The growth rate of chicks deficient in vitamin K is not directly affected until the hemorrhagic condition becomes severe (Almquist and Stokstad, 1930). On the other hand, in the vitamin A deficient mammal, there is a decreased food intake which can be restored by feeding this vitamin (Guilbert et al., 1940). Vitamin D deficiency also deareases the food intake of chicks (Baird and Greene, 1935). The administration of the vitamin D to deficient rats, however, increases or decreases food intake depending on the character of the rachitic diet fed. On a low phosphorus rachitic diet, vitamin D may decrease food intake of rats (Schneider and Steenbock, 1939); on a low calcium rachitic diet, the vitamin increased the food intake (Zucker et al., 1938; Krieger et al., 1940). Vitamin E seems to have little effect on food intake. Food consumption (in terms of calories) in chicks is the same on diets with or without vitamin E. Growth, too, is the same whether or not the ration is deficient in this vitamin (Pappenheimer et al., 1939). However, rats on a vitamin E deficient diet show a decreased growth rate a t the tenth week and by the eighteenth week the growth curve flattens out at subnormal weights. It is not known whether this effect may be attributed solely t o a decrease in food intake (Evans, 1928).
Ix. THE ROLEO F
ESSENTIAL
MINERALSON FOODINTAKE
1 . Phosphorus
Decreased and depraved appetite, osteophagia, and coprophagy have been noted in cattle on low phosphorus diets (Green, 1925). McCollum el al. (1939) described phosphorus deficiency in rats. Weaned animals restricted to a phosphorus deficient diet grew slowly for 2 4 weeks, then declined and died after 7-9 weeks. Except for the effect of magnesium deficiency these rapid and lethal effects of phosphorus deficiency are without parallel. During deprivation phosphorus is transferred from the bones to the soft tissues. Interestingly, the animals show fairly good appetites throughout the first half of the survival period; no
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symptoms of pica (abnormal appetite) on low phosphorus rations were noted. Forbea (1937) summarized the abnormalities which result from the feeding of low-phosphorus rations, some of which are: (1) the lowering of the inorganic phosphorus of the blood; (2) poor utilization of feed and storage of energy; (3) a loss of appetite. He further stated, “TOspecify the function of phosphorus in animal metabolism is to enumerate the vital processes of the body; the integrity of these processes being maintained by safety provisions of various kinds, including stores of phosphorus, mainly in the skeleton, of extents commensurate with the demands likely t o be made upon them, and also with the supreme importance of the animal economy as a whole.” d . Calcium Calcium deficiency in young rats results in poor growth due to decreased food consumption (Boelter and Greenberg, 1941) and the animals show decreased reactivity and sensitivity to noises. The skull and brain of the rats which died in the cages were eaten by the surviving animals, indicating a craving for a certain type of nutrient, presumably present in the parts of the carcass consumed.
3. Magnesium Magnesium deficiency in rats causes a sharp drop in food intake and a greatly reduced eflsciency of food utilization (Kleiber et al., 1941), but the activity of the rat is not appreciably affected. One of the outstanding symptoms of this deficiency is a hyperirritability of the nervous system characterized by convulsions. Even the hissing of an air blast or the sound of rapidly running water throws a magnesium-deficient animal into convulsions. By the use of drugs, the lesion concerned with hyperirritability has been localized in the midbrain or pons (Greenberg and Tufts, 1938). Feeding magnesium to the deficient rats increased their food intake and the efficiency of food utilization (Tufts and Greenberg, 1938).
4.
Sodium
In one study on the effect of sodium deficiency it has been shown that this decreases the food intake and the efficiency of food utilization (Kahlehberg et al., 1937). 6. Potassium
Rats on low potassium diets grow slowly but steadily. The slow growth rate is the result of a decrease in food intake. The rats exhibit a striking alertness and a peculiar pica indicating a craving for some nutrient.
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They appear to be constantly searching for something and licking each other’s genitals (as well as their own), especially immediately after urinating. Coprophagy is not observed (Orent-Keiles and McCollum, 1941). 6. Chlorine
Chlorine deficient rats grow more slowly than controls, though their food intake is the same or greater. The greater efficiency of food utilization of the chlorine-fed rats enables them to make a greater gain with a lower food intake (Greenberg and Cuthbertson, 1942). When the chlorine deficiency is made more severe the efficiency of food utilisation decreases still further and the food intake also decreases somewhat (Cuthbertson and Greenberg, 1945).
7. zinc In a single study of sinc deficiency it has been found that the food intake and the efficiency of food utilisation decreased (Stirn et at., 1935). 8. Cobalt Cobalt deficiency sharply reduces the food intake of ruminants (McCollum el al., 1939). Since cobalt is without effect when it is injected, it would appear that the cobalt acts not directly on the host, but upon some of the organisms in the nunen. This perhaps explains why horses do not suffer from cobalt deficiency (McCance and Widdowson, 1944). 9. Manganese
Rats on manganese deficient diets show no difference in growth and appearance. It may be assumed, therefore, that there is also no difference in their food intake (Orent and McCollum, 1931). On the other hand, manganese deficiency in chicks retards growth and this is presumably due, at least in part, to a decreased food intake (Wilgus, 1939). 10. Iodine
Iodine deficiency in rats produces no changes in food intake and no pathological changes have been noted (Jackson and P’an, 1932). On a goiter-producing ration, however, iodine prevented thyroid enlargement and caused an increme in the iodine content of the thyroid of rats though it had no effect on the body weight (Levine et al., 1933). 11. S e h u m Voluntary inanition is caused by selenium. The intake of a diet containing seleniferoue corn was reduced approximately by half (Franke, 1934). €tats fed diets with varying amounta of selenium (as sodium
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selenite or as selenium in organic form) distinguished between food containing small increments of selenium (Franke and Potter, 1936). I d . Fluorine Fluorides, when fed to animals, retard growth and lower the food intake (Kick et al., 1935).
X. ENDOCRINES AND FOOD INTAKE Not only do the nutrients present in diets influence the food intake of animals, but the endocrines are also of utmost significance in this matter. Much information concerning the effect of the endocrines on food intake has been obtained by removing the glands and studying the effect of the addition of the missing hormone to the diet of the experimental animal (replacement therapy). Deficiencies and special needs produced by glandular removal are definitely reflected in changes in the intake of nutrients. The operated animal is somehow guided to ingest nutrients which help to correct the deficiencies as well as to maintain normal growth and health (Richter, 1942). This technique of investigation has been used effectively in obtaining information on the effect of the endocrines on food intake by allowing the operated animals a wide choice of diets and determining the preferred nutrient associated with the removal of the particular endocrine gland. Injection of anterior pituitary hormone into hypophysectomized rats causes an increase in the food intake and a more efficient food utilization, since injected animals gain more weight than the controls on the same food intake. The increased gain was associated with a retention of nitrogen (Lee and Schaffer, 1934). The food consumption of rats decreased following removal of the hypophysis. Paired-fed controls lost weight less rapidly than did the hypophysectomized rats during the first week, but afterwards they lost weight a t about the same rate. Hypophysectomy also changed the character of the metabolism of the rats. When all the rats were placed on a sub-maintenance level of food intake, the control rats lost 262’ g. or 60% of their initial body fat, but retained their fat-free dry tissue weight including their body nitrogen. The level of food consumption was sufficiently low to cause a marked depletion of body fat stores, but not so low 8s to necessitate utilization of the body nitrogen for energy purposes. On the other hand, the hypophysectomized paired-fed mates lost only 121 g. or 28.3% of their initial body fat, They also lost 17.3 g. or 18.6% of their original body nitrogen as well (Lee and Ayres, 1936). The hypophysectomized rats cannot conserve their body protein nor utilize their body fat as well as normal rats.
* This figure Beem high for a rat.
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Adrenalectomired rats show a much greater intake of salt than normal animals (Richter, 1936). Injection of desoxycorticosterone acetate in adrenalectomized rats caused a decrease in intake of salt associated with better retention. Injection of desoxycorticosterone acetate into normal rats caused an increased salt intake also associated with increased salt retention but a decreased food intake (Rice and Richter, 1943). The intake of calcium by parathyroidectomized rats was increased but returned to normal when parathyroid was implanted in the anterior chamber of the eye (Richter and Eckert, 1937). In parathyroidectomized rats an increased retention of phosphorus was observed when the animals were fed a low-calcium, high-phosphorus diet. Apparently such animals cannot eliminate excess phosphorus without calcium. When the phosphorus concentration in the body reaches a certain level the animals refuse to eat low-calcium-high-phosphorus-containing diets. The animals partake of a small amount of such diets, then suddenly stop eating it. “That the mechanism ‘may be’ neurogenic is suggested from the fact that the animals taste the food first and then stop eating.” Incorporating calcium in the diet, however, makes the food acceptable because it enables the parathyroidectomized animal to excrete the excess phosphorus (Shelling, 1932). Depancreatized rats eat little sugar and much fat. When forced to eat carbohydrate, the food and water intake of the rats increases because sugar and water are being lost in the urine and the increased food and water intake is probably an attempt to compensate for these losses. Yeast and liver powder intake are also increased in such animals indicating that some vitamins may also be involved in this condition (Richter and Schmidt, 1941). Insulin seems to increase food intake. “The study indicates that insulin does affect the hunger drive as measured by the rate and number of feeding responses. The effect may be a depression or a facilitation depending upon the time relations between injection and feeding” (Morgan and Morgan, 1940). “The dietary control of insulin is interesting. On heavy feeding, the Islets of Langerhans hypertrophy, producing more insulin to take care of the excess sugar. However, this often sets up a vicious cycle: the more food, the greater the hyperinsulinism, and the greater the hyperinsuliism the greater the desire for food. This leads to obesity and frequent degeneration of the Islets of Langerhans due to overwork” (Brody, 1945). Gastric motility in man has been shown to be increased by insulin injection. Insulin sensations, “especially hunger, parallel rather closely the degree of gastric motility. This parallelism may provide a rationale for the use of insulin for the relief of anorexia” (Quigley et al., 1929).
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Thyroidectomiaed rats have a smaller food intake (Warkentin et al., 1943) while an excess of thyroid brings about an increase in food intake (Samuels, 1947).
XI. THEROLE)OF DELETERIOUS COMPOUNDS ON VOLUNTARY FOOD INTAKE The presence of deleterious substances in foods is more common than generally has been supposed. Animals seldom, if ever, consume poisonous plants when good forage is available. In its absence, they will eat poisonous plants (Sampson and Malmsten, 1935). The glucosidal alkaloid, solanine, is found in the potato. It is distributed throughout the plant but only very small quantities are found in the pulp of the tuber. It is concentrated in the peel, especially in very young and sprouting potatoes and is also found abundantly in greencolored skin and flesh. The amount of solanine found in potatoes is ordinarily too small to be harmful. Under conditions not yet understood, however, its content may rise to toxic levels (Sollmann, 1942). Cereals contain phytic acid which interferes with the absorption of calcium. The resulting calcium deficiency may in turn decrease food intake. Whether or not the decreased absorption of calcium caused by the phytic acid is ordinarily suflicient to affect food intake is a moot question (McCance and Widdowson, 1942). Flour, improved and bleached by NCL, the “agene” process, contains a toxic compound causing canine hysteria or running fits in dogs. Untreated flour does not cause this disturbance. Affected dogs returned towards normal when the treated flour W L ~ Breplaced by the same flour which was not treated (Mellanby, 1946). (See Addendum, p. 148.) Some foods contain compounds which are injurious for some species of animals but not for others. Rye seems to have a deleterious effect on chich, causing “sticky” droppings, a condition which occurs only in young chicks. If the chicks are kept on a good ration for about 4 weeks and then fed rye, no such difficulty is experienced (Halpin et al., 1936). Dehydrated alfalfa when fed free-choice to chicks is immediately unacceptable, the chicks refusing to eat it (Dove, 1935). The reaaon for this is unknown. Cottonseed and linseed meal are protein concentrates of great interest in this connection because both contain deleterious compounds exerting different effects on different animals. Cottonseed contains gossypol which depresses food intake and growth in pigs, but linseed meal does not. In chicks, on the other hand, cottonseed is well utilieed but linseed depresses food intake and growth. Both protein concentrates are equally well utilized when fed on the same protein-brtsis to rats (Bethke et al., 1928).
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Rats are known to be resistant to gossypol poisoning. When fed in excessive amounts, however, a decrease in food intake occurs, which is the characteristic symptom of gossypol intoxication (Schwartae and Alsberg, 1924).
Buckwheat contains a fluorescent dye which sensitizes animals with fair skins to light and causes “buckwheat itch” when eaten. Albino animals (guinea pigs, rabbits) quickly die if they are fed liberally on buckwheat and then exposed to sunlight. Pigmented animals, on the other hand, are not seriously affected (McCollum et al., 1939). Rats fed a ration in which all the proteins and B vitamins are supplied by the mycelium of Aspergallua sydowi fail nutritionally due to the presence of a toxic compound of unknown composition (Woolley et at?.,1938). Some food toxins exert their action indirectly by destroying some essential nutrient. Certain raw fish contain an enzyme which destroys thiamine, indirectly causing thiamine deficiency. This induced deficiency can be corrected by feeding a sufficient quantity of the vitamin (Green and Evans, 1940). Another such example is 3, 3‘-methylene-bis-(4-dihydroxycoumarin) which occurs in spoiled sweet clover. This compound interferes with prothrombin formation causing a lowered prothrombin content of the blood and hemorrhages. Vitamin K counteracts the hemorrhagic tendencies of this compound (Link, 194344). Rancid fats contain injurious compounds that act, in part, by destroying fat-soluble vitamins in food, especially vitamins A and E. The destructive action of the rancid fat often takes place in the intestinal tract of the animal. In some herbivorous animals, the destruction of vitamin E through the action of rancid fats is manifested as muscular dystrophy (Madsen et al., 1938 ;Mattill and Golumbic, 1942). Rancidity occurs most frequently in fats having fatty acids of a high degree of unsaturation. Vitamin E deficiencies are generally believed to be due largely to the destruction of a-tocopherol in the diet. However, this is disputed by some who contend that, e.g., encephalomalacia in chicks is caused by the fatty acids themselves (highly unsaturated fatty acids from lard), and not by the destruction of the vitamin E in the diet. The damage must, therefore, be caused by the fatty acids entering the tissues and is prevented by tocopherol (vitamin E) (Dam, 1944). The consumption of corn has long been associated with pellagra and has been considered a causative agent. Recently a “pellagragenic” factor in corn has been reported and it has been found that nicotinic acid counteracts its effect (Woolley, 1946). Perhaps the most important and most widely studied foodstuff containing injurious substances is the soy bean. It has been widely used aa a
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food in the Orient, where, however, it is seldom consumed in “unprocessed” form as are other legumes. Digestive disorders have been reported to occur when unprocessed soy beans are used (De et al., 1945). Many Americans who were Japanese prisoners in World War I1 reported that soy beans were unpalatable and refused to eat them, even when starving, because of the nausea, vomiting, and diarrhea which followed their ingestion (Cartwright and Wintrobe, 1946). It is possible that the digestion and utilization of unprocessed soy beans depends, at least in part, on the nutritional state of the subject. This is suggested by the results of an experiment with 2 well-nourished subjects who complained of flatulence when fed either raw or autoclaved soy beans but no other gastrointestinal symptoms were observed. In the subjects fed the heated beans there was about 20% greater nitrogen retention (Lewis and Taylor, 1947). More work along these lines is necessary. Soy beans also possess a goitrogenic factor which causes an enlargement of the thyroid. This effect is counteracted by iodine administration. The depressing effect on egg production and reproduction in domestic fowl, caused by a simplified ration containing soy bean oil meal, however, is not overcome by iodine (Wilgus et aE., 1940). Navy beans contain an antiamylase which inhibits the digestion of starch (Bowman, 1945). During dehydration and storage, most foodstuffs deteriorate gradually and lose their palatability. The reactions involved in such food deterioration have been extensively studied during the past few years. Many of these involve a reaction between amino acids (or the nitrogen groups of proteins) and some other reactive compound such as reducing sugars (Maillard reaction). Changes in color, texture, and flavor take place in the deteriorated products. The color is often dark brown, or even black. Apparently the darkening is nonenzymic in character (Weast and Mackinney, 1941).2 The nature of the compounds formed during these deteriorations and their role in nutrition need to be studied further. It is possible that some of the compounds formed during the deterioration are injurious and may decrease food intake (loss of palatability).
XII. PROTECTION AGAINSTDELETERIOUS COMPOUNDS Considering the wide distribution of deleterious substances, it is remarkable that most of them have escaped recognition for so long. There are perhaps many reaspns for this. First, many such compounds are destroyed in the processing of raw foods. Then, some compounds are counteracted by other nutrients present in the diet. In still other cases, the organism adapts itself to the effects of these compounds. For a complete review of the literature, aee E. Stadtman, this volume.
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I. Elimination of Deleterious Compounds from the Diet a. Heat. Compounds of a protein nature are destroyed by heat coagulation during canning or cooking. Among the compounds in this class are the antitrypsin of soy bean (Osborne and Mendel, 1917) and of egg (Balls and Swenson, 1934), the avidin of egg white (Eakin et al., 1941), the toxic principle of the mycelium of Aspergillus sydowi (Woolley et al., 1938), and the thiamine-destroying enzyme present in fish (Green and Evans, 1940). b. Fermentation. Fermentation will of ten destroy deleterious substances. Much of the phytin in whole wheat bread is destroyed during fermentation (McCance and Widdowson, 1942). c. Germination. Germination has been shown to improve the nutritional quality of the soy bean, and this is presumably a consequence of the destruction of some of the deleterious compounds (Everson et al., 1944). d . Chemical Treatment. Mild hydrolysis will destroy the toxic compoiind in the myoelium of Aspergillus sydowi (Woolley et al., 1938). Sometimes a very simple treatment will eliminate injurious compounds; e.g., linseed meal can be detoxified simply by soaking it in water for about 12 hours and subsequent drying (Kratzer, 1947). e. Destruction in the Digestive Tract. Deleterious compounds are sometimes destroyed during digestion, either by the enzymes contained in the food itself or by enzymes secreted into the intestinal tract or by the enzymes furnished by intestinal bacteria. About one-half, and sometimes even much more, of the phytin fed to man disappears during passage through the gastrointestinal tract (Cruickshank et at., 1945). f. Conversion into Nontoxic Compounds. Toxic compounds can be converted chemically to nontoxic compounds. Gossypol combines with iron, whereby its toxic action is largely dissipated (Withers and Carruth, 1917). Benzoic acid is rendered nontoxic by its physiologic conversion into hippuric acid in the body. 9. Adsorption. Lloyd’s reagent, when fed with the mycelium of Aspergillus sydowi, renders it nontoxic (Woolley et al., 1938). Presumably the compound is so strongly adsorbed by this reagent that it is eliminated in the feces. 2. Counteraction of Deleterious Compounds by Specific Nutrients
Many injurious compounds exert their action by producing a deficiency of one or more essential nutrients. Usually the diet contains the particular nutrient in sufficient excess so that the deficiency does not develop. But if the conditions are such that the deficiency does develop, it can be
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overcome by adding the necessary nutrient to the diet. Some of the deficiencies known to be produced by such compounds are: 1. Biotin deficiency from the ingestion of raw egg white. This deficiency has been produced experimentally and it can be prevented by the addition of biotin (du Vigneaud et al., 1940). 2. Calcium dejiciency. Since phytin inhibits the assimilation of calcium, this defect can be overcome by adding additional calcium to the diet (McCance and Widdowson, 1942). 3. Methionine deMenq. Raw soy beans cause a methionine deficiency which can be corrected by feeding additional methionine (Hayward and Hafner, 1940). 4 . Thiamim dejiciency. Certain fish contain a special enxyme which destroys thiamine during digestion, thus causing a vitamin deficiency. The addition of thiamine equal to, or in excess of, that destroyed will either correct this deficiency or will prevent its occurrence (Green and Evans, 1940). Injurious compounds may be counteracted by agents which are not ordinariIy considered to be nutrients; e.g., selenium poisoning is counteracted by arsenic (Dubois et al., 1940). Feeding proteins will also counteract selenium toxicity (Gortner, 1940). It is not known whether the protein furnishes a specific nutrient or counteracts the selenium in some other obscure manner. 3. Physiological Adaptation to Deleterious Compounds The animal organism can sometimes adapt itself (within certain limits) to adverse dietary conditions. For example, the rat adapts itself to dietary fluorine by progressively increasing the elimination of fluorine in its feces and urine (Lawrena et al., 1940). Also, when forced to live under conditions of restricted water intake, rats will adapt themselves to living on less water. When rats are allowed just enough water to maintain their body weight, their daily water consumption necessary to maintain their weights decreased from 8 to 4 ml. (Jackson and Smith, 1931). In this connection it is interesting to point out physiological reactions which occur when soy beans are fed to chicks. The pancreas of chicks fed raw soy beans hypertrophies and feeding methionine does not alter this condition. But when properly heated soy beans are fed, the weight of the pancreas is restored to normal. The amylase content of the pancreas of the chicks fed raw soy beans decreases. The feeding of methionine or of properly heated soy beans greatly increases the amylase content of the pancreas of the chicks. Methionine may play a definite role in determining the amylase content of the chick’s pancreas (Chernik et al., 1947).
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One of the most interesting demonstrations of adaptation is that of man to the phytin present in high extraction bread (Walker et d.,1946). When human subjects were fed a high extraction bread (containing considerable phytin) they showed a negative calcium balance which slowly decreased until calcium equilibrium was reestablished. Still later the subjects showed positive calcium balance and regained most of the calcium lost during the previous period. The mechanism of this remarkable adaptation is unknown.
4. Decrease in Food Intake as a Protection against Deleterious Compounds Perhaps the most frequent defense used by animals against toxic compounds is to limit their food intake so that the ingestion of the compound is reduced to nontoxic levels. The difficulty with this mode of adaptation is that the need for calories to sustain the life of the animal is critical. This results in a physiological struggle between the factors stimulating an increase in food intake (need for calories) and those decreasing food intake (defense against toxic compounds), Under some conditions, the animal seems almost rational in its reactions; e.g., when rats are confronted with diets containing varying amounts of selenium, they will choose the diet with the lowest selenium content. When that diet is removed, the rats will still choose the diet containing the least selenium from among the remaining selenium-containing diets (Franke and Potter, 1936). The important question atill remains unanswered: What mechanisms enable the animal to choose its food so accurately, so often? XIII. PHYSIOLOGICAL MECHANISMS DETERMINING FOODINTAKE The mechanisms which control food intake in the normal animal are extremely well balanced. “Work output and heat production are controlled in such a way as to maintain an equilibrium between energy intake and energy output; as a result, the amount of energy stored as fat remains almost constant from day to day and from year to year, even though the organism must submit to variations in diet, in work and in environmental temperature. The precision with which this equilibrium is maintained suggests that there must be some quantitative regulation and integration of the variable factors upon which this equilibrium rests, i e . , of food intake, work output and heat loss.” (Brobeck, 1946a). The regulation of energy intake (food intake) must be regarded as one of the most important functions of a normal animal. 1 . Food Intake and Caloric Output An increased food intake is generally associated with an increased caloric expenditure caused by increased work, a cold environment, or thyroid
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feeding. Under certain conditions, increased caloric expenditure is not balanced by increased food intake due to some failure in the regulatory mechanism of energy exchange and the animal loses weight. Such is the case with rats kept at excessively high temperatures when the caloric expenditure increases without a compensating increase in food intake. The rats drink large quantities of water but their food intake does not increase and may even decrease (Brobeck, 194613). A nutritional deficiency which is associated with an increased caloric output has been studied in animals fed thyroxine or desiccated thyroid. The animals lose weight because of insufficient food intake caused by intervening deficiencies of thiamine, pyridoxine, and pantothenic acid. Addition of these vitamins brings about an increase in food intake, and the animals regain lost weight (Drill, 1943). Under some conditions of thyroid feeding a deficiency due to the absence of an unknown nutrient also develops in addition to the deficiencies of thiamine, pyridoxine, and pantothenic acid already mentioned. This obscure deficiency has been studied by Ershoff (1947) who found that liver stimulates the food intake. Liver, therefore, contains an unknown nutrient which increases food intake under these conditions. These studies suggest that when we consider the relation of caloric output to food intake, the possibility of the existence of intervening nutritional deficiencies must not be overlooked. 2. Growth and Food Intake
Increased growth is associated With increased protein utili~ationor retention. Protein retention is accompanied by an increase in caloric intake (Barnes and Bosshardt, 1947). 3. Internal Environment and Food Intake
The maintenance of a constant internal environment is frequently referred to as one of the powerful determinants controlling food intake (Young, 1941). It has been suggested that the amount of water in the tissues is related to food intake, which is decreased by dehydration and increased by hydration. The large food intake which is associated with obesity has been attributed to a general hydration of the tissues (Hoelzel, 1945). The decrease in food intake caused by dehydration has already been discussed (Jackson and Smith, 1931). Changes in the internal environment, following the removal of endocrine glands, are followed by sharp changes in the intake of specific nutrients, such as sodium chloride in the adrenalectomized rat (Richter, 1936), or calcium (Richter and Eckert, 1937), and phosphorus (Shelling, 1932) in the parathyroidectomized animal. The maintenance of the glucose content of the blood and tissues, and
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its metabolism, seems to be closely linked with food intake. Glucose is a key compound in the energy metabolism of the brain. “A considerable amount of evidence has been accumulated from various sources, which indicates that glucose is the main, if not the only, source of energy for the brain” (Mann, 1944). The major symptoms of hypoglycemia indicate that the functions of the central nervous system are primarily affected by an inadequate supply of glucose.
4. The Possible Role of Enzymes The utilization of proteins and the metabolism of glucose involve complex enzyme systems. The enzyme systems through which the energy of glucose is made available for synthetic and other metabolic reactions may be of importance in the control of food intake. Some of the centers influencing the food intake are in the brain (Brobeck, 1946a), and the brain depends on glucose for energy (Mann, 1944). It can, therefore, be expected that disturbances in carbohydrate metabolism would produce changes in the brain which could affect the food intake. Thiamine deficiency is an example of a disturbance in carbohydrate metabolism which is immediately reflected in a decreased food intake (Cowgill, 1934). The metabolite, pyruvic acid, cannot be properly metabolized (Peters, 1936) resulting in a biochemical lesion associated with a sharp decline in food intake. Addition of thiamine to the diet causes an immediate increase in the food intake, which is probably a consequence of the restoration of normal carbohydrate metabolism. Phosphorylation of glucose is a process essential for its conversion to glycogen or fat, or for its oxidation to supply energy needed by the living organism (Cori, 194546). Some of the reactions of glucose which are made possible by its phosphorylation are shown below: glycogen hexokinase
TL
+ PO4
glucose ____ .+ glucose - 6P04 A.T.P.
Pyruvate
+ POc
From this it can be seen that phosphate is essential to phosphorylate glucose and that phosphate is liberated when the phosphorylated glucose is converted to glycogen, or when pyruvic acid is formed. Pyruvic acid is then metabolized and presumably either is converted to fat or amino acids, or is oxidized. Phosphate, therefore, plays a vital role in the metabolism of carbohydrates and its concentration in the cells should
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have a decisive influence on the equilibria of the metabolic reactions of glucose. The following speculation may be made here. If the concentration of phosphorus in the cell is too low, glucose cannot be efficiently phosphorylated and it cannot, therefore, serve as a source of energy. If the concentration of phosphorus in the cell is too high, the equilibria involving the liberation of phosphate are disturbed and the phosphorylated carbohydrates (or their derivatives) liberate phosphate with difficulty, which interferes with the formation of such compounds as glycogen and pyruvic acid. Accordingly, excessive phosphate concentration in the tissues should interfere with carbohydrate metabolism. Interference with carbohydrate metabolism should interfere with the energy exchange of the animal and thereby decrease the food intake. Low or excessive phosphate concentration in the tissues would affect all phosphorylating enzymes and it is therefore possible that food intake would be affected by metabolic disturbances other than carbohydrate metabolism. There is considerable evidence showing that a low phosphate intake decreases the total food intake: 1. A low phosphate diet decreases food intake; the addition of phosphorus causes an increase in food intake (Kleiber et al., 1936). 2. Vitamin D deficient rats kept on low phosphorus diets do not grow when the diet is supplemented with vitamin D, even though there is mineralization of the bones (Zucker et d.,1941). It has been shown that in such cases the weight of the animals may even decrease (Schneider and Steenbock, 1939). Studies of calcium and phosphorus balances and tissue analyses revealed that the vitamin D supplement induces utilization of phosphorus by the bone, thus depriving the soft tissues of phosphorus. The inhibition of growth may be attributed to the lowered phosphorus content in the soft tissues causing a decrease in the food intake. 3. In rickets the absorption and retention of calcium and phosphorus is decreased. It may be expected that a lowered concentration of phosphate exists in the cells of rachitic rats which is reflected in a fall in blood phosphates. In the rachitic chick the food intake decreases and this is accompanied by a disturbance in carbohydrate metabolism (Baldwin et al., 1928). Thus, at least in the caR of the rachitic chick, it has been shown that there is an association between a decrease in the phosphorus concentration of the blood and a disturbance in carbohydrate metabolism. A normal animal excretes phosphorus, ingested in excess, regardless of the calcium intake. But a parathyroidectomized animal is unable to do this unless a suflicient amount of calcium is also present in the diet. In the absence of a sufficient calcium intake, the parathyroidectomized rat retains inorganic phosphorus in its tissues and there is also an increase in the inorganic phosphate content of the blood. Under these conditions,
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rats stop eating a low-calcium, high-phosphorus diet. When the rats are fed an optimal dietary regime, they regard it suspiciously at first, but having once tasted the new diet they continue to eat it. In this instance, the rat must apparently depend on a taste mechanism in order to distinguish so quickly between a low-calcium high-phosphorus diet and a normal diet. On the other hand, if the rats are shifted from a normal to a low-calcium high-phosphorus diet, the decrease in food intake occurs gradually as the retention of phosphorus increases in the tissues. That dietary phosphorua is responsible for a decreased food intake in the parathyroidectomized rat is substantiated by the finding that it occurs only when the dietary phosphorus is utilized. This is obvious from the fact that orthophosphates and pyrophosphates cause a decreased food intake while metaphosphates and hypophosphites do not, since the latter compounds are either not absorbed or are excreted unchanged. The addition of calcium makes the diet acceptable because it enables the animal to get rid of the excess phosphorus. Excess phosphorus might also exert its effect on food intake by affecting phosphorylating enzyme systems other than those participating in carbohydrate metabolism. Methionine plays an interesting role in food intake. Proteins containing an inadequate amount of methionine do not promote growth. Addition of methionine greatly increases growth and food intake (Jackson and Block, 1932). On the other hand, an excess of methionine decreases growth and food intake (McKittrick, 1947). Methionine may exert its effect on food intake either through its action on some enzyme system or in some other unknown manner. 6. The State of Tissue Irritability and Food Intake
Animals belonging to the lower forms of life start and stop eating rather abruptly. What determines whether they eat or not? In protosoa depletion of the ingested food materials results in an increased motility and in an increased rate of food intake. These phenomena indicate that a condition of increased cell excitability may be responsible for feeding habits. “A state of hunger in protozoa is a state of increased excitability” (Carlson, 1916). A similar association of a state of hunger with increased excitability has been demonstrated for the rat (Richter, 1927). The increased excitability in the rat is reflected in a spontaneous activity which has approximately the same periodicity as that which physiologists have demonstrated for hunger contractions in the stomach. If the animals are kept under constant conditions and away from external stimuli, the activity is regularly terminated by feeding. Such a state of increased excitability could account for the increased sensitivity to food odors in the hungry individual.
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6. The Gastrointestinal Tract and Food Intake
The gastrointestinal tract, and especially the stomach, has been studied to determine its role in regulating food intake. In spite of extensive investigations, very little progress has been made along this line. More exact information is needed of the effect of food on the flow and composition of the gastrointestinal secretions. Voluminous data on this subject are available (Carlson, 1916; Pavlow, 1910; Babkin, 1944), but its relation to the question of food intake is not clear. Perhaps the gastro-intestinal tract is less important in the control of food intake than has hitherto been supposed. In thiamine deficiency, food leaves the stomach slowly, if at all and the animals eat very little food (Samuels, 1947). Force-feeding thiaminedeficient rats by stomach tube results only in distending the stomach and in early death. It would seem, therefore, that when the stomach is full of food, there should be no desire for additional food. This is not the case, however, since vagotomired rats continue to eat after their stomachs are full of food and they often pack sufficient food in their stomachs to die of suffocation (Shay, 1947). The desire for food is perhaps influenced by other factors as well as the amount of food in the stomach. Holinger and collaborators (1932) reported an interesting observation along these lines. A dog fed through a jejunal fistula had no desire to eat by mouth. The diet fed through the fistula w w complete insofar as its vitamin content was concerned, and further addition of vitamins did not create any desire to eat by mouth. But when the vagi were sectioned, the animal developed an excessive desire for food by mouth. The food eaten by the dog could not be utilized since the phylorus opened to the outside, yet “the excessive appetite of the dog has persisted unabated for the past 5 months and might be compared to that of a normal young dog starved for 48 hours. The only interpretation of this sudden appetite that has occurred to us is that section of the vagi severed the pathway of the impulses responsible for the production of anorexia.” There is also other evidence to indicate that the stomach does not play a very critical role in controlling food intake. Patients from whom the stomach has been removed still experience sensations of hunger and have a desire for food (Carlson, 1916). It seems probable, therefore, that the impulses controlling food intake in part ofiginate in the systemic metabolic reactions.
7. Intestiinal Bacteria and Food Intake It is possible that intestinal bacteria play an important role in controlling food intake. Bacteria may do so by their digestive action on
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crude fiber and other carbohydrates in the gastrointestinal tract, thus increasing or decreasing the stool bulk. Stool bulk is increased not only by undigested crude fiber but also by chemical compounds such as the lower, volatile fatty acids arising from the fermentation of carbohydrates by the intestinal bacteria (Williams and Olmstead, 1936). The effect on stool bulk produced by the action of intestinal bacteria on crude fiber has been studied by Hoppert and Clark (1945). Crude fiber from different sources behaved differently in the extent to which it was digested or affected the stool bulk. Wheat bran which is often used as a laxative does not uniformly promote laxation, and sometimes is constipating. It becomes a laxative only when acted upon by intestinal bacteria (Reyniers, 1946). This observation was made possible by the use of germ-free animals. In the germ-free animal little laxation was obtained with bran and, in most instances, it caused constipation. “When bran was treated in vitro with microorganisms from the caecum of a normal animal, the bran could be activated to exert a laxative effect . . . l l (Reyniers, 1946). Intestinal bacteria may also control the food intake in a manner which is not connected in any way with stool bulk. Bacteria may synthesize essential nutrients which, in turn, may affect voluntary food intake. A striking example of the possible importance of intestinal bacteria arises from the demonstration that cellulose is an essential nutrient for the guinea pig (Woolley and Sprince, 1945), although it is known that cellulose cannot be digested by the enzymes in the gastrointestinal tract of the guinea pig. The cellulose is probably acted upon by intestinal bacteria, and the product or products synthesized may contain among them nutrients essential for the guinea pig. The possibility that the fed cellulose contained an essential nutrient as an impurity seems very remote since purified cellulose was also found to be active. 8. Role of Flavors in Food Intake Natural flavors of foods may influence the food intake. Spices and condiments are used widely to stimulate food intake but little is known of how they function. Some of them undoubtedly stimulate the flow of gastrointestinal secretions, but their exact relation to food intake is not clear. Flavoring substances may possibly act in stimulating food intake by taking part in metabolic reactions. The work of Haagen Smit et al. (1945) on the flavor components of pineapple lends probability to such a suggestion. One of the flavoring substances of pineapple is closely related structurally to methionine. Since methionine has such a pronounced influence on voluntary food
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intake, this structurally related compound from pineapple may also affect food intake. Knowledge of the chemistry of flavoring substances and their metabolism would be of much importance in understanding food intake. Flavors may influence food intake in another way. Animals seem to possess only limited ability to recognize such nutrients as proteins (Scott, 1946b), or pantothenic acid (Scott and Quint, 1946) because they do not seem to possess any characteristic taste which would facilitate their recog-
CHs
CH,
I
I
S
S
I CH2
I CHa
I
CHNHZ Flavor component of pineapple B-methylthiolproprionate
I
COOH Methionine
nition by the animal. Easily recognizable flavors incorporated with these nutrients would enable animals to associate the sense of satisfaction and well-being obtained from the difficultly recognizable proteins and pantothenic acid with the easily recognizable flavors. The flavors would help animals choose such needed nutrients so long as they were associated with them. This possibility has been demonstrated experimentally with pantothenic acid (Scott and Quint, 1946) and with thiamine (Harris et al., 1933). 9. Appetite Centers and Food Intake
Whatever the processes controlling food intake may be, the impulses which cause the animal to consume food must somehow be transmitted to the anatomic areas responsible for the ingestion of food. Some of these impulses are probably transmitted through the vagi since sectioning these nerves results in an increased food intake (Shay, 1947; Holiiger et al., 1932). This presumably occurs because impulses regulating the food intake cannot reach the stomach or other centers regulating food intake. It is not known where these impulses originate but there is good evideace that some of them arise in the hypothalamus (Brobeck et al., 1943), since injury of this organ at the appropriate site results in a greatly increased food intake. It may be supposed that a center which regulates food intake has been destroyed by the injury.
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A lesion in the hypothalamus also deprives the rat of the ability to respond to changes in the caloric content of food. Normal rats increase their food intake if its caloric density is decreased and decrease it when the caloric density is increased. Rats with hypothalamic injury will ingest food without regard to its caloric density. They become very obese on diets of high caloric density but they can barely maintain their weight (and even lose weight) on diets of low caloric density (Brobeck, 1947). These investigations of hypothalamic injury have disclqsed that centers exist which decrease food intake when the food needs of the animal are satisfied. Are there, perhaps, also centers which cause an increase in the food intake of animals? But if such centers exist, very little is known about them. 10: Taste Mechanisms and Food Intake
Taste is of great importance in controlling food intake. If food tastes good, it is eaten; if it tastes bad, it is not eaten. The problem is what makes a food taste good or bad. The concentration of a nutrient in the tissue fluids must have a decisive influence on the taste of the nutrient. It is known that in adrenalectomized rats there is a sodium deficit throughout the body. This deficit influences the taste mechanism in the mouth, presumably located in the taste buds. Such adrenalectomized animals can taste one part of salt in 33,000parts of water, whereas normal animals cannot taste the salt except in muchhigher concentrations of 1 part salt to 2,000parts of water. A sodium deficit in the tissues, resulting from adrenalectomy, would, therefore, seem to enable the animal t o taste salt in concentrations too low to be detected by normal animals. A salt deficit in normal animals, arising from a low salt diet, might give different results, but the problem has not been studied from this angle. Under the conditions of adrenalectomy, the intake of salt is increased. Could this be interpreted to mean that salt tastes good to adrenalectomized rats? (Richter, 1936). While the taste mechanism may be important in controlling food intake, its action is not direct. Actually, impulses from the mouth enter the central nervous system, perhaps even some specialized taste center, from which efferent impulses stimulate the animal to ingest food. At any rate, the adrenalectomized rat does not differentiate between salt solution and pure water and may even die from an insufficient salt intake, if the taste nerves are sectioned (Richter, 1942). In the parathyroidectomized rat the calcium concentration of the tissues is decreased. Such rats voluntarily increase their intake of calcium (Richter and Eckert, 1937) and decrease their intake of phosphorus on low-calcium high-phosphorus diets (Shelling, 1932). Could this also be
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interpreted to mean that calcium tastes good and phosphorus tastes bad to parathyroidectomized rats? Human subjects were studied to determine the preference for sugar solutions in relation t o the blood glucose level (Mayer-Gross and Walker 1946). Blood glucose was regulated by insulin administration. The subjects were offered the following solutions during the normal and hypoglucemic states: 30% sucrose, saccharine with the sweetness of approximately 3oy0 sucrose, 5% sucrose and o.5y0 salt. They were asked to pick the solution they preferred for a “long drink.” “The great majority of subjects whose blood glucose level was over 50 mg. per 100 ml. rejected the 30 per cent sucrose solution as too sweet.” But very few subjects, whose blood glucose was below the 50 mg. % level, rejected the solution as too sweet and most of them actually preferred it. Apparently the taste of sugar is determined t o a large extent by the concentration of glucose in the blood and tissues. Of special interest in this study was the reaction of the subjects to saccharine (Mayer-Gross and Walker, 1946). While a large number of subjects preferred saccharine to 30% sucrose solution when their blood glucose was above 50 mg./100 ml., only a negligible number preferred it when their blood glucose was below 50 mg./100 ml. Some of the hypoglucemic subjects denied that saccharine even possessed any sweetness at all. Thus, hypoglucemic patients can discriminate between 2 compounds of equal sweetness, where one is able to replace the loss of glucose metabolically and the other (saccharine) not. Apparently, the taste perception for sweetness was depressed in the hypoglucemic patients since only half of the subjects with blood sugars below 50 mg. yo could tell that 5% sucrose solution had any sweet taste. The depression of taste perception affected only the sweet taste while salt and bitter tastes appeared to be undisturbed. Attempts to deceive the patients with flavors failed. The patients were offered peppermint, strawberry, anise seed, cinnamon, and clove flavors made up in 2.5% sugar solution. In many instances, the subjects tested had a strong liking for one or another of these flavors. In the hypoglucemic state, however, they rejected the preferred flavored solution in favor of a 30% sucrose solution. The blood sugar level evidently plays an important role in determining the sweetness of a sugar solution or its acceptability (Mayer-Gross and Walker, 1946). Additional evidence relating to the important role of the central nervous system in controlling the intake of food comes from other studies made with hypoglucemic patients. Spontaneous movement of the mouth and face in hypoglucemia are said to be produced by the excitation of areas in the central nervous system connected with food intake. Apparently, centers in the brain regulating food intake respond to low blood sugars in such a way as to cause an increased food intake (Mayer-Gross, 1941).
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SUMMARY
1. Palatability of Food Intrinsically, the various nutrients are not necessarily palatable or unpalatable. The same nutrient may be palatable or unpalatable depending, in part, on the chemical composition of the body’s internal medium. The composition, in turn, depends upon many factors among which the continuous activity of the enzyme systems is of decisive importance. Changes in the internal medium may also be caused by: 1. Differences in caloric expenditures due to work, heat, cold, or variations in basal metabolic rates; 2. Metabolic disturbances arising from: (a) Nutritional deficiencies, (b) Endocrine disturbances; 3. Imbalance among nutrients. Changes in the internal medium are accompanied by changes in acceptability of foods. The following are examples: 1. Concentrated sugar solutions may be unacceptable to subjects with a normal blood sugar level, but may become highly acceptable t o the same individual with a low blood sugar. 2. Glucose may be acceptable to normal rats but becomes unacceptable to the same rats when they are deficient in thiamine. 3. The acceptability of salt aa measured by the amount ingested is greatly increased in the adrenalectomized rat. 4. Diets containing low calcium and high phosphorus, which are normally accepted by rats, become completely unacceptable to parathyroidectomized rats. 5. The acceptability of low methionine diets is increased by the addition of methionine but only up to a certain point, beyond which further addition of methionine decreases the acceptability of the diet. Thus an essential nutrient may, under certain conditions, become an injurious factor. Acceptability of a food is also greatly influenced by deleterious compounds which are not nutrients. Defense mechanisms against such compounds have been discussed. 3. Possible Mechanisms Controlling Food Intake There is apparently an elaborate mechanism through which an increase or decrease in food intake is effectuated. The mechanism is not known in its entirety but the following components can be recognized: 1. Sensitive cells or “appetite centers” in the central nervous system. 2. Nerves (the vagi and others) which conduct the impulses to and from the central nervous system. 3. The gastrointestinal tract (the stomach), 4. The taste buds in the mouth.
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Just how these act to change food intake is not known; it cannot be vouchsafed that they are involved.
3. Variability in the Reaction to Nutrients Whatever the mechanism which enables the animal to respond by changing its food intake, the ability of the animal to respond varies with the nature of the nutrients. Roughly, nutrients may be differentiated into 3 classes according to the animal’s response: 1. Nutrients towards which there is an immediate response by the animal which is deficient in them. These nutrients include glucose and minerals, such as sodium and phosphorus. Responses to these compounds are immediate, suggesting that the taste mechanism may be the chief means by which they are recognized and accepted. 2. Nutrients to which there seems to be a delayed response. Certain members of the vitamin B complex, especially thiamine and, to a lesser extent, riboflavin and pyridoxine belong to this class of nutrients. These nutrients seem to bring relief from a distressing phyaiologica1 experience and this is presumably aseociated with a reaction in the taste mechanism to the deficient nutrient. 3. Nutrients to which there is little or no response by the animal which lacks them. These nutrients include proteins and vitamins such as pantothenic acid. These nutrients may cause a marked physiological effect on the animals, yet animals deficient in these components do not select diets containing them. Perhaps their ingestion gives no recognizable sense of physiological relief or more likely the nutrients do not have the chemical make-up to stimulate the taste mechanism. Combining these nutrients with characteristic flavors has heIped animals to choose diets containing them.
4. Glucose and Energy Exchange Perhaps the greatest impetus which motivates animals to eat or to stop eating is derived from the energy exchanges of the animal. Changes in food intake, however, do not always parallel energy expenditures : 1. Increased caloric expenditures resulting from the ingestion of thyroid are not balanced by equivalent increases in food intake, and the animals lose weight. This has been found to be due to an intervening nutritional deficiency which, when corrected by the addition of the missing nutrients to the diet, is followed by a much greater increase in food intake. 2. Increased caloric expenditures resulting from excessive heat are followed by decreases in food intake but the cause of this has not been determined. A consideration of the relation between energy exchange and food intake
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indicates that both glucose and phosphate metabolism play a major role in determining the acceptability of food. 5. Differentiation of Psychological from Physiological Factors i n the Con.tro1 of Food Intake While without doubt physiological factors play a decisive role in the control of food intake, psychological factors are also important and under certain conditions may even be the predominating factors. So far as the food intake of large populations is concerned the question of paramount significance is the interrelationship between strictly physiological and psychological factors, or where one stops and the other comes into play. It is generally assumed that animals can choose nutritionally correct foods more readily than man. Is this due to a loss of faculties by man but retained by animals, or, is it possible that both animals and man are equal in this respect, but man's choice of food is influenced by psychological factors which do not affect animals? In so far as man is concerned, appetite levels and food intake are probably under the control of physiological forces, which can, however, be overcome by psychological and other factors. AcmowmnQMx" The writer wishes to acknowledge with thanks the help received from Dr. 9. Morgulis who carefully read the manuscript and made numerous valuable and constructive suggestions and changes. He also wishes to thank Dr. R. H. Barnes for many constructive suggestions during the course of the work.
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Shelling, D. H. 1932. Calcium and phosphorus studies. I. The effect of calcium and phosphorus of the diet on tetany, serum calcium and food intake of parathyroidectomized rats. J. Biol. Chem. 96, 195. Silber, R. H. 1944. Studies on pantothenic acid deficiency in dogs. J. Nutrition 27, 425. Sollmann, T. 1942. A Manual of Pharmacology. 6th ed., Saunders, Philadelphia, pp. 207, 559. Spector, H., and Mitchell, H. H. 1946. Paired feeding in the study of the counteraction by nicotinic acid and tryptophane of the growth-depreming effect of corn in rats. J. Biol. Chem. 166, 37. Stirn, F. E., Elvehjem, C. A., and Hart, E. B. 1935. The indispensability of zinc in the nutrition of the rat. J . Biol. Chem. 109, 347. Street, H. R., Cowgill, G. R., and Zimmerman, H. M. 1941. Some observations of vitamin Ba deficiency in the dog. J. Nutrition 21, 275. Sumner, E. E.,and Murlin, J. R. 1938. The biological value of milk and egg protein in human subjects. J. Nutrition 16, 141. Sure, B.,and Dichek, M. 1941a. The sparing action of thiamine on body tissue catabolism. J. Nutrition 21,445. Sure, B.,and Dichek, M. 1941b. Riboflavin tu a factor in the economy of food utilization. J. Nutrition 21, 453. Tufts, E. V., and Greenberg, D. M. 1937-38. The biochemistry of magnesium deficiency. I. Chemical changes resulting from magnesium deprivation. J. Biol. Chem. 122, 693. Voris, L., Black, A., Swift, R. W., and French, C. E. 1942. Thiamine, riboflavin, pyridoxin, and pantothenate deficiencies as affecting the appetite and growth of the albino rat. J. Nutrition 23, 555. Waisman, H. A,, McCall, K. B., and Elvehjem, C. A. 1945. Acute and chronic biotin deficiencies in the monkey (Macacn mulatta). J. Nutrition 29, 1. Walker, A. R. P., Irving, J. T., and Fox, F. W. 1946. Nutritional value of high extraction wheat meals. Nature 167, 769. Warkentin, J., Warkentin, L., and Ivy, A. C. 1943. The effect of experimental thyroid abnormalities on appetite. Am. J. Physiol. 139, 139. Weast, C. A., and Mackinney, G. 1941. Non-enzymic darkening of fruits and fruit products. Ind. Eng. Chem. 33, 1408. White, A., and Sayers, M. A. 1942. Accelerated growth rate on dietary nitrogen obtained from the pancreas. PTOC.SOC. Exptl. Biol. Med.61,270. Wilgus, H. S.,Jr. 1939. The role of manganese in poultry nutrition. Proc. World’s Poultry Congr. 7th Congr. Clevrland, Ohio, p. 171. Wilgus, H. S.,Jr., Gaasner, F. X., Patton, A. R., and Gustavson, R. G. 1940. Thc goitergenicity of soy beans, Poultry Sci. Abstr. 19, 366. Williams, R.D., and Olmstead, W. H. 1936. The manner in which food controls the bulk of the feces. Ann. Intern1 Med. 10, 717. Withers, W. A., and Carruth, F. E. 1917. Iron tu an antidote to cottonseed injury. J. Biol. Chem. 32, 245. Wooley, J. G., and Sebrell, W. H. 1945. Nicotinic acid, an essential growth factor for rabbits fed a purified diet. J. Nutrition 29, 191. Woolley, D. W. 1946. The occurrence of a “pellagragenic” agent in corn. J. Bid. Chem. 163,773. Woolley, D. W., Berger, J., Peterson, W. H., and Stcenbock, H. 1938. Toxicity of Aspergillus sydowi and its correction. J . Nutrition 16,465.
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Woolley, D. W., and Sprince, H.1945. The nature of some new dietary factors required by guinea pigs. J. Biol. C h m . 167,447. Young, P. T. 1933. Food preferen- and the regulation of eating. J. C m p . P8ychol. 16, 167.
Young, P. T. 1941. The experimental analysis of appetite. PsychZ. Bull. 88, 129. Zucker, T. F., Hall, L., and Young, M. 1938. Growth-promoting effect of vitamin D on low calcium diets. Abatracts, 96th meeting Am. Chem. Soc., Div. of Biol. Chem., p. 17. Zucker, T. F., Hall, L., and Young, M. 1941. Growth and calcification on a diet deficient in phoaphate but otherwiee adequate. J. Nutrition 11, 139. Addendum to Section XI, paragraph 4, page 124 Rabbits, cats and ferrets react similarly while rats and monkeys show no convulsions and little other gross evidence of int,oxication. Chickens, guinea pigs and hamsters evince no toxic reaction, nor has any toxic reaction 80 far been demonstrated in the human ingcsting “agenized” flour (Boudreau, 1947).
Biochemical Factors Influencing the Shelf Life of Dried Whole Eggs and Means for Their Control BY HOWARD D . LIGHTBODY* AND HARRY L. FEVOLD* Bureau of Agricultural and Industrial Chemietry. AgFieu2tural Research Administration. U S. Department of Agriculture
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CONTENTS
I. Introduction . . . . . . . . . . . . . . . . I1. Chemical Composition of Whole Egg . . . . . . . . . 111. Variation in the Composition of Liquid Egg . . . . . . . Variations Attributable to Flo:k Rations . . . . . . . IV . Variations of the Raw Materials Related to Egg Storage Qualities . V. Physical Properties of Whole Egg Powders . . . . . . . VI Criteria of Quality and Dcterioration . . . . . . . . . 1 Palatability . . . . . . . . . . . . . . . 2. Fluorescence . . . . . . . . . . . . . . . 3. Volatile Products . . . . . . . . . . . . . 4.pH . . . . . . . . . . . . . . . . . 5. Solubility . . . . . . . . . . . . . . . . 6. AerationPower . . . . . . . . . . . . . . 7 Thickening and Emulsifying Power . . . . . . . VII Chemical and Physical Changes Associated with Deterioration . . 1. Proteins . . . . . . . . . . . . . . . . 2 Lipids and Lipid Solubles . . . . . . . . . . . 3. Lipoproteins . . . . . . . . . . . . . . . 4. Enzyme Action . . . . . . . . . . . . . . VIII. Quality Retention Measures . . . . . . . . . . . . 1 Low Moisture . . . . . . . . . . . . . . 2. Storage Temperature . . . . . . . . . . . . 3 GssPacking . . . . . . . . . . . . . . . 4. Acidification . . . . . . . . . . . . . . . Referencea . . . . . . . . . . . . . . . . .
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I. INTRODUCTION Quantity production of dried whole egg powders is chiefly an accomplishment born of a subsistence need of the armed forces and civilian populations during the war . Since the product was prepared for consumption in the form of scrambled eggs or omelettes primarily, rather than as a constituent of bakery products, a major peacetime use, powder preparations which simulated fresh shell eggs in flavor when the products reached the ultimate consumers were required. The objectives of researches car-
* Present Addreas-Quartmmsster Food & Container Institute for the Armed Forces. 1849 W Perahing Road. Chicago. Illinoie 149
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ried on during the war were, in consequence, primarily to retain palatability rather than those properties which ensure useful bakery products. After the emergency arose, it soon proved to be possible with control of drying equipment already designed to obtain powders with moisture contents of 5% or even lower which possessed acceptable flavors when reconstituted. With this accomplished, the problem then became one of retention of palatability under the very severe conditions of transportation and storage required in maintaining a supply for the armed forces wherever located. Solution of the problem was sought through both empirical and basic research procedures. This review represents an endeavor to assemble and relate not only the results of the investigations which have been reported in the technical literature, but more especially to collate the results of those researches into the nature of the chemical, physical, and biological changes which result in quality differences and deterioration before and during processing and storage.
11. CHEMICAL COMPOSITION OF WHOLE EGG’ Eggs vary markedly in size but the ratio of yolk to white in fresh eggs from mature hens is quite constant though, less than that of eggs from pullets (Morris, King, and Nestler, 1935). The liquid contents of averagesized shell eggs of about 56 g. weigh 49.8 g. The white and yolk account for approximately 65% and 35% of the mixed egg liquid, respectively. The white contains about 88% and the yolk 49% water. Of approximately 25.6 g. of solids in 100 g. of liquid egg mixture, 7.8 g. are white and 17.8 g. are yolk. The major organic constituents of the whites are a mixture of seven proteins (or protein groups) ; i.e., ovalbumin, ovomucin, conalbumin, ovomucoid, and the ovoglobulins Ci,Gz (lysozyme), and GO. These together make up 82.8% of the egg white solids. The nonprotein solids of dry white consist of small quantities of glucose (2.8y0), lipids (1.4Y0),and ash (5.6%). Of the yolk solids approximately 62y0 are lipids, 32.7% proteins, and 2.9% ash. Carbohydrates are absent or, a t most, present in very small quantities. It is generally stated that egg yolk contains two proteins; i.e., vitellin and levitin in proportions of 4:l. Vitellin exists in combination with phospholipids as the lipoprotein lipovitellin. Recent work (Fevold and Lausten, 1946a, 1946b) has shown that 2 lipoproteins are present in egg yolk, one identical with lipovitellin as described in the literature and a second, lipovitellenin, which contains Reliance has been placed upon the aummary, “The Chemical Composition of the
Egg,” by Dr. E. M. Cruickshank (1940),“Eggs and Egg Products,” U. S. Dept. Agr. Circ. NO. 683 (1941),and “Handbuch der Eierkunde,” by J. Grossfeld. The analytical values for powders given here were usually obtained by recalculating results obtained from t h e literature on the &-weight baais.
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a greater amount of lipid in proportion to protein. The protein, vitellenin, contains less than one-third as much phosphorus as vitellin and comprises roughly one-third of the total protein in the lipoprotein complex usually designated lipovitellin. Evidence of the presence of an additional phosphoprotein, heretofore unidentified, is to be reported (Mecham and Olcott, 1946). This phosphoprotein is characterized by its unusual phosphorus content of approximately 10%. Recent cataphoretic evidence (Lundgren and Ward, 1946) also indicates livetin to be a complex of 2 or more proteins, some of which (Lineweaver and Kline, 1946) exhibit enzymic activity under controlled conditions. Free amino acids are present in small amounts (Wohlgemuth, 1905). The lipids, i.e., ether extracts of egg yolk, are complex mixtures of glycerides and phosphatides. The latter make up about 16% of the total ether extract. The fatty acids identified in the yolk are palmitic, stearic, oleic, linoleic, and linolenic acids. The lower fatty acids have been shown to be absent from egg fat (Riemenschneider et al., 1938). The saturated acids together form about 31% of the total. The unsaturated acids, oleic, linoleic, and linolenic, account for approximately 50%, 20%, and 3% of the total, respectively. Other more highly unsaturated acids such as arachidonic and clupanodonic, have been identified as probably occurring in the phospholipids. Two groups of phospholipids have been identified; namely, lecithins and cephalins. The first of these appears to be largely in combination with proteins. Complete separation and characterization of the phospholipids do not appear to have been accomplished. Chargaff et a?.(1942) found 42% of the total nitrogen of egg phosphatides to be amino nitrogen. Since they were unable to detect more than very small quantities of amino acids in their preparations, this percentage appears to indicate the distribution of cephalin and lecithin in the egg phosphatide mixture, ie., 42% cephalin and 58% lecithin. As already indicated, the ash content of dry white is approximately twice that of yolk. The relative contribution, however, of solids by yolk and white to dried whole egg powder is in the approximate ratio of 2:l. The total ash of whole egg powders is derived about equally from the yolk and the white. Something more than one-half of the total ash is made up of sulfur, phosphorus, potassium, and sodium. Metals which may serve as oxidation catalysts, i.e., copper, iron, and manganese, are present in low concentrations. Dry whole egg powders contain about 7.5 parts copper, 350 parts iron, and 1.3 parts manganese/million. There appears to be no direct evidence available concerning the significance of these elements, as egg contituents, in oxidative deterioration of whole egg powders. Some of the quantitatively minor constituents of eggs, such as pigments
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and vitamins, contribute notably to consumer acceptance and to the nutritive value of powders and are, therefore, of interest in connection with quality deterioration. Because of the marked variations in contents of these constituents, with conditions of egg production, their relations to the general problem will be discussed in the section following.
111. VARIATIONIN THE COMPOSITION OF LIQUIDEGG The raw materials to be dried are by no means chemically uniform even though care has been taken to avoid predrying deterioration. Egg composition has been shown to vary, especially with the age of the laying hens, and with the feed supplied the flocks. Variations, due to flock feeding practices, have been observed to be related to the season and region of production, and provision has been made for such variables in the U. S. War Food Administration (1944) purchase specifications.
Variations Attributable to Flock Rations The manganese content of eggs has been shown to be correlated positively with the manganese content of the diet (Lyons and Insko, 1937). ‘Likewise, the amount of iodine present is dependent upon the amount and form of the iodine ingested (Wilder et al., 1933;Asmundson et al., 1936). The characteristics of body or depot fat of birds, as for other species, have been shown to be closely related to the composition of the diets of the fowl. Evidence, however, to indicate that body fat is not a significantly important source of yolk fat is available (Almquist et al., 1934). It appears that, within limits, the properties of body fat and those of egg fat may be altered by dietary regimen but quite independently of each other. Feeding highly unsaturated hemp seed oil to hens initially producing egg fat containing 31% solid acids, 47-51% oleic acid, 1519% linoleic acid, and 2-3Y0 linolenic acid, has been found (Cruickshank, 1934) to increase the iodine value of egg fat about 50% within 2 weeks. Upon feeding mutton fat, however, in the same series of experiments, no decrease in iodine value was observed over a 10-week period. Cottonseed meal, when fed in quantities amounting to Sq/Z% or more of the feed mix, has been shown to have decided influence on the storage life of shell eggs (Thompson, 1918; Shenvood, 1925; Sipe, 1931; Upp, 1932; Smith, 1937). Schaible et al. (1946) have reported cottonseed oil to contain a factor which causes relaxation of the vitelline membrane. Reduction of the fat content of the diets from a normal value of to 0.0S70, has been observed to cause a decrease in iodine value of the egg fat from 65 to 53 (Russell el al., 1941). The writers are not aware of reports of studies concerning the ability of fowls to synthesize linoleic or linolenic acids such as those by Bernhard et al. (1942), for rats. Using deuterium, supplied
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as heavy water, as an indicator, and a fat-free diet, these authors concluded from analyses of the body fat, that rats are unable to synthesize these unsaturated fatty acids. As will be discussed later in this review, the composition and properties of the egg lipids may be significant factors in quality retention of the dried products. Changes in quantity or composition of egg proteins, attributable to changes in diet, have not been definitely demonstrated. Feeding 4 different diets, characterized by differences in the protein sources, and analyzing the eggs produced for total and amino nitrogen, tyrosine, tryptophane, and cystine, failed to reveal significant protein differences (McFarlane et al., 1930). Data from another series of similar experiments reported (Titus et al., 1939) show small but significant differences in the amount of protein present in the dry matter of yolk and white. For example, a crab meal diet gave 32.0% and a standard diet 31.4% protein in the dry yolk, Examination of the proteins of eggs produced, during consumption of diets containing wheat middlings, corn, and soybean as protein sources, showed no marked differences in composition as indicated by analysis for total amino and amide nitrogen, sulfur, tryptophane, cystine, arginine, histidine, and lysine (Calvery and Titus, 1934). Because of the established relations existing between vitamin content of feed and egg hatchability, the vitamin content of eggs has received much attention by workers in poultry nutrition. Measurement of the increases in the vitamin A content of eggs, resulting from supplements of vitamin A in the diet, was reported as early as 1933 (Thomas and Quackenbush, 1933). Extensive investigations (Sherwood and Fraps, 1934; DeVaney et al., 1935; Bearse and Miller, 1937; McClary et al., 1938; Baumann et al., 1939), in which the feed vitamin A was supplemented by alfalfa and fish oils, indicate that the vitamin A of eggs may be readily increased by two times the content of eggs produced by feeding practical or more usual diets. The vitamin content was estimated by bioassay and spectrophotometric methods. A somewhat similar situation exists in regard to increased vitamin D content of yolks after increasing the vitamin content of the feeds by use of irradiated ergosterol or fish oils (Branion, 1934; Bethke et al., 1937; Murphy et al., 1936). The absolute yolk increases in vitamin D content appear to be much greater than those of vitamin A and are reported to be as great as 200-fold. The amounts of the water-soluble vitamins, riboflavin (Lepkovsky et al., 1938; Hunt et al., 1939; Klose et al., 1946), thiamine (Klose et al., 1946) and pantothenic acid (Snell et al., 1941) in eggs are also related to the contents of the same vitamins in the feeds, and each may be made to vary several fold by controlled feeding. The pigmentation of egg yolk has been the concern of many investiga-
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tors (Bethke et al. ,1927; Ellis, 1933; Koenig et al., 1935; Russell and Taylor, 1935; Gillam and Heilbron, 1935; Heiman and Wilhelm, 1937a, 193713; Wilhelm and Heiman, 1937; Hughes and Payne, 1937) largely because of the relation between color, consumer acceptance, and vitamin content. Eggs, discolored because of the ingestion of some kinds of weeds and thus rendered unsuitable for drying, are a problem in some areas (Smith, 1937; Temperton, 1944). Based on the results of chromatic studies of grassland silage, Bolton and Common (1941) suggest that the seasonal incidence of olive-tinted yolks is due to the special richness of the early summer pastures in carotenoids, and the simultaneous existence of conditions which increase the lability of the carotenoids with the production of blue or blue-green pigments. One such factor might be acidity, present alike in grass eilage and in the fowl’s crop. Schaible and Bandemer (1946) attribute the off-colors observed in eggs from flocks fed cottonseed oil, t o a diffusion of conalbumin from whites through the vitelline membrane where it comes in contact with the yolk iron to form a pink complex. The olive color of yolks from hens fed cottonseed meal is due, according to Swenson et al. (1942), to a reaction between gossypol and iron released from yolk protein combination by ammonia. Because of the changes in colors of whole egg powders during storage, pigmentation of the raw materials used for drying is of considerable interest in relation to the subject of this review. Color analyses of powders may be useful as criteria of changes that have taken place. The changes are the result of destruction of natural pigments and the formation of other pigments. Color has been demonstrated to be by no means a reliable index of vitamin content. Although it is generally true that highly colored eggs are rich in vitamin A, this is so only because carotene in natural foods is usually associated with pigments which are deposited in the egg yolk. The reverse statement that low-colored eggs are lacking in vitamin A need not be true, since vitamin A from fish liver oils is deposited in the egg yo&, even though the feed is lacking in pigments which increase the color of the egg yolk.
IV. VARIATIONS OF
THE &AW
MATERIALS RELATED TO
EGGSTORAGE QUALITIES
Exact information relating quality of raw materials to quality and keeping properties of the finished products is scanty or unavailable. The relations between grade, geographic area, and season of production of eggs to powder yields and quality, as estimated by objective criteria, have been studied by Pearce et al. (1946). Grade A large eggs were found t o have the lowest average solids content (25.6%) of the several grades studied, and Grade C eggs the highest (26.6%). The solids content increased
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about 0.5% during the period from December, 1944 to July, 1945. The average potassium chloride value (a solubility criterion) of powders produced from Grades B and C was greater than the value for Grade A medium eggs. Powders produced from Grade C eggs were inferior in quality to powders from other eggs as measured by fluorescence. As the season progressed, decreases in fluorescence value and pH and increases in potassium chloride value and foaming value were observed. The authors suggested that the changes may be attributed to feeding practices and to increased age of the hen. Thistle e2 al. (1945), using the potassium chloride value (cf. Sections VI-2 and VI-5) and fluorescence, were unable to detect any difference in rate of change during storage which could be related to the initial quality of the powders examined, provided the moisture contents of the samples were similar. Deteriorative changes initiated in the raw material before drying may be expected to be reflected in the functional properties of powders, and in consequence it is the practice to control shell egg quality by inspection in the breaking rooms. The control measures used are largely for purposes of insuring sanitation and protection of the public health. A discussion of the inspection procedures and their relation to the biochemistry of deterioration is not within the scope of this review. The technical literature reveals reports of a number of studies concerning the nature of changes taking place in shell and frozen eggs which may be expected to influence the properties of powders prepared from them. Mitchell (1932), in an extensive series of analyses, found that during storage of shell eggs, the solids increased about 1% on an average and those of yolks decreased about 3.9%. In the same sene8 the solids content of the mixed edible portions was found to have increased 0.46%. Dextrose and water-soluble nitrogen content remained unchanged during storage. The diffusion of water from the white into the yolk is accompanied by a change in the texture of the white from thick to thin. This movement of water is not, however, a direct result of the changes in the white (Holst and Almquist, 1931). Structural changes in both white and yolk contribute to decreases in aerating power. These changes are especially important t o the baking industry, and adding salt, sugar, or glycerin to frozen eggs has been found to be a useful empirical procedure for retarding such deteriorative changes. When egg white is held a t temperatures below freezing and then thawed and allowed to stand, the watery fraction is observed to be considerably greater than that of fresh white. Yolks frozen at low temperatures develop a pastelike or rubbery consistency; the mechanisms of this change are not understood. A study of the relations between the sanitary conditions of flock environ-
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HOWARD D. LIQRTBODY AND HARRY L. FEVOLD
ment and storage life of shell eggs has been made by Thompson et al. (1930). Dirty, moist nests and water condensation on egg surfaces are factors in determining microbial passage through the shells (Stuart and McNally, 1943). Mallman and Davidson (1944) report eggs as laid to be relatively free from microbiological contamination, and propose a process preventing contamination by dipping fresh eggs in mineral oil containing an antibiotic agent. McFarlane et a2. (1945) have recently reviewed plant practices as they affect the microbiological conditions of liquid, frozen, and dried eggs. Gross bacterial contamination of mixed egg liquid may result in lowered pH in the reconstituted powders. Lovern (1946) attributes this effect to lactic acid formation. There is no evidence that the sugar-splitting bacterial enzyme is active in the dried powder, inasmuch as lactic acid does not appear to increase in the powders. That the bacterial count may be correlated with insolubilization of powders, under some conditions, is indicated by the work of Stuart et a2. (1945). Stuart et al. (1942) find bacterial death rates greater in powders containing 5% moisture or less than in powders containing larger amounts of water. Above 5% there is an increasing survival time with increasing moisture until a sufficient amount is present to permit microbial growth, that is, about 10%.
V. PHYSICAL PROPERTIESOF WHOLEEGG POWDERS On examination of photomicrographs of spray-dried whole egg powders the particles are found to be hollow spheres, characteristic of particles produced from filming nozzles as described by FoIger and Kleinschmidt (1938). Jones (1946) (see also Shaw et al., 1946) has measured the diameters of particles of a number of commercial samples and found the particle size to vary over a range from 7p-12Op. Reeve (1946), by use of staining techniques, has investigated the structure of the particle walls and observed minute fat globules dispersed throughout the films. Shaw et al. (1946) have estimated the specific surface areas of several spraydried and lyophilized egg powders by calculations from the low-temperature adsorption isotherms for nitrogen and argon. They found the specific surfaces of spraydried particles, expressed as sq. m./g. powder, to range from 0.110.41. Powders produced by drying from the frozen state, which appear sponge-like under the microscope, were found to have specific surfaces ranging from 0.48-0.83 sq. m./g. Efforts by Jones (1946) to correlate these physical properties of spray-dried powders with aeration properties and deterioration rates have not been successful. Stitt and Kennedy (1946) have investigated the mean specific heats of whole egg powders for the temperature interval 0°C. (32°F.) and 66°C. (150.8"F.). The heat capacity waa found to exceed the heat capacities
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of equivalent quantities of dry solids and water by an amount which varies with the moisture content. They suggest that the heat capacities of eggs may be differentially additive above a moisture content of 15%, and offer an explanation of the observed results based upon structures which permit a penetration of water into the interior of the particles. The relationship between the water content of whole egg powders and vapor pressure of water over a range of conditions likely to be encountered in storage was investigated by Gane (1943). His data show the equilibrium and minimum water content of powders held at absolute humidities of 0.05 and 0.7 and a t temperatures between 10OC. (50'F.) and 80°C. (176°F.). As the storage temperature was increased, an increase in the active water, measured as the equilibrium relative humidity, was observed. This effect is especially pronounced a t the higher water contents and a t temperatures above 37°C. (98.6"F.). A study of the relations between moisture content and relative humidity of yolks and whites, separately dried, showed that the moisture retention by whole egg powders can be accounted for by the proteins and salts present in the mixture. Extensive protein denaturation was observed to exert only a slight effect on the water relations. Powders prepared by drying from the frozen state did not differ from spray-dried preparations in these respects. Makower (1945) extended the data of Gane a t moisture contents ranging from 0.5-5.5%. He found protein denaturation to be an important factor in moisture equilibrium at low moisture (O.6yO)levels where it is of the same order of magnitude as the absolute value of the vapor pressure. However, the rate of change in vapor pressure of low-moisture powders with heating is very slow. The degree of denaturation occurring in spray drying where excessive heat is avoided is not sufficient to affect, appreciably, vapor pressure relations. The value of the ratio of heat of adsorption of water by egg powders to the heat of condensation of water vapor at the same temperature was found to increase slowly from 1.1-5.5y0 water to 2.1 at 0.5%. Thus the heat of vaporization of water from powders containing 0.5% water is more than twice the heat of vaporization of pure water at the same temperature. Holding powders a t 70°C. (158°F.) resulted in a pressure increase with time. The cause of this increase waa identified with the liberation of carbon dioxide, and presumably was effected by some decomposition process.
VI. CRITERIAOF QUALITYAND DETERIORATION Those properties of eggs which determine value are the distinctive odor and flavor, aerating or leavening power (sponge and angel cakes), coagulation or thickening power (custards), emulsifying power (salad dressing), enrichment (moistening and prevention of staling), color, and nutritive
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value? Of these properties, palatability, including flavor, odor, and texture, aerating and thickening power, are of major usefulness in determining the value of whole dried eggs as presently used. It is generally recognized that natural egg flavor is greatly to be desired in custards, omelettes, and scrambled eggs. Variations in flavor of egg powders are minor factors in culinary preparations subjected to baking temperatures, but retention of beating or aerating power is of major importance in determining the value of powders used in some baked products. Palatability and aerating properties do not appear to be interdependent, and it is possible to prepare powders of excellent flavor but of very inferior aerating power. Spray drying usually results in marked diminution of aerating power even though palatability is retained. On the other hand, powders prepared by drying from the froBen state readily retain the aeration power as well as palatability of the emulsions from which they are prepared, though these properties may be lost rapidly during storage. Thickening power also appears to be independent of palatability, since thickening may be retained during storage of powders containing added sugar, while palatability has deteriorated markedly (Stewart, 1946). 1. Palatability
Since whole egg powders prepared for military or civilian use during the emergency of war were consumed largely as scrambled eggs, flavor was considered by purchasing agencies as a major quality criterion. As is common with all subjective tests, flavor is extremely difficult to estimate and to obtain agreement upon between tasting groups. Especially is this true of off-flavors developed during storage as a result of chemical changes taking place in the powders. Off-flavors produced during processing, such as “burnt” (resulting from drying conditions that are too severe), “fishy” (associated with copper contamination), “sour” (attributable to bacterial action of the liquid egg prior to drying), and “other flavors” (traceable to storage in non-airtight containers in proximity to articles with pronounced odors), have been troublesome in the development of the industry. These will not be given further consideration here. Such off-flavors may be regarded very largely as “all-or-none” flavors in contrast to the slowly developing and poorly characterized flavors of storage deterioration. Small texture differencesare perhaps even more difficult to estimate than flavor. Unless very marked, as may be the case in over-heated powders, “collector dust,” or when “creaming” has occurred, texture is not likely to be the determining factor of palatability of the reconstituted preparations. *Animal feeding experimentg have indicated no loss of nutritive values of whole egg powders during storage, except loases attributable to decreases of some vitamins. This is true even though deterioration aa estimated by the usual criteria has rendered the product unacceptable for human food (Klose and Fevold, 1946).
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A procedure for flavor evaluation was devised early in the development of the whole egg drying industry a t the Low Temperature Research Station in England (Bate-Smith et al., 1943). The procedure specifies exact conditions for reconstituting the powder and preparing a test scramble. The palatability estimations are made according to the method, by a test panel of 5 or 6 experienced judges. The flavor value is expressed by scoring spray-dried eggs on points from 0-8 inclusive. Fresh shell eggs prepared and evaluated under the same conditions are usually given scores of 9 or 10. This original method has been carefully studied, improved by modification, and its usefulness and accuracy evaluated (Boggs, 1946). With carefully selected and trained panel personnel and under carefully controlled conditions, the results appear to be reliable to %--% point of the numerical flavor scoring scale. As a research tool for studying the causes and conditions of flavor change during storage, it is very useful, and a t present it is the only available method which can be relied on to give satisfactory results. I n such studies it is desirable to avoid all possibility of complicating the off-flavor storage changes by excluding the “all or none” type of flavors, an end that can be attained by using egg powders, prepared from shell eggs of uniform high quality, dried from the frozen state. Palatability tests are used in this country by several purchasing agencies as a means of assessing the quality of products. There are, however, obvious disadvantages to the general use of the method. The time required for evaluating routinely large numbers of samples is great. An appreciable number of individuals must be available from which to select a satisfactory taste panel. Environmental factors must be carefully controlled, and, since such factors are not uniform from one control laboratory to another, agreement between evaluating groups is likely to be poor. The cost of conducting taste panels is yet another disadvantage. Because of these disadvantages, repeated efforts have been made to devise objective methods of evaluation that would show close correlation with palatability measurement, and could be used in place of, or supplementary to, the latter if desired. Discussion of a number of such methods follows. 8. Fluorescence The most successful and generally used of the proposed objective methods is the fluorescence test as adapted by Pearce and Thistle (1942), from a method of estimating age and storage treatment of shell eggs, devised by Dingemans (1931, 1932). The method was later revised by Pearce el al. (1943). Pearce (1942) presented evidence of excellent correlation between fluorescence value and other quality measurements when the comparisons were made on inferior powder samples. Other quality estimates included in this comparison were KC1 and water values (solubility tests).
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pH, beating value, and palatability. The authors stressed correlation of fluorescence values and palatability. Thistle el aZ. (1943), in a comparative study of the relative merits of fluorescence,volatile materials, bacterial count, beating value, pH, water value, KCl value, and palatability, concluded fluorescence measurements to be more precise and to distinguish more sharply between powders from different drying plants than do other available quality tests. Correlation coefficients between fluorescence and palatability for prime quality samples was found to be -.310 and the similar value for dust collector samples - .854. Stewart el aZ. (1943)point out that in spite of the very high correlation coefficient, deviations are sometimes large, amounting to 1-2 points of the flavor score. They express the opinion that all defects in flavor are not accompanied by changes in fluorescence. Marked deviations between flavor score and fluorescence values have repeatedly been observed, especially in powders stored for long duration a t low temperature (Fevold et al., 1946). There is also evidence that the excellent correlation between salt-water soluble fluorescence and palatability, usually found in powders containing relatively high-moisture content, may not be attained when the tests are applied to low-moisture samples. For example, egg powders of 4% moisture, whose palatability has been lowered from 8.5 to 6 during storage, generally have a fluorescence value of approximately 36. Egg powders with 2% moisture or less, however, whose palatability has been reduced to the same value, show only small, and in some cases, negligible increases in fluorescence value; and if storage is continued until the fluorescence value reaches 36, the palatability is reduced to 4 or below. Additional evidence of failure of fluorescence and palatability to correlate satisfactorily is supplied by experiments designed to reduce the quantity of basic groups present in whole egg powders by absorption. Whole egg powders (moisture 2.6y0) prepared by lyophilization, after addition of an absorbent clay (“Volclay”) to the egg emulsion and storing the dried powder at 36.5”C. (99.2”F.) for 4 weeks gave results indicating wide divergence in the two criteria values. In the presence of “Volclay” the fluorescence values increased only half as much as did those of the control. The retarding action of the clay on the development of off-flavor was not significant. The clay served partially to control the fluorescence changes (a result to be expected in view of its base-absorbing properties) but not to retard palatability changes. Data to be discussed later in this review indicate that the major flavor changes are due to changes in the egg lipids, whereas salt-water solublefluorescing substances a.rise from protein changes, primarily in the egg white. Any correlation of the two is, therefore, incidental, and it is not surprising that they do not correlate under all conditions.
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Boggs et al. (1946) have investigated the relation of the fluorescence of ether extractables to palatability and compared the results to the fluorescence values of the salt water extract obtained by using the method published by the Canadian workers. This investigation was based on experimental data, previously collected, which had shown that the major storage flavor change takes place in the ether-soluble fractions. Dutton and Edwards (1945) have shown that cephalin may react with aldehydes ta give ether-soluble, brown, fluorescing materials; and it appears that this reaction might account in part for the discoloration and fluorescence in egg lipids during storage of the powder. It was, therefore, possible that measurement of the lipid fluorescence might serve as a better index of flavor change than salt-soluble fluorescence. The data show that moisture content and storage temperatures markedly influence the development of salt-water soluble fluorescing substances. The changes responsible for loss of palatability and those responsible for the production of salt-soluble fluorescing substances take place simultaneously and correlate very well (correlation coefficient +0.97) when spray-dried powders containing 4 4 % moisture or above are used in storage experiments. The lipid fluorescence $nd palatability values obtained on the same series of samples correlate with a coefficient of +0.98. With good quality egg powders of 1-201, moisture, the rate of development of salt-water fluorescence is lowered to a greater extent than is the rate of development of off-flavors, and the excellent correlation formerly observed, is not then obtained. I n many cases the changes in salt-water fluorescence value during storage are observed to be slight even though the flavor changes are marked. A similar failure of correlation between flavor and lipid fluorescence is not observed when low moisture storage powders are studied. Boggs et al. (1646) conclude with a note of caution that though lipid fluorescence has several advmtages, it may be incidental, as is true in the case of salt-water fluorescence, to the changes resulting in off-flavors. Fryd and Hanson (1944, 1945) criticize the fluorescence (salt-water soluble) method, believing the problem to have been over-simplified, and suggest that the Pearce and Thistle (1942) data indicate very strongly that some factor other than direct flavor-fluorescence correlation is in operation. These investigators have devised a simple and direct method of estimating fluorescence. A slide is prepared consisting of a layer of powder 1.2 111111. thick between two microscope slides. The slide is placed in a photofluorometer and the fluorescence is determined by use of a filter for the incident light. The authors developed a formula by which the flavor score can be predicted using fluorescence and solubility values (refractometric), glucose, free fatty acids, and moisture contents. The authors lay claim to an accuracy of f1.2 flavor unit and an applicability
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to powders with flavor scores of 7 or less. They recommend the formula as of value in reinforcing the opinions of flavor-tasting panels.
8. Volatile Products It is obvious, from the changes in odor which develop during unfavorable storage, that deterioration of whole egg powders may be accompanied by the formation of volatile products. This change can also be demonstrated by including adsorbent charcoal in sealed compartmented packages of the powders (Conquest and Turner, 1943). Good grades of powder may be held for a long time without noticeable changes in odor in the presence of the adsorbent in such compartmented and sealed packages. Also, charcoal may be used to deodorizie powders that have developed unpleasant odors. Powders that have been stored in the presence of charcoal, however, develop off-odors very quickly after removal of the adsorbent, So far as the reviewers are aware, no successful attempt to elute the odoriferous materials from the adsorbent and identify them has been reported. Steffen et al. (1943), working on the premise that a progressive protein denaturation or decomposition might increase either the lability or the number of sulfhydryl groups, modified the Eber test for hydrogen sulfide and investigated the usefulness of the test as a criterion of powder quality. By comparing the results of the HzS test and palatability scores, they were able to differentiate the adsorbed and other off-flavors. They reported satisfactory correlation between such off-flavors as sour, scorched, fishy, soapy, oily, an undefined off-flavor, and palatability. It is not clear from the report whether off-flavors resulting from storage were correlated with HzSformation. They found that volatile acids did not increase during storage. Variable amounts of volatile acids, 8-17ojO of the free fatty acids, were found in commercial powders, and presumably indicated breakdown prior to drying. The pH of reconstituted whole egg powders has been investigated by several laboratories as a possible means of estimating quality. Powders which reconstitute to give a pH of 7 or below are generally observed to possess unsatisfactory palatability scores, even though storage changes do not involve flavor. Though acid and cheesy flavors may not be observed, the major cause of lowered score accompanying unfavorable pH appears to be due to changes in texture. Thistle et al. (1943) found only slight correlation between pH and KC1 value when prime quality samples were used. Poor correlations were also found to exist between pH and fluorescence and between pH and palatability. The changes in pH values of stored powders become increasingly
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pronounced as the moisture concentration increases (Stuart, Hall, and Dicks, 1942). The results of a study of the causes of pH changes in eggs during drying and storage were reported by Brooks and Hawthorne (1948b). The pH of 7 freshly mixed pulps prepared from 1- to 2-day old eggs averaged 7.53 a t 15°C. (59°F.). Because of loss of COZ,the pH of liquid whole egg mixtures increased when stored in atmospheres containing less than 5% COZ. The usual pH (8.5-8.9) of reconstituted powders is apparently due to equilibration of the powders during drying with the COz of the drier air, i.e., between 0.03 and 0.0003 atmospheres partial pressure of COZ. The buffering capacity of reconstituted whole egg powders in equilibrium with air was found to be constant between pH 8.6 and 6.8 when titrated with hydrochloric acid. Through this pH range the major part of the buffering is attributable to the proteins and possibly to unknown buffering systems derived from the yolk. The phosphate present can account for about 10% or less of the total buffering action of the system. A quantity of oleic acid equivalent to 10 ml. of 0.5 N sodium ethylate/g. of ether extract wm required to lower reconstituted liquid pH from 8.6 to 7. The pH of reconstituted powders decreased, depending on the water content of the material, temperature, and length of storage. Below 4.5% water there was little change in pH or free fatty acids during storage at 20°C. (68°F.) for 40 weeks, and the pH agreed with values calculated from the free fatty acid content. When the water contents and temperatures of the stored powders were high, however, the amount of fatty acid liberated was not large enough to account for the observed decreases in pH. Under these conditions increases in orthophosphate and acid-soluble phosphorus are found (Brooks, 1943). In commercial samples that have a low pH (5.9-7) these authors consider proteolytic breakdown, liberation of phosphoric acid from lecithin, and enzymic breakdown of the carbon chains of fatty acids as possible sources of acids. Determinations of nonprotein nitrogen, iodine values of extracted fats, orthophosphate, and volatile acids yielded little or no evidence that such postulated changes could account for the decreases in pH. It was found, however, that most of the samples of low pH contained large amounts of lactic acid, in some cases as much as 1% of the powder. By identification of lactic acid by isolation of zinc lactate from the low pH powders, and by relating pH change to lactic acid titration, together with extensive hydrolytic breakdown the authors account for pH values of 7 or less in commercial powders. They suggest that the most probable explanation of the low pH of some commercial samples is bacterial spoilage, but are unable to relate the spoilage to conditions existing before or after drying. The commercial samples used in the study were of usual water content and had not been held at high temperatures.
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Similar results could not be obtained by powders prepared from good quality pulps held under similar conditions. Evidence that lowered pH does not of itself necessarily contribute to the loss of properties used to estimate value of the egg powders comes from two sources. Kline and Stewart (1947) applied results of studies of the rate of the reaction between amino acids and sugars as influenced by pH in a study of the effect on the rate of insolubilization of dried whites. They found that the rate of insolubilization was markedly retarded by adjusting the pH of liquid white to 4.8 before drying. The acidified product (9.1% moisture) possessed a storage life 4-5 times that of a normal dried white (moisture 10.1%) when each was stored a t 6OOC. (140OF.). Boggs and Fevold (1946) adjusted the pH of mixed whole egg liquid to 5.5 (the pH of egg yolk), and observed the pH change to effect approximately a 2.5-fold increase in the time powders prepared from the acidified liquid retained acceptable flavor properties. 6. Solubility Solubility of egg powders has been extensively studied in order to correlate the results obtained with functional properties such as palatability and cake baking. A number of procedures have been recommended for estimating “solubility” of whole egg powders. All measure essentially the solubility, or change in solubility, of the egg proteins. The methods are empirical, since solvent, conditions of manipulation, time of contact of powder and solvent, temperature, pH of extraction, and method of estimating the solids extracted, all influence the sohbility values. Since the tests are arbitrary, major consideration has been given to convenience and standardization of procedure in order that solubility values may be readily obtained and that they may be comparable. Such tests when adopted by processors and buyers have proved useful as a means of estimating collectively (1) heat damage during processing, (2) inclusion of collector powders, and (3) rate of cooling of powders subsequent to drying. Application of the tests for estimating processing mistreatment should be made as soon as possible after drying to avoid storage complications. Three general procedures have received group acceptance and are in general use: the water solubility method of Stuart et al. (1942); the KCl method of Thistle et al. (1943); and the NaC1-refractometric method of Haenni (cf. Hawthorne, 1944). The solubility test devised by Stuart, Grewe, and Dicks (1942) depends on the use of distilled water as the solvent, and measuring either the volume of a centrifugate of the extracted, heat-coagulated proteins or the volume of a picric acid precipitate. The volume of the centrifuged heatcoagulate is known as the “Stuart Index.” The authors report the results
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of a study of the relation of this index to scrambling quality and suggested a minimum index value for acceptability of powders. The textures of the coagulates made from recently prepared powders and those from stored powders were observed to be quite different and behaved differently when centrifuged. They also investigated the baking quality (sponge cake) and solubility index correlation and again observed a difference in behavior between recently processed and stored powders. They suggested two kinds of insolubility: process insolubility and storage insolubility. Stuart, Hall, and Dicks (1942) show that storage solubility changes are much more pronounced at powder moisture contents of 5% or greater than when the moisture is less than 5%. Stuart et al. (1945) associate change in powder solubilities with sanitary history as estimated by direct microscopic counts. Reference has already been made to the KC1 value devised by Thistle et al. (1943). The test, as described by these authors, is made by suspending 2.2 g. of powder in 100 ml. of 10% KCl solution and, after shaking for 1 hour, filtering through No. 1 Whatman filter paper. A 20-ml. aliquot of the filtrate is coagulated and dried at 110OC. (230°F.) for 16 hours and the residue weighed. After deducting the weight of the dissolved potassium chloride, the results are expressed as percentages of the original weight of the powder sample. A similar procedure using water as the solvent gives a result termed “water value.” The two values are in fair agreement when “prime quality” samples are used. The water value is notably greater than the KCl value when dust-collector samples are compared. The powders used in their investigations contained 3.08 and 6.78% moisture. The correlation of the KCl value with palatability did not differ significantly with that obtained with fluorescence measurements and palatability. The authors point out that the quantity of material passing through the filter is greater than the sum of the nonlipid components of the powders and assume that some lipid-protein complex is rendered filterable by the salt solution. They conclude that processing or storage treatments alter this complex and result in low values. As is true of the fluorescence test, no correlation of KCl values with palatability was observed when prime quality powdem are used. The authors ascribe the poor correlation of KC1 value and fluorescence (“prime quality” samples) with palatability to the weaknesses inherent in the palatability technique. It is more probable that since the changes measured are not those directly responsible for changes in palatability, differences in solubility and fluorescence may be expected to occur without concomitant changes in palatability. Hawthorne (1944) has reported the results of a comparative study of the several solubility methods in general use. The centrifuged, heatcoagulable solubility index and the Esbach sedimentation index (Stuart,
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Grewe, and Dicks, 1942) were examined. Only a weak correlation was found between solubility in potassium chloride and either index when commercial samples were used, though good correlation between the 2 methods was obtained by using mixtures of soluble and completely insoluble powders. The water value and potassium chloride value procedures described by Thistle et at?. (1943) were also used in the study. Comparative results are not reported, but Hawthorne observed an invariably greater solubility in potassium chloride than in water, a finding contrary to that published by the Canadian workers. Hawthorne accepts the chemical method as the best for determining the percentage of soluble protein in the egg sample. In brief, the procedure is: first, determine the total nitrogen of the sample; second, extract the soluble nitrogen with 10% potassium chloride and filter; and finally, estimate the total nitrogen in the filtrate. The results are expressed as the % of the total nitrogen extracted by the salt solution. The solubilities of carefully prepared samples of spray-dried powders range from 95-9870. Because the chemical procedure is too time consuming to be serviceable in control laboratories, Hawthorne sought a physical method which would most nearly yield results equal to those obtained by the chemical method. A physical method which correlates best with the chemical method is that devised by Dr. K. D. Haenni and described by Hawthorne. The method makes use of a refractometer and a 5% sodium chloride extract (1 g. powder in 5 ml. of salt solution). The results are expressed as the “solubility index by refractometer,” defined as: SI. = NDZ5(test solutions) - ND25 (solvent) X 1000. This procedure eliminates the need for refractometer corrections and minimizes errors due to small temperature variations. White and Grant (1943) recommend defatting powders with petroleum ether, followed by extracting for a period of 2 hours with a 5% solution of sodium chloride before estimating the refractometric value. The value determined by use of the defatted salt-water extract was found to be linearly related to the content of water-soluble nitrogen of the whole powder and to the potassium chloride value. A curvilinear value was obtained with the content of crude albumin nitrogen and with the potassium chloride value of whole egg powder. They found that the potassium chloride values of whole egg powder of approximately 35% and higher give a measure of both the amount of soluble protein and of the fat-protein complex. Values below 35% are approximately the same for both whole and defatted powder, indicating the potassium chloride value in this range to be dependent only on the amount of soluble protein present. They remark, “for the most part, the potassium chloride value gives a measure of the overall quality rather than of solubility alone.” Stuart, Grewe, and Dicks (1942) prepared pound cakes using powders
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possessing markedly different solubilities (Stuart index ranging from 0.11.3) and correlated solubility with texture, grain, taste, color, and volume. Their results show a direct relation between solubility index and cake quality. Of the factors defining cake quality, “cake volume” shows the most consistent relation to solubility or solubility index of egg powders. They suggest that factors other than solubility exert profound influence on the properties measured, especially on grain and texture. Preparation of Madeira cakes has likewise shown a correlation between the baking quality and powder solubility as estimated by a modified Haenni method (Grover and Hawthorne, 1945). 6. Aeration Power
Aeration power, also termed foaming power and whipping power, is a criterion of quality adopted from the egg white evaluation procedure. The property of retaining air, introduced during vigorous whipping, to form a more or less rigid structure (leavening), maintained during baking, is one of importance in imparting lightness and characteristic texture to some cakes. It is used as a substitute for the more time-consuming baking tests and the conditions of time, temperature, rate and manner of whipping have been adapted largely from the accumulated experience of the dried egg white industry. The quantity and stability of foams, prepared under standard conditions, serve as an estimate of the usefulness of the powders in the baking industry. Bennion et al. (1942) appear to have been the first to report the results of a study of the relation of aeration power of whole dried egg powders to baking quality. They applied the experimental procedure and method of expressing results (“percentage increase in volume”) derived by Henry and Barbour (1933) to a series of estimations of the beating properties of commercial spray-dried whole egg powders. No sample of commercial spray-dried powder examined gave foam of suflicient volume and stability to allow a measure of foaming or of foaming power. By increasing the quantity of reconstituted mixtures used in the tests, adding sugar (75 ml. of egg and 60 g. of sugar) and carrying out the beating operation in a constant-temperature room at 18.3”C. (65°F.) under standardized conditions of time and beater speed they were able to form measurable foams which correIated in voIume with the grades of sponge cakes prepared from the powders. Evidence of deterioration of aerating power and of baking properties were observed in stored powders. Commercial powder samples were always found to be inferior to samples freshly prepared in the laboratory. Hawthorne and Bennion (1942) later revised the test by beating at 40°C. (104°F.). The modification results in increased diflerence of volumes between samples, and thus in more accurate grading. Beating a t higher
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temperatures was found to give even greater foam volumes, but was impractical because of the difficulty of maintaining the temperature during the test. The authors suggest that it should be possible by a suitable choice of conditions to prepare first-quality sponge goods from almost any sample of spray-dried egg powder, and that the optimum conditions of use for a given sample of powder may be determined by preliminary small-scale tests. Reid and Pearce (1945) have reported the results of a study of objective tests as means of estimating baking quality. Quality was estimated by measuring loaf volume of cakes prepared by a standardized procedure. Foam volume was measured after beating a mixture of sugar, egg powder, and water, maintained at 42.8'C. (109°F.) for 10 minutes. They conclude that the potassium chloride value and salt-water soluble fluorescence of untreated powder are about equally useful in predicting foam volume. Closest correlation, however, was obtained between foam volume and loaf volume.
7. Thickening and Emulsij&g Power Reports of investigations of the nature of the chemical and physical changes that may cause losses in thickening and emulsifying powers are not available. The storage lives of 5 commercially prepared spray-dried whole egg powders have been investigated by Dawson el al. (1945), who used subjective criteria. The 3 4 % moisture powders were stored in hermetically sealed vessels for 1 year at temperatures of o", 7.2", 20°, 23.9", 30°, and 43.3"C. (32", 45", 68")7 5 O , 86", and 110°F.). The degree of quality retention by the 2 samples possessing best original quality was measured by evaluating the powders in 5 food preparations. Thickening power (custards) was retained without serious loss for 1 year at 7.2"C. (45°F.). The storage life of powders was reduced to 20 weeks by storage a t 23.9"C. (75°F.). Solubility (Stuart index) was reported to correlate closely with functional properties in baked custards. The emulsifying power (mayonnaise) of the same samples was retained for 1 year while stored at 20°C. (68°F.))but was lost after 31 weeks at 23.9"C. (75°F.) Tracy et al. (1944) found that the usefulness of spray-dried yolk for improving whipping of ice cream mixes and imparting desirable body properties of the frozen product was retained even though the powders were stored under unusually severe conditions of time and temperature.
VII. CHEMICAL AND PHYSICAL CHANGES ASSOCIATEDWITH DETERIORATION During the period of intensive study necessitated by the emergency imposed by war, the objectives of the researches were clearly to supply
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“know-howl1 of processing and storage. Since basic information was not available for application to the solution of problems arising in a new and rapidly expanding industry, and since the need was urgent, research was conducted very largely by the empirical or trial and error method. Nevertheless, a11 who were engaged in the effort to supply the industry with guiding information were probably aware that satisfactory solutions to the many problems could be attained only through comprehensive knowledge of the nature of the chemical and physical changes taking place in the raw materials and the finished products. Most groups engaged in the work undertook to investigate the fundamental nature of the changes as time and facilities permitted. Powdered whole eggs consist of a diversity of components such as proteins, lipoproteins, lipids with an unusually high percentage of phospholipids, carbohydrates, and pigments. During homogenization and drying, all these components become intimately mixed and all organization of the original egg is lost. The problem, therefore, becomes the more complex, due to the increased opportunity for components to react with one another in addition to the usual deteriorative reactions of the components themselves. Both kinds of reactions have been found to take place, but basically the problems are not new, being mainly those of lipid and pigment oxidation and protein denaturation changes. In addition, however, certain changes, which may be ascribed to the unusual composition of the product and are peculiar to po\;rdered egg, necessitate new procedures for their control. 1. Proteins
The proteins of egg white have probably received as much attention as any other group of proteins. The amino acid composition of some of them is as well established as the presently available methods for amino acid analyses will permit. The component proteins of egg white have been identified electrophoretically (except for mucin) (Longsworth et al., 1940) and some of the individual proteins have been isolated. Their biological properties have been given much attention, but can scarcely be said to have been thoroughly explored, as witness recent reports on lysozyme (Alderton et al., 1945, 1946), conalbumin (Alderton et al., 1946), and antitrypsin (Lineweaver and Murray, 1946). Yolk proteins have not been investigated as thoroughly as have those of the white; only recently evidence indicates both vitellin (Fevold and Lausten, 1946b) and livetin (Lundgren and Ward, 1947) are protein groups rather than entities. There appears to be little information available concerning changes in the proteins that occur in eggs before they reach the driers. The causes of viscosity changes resulting in thin white are still without adequate
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explanation though the matter has been a subject of much research. Enzyme activity does not appear to offer an adequate explanation (Balls and Hower, 1940). These authors found that the total mucin content did not vary but the liquefaction of thick egg white is due to a physical change in the mucin fibers. This change can be retarded by maintaining the pH near 8 (Sharp, 1937). St. John and Caster (1944) estimated the bound water of thick and thin egg white by a calorimetric method. A1 though marked variations were found to exist between individual eggs, in the average % ’ of bound water they were unable to correlate variations with the “wateryness” of the white. Much the same state of knowledge exists in relation to protein changes taking place in frozen (or thawing) eggfi. It is customary to refer to the changes as alterations in the colloidal state. Perhaps the peculiar behavior of the recently isolated lipoprotein, lipovitellenin (Fevold and Lausten, 1946a), which undergoes changes in solubility (denaturation) when frozen (or during thawing), may contribute to an explanation of the alteration in properties that are commonly observed in frozen yolks. Changes in the egg proteins during spray drying are those of decreased solubility due to excessive heating. Under good operating conditions the protein solubility compares favorably with that of the lyophilized product, and hence is of minor importance in relation to most of the functional properties of spray-dried egg powders. The aerating properties of spraydried egg powders, however, are always markedly inferior to the fresh whole egg or the lyophilized product because of changes not understood at the present time. Aeration power does not apparently correlate with solubility. The accumulated information resulting from studies of changes taking place in stored dried egg white appears to be directly applicable to the protein changes taking place in stored whole egg powders. The changes are manifested in three ways: (1) a change in solubility of the proteins; (2) a discoloration of the product because of the development of a brown pigment; and (3) the development of fluorescing substances solubIe in salt water. All of these changes apparently develop simultaneously and seem to be controlled by the same factors. Although reactions responsible for loss of solubility, development of color, and appearance of salt-water soluble fluorescing substances will be discussed later, it should be stated here, for the sake of clarity in the discussion that follows, that they are believed to be due to the same or to closely related reactions. Balls and Swenson (1934) suggested that the darkening of trypsintreated unfermented egg white might be the result of condensation of reducing sugars with the amino groups of proteins and protein hydrolytic products. This reaction had been shown by Maillard (1916) to result in
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the dark-colored products (humins) often associated with proteinaceous products. It has later been shown by various investigators that sugars are involved in the darkening of egg white and egg powders. As a result of an investigation of the effect of fermentation of liquid egg white upon the stability of the dried product when stored, Stewart and Kline (1941) found that flake albumen freed from dextrose by bacterial action was color stable for many weeks at 40°C. (104°F.) when the pH of the liquid white ranged from 6.3-8.5 before drying. Furthermore, the addition of as little as 0.1% dextrose to the sugar-free white before drying caused loss of solubility and development of pigment during storage for 12 weeks a t 4OOC. (104°F.). Storage deterioration rate of dried albumen, as measured by solubility and fluorescence, was found to be retarded by prefermentation (Stewart et al., 1943). Using yeast to remove the sugar from whole egg pulp, Hawthorne and Brooks (1944) demonstrated an improvement in the storage life of the dried products (moisture approximately 5%). Storage improvement was shown by lowered rate of denaturation and browning, retardation of pH changes, fluorescence, creaming, and loss of beating power. They compared the effectiveness of removal of reducing sugar (glucose) by fermentation and the addition of nonreducing sugars such as lactose or sucrose (10-15’%) as methods of retarding deterioration, and concluded that the 2 procedures were about equally effective in preventing development of fluorescence and pH changes. Powders prepared from yeast-treated pulp retained such beating power as they possessed a t the initiation of storage, but the beating power of the freshly prepared material was little, if any, better than the ordinary spray-dried product. Since yeast fermentation introduced foreign flavors, it was found impossible to estimate satisfactorily off-flavors which may have developed. Bate-Smith and Hawthorne (1945) investigated the quantitative changes in reducing sugar and amino acid nitrogen of stored dried egg products, and correlated the observed changes with losses of solubility, i.e., % of total nitrogen soluble in 10% potassium chloride solution or water. In whole egg powders the loss of glucose and reduction in solubility were found to take place simultaneously. A considerable time lapse was observed between disappearance of sugar and loss of solubility in dried whites. The presence of lactose or sucrose (10-15010) in whole egg powders did not prevent the loss of glucose, but did retard the rate sharply, and resulted in a lag period during which there was a loss of glucose without loss of solubility. A similar lag in solubility change, without retarded glucose disappearance, was observed when pulps were adjusted to pH 6 with lactic acid before drying. Changes in amino nitrogen content during storage were observed by use of the Van Slyke method. Marked reduction in
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HOWARD D. LIQHTBODY AND HARRY L. FEVOLD
free amino nitrogen was observed with egg whites, sugar-dried egg, and whole egg powder prepared from pulp adjusted to pH 6. The free amino nitrogen losses parallel the losses of glucose but during the first few days’ storage, no changes in solubility occurred. With whole egg powder unmodified by sugar addition or pH adjustment the decreases in sugar, amino nitrogen, and solubility occurred at the same time. The authors’ data show that glucose disappears at apptoximateIy the same rate from norma1 (pH 8.5) whole egg and acid powder, and this similarity of rate is taken to indicate that the same basic reaction occurred in each case. By adding varying quantities of glucose to fermented pulp, Bate-Smith and Hawthorne (1945) found the amount of sugar normally present is sufficient to produce maximum insolubility. Replacing glucose with sorbitol or methyl glucosides resulted in retention of solubility. The addition of 5-10% glycine or alanine to unfermented pulps before drying served to retard the development of insolubility, but browning and fluorescence were not favorably dected. Cysteine gave better results with respect to fluorescence and color, and was effective at lower concentrations. They suggested that removal of glucose is unnecessary, since it is sufFicient to prevent the reaction of glucose with the proteia amino group^, and the best method of doing this would appear to be to add an amino compound to the egg pulp before drying. The amino compound must be so selected as not to result in formation of products contributing to off-flavors. Kline and Fox (1946) make a similar suggestion with regard to prevention of development of color and insolubility in stored dried egg white. Since cysteine reacts with glucose to form a relatively stable, colorless thiazolidine carboxylic acid, they suggest that this amino acid may have special virtue in rendering glucose of dried egg white nonfunctional. The storage properties of egg powders prepared from fermented liquid whole egg to which monosaccharides were added were also investigated by Bate-Smith and Hawthorne.(1945). The sugars were added in quantities to effect reducing power equivalent to that attributable to glucose in unfermented pulps; namely, about 0.4 g./lOO ml. Addition of glucose, mannose, fructose, and arabinose resulted in each case in insolubility. The action of arabinose was particularly marked. It seems of interest to note that Ball et al. (1943) found these sugars to fall in the same order as judged by decreasing protection against sulfhydryl formation during heat coagulation of egg albumin. Ball et aE. found L-arabinose to be much more effective than D-glucose, D-fructose, or D-mannose in preventing heat coaguIation of egg albumin in solution at 70°C. (158’F.) for 15 minutes. After heating and removing the coagulum by filtration, the sugar protein mixtures were dialyzed and adjusted to pH 4.8. Coagulation was then observed, indicating that denaturation had occurred in the filterable albu-
SHELF LIFE OF DRIED WHOLE EGQS
173
min. Hardt et al. (1943) reported electrophoretic evidence for the inhibition, by glucose, of formation of the “C” complex of Van der Scheer et al. (1941) appearing in heated horse serum. To explain their results, Bate-Smith and Hawthorne suggest that the process which results in loss in solubility takes place in two stages. The first reaction consists of condensation between reducing groups of the glucose and free amino groups of the proteins. The products of this reaction must then undergo further changes which result in the protein becoming insoluble. These workers showed that the insolubilizing reaction is inhibited by adding lactose or sucrose, or by lowering the pH. The second, i.e., insolubilization reaction, may be due to a rearrangement of n-glucoside compounds with the formation of isoglucosamines, since the latter possesses CZcarbonyl groups. This hypothesis was tested by adding a number of methylated glucoses and observing solubility changes. The results indicated that blocking the hydroxyl group on Cf of the glucose did not prevent loss of solubility. Even when all the hydroxyl groups were blocked by methylation, loss of solubility was observed, though at somewhat slower rate. They suggest that the lowered rate is due to decreased activity of the reducing group. The development of color and fluorescence in an aqueous solution of glucose and glycine, buffered at pH 7.3 and held at 50°C. (122”F.), was studied by Olcott and Dutton (1945). The curves representing optical density and fluorescence were found to be superimposable over the time range of 50-300 minutes. In parallel experiments the same authors determined the amino nitrogen loss in solutions (pH 8) of glucose and egg albumin (crystalline), egg-white globulin, livetin, lipovitellin, and casein held for 7 days at 5OOC. (122°F.). Losses of amino nitrogen ranged from 3444%. The rate of browning of glucose and glycine was increased with increasing moisture content up to lo%, and by increasing pH. Frankel and Katchalsky (1937) found that the reaction between amino acids and peptides with aldoses had a pH optimum. Although the optimum varied somewhat, depending on the reactants, it appeared to be rather fairly consistently near pH 8, and falling off to near zero at pH 4 and 10. These experiments indicate rather clearly that fluorescence as well as pigment formation in dried egg is the result of the glucose-amine reaction. 2. Lipids and Lipid Solubles
The considerable quantity of lipids, present in whole egg powders, led to early consideration of the deteriorative changes they may undergo during processing and storage. Much confusion arose during the time of intensified research regarding the occurrence of fat changes during storage, and the reliability of quality criteria based on the usual rancidity tests.
174
HOWARD D. LIGHTBODY AND HARRY L. FEVOLD
Tests of stored powders for peroxides, aldehydes, free fatty acids, and liberated phosphorus do not show correlating increases with quality losses, as the changes are measured by the usual methods (Bate-Smith, 1942). Significantly, the British investigators observed a drop in the peroxide number to zero during storage in nitrogen. A small decrease in the oxygen of the atmospheres of hermetically sealed cans containing whole egg powders was observed. The absorbed oxygen was reported to have a great effect on flavor. That oxidation is a significant factor in deterioration, is evidenced by the prolonged shelf life of powders packaged in inert gases to insure maximum exclusion of oxygen (see last section of this review). Makower and Shaw (1946), using whole egg powders prepared by lyophilization and a constant volume respirometer ("Summerson" type) , determined the rate of oxygen absorption, temperature coefficient, and variation of the rate of absorption with the moisture content. Measurements were made at 7 different moisture levels varying from 0.25%-7.070 and a t 22", 30.2", and 39.9"C. (68", 86", 104°F.). The rate of oxygen absorption was found to be approximately doubled as the moisture was increased from the minimum to the maximum values used on the experiments. The temperature coefficient per 10°C. (50°F.) appears to be approximately 3.1. Vitamin A and the carotenoids are among the egg powder components most suspect of ready destruction by atmospheric oxygen. Klose et al. (1943), Hauge et al. (1944), Denton, et al. (1944), and Cruickshank et al. (1945), have reported losses of vitamin A in powders during storage. Low storage temperatures increase the vitamin retention. The latter authors found no loss in a nitrogen atmosphere at 15°C. (59°F.) for 5 months. Similar beneficial results of nitrogen storage were obtained by Fevold and Shapiro (1946), who also found the rate of vitamin destruction to be independent of the moisture content in samples ranging from 1.0-9.2% moisture stored for 6 weeks a t 37°C. (98.6"F.). Klose et al. observed a retarded rate of loss during storage in sealed containers, and Bohren and Hauge (1946) found that powders, sealed in tin cans, retain about 75% vitamin A potency regardless of storage temperatures. The rate of destruction of carotenoids roughly parallels that of vitamin A loss. Spectrophotometric methods of pigment analysis have been used by Hauge et al. (1944) and by Dutton and Edwards (1945). The former authors reported carotenoid losses, amounting to 29% of the total, during storage for 0 months at room temperature, while the latter found the loss to amount to 26y0 during 6 months' storage in barrels at 37°C. (98.6"F.). Dutton and Edwards found no pigment destruction, during the same storage time, when storage temperatures were held a t -9.4"C. (15°F.). The relation of formation of free fatty acids to storage and quality
SHELF LIFE OF DRIED WHOLE EGGS
175
changes was investigated by Brooks (1943). He estimated the acids by using the association of official agricultural chemists (A.O.A.C.) method and expressed the results as ml. of 0.05 N sodium ethylate solution necessary to neutralize the acids from 1 g. of ether extract. The preliminary drying of the powder recommended by the A.O.A.C. was deemed unnecessary and omitted. Brooks concluded that freshly dried egg normally contains small quantities of ether-soluble acid (1.30-1.56 ml. sodium ethylate/g. ether extract), and found increases during storage, depending on water content and temperature. Maximum increases were held to be of little significance in relation to quality change. Fryd and Hanson (1944)) using the same method of estimation and likewise omitting drying, concluded the free fatty acids to be important in flavor and included a factor, which is a function of free fatty acids, in an equation recommended as a means of predicting flavor scores. The factors influencing estimation of free fatty acids in dried egg powders, were examined experimentally by Kline and Johnson (1946). They showed that the use of the A.O.A.C. method (1945), because of incomplete extraction of fatty acids (added to liquid eggs prior to drying), results in low absolute values. The error can be eliminated by reconstituting, acidifying to pH 4.5, and drying from the frozen state under vacuum, prior to extracting the oil. These authors also found marked differences in the acidity of ether extracts prepared from samples of whole egg powder equilibrated to moisture levels ranging from 0.314.99%. A study of the extracted materials showed that approximately 70% of the acidity of the ether extract from fresh egg powder resides in the phospholipid (acetoneinsoluble) fraction of the oil. Since ethanolamine-cephalin acts as a monobasic acid which is completely titrated to phenolphthalein in solvents of low dielectric value (benaene is used in the A.O.A.C. method), these authors assumed that the high values were attributable to cephalin, and the variable results to variable extraction of cephalin with changes in moisture content. By comparing the alkali-binding capacity and the amino nitrogen of the acetone-insoluble fractions of ether extracts, these authors were able to confirm the relation of cephalin to the titration values. Cephalin nitrogen is about 30% of the total nitrogen in the phospholipid mixture from unstored spray dried powders, and a somewhat greater percentage from the powders prepared by lyophilization. Cephalin, in the latter case, accounts for about 38% of the total acetone-insolubles of the ether soluble fraction. Van Slyke values on the unhydrolyzed phospholipids are lower than those on the hydrolysed phospholipids, and the sodium ethylate equivalence on the unhydrolyaed preparations from stored powders, is probably due to the ease of hydrolysis by alkali of an amide formed during storage.
176
HOWARD D. LIGHTBODY AND HARRY L. FEVOLD
Johnson and Kline (1946) proposed a method of determining the free fatty acids in whole egg powders. The method makes use of the differential solvent power of cold acetone as a means of extracting the glycerides and free fatty acids from the powders, and uses an alcoholic magnesium chloride solution to remove traces of the phospholipids extracted. The proposed method does not require preliminary drying since the differences in cephalin extracted at varying moisture levels are eliminated. Further experimental attention was given to the difficulty of obtaining reproducible results on the same lot of egg powders at the same moisture level, a difficulty which is due to inconsistent extraction of the fatty acids unless the pH be adjusted to 4 prior to drying. Although it was found that acetone did not completely recover oleic acid, added to or present in liquid egg mixtures prior to drying and without adjusting pH, extraction of the acids was complete from powders which had developed adidity while in the dry state. The failure of the solvent to remove the acid added to the liquid mixture before drying, is attributed to combinations formed with bases in the egg mixtures. Oleic acid added to separate yolk and white resulted in only 4% recovery from whites and 81% from yolks at pH 8, while at pH 4, oleic acid recovery from the yolks was 84% and from the whites 96%. These authors suggest that the basic proteins, particularly lysozyme, may be responsible for acid retention through salt formation. It is apparent from these results that, because of the limitations in the analytical method generally used, manifold increases in free fatty acid content of stored egg powders have been masked by the presence of cephalii in the acid extracts. Unfortunately, reports of studies of the relation of free fatty acid content t o storage changes and quality deterioration are not now available. An investigation, by Edwards and Dutton (1945), led to the suggestion that the cephalin amino group may contribute to powder discoloration by reacting with aldehydes. A brown product was prepared in concentrated form by extracting with ether, a powder sample, that had been stored for nine months at 37°C. (98.6"F.). The light absorption properties were compared with a reaction product of cephalin and acetaldehyde and the 2 absorption curves were found to be closely comparable through the wave length range to 250-550 p . Reactions of ethanolamine (the nitrogenous constituent of cephalin) with acetaldehyde gave a brown product which possessed adsorption properties similar to those of the cephalin-aldehyde and the material which was separated from stored powders by ether extraction. The isolated and synthetic products were strongly fluorescent. The authors suggest that the failure to detect aldehydes in egg fat extracted from stored powders of low quality, may be due to the prompt reaction of these compounds with amines. They suggest also that the
SHELF LIFE OF DRIED WHOLE EQQB
17Y
formation of lipid amine-aldehyde reaction product, implies oxidation since aldehydes have been shown to develop during the oxidative breakdown of fats. In a second paper, Dutton and Edwards (1946), reported the results of a spectrophotometric and fluorometric study of the ether extract of stored whole egg powders, and of fractions prepared by saponification, and by acidified ether and dilute acid (aqueous) extractions. The powders used were spray dried, contained about 5% moisture, and had been stored in barrels at 36.7", 21" and -9.4"C. (97", 70", and 15°F.). They were examined at the end of 1, 3, 6 and 9 months of storage. The absorption (A-270 p ) by the saponified dilute acid (aqueous) fractions, (lipidamine reaction products) showed a high reaction rate which decreased with time. A t the intermediate storage temperature the reaction proceeded more slowly and at a nearly constant rate. A similar reaction product was not found in powder stored at -9.4OC. (15OF.). The absurption, a t A-270 p, by the saponified acidether fraction from powders stored at the 2 higher temperatures, likewise showed increased intensification with time. Evidence of increases in fatty acid conjugated double bonds, found in absorption spectra, was interpreted as increases in triene and tetraene conjugation. These authors point out, however, that the observed increases in absorption can be equally well explained by assuming the development of generally absorbing polymerization products, and suggest polymerization to be the more likely explanation. A study of the fluorescence properties of the fractions revealed the total fluorescing substance to be in the acid aqueous fraction. It appears, therefore, that the fluorescing materials of the total lipid extracts are released from their lipid combinations in the stored egg powders, by saponification. Since the degree of fluorescence of the total lipid extracts and the acid aqueous fraction parallels the increasing absorption at A-270 p, the identity of the fluorescing and absorbing substances is indicated (i.e., lipid amine-aldehyde reaction products). A report regarding the correlation of the lipid soluble fluorescence with palatability, has already been discussed in the section on criteria of quality and deterioration. An investigation of the properties of cephalin prepared from egg powders has been made by Kester (1946). He finds that use of the present available methods does not enable the separation from eggs of cephalin which, by analysis, meets the requirements of a glyceride containing 2 fatty acids and 1 phosphoethanolamine. This experience appears to be universal among workers who have attempted isolation and purification of cephalin from any source. Cephalin prepared from egg powders does not show significant evidence of changes during storage as indicated by nitrogen and phosphorus contents or N/P ratios. Amino nitrogen of
178
HOWARD D. LIGHTBODY AND HARRY L. FEVOLD
cephalin preparations from slightly deteriorated powders is lower than that from fresh powders, but is restorable to a considerable degree by acid hydrolyses. For example, the amino nitrogen of a cephalin fraction from a sample of freshly prepared powder had an amino nitrogen content of 0.92%) and that from a mildly deteriorated powder only 0.53%. After acid hydrolysis 0.93y0 and O.Sl%, respectively, of the 2 samples appeared as amino nitrogen. Amino nitrogen in extensively deteriorated cephalin, however, was markedly lowered and was restorable to only a minor extent by acid hydrolysis. I n this case acid hydrolysis increased the amino nitrogen to 0.55% of a preparation that was found before hydrolysis to contain but 0.35% of amino nitrogen. Kester (1946) has confirmed the fact, repeatedly observed by previous investigators, that fatty acids isolated from phospholipids (particularly cephalin) contain non-removable nitrogen (and also phosphorus) amounting to 4-67’0 of the total nitrogen of the unhydrolyzed compound. Cleavage of unsaturated fatty acid chains with the formation of aldehydes, probably occurs late in the chain of reactions. This results from the initial formation of peroxide compounds, caused by oxidation. He suggests that following the rearrangements of the peroxide to form alkenols and epoxides, the latter may be expected to react with the amino group of the molecule to form substituted alkylol amines. These may account for loss of amino nitrogen during cephalin deterioration, as well as the chemically bound nitrogen in the fatty acids. Aldehyde production (Kreis test) in egg oil undergoing oxidation under a variety of conditions has been studied by Edwards el al. (1946). The acetone soluble egg fat stored alone, rapidly develops aldehydes and rancid odors. Aldehydes do not develop and rancid odors do not appear in the same fat containing added phospholipids. Added livetin or egg white also retard the appearance of aldehydes. Finally, egg fat which has been allowed to become rancid gives a decreased Kreis test after storage with added phospholipid or livetin. It appears probable from these results that either phospholipids prevent the positive response to the Kreis test by combining with the aldehydes, or, because of the ease of oxidation, prevent the formation of aldehydes by some reaction such as that suggested by Kester. Since the egg proteins can scarcely act as “antioxidants” by removal of oxygen, it seems likely that the effect observed in the presence of proteins is due to an aldehyde-amine reaction. The inhibition of aldehyde formation by phospholipids and proteins, as indicated by the Kreis test, appears to offer an explanation for the failure of air-stored egg powders to respond to the usual rancidity tests. In order to identify the component(s) of whole egg powder which undergo changes, resulting in off-odors and off-flavors, storage studies with whole egg powders and certain fractions of such powders were carried out
179
SHELF LIFE OF DRIED WHOLE EGG8
TABLFJ I Eflect of Storage of Whole Egg Powder, Yolk Powder, Egg White Powder, Crude Lipovitellin and the Fat-Livetin Fraction of Yolk on Palatability and Fluorescence Values of Reconstituted Whole Egg Powders"
Storage temp., "C. Powders used *
-34 WE
....
WE
........
WE
Y&W
y,w
....
Y&W
Y
W
Y&W
W
Y
Y&W
........
y, w
LV, FL, W
LV, FL, W
....
LV,FL, W LV,w LV, FL, W
w
- -
+36.5
WE
LV, FL, W FL, W
Scoresc after storage
LV FL FL, LV
0
P SWF FSF P SWF FSF P SWF FSF P SWF FSF P SWF FSF P SWF
FSF P
SWF FSF P SWF FSF P SWF FSF P SWF FSF
2 4 8 weeks weeks weeks
-
9.0 17
8.7 17
8.9 17
9.0 17
6.0
22
5.3 25
9.0 16
9.0 16
9.3 14
9.0 16
8.9 20
9.0 20
9.0 16
7.6 15
6.5 15
9.0 16
7.4 18
6.1 20
8.8 14
8.8 14 7 8.7 14 8 7.2 15 14 7.0 15 15
8.9 14
..
.. ..
..
..
.. ..
8.8 14
..
8.8 14
..
8.8 14
..
--
..
..
..
..
..
..
*.
..
..
..
..
..
..
8.5 14
..
6.4 16
..
6.0 16
..
9.1
..
4 4.4 29 49 9.1 13 4 8.6 21 4 5.1 15 33 5.0 23 31 8.9 14 7 6.0 14 11 4.5 21 38 4.6 19 33
-- -
16 weeks 9.0 17 4 3.7 45 67 9.0 16 4 8.6 32 5 5.0 20 50 4.9 37 47
.. .. ..
.. .. ..
..
..
.. .. .. ..
Fevold et al. (1946). Moisture contents were: egg powder 1.8%, yolk powder 1.3%, egg white 4.2%, Lipovitellin 3.6%, fat-livetin 1.8%. Y = yolk, W = white, LV = Lipovitellin, FL = fat-Livetin, WE = whole egg powder. Whole egg, egg white, and yolk powders are in vapor pressure equilibrium with one another at 2.6, 1.3, and 4.2% moisture, respectively, at 10°C. (50°F.). Makower reporta the corresponding value for whole egg powder to be 2.0. c P = palatability, SWF = salt-water fluorescence, FSF fat-soluble fluoreecence. 0
b
-
180
HOWARD D. LIOHTBODY AND HARRY L. FEVOLD
by Fevold et al. (1946). Yolks and whites were separated, and the yolk further divided into 2 parts designated as lipovitellin (85% lipoprotein and 15% “fat”) rnd fat-livetin. Whole eggs and each of the 4 fractions were dried from the frozen state. The whole egg powder and each of the fractions were stored at -34°C. (-29.2’F.) and 36.5”C. (97’F.) for 16 weeks. The products stored at the low temperature served as controls. Reliance was placed upon palatability (off-flavor and off-odor) as the most suitable criterion of deterioration, though salt water soluble fluorescence and lipid fluorescence determinations were also made. To estimate deteriorative changes, each of the products and fraction mixtures was reconstituted with control fractions to simulate whole egg composition and t,hen scrambled. A summary of the authors’ results is given in Table I. These authors concluded, from their data, that palatability changes were quite independent of the measured changes in the proteins, and that off-flavors were present only when the tested materials contain lipids which had been stored at the high temperature. Salt-water soluble fluorescence values failed to show a correlation with flavor changes; this is to be expected since the fluorescing materials, extracted by salt solution from powders after defatting with an organic solvent, are most likely of protein origin. The observed correlation between lipid fluorescence and palatability was very good. It is still questionable, however, whether the same chemical reaction is responsible for both lipid fluorescence and off-flavors. The egg components responsible for flavor changes were further traced by fractionating the “fat-livetin” mixture with organic solvents. Because of the difficulties encountered in freeing the fractions of solvent, it was necessary to use a modified palatability testing procedure which gave only qualitative results. Storage of livetin resulted in no development of offflavor. When the stored phospholipids were added to whole fresh egg powders the resulting products were found to have the off-flavor characteristic of storage deterioration. Studies using the third fraction (acetonesoluble fats, containing about 10% of the original phospholipids) yielded inconclusive results due to solvent retention and adverse effects upon texture when added to the control. The major flavor change in this fraction was not, however, characteristic of stored whole egg powders, but was due mainly to rancid odors and flavors. The authors concluded that the major off-flavor characteristic of stored whole egg powder arises from changes in the phospholipids. They call attention to the well-established protection afforded other fats against oxidation by the presence of phospholipids, and suggest that the usual rancidity changes are not observed in egg powders because of the antioxidant activity of the phospholipids. It appears, however, that the phospholipids are themselves oxidized and the oxidation products contribute to the flavor changes.
BHELF LIFE OF DRIED WHOLE EGGS
181
The usefulneea of some of the more common antioxidants in vitamin A preservation has been investigated by Fevold and Shapiro (1946). Hydroquinone, diphenylamine, gum guaiac, d-isoascorbyl palmitate, Z-ascorbyl palmitate, tocopherols, and wheat germ oil were added to liquid egg mixture before drying. Powders containing added a-tocopherol (0.02%) showed improved vitamin A retention about equivalent to the retention observed in nitrogen packed samples. Compression of powders into coherent blocks serve to retard the rate of vitamin A loss, and by compressing powder containing tocopherols, it is possible to attain complete retention of the vitamin during storage for 6 weeks at 37OC. (98.6OF.). So Specisc gravity of whole egg foam storage, weeks Dried materiala used and tested
+
Yolk whites Yolks stored at 34.4"C.(94°F.) whites stored at -34.4% (-30'F.) Yolka stored at -34.4"C.(94'F.) whites stored at -34.4"C.(-30°F.) Yolks stored at -34.4"C. (94°F.) whites stored at -34.4'C. (-30°F.) Yolks stored at 34.4%. (94°F.) whites stored at 34.4"C.(-30°F.)
+
+ +
+
a
0
12
16
-
-
0.31
0.62
0.23
0.25
0.23
0.25
0.32
0.68
Moisture content of yolk, 1.3% and of whitee, 4.2%.
far as the writers are aware, a systematic investigation of the usefulness of antioxidants as retardants of off-flavor development has not been reported. Olcott (1946) compared the antioxidant activity of 16 amino acids and derivatives with hydroquinone when added to egg-glycerides from which the phospholipids had been removed by acetone and magnesium oxide precipitation. Proline possessed the highest activity of the series. The inactivity of acylated amino acids suggests that the antioxidant action is dependent upon the presence of a basic group. The addition of proline (equivalent to 0.25% of the dry weight) to homogenired whole eggs was found to be of no value in retarding the loss of palatability. Information which indicates that deterioration of the lipids of the egg may have some bearing on retention of beating power of egg powders comes from dserent sources. Bennion et al. (1942) observed that the addition of surface active agents to reconstituted egg before whipping, caused increase in volume and stability of foam. The presence of the egg
182
HOWARD D. LIQHTBODY AND HARRY L. FEVOLD
however, depressed the foaming power of the surface active agent. They attributed the depressing action t o the egg lipids, and remarked that attempts to produce dried egg of improved baking quality must aim at preserving the lipid constituents unchanged (physically and chemically). Brooks and Hawthorne (1944) suggest that spray drying in the absence of sugar liberates some component, presumably lipid in nature, which inhibits foaming. They found that cold petroleum ether extracted 24% of the phosphatides and all the fat of stored dried egg. It extracted none of the phosphatides from fresh liquid or sugar dried egg and only 270 and 701, of the fat of these two, respectively. Boggs (1946) stored dried yolks (to which 20% sucrose had been added prior to drying) and dried whites (without added sugar) at 34.4"C. (94°F.) and -34'4°C. (-30°F.). Various combinations were made, rehydrated and beaten at 25°C. (77°F.). The results are shown on page 181 and indicate that the deterioration which causes reduction in aerating power takes place in the yolk.
3. Lipoproteins The relationship of lipoproteins to deterioration of egg powders has received virtually no attention. Maintenance of a lipoprotein complex is essential for the emulsifying action of egg yolk in mayonnaise production (Sell et al., 1935). The solubility properties of the lipoproteins change on storage, and this is especially true of lipovitellinin (Fevold and Lausten, 1946a). The relationship of this change to changes in the functional properties of the egg powder is, however, not known. Apparently the lipoproteins do not contribute markedly to loss of palatability (Fevold et al., 1946).
4. Enzyme Action As is true with other foods preserved without subjection t o heat, the deterioration action of enzymes in stored whole egg powder is quite possible. The formation of free fatty acids and phosphates has been suggested by Brooks (1943) to be due to action of lipase and lecithinase in comparatively low moisture products. It seems probable that the raw egg material entering driers may be variable in enzyme content. Commercial liquid egg mixtures are made up of fertile and infertile eggs. Embryonic development may have been initiated but not progressed to the state where detection makes elimination of such eggs possible. Bacterial contamination of the pulp during predrying handling is, of course, variable in quantity and types of microorganisms. The natural defense of egg white against air-borne organisms made possible by lysozyme appears to be largely lost when yolk and white are mixed (Berry, 1946). The survival of bacteria in the powders is dependent upon rates of cooling, moisture
SHELF LIFE OF DRIED WHOLE EGGS
183
content, and temperature. The sensitivity of proteins to denaturation during preheating set such limitations on temperature and time that little assurance can be given that enzymes of bacterial or other origin can be heat inactivated before the raw material reaches the driers. Much of the research on egg enzymes, as distinguished from microbial enzymes has been related to the development of the embryo as a part of the broader field of comparative morphology. Needham (1931, 1942) has reviewed the work on egg enzymes. Lipase, lecithinase, cholinesterase, procainesterase, amylase, catalase, histozyme, hippuricase, salicylase, “dopa” oxidase, carotenase, lysozyme, ovomucoidase, and protease have been reported to be present in unincubated eggs. Glycolysis (Needham, 1931) has been reported to occur in fresh eggs, while respiration of neither the whole egg nor the egg parts has been detected (Smith, 1931). Some of the results reported have been contested. The differences may be due in part to the low enzyme activities found in eggs compared with other sources usually studied. Faulty and, in part, obsolete techniques, however, have been used for identification and for estimations of the enzyme activity. Lineweaver et al. (1946) have recently reexamined the enzyme content of unincubated hens’ eggs, with due attention to age of eggs, sensitivity of assay methods, actions of microorganisms, and unstable substrates. These workers have examined eggs for activities of esterases (lipase, lecithinase, phosphatase, etc.), catalase, peroxidase, oxidases, and proteases. Since quality deterioration of whole egg powders is accompanied by changes in the lipid fractions, and since increases in fatty acids and organic and inorganic phosphorus have been observed by Brooks (1943) to be due to actions of esterases and lecithinase, these enzymes have received special attention. The possibility of the occurrence of other enzymically catalyzed reactions, however, is of basic importance in the over-all problem. Esterases have long been recognized to be present in eggs though complete agreement as to the quantities present does not exist. Wohlgemuth (1905) observed that free fatty acids were formed during aseptic autolysis of eggs. Koga (1923) interpreted increases in acidity of egg yolk used in his experiments as due to lecithinase action, and showed that ethyl butyrate was hydrolyzed by an enzyme in egg yolk. Curiously, Koga found no tributyrinase (presumably, therefore, the increase in acidity was due to lecithinase rather than to lipase), whereas Ammon and Schutte (1935) found considerable amounts in the yolk. Lineweaver et al. (1946) have confirmed Ammon and Sshutte. Since tributyrinase does occur in the yolk, the acidity increase observed by Koga might be interpreted as being due to either lecithinase or lipase. On study of Koga’s method, however, it was found that the increased acidities were almost certainly the result
184
HOWARD D. LIOHTBODY AND HARRY L. FRIVOLD
of microbial contamination. Koga determined 24-hour increases in acidity in diluted egg yolk containing 2% toluene. Experiments carried out by his technique revealed no increase in acidity in 8 hours, but a large increase in 24 hours. It was then shown that, in spite of the toluene, gross microbial contamination was consistently evident in 24 hours (but not in 8 hours). When microbial activity was suppressed with merthiolate no increase in acidity occurred even in 48 hours. Merthiolate in the concentration used does not inhibit egg tributyrinase. Further study of the egg lipolytic esterases, which appear in the livetin fraction of the yolk, showed that triacetin and tripropionin as well as tributyrin were readily hydrolyzed but that tricaproin and higher lipids were not attacked by egg esterases (ie., the experimental error was such that the rate was at most 1/1000 the rate of tributyrin). Since eggs contain only lipids with 16 or more carbon fatty acids (Riemenschneider et al., 1938) it is evident that uncontaminated eggs do not contain enzymes (either lipase or lecithinase) capable of hydrolyzing egg lipids. This conclusion removes the support from the suggestion of Brooks that lipolytic enzymes may be responsible for the increases in free fatty acids in eggs. Whereas, these results do not support the lipolytic enzyme origin of free fatty acids in eggs, such origin can only be eliminated from consideration by identiflcatio. of a nonenzymic agent or agents that quantitatively account for the observed increases in free fatty acids. The egg lipids may be sufficiently unstable a t the alkalinity of dried eggs to account for the observed formation of free fatty acids during storage (Lineweaver et aE., 1946). It is unlikely that the small increases in free fatty acids that occur at low moisture levels contribute in any important way to dried egg deterioration. The data of Brooks (1943) show that the increase in acidity at 2.5% moisture is very low, about 0.5 mg. as oleic acid/month/g. of egg at 37°C. (98.6'F.). This is about 25% increase in the usual free fatty acid value for freshly dried eggs. Thompson (1943), using phenylphosphate as substrate, reported the presence of an alkaline phosphatase in egg yolk. Lineweaver et al. (1946) repeated Thompson's work, also using glycerophosphate as substrate and found evidence of very small, but measurable, quantities of the enzyme. The livetin fraction accounts for only about one-fifth the total quantity of the phosphatase present in yolk. This contrasts with the distribution of other yolk enzymes which appear to be concentrated in the livetin. The possibility that inactivation of phosphatase occurs during the preparation of livetin was not eliminated. Although numerous investigators have found catalase to be present in eggs, Koga (1923) failed t o find it. Lineweaver et al. confirmed the results of former workers and in particular the report by Pennington and
SHELF LIFE OF DRIED WHOLE EGGS
185
Robertson (1912), which showed that catalase varies markedly from egg to egg, and is largely (about 85%) present in the white. Lineweaver et al. demonstrated that egg white catalase is heat labile (95% being destroyed at 63°C. (145.4"F.) in 100 minutes). This temperature is far below the usual outlet temperatures of spray driers. Peroxidase was not present in detectable quantities in either yolk or white (Lineweaver et al., 1946). The presence of oxidases in egg white capable of oxidizing catechol, dihydroxy-phenylalanine,and adrenaline, to brown colored substances was reported by Koga (1923). Tyrosine was not oxidized. Koga's experiments were repeated by Lineweaver et al. and his observations confirmed, but additional investigations revealed the changes observed by Koga are probably not due to enzymes but to autooxidation of the phenol which proceeds readily at the pH of egg white. He compared the color development when these substrates were added to raw and to heat-treated egg white, Lineweaver et al. confirmed his observation that autooxidizable substrates develop color rapidly with raw egg white but only slowly with heated egg white. They showed, however, that heated egg white (and heated egg albumin) prevented the color development when added to an autooxidizing mixture. The presence of more than traces of aerobic oxidases in fresh eggs is ulilikely, as may be inferred also from the absence of oxygen uptake by whole eggs (Smith, 1931). The presence of carotenase, reported by Wohlgemuth (1905), was inferred from an extended autolysis experiment. No attempt has been made to repeat the 2 4 4 8 hour experiment from which Koga concluded that salicylase (salicylaldehyde oxidase) is present in unincubated egg yolk. The presence of an a-amylase largely concentrated in the yolk, and similar in properties to salivary amylase, exhibiting optimum activity at about pH 7, as reported in the older literature reviewed by Needham, was codrmed by Lineweaver and associates. The yolk amylase appears in the livetin fraction. Proteolytic enzymes, both the proteinase and ereptase types, have been reported to occur in eggs. A tryptic proteinase was reported to be present in egg white by Balls and Swenson (1934), but later results (van Manen and Rimington, 1935, and Balls and Hoover, 1940), indicated the absence of p r o t e h e in egg white. Thompson (1943) reported both peptic and tryptic proteinase in egg white, but none in yolk. In view of the careful work of Balls and Hoover (1940), however, and the methods used by Thompson, the reviewers do not feel that the presence of peptic and tryptic proteinaae in egg white has been satisfactorily established. Several authors have reported erespin to be present in eggs (Koga, 1923; van Manen and Rimington, 1935; also see Needham, 1931 and 1942 for a review).
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HOWARD D. LIGHTBODY AND HARRY L. FEVOLD
The amounts reported are so small that the microbial factor, as in the case of Koga’s lecithinase-lipase results, must be carefully evaluated. Cholinesterase(s) is present in both yolk and white of egg (Ammon and Schutte, 1935). Lineweaver et al. (1946) showed that it appeared in the livetin fraction of the yolk. Both benzoylcholine and acetyl+methyl choline were hydrolyzed, indicating the presence of specific and nonspecific cholinesterase (Mendel and Rudney, 1944). An enzyme or enzymes capable of hydrolyzing methylbutyrate, earlier reported t o be present in both yolk and white, and benzoylbutyrate, were found in the livetin fraction by these workers. The possible relation between microbial action in egg mixtures before drying, surviving organisms subsequent to drying, and active microbial enzyme systems, t o rate of deterioration of powders has not been extensively investigated. Stuart, Hall, and Dicks (1942), reported the results of microbial and chemical analysis of a composite sample of commercial spray-dried powder stored at 30°C. (86°F.) for 60 days, and at 13 humidities, from 20% (moisture content 1.94%) to 100% (moisture content 33.7%). Lipolyt,ic organisms were found to survive storage to a much greater extent than did others included in the total plate counts. It should be noted, however, that the relation between moisture contents and vapor pressures reported by these authors is in marked disagreement with similar data reported by Gane (1943) and Makower (1945). Stuart el al. found about one-half the moisture a t a given vapor pressure as did Gane and Makower. The resazurin reduction test (“reductase test”) was used by Hirschmann and Lightbody (1947a) to investigate the effects of dehydration (dried by lyophilization to about 2% moisture), and of storage on the rate of dye reduction by inoculated (Pseudmonas jluorescens) and uninoculated egg emulsions in relation to viable count. They found a general relationship between plate count of inoculated liquid egg and dye-reducing time. This relationship did not hold for samples dried by lyophilization or those which had been stored. The dye-reducing action of reconstituted preparations made up from inoculated samples, remained relatively high although plate counts were reduced 99% or more by drying. Holding inoculated egg liquid at 65°C.for 20 minutes before drying resulted in 99.9% reduction in viable organism and a similar loss of dye reducing activity. After storage in air for 12 weeks at 30°C., the reducing activity correlated well with plate counts of the emulsions from which the powders were prepared, but bore no relation to the plate counts of the powders. In a second communication, the same authors (Hirschmann and Lightbody, 1947b), reported the results of deterioration studies of similarly inoculated preparations using palatability, salt water soluble fluorescence, lipid fluorescence,
SHELF LIFE OF DRIED WHOLE) EQQS
187
free fatty acid content, and pH as quality criteria. The powders were stored in air at -29°C. (-20.2"F.) and +3OoC. (86'F.) for a total of 12 weeks. Inoculation caused increase in salt-soluble fluorescence of unstored powders and the initial difference due to the microorganisms was maintained during storage at each of the temperatures used. The initial lipid fluorescence was not affected by the organisms added, nor by storage a t the low temperature. The lipid fluorescence, however, was found to have increased approximately 13-fold during storage at 35°C. (95°F.) though the plate count (per g. powder) had decreased from an initial value of 55 X los to < 100. Uninoculated controls also showed marked increases in lipid fluorescence at the higher storage temperature. The rate of increase was, however, much less. The free fatty acid content of the inoculated high temperature storage powder (expressed as oleic acid) was found to be approximately 9 times that of the similar powder stored at -29OC. (-20.2"F.), and 25 times that of the uninoculated low or high temperature stored controls. There was no significant difference between the free fatty acid content of the uninoculated powder stored at -29°C. (20.2"F.) and at 35°C. (95°F.). A close correlation between lipid fluorescence, fatty acid content, palatability loss, and dye color change was observed. The authors suggest that deterioration, as estimated by the criteria used in their experiments, is related to the activity of nonreproducing bacteria, i.e., to cell components (presumably enqmes) that remain active after the death of the organisms.
VIII. QUALITY RETENTION Mmsmtns The objectives of all research relating to dried eggs are obviously to devise methods by which the product may be prepared economically and delivered to the consumers with retention of all the attributes which assure maximum usefulness and widest acceptance. These properties are palatability, absence of health hazards, unimpaired nutritive value, and the several functional properties. In the preceding sections of this review, an attempt has been made to call attention to the variables in the raw materials and processing and storage procedures, that relate to the biochemical factors which may influence the shelf life of the finished products. In order to insure complete quality control, it appears desirable to extend control to flock nutritioh since a t least some of the changes observed in deteriorating powders may be associated with the properties of those egg components which have been shown to be closely dependent upon feeding practice, namely, the lipids. So far aa the writers are aware, the possible relationship between diet and powder stability has not been investigated. To attain best control of biochemical changes initiated by contaminating microorganismS, due attention must be given to flock mnitation, exclusion
188
HOWARD D. LIGHTBODY AND HARRY L. FEVOLD
of t(dirties,JJtemperature, and moisture control, during storage of shell eggs, thawing of frozen eggs, and holding of liquid egg under sanitary conditions, and elimination of sources of contamination in plant equipment (Zagaevsky and Lutikova, 1944). Handling eggs for drying has been discuised by Goresline and McFarlane (1944). Microbiological control has recently been reviewed by McFarlane et a2. (1945). Consideration of hazards to the health of the consumer has arisen through detection of Salmonella organisms in eggs. The occurrence and distribution of Salmonella in dried eggs, prepared in Canada, have been investigated by Gibbons and Moore (1944). An investigation of the source and mode of entry of SalmoneIla organiams in spray-dried whole egg powders has been reported by Soloweyet al. (1946). Soloweyet al. (1947)have identified 52 Salmonellatypes in isolatesfrom 1,810 of 5,198 egg powder samples examined, which were obtained from the products of 100 dehydration plants. Pasteurization techniques, applicable to liquid whole egg and their effectivenessin extending keeping time, have been investigated by Winter et al. (1946). A study of the destruction of Salmonella in liquid whole eggs by pasteurization, has been reported by Winter, et al. (1946). Temperatures and times necessary to destroy 164 strains, representing 16 types commonly found in eggs, were determined. Gibbons et al. (1946) have reported that the conditions of time and temperature required for vat pasteurization, i.e., 60°C. (140°F.) for 30 minutes, do not significantly change powder quality, either after drying or after storage. Fluorescence, KC1 value, Haenni solubility, foam volume, creaming, flavor, and pH measurements were used to estimate quality. The pasteurized preparations showed some tendency to pack more densely and had slightly l o w e d solubilities. 1 . Low Moisture The earlier efforts to improve shelf life were by lowering the powder moisture content. When drying whole egg first assumed importance, it was found that at 5% moisture, or below, microorganisms did not increase in numbers during storage. Subsequently, it was found possible to reach lower moisture values in the driers and to maintain egg powders at moisture contents of near 5% in equilibrium with moisture of air under the condition encountered in temperate climates (Gane, 1943). Maintenance of lower moktures, especially in shipments deatined for tropical areas, required packaging in hermetically sealed containers. The relation between moisture content and rate of deterioration has been investigated in several laboratories (Stuart, Hall, and Dicks, 1942; Brooks, 1943; Hawthorne, 1943; White and Thistle, 1943a, 1943b; Stewart et al., 1943; Gibbons and Fulton, 1943; Thistle et al., 1944; and Boggs and Fevold, 1946). The following table, reproduced from the report of Hawthorne (1943) is illus-
=ELF
189
LIFB OF DRIED WHOLE EGG8
trative of the effects of moisture content. The samples used in this study were packed in air-sealed tins. Tasm I1 The Effectof Moislurs Content on Dsteriorath of Dried Whole Egg [@ w e e k at 2PC. (68°F.)]"
% Deterioration
a
Sample
Moisture, %
AS1 M 10 M 13 M 9 M 14 M 12 M 6 M 4 M 8 M 3 M 16 M 2 M 17 M 15
2.3 2.6 3.3 3.6 3.6 4.3 6.0 6.6 6.9 6.3 6.4 6.6
6.7 6.8
Solubility
6 12 9 11
17 27 24 22 24 26 26 31 29
Flavor
Beating Power
21 21 23 33 28 38 36 40 60 40 49 37
20 27 13 40 61 47 62
-
66 75 65 66
50
79
29
68
Hawthorne (1943).
Boggs and Fevold (1946), applying palatability criteria to powders prepared by drying from the frozen state, found that progressive decrease of the water content between 6.0 and 0.5% resulted in a progressive increase in shelf life. The increase was roughly proportional to the amount of water removed. (Fig. 1.) Several modifications in drier design and plant operations, have been devised in order t o attain low moisture products without powder insolubilization. These are reviewed in the interim reports of the National Committee on Poultry Products Research, Coordinated Dried Egg Research Program (Stewart, 1944a, 1944b), and also by Stateler (1945). Low moisture content may be obtained by using belt-type redriers which carry the powder through dehumidified heated air, or inert gas, secondary cyclone redriers with inlet temperatures of 1O7-12l0C. (213.8-249.8OF.) , and in certain types of spray driers by preheating the egg liquid just prior to drying. The decreased viscosity and surface tension, brought about by preheating, materially help to attain better atomization at spray nozzles and more rapid rises in egg-particle temperatures during drying. Payawal
190
HOWARD D. LJQHTBODY AND HARRY
L. E'EVOLD
et aZ. (1946), have found the rate of coagulation of liquid whole egg, as measured by apparent viscosity, to increase slowly but regularly with rise
i
8-
I
I
2
WATER
I
4
CONTENT 0
I
6
Fig. 1. Relation of water content of egg powders to palatability during storage. (Bogga and Fevold, 1946).
in temperature ("instantaneous heat treatment") from 56"-66"C. (132.8"15023°F.). A drop in viscosity was observed over the temperature range 67"-68"C. (152.6"-154.4"F.). At 69"-70"C. (156.2"-158"F.) the viscosity again increased and coagulation set in just above 73°C. (163.4"F.). By holding a t constant temperatures, they found that the apparent viscosity increased as a linear function of the time of heating. The rate of viscosity increase was observed to be markedly increased by elevating the holding temperature from 62.5"-66"C. (144"-150.8"F.). 2. Storage Temperature
The relative stability of whole egg powder stored a t different temperatures is not now entirely predictable. The effect of storage on flavor and
191
SHELF LIFE OF DRIED WHOLE EGGS
cooking quality of spray-dried whole egg has been investigated by Dawson et al. (1945). Commercial spray-dried powders containing 3-5% moisture, packaged in air, were stored a t temperatures ranging from 0°43.30C. (32"-llOoF.) for 52 weeks. The usefulness of the preparations were then estimated for preparing scrambled eggs, baked custards, popovers, mayonnaise, and foundation cake. The authors concluded that dried whole eggs containing 3-5% moisture should be stored at temperatures of 15.6OC. (60°F.) or lower in order to maintain quality for longer than 6 months.
-20.C. 4%
20.C.
4-
1
I
I
I
It has been the more general practice to conduct storage tests at temperatures comparable with those prevailing under the more severe conditions of storage and transport, incident to military use in tropical areas or under even higher temperatures of "accelerated" storage. Information is meager regarding "shelf life" of good quality products, dried and packaged under most favorable conditions as at present indicated, and stored at temperatures readily available in refrigeration practice, or even a t room temperature, during long time experiments. There is some evidence that the deteriorative changes encountered when the products are stored a t lower temperatures are not identical with those observed in products stored a t higher temperatures. Since the quality criteria more generally used me
192
HOWARD D. LIGHTBODY AND HARRY L. FEVOLD
those dependent upon chemical alteration of the protein, it seems likely that only partial evaluation of the stored powders has been attained. Reports of investigations of storage temperature and quality relationship have been made by White and Thistle (1943a, 1943b); White et al. (1943); Gibbons and Fulton (1943) ;White and Grant (1944); Stewart et al. (1943); Thistle et al. (1944) ;Hay and Pearce (1946);and Boggs and Fevold (1946). Thistle et al. (1944) report detection of deterioration by use of objective tests, even in samples stored in air at -40°C. (-40°F.). Boggs and Fevold (1946) were unable to detect changes in palatability during storage at -20°C. (-4°F.) or at 4°C. (39.2"F.) for 8 months. Figure 2 illustrates the results obtained using air storage and palatability as criteria. The results show that powders of high initial quality, containing 2% moisture, may remain suitable for scrambling (palatability score 6 or greater) when stored in air for 8 months at 20°C.(68°F.). 8. Gas Packing
The available results of early investigations of the protective action of packaging in inert gases, nitrogen and carbon dioxide, are difficult to compare and evaluate, very largely because of difference in sample moisture content, storage temperatures, and evaluation criteria. In general, slight if any benefits appear to be derived by packing in an atmosphere of nitrogen when quality is measured by objective tests (White el al. 1943). However, Boggs and Fevold (1946) report improved palatability retention when powders are prepared by lyophilization and packed in nitrogen, care being taken t o prevent contact with air during the processing. The protective action of carbon dioxide packaging is clearly established by the work of White et al. (1943),who used objective criteria, Pearce et a2. (1946), using both objective and subjective tests with both high and low moisture samples, and by Boggs and Fevold (1946) who used palatability and reduced the moisture content of their samples below 2%.
4. Acidi$cation It is difficult to separate inert gas (oxygen exclusion) and pH effects when powders are stored in an atmosphere of COz. Stewart et al. (1943) appear to have been the first t o report the results of acidifying (with lactic acid) liquid whole eggs before drying upon the storage life of the dehydrated products. The liquid egg preparations of pH 7.5, 6.5 and 5.5 were dried to 5.5y0 and 0.6% moisture and stored in sealed tubes at 50°C. (122°F.) for 15 days. Solubility and salt water soluble fluorescence measurements were used to estimate quality. The authors conclude that lowering the pH had a marked beneficial effect on solubility and a small adverse effect on fluorescence. Palatabilities were reported to be unsatisfactory
193
SHELF LIFE OF DRIED WHOLE EQQS
because of marked alteration of flavor and texture, a deviation from the norm that is usually objectionable to the judges. Pearce et aE. (1946a) adjusted the pH of reconstituted powder to 6.8, redried to 1.7% moisture, and packed in air in tin plate containers. The products were stored at 27°C. (80.6°F.) and 38°C. (100.4"F:) for 64 and 16 weeks respectively. A portion of the spray-dried powder subsequently reduced to 1.7% content by
I
I
4 STORAGE
pH
I
8.5
I
I
12 16 T I M E ( W E E K S A T 36.5.C.) 8
I
Fig. 3. Comparative effects of acidification, gas packing, and acidification plus gas packing on palatability in egg powders during storage. (Boggs and Fevold, 1946.)
low temperature vacuum drying, served as control. Samples of acidified and untreated products were stored in nitrogen and also in carbon dioxide. Both objective and palatability tests were used t o evaluate the stored products. Acidification (pH 6.7) of liquid egg prior to drying, was found to have no significant effect, other than to retard the natural reduction in pH observed in untreated powders. Boggs and Fevold (1946) adjusted the pH of whole mixed liquid egg samples to the natural pH of mixed yolk, namely, 5.5 and also to pH 4.5 and 6.0, dried the mixtures from the frozen state to 2% or less moisture, added sufficient dry sodium bicarbonate to the samples to restore material to pH 8.5 (when reconstituted). The samples were stored in air, nitrogen, and carbon dioxide at 36.5"C. (97°F.) for 8 to 16 weeks. Fig. 3, reproduced from their article, summarize their results.
194
HOWARD D. LIGHTBODY AND HARRY L. FEVOLD
The data show that acidification of egg emulsion to pH 5.5, before drying, effects approximately a 2.5-fold increase in the time during which the powders may be stored in air and remain acceptable for scrambling. Nitrogen storage resulted in very little additional increase in quality retention. Storage in CO, markedly increased quality retention, and the storage life of the samples was approximately 4 times that of controls stored in air. By combining acidification with either nitrogen or carbon dioxide storage, the acceptability of the powders was increased more than 4-fold, though carbon dioxide storage gave slightly superior results. Conrad (1946), using commercial eggs and spray drying, has reported comparable extension of shelf life of the products when palatability was used as the criterion, but did not observe beneficial effect of acidification on sponge cake volume either alone or in combination with sugar. He observed a detrimental effect of acidification when sugared eggs were stored in air, and no retardation of off-flavor changes when similar eggs were gas packed. Sugared eggs seem to be much more susceptible to oxidative changes than are unsugared eggs. So far as the writers are aware there is no adequate explanation of the beneficial effects of acidification. The retarding effect of lowered pH on the rate of formation of aldehyde-amine products, and on the rate of formation of free fatty acids appear of interest. Other factors of possible interest in this regard are of a more general nature, e.g., the relation of protein, carbohydrate, and lipid stability to pH.
REFERENCEB Alderton, G., and Fevold, H. 11. 1946. Direct crystallization of lysozyme from egg white and some crystalline salts of lysozyme. J. Biol. Chem. 164, 1-5. Alderton, G., Ward, W. H., and Fevold, H. L. 1945. Isolation of lysozyme from egg white. J . Biol. Chem. 167, 43-58. Alderton, G., Ward, W. H., and Fevold, H. L. 1946. Identification of the bacteriainhibiting, iron-binding protein of egg white as conalbumin. Arch. Biochem. 11, 9-13. Almquist, H. J., Lorens, P. W., and Burrnester, B. R. 1934. Relation of depot to egg yolk fat in laying hens. J. Biol. Chem. 106, 365-371. Ammon, R., and Schutte, E. 1935. uber daa verhalten von Enzymen im Htihnerei wahrend der Pebrutung. (The enzyme content of hen’s eggs during incubation.) Biochem. 2. 276, 216-233. Asmundson, V. S., Almquist, H. J., and Klose, A. A. 1936. Effect of different forms of iodine on laying hens. J. Nutrition 12, 1-14. Assoc. Official Agr. Chem. 1945. Official and Tentative Methods of Analysis. 6th ed., Washington, D. C., p. 354. Ball, C. D., Hardt, C. R., and Duddles, W. J. 1943. The influence of sugars on the formation of sulfhydryl groups in heat denaturation and heat coagulation of egg albumin. J. Biol. Chem. 151, 163-169. Balls, A. K., and Hoover, 5. R. 1940. Behavior of ovomucin in the liquefaction of egg white. Ind. Eng. Chem. $2, 694-596.
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Balls, A. K., and Swemon, T. L. 1934. Proteolysk in stored eggs. Znd. Eng. Chem. 20, 570-572.
Bate-Smith, E. C. 1942. Part I. Dried eggs: Summary of work a t low temperature station, Cambridge, England. Bate-Smith, E. C., Brooks, J., and Hawthorne, J. R. 1943. Dried egg. I. Preparation, examination and storage of spray-dried whole egg. J. SOC.Chem. Znd. 62, 97-100.
Bate-Smith, E. C., and Hawthorne, J. R. 1945. Dried eggs. X. The nature of the reactions leading to loss of solubility of dried-egg products. J. SOC.Chem. Znd. 04, 297-302.
Baumann, C. A., Semb, J., Holmes, C. E., and Halpin, J. G. 1939. The determination of vitamin A in the hen's egg. Poultry Sci. 18, 48-53. Bearse, G. E., and Miller, M. W. 1937. The effect of varying levels of vitamin A in the hen ration on the vitamin A content of the egg yolk on hatchability and on chick livability. Poultry Sci. 10, 39-43. Bennion, E. B., Hawthorne, J. R., and BateSmith, E. C. 1942. Beating and baking properties of dried egg. J. SOC.Chem. Znd. 61, 31-34. Bernhard, K., Steinhauscr, H., and Bullet, F. 1942. Deuterium as an indicator for investigating fat metabolism. I. Essential fatty acids. Helv. Chim. Acta 26, 1313-1318.
Berry, J. A. 1946. Western Regional Research Laboratory, private communication. Bethke, R. M., Kennard, D. C., and Sassaman, H. L. 1927. The fabsoluble vitamin content of hen's egg yolk as affected by the ration and management of the layers. J . Biol. Chem. 72, 695-706. Bethke, R. M., Recvd, P. R., Wilder, 0. H. M., and Kennard, D. C. 1937. The comparative efficiency of vitamin D from cod-liver oil and irradiated cholesterol from laying birds. Poultry Sci. 16, 438441. Boggs, M. M. 1946. Western Regional Research Laboratory, private communication. Boggs, M. M., and Fevold, H. L. 1946. Dehydrated egg powders. Factors in palatability of stored powders. Znd. Eng. Chem. 38, 1075-1079. Boggs, M. M., Dutton, H. J., Edwards, B. G., and Fevold, H. L. 1946. Dehydrated egg powders. Relation of lipide and salt-water fluorescencevalues to palatability. Znd. Eng. Chem. 38, 1082-1084. Bohrcn, B. B., and Hauge, S. M. 1946. Vitamin A retention in dried eggs as affected by compression and packaging in tin cans. Food Research 11, 39-40. Bolton, W., and Common, R..H. 1941. Carotenoids of grass silage. Nature 148, 373. Branion, H. D. 1934. The vitamin D content of eggs. Can. Pub. Health J . 26,171-174. Brooks, J. 1943. Dried egg. 111. Relation between water content and chemical changes during storage. J. SOC.Chem. Znd. 02, 137-139. Brooks, J., and Hawthorne, J. R. 1943rt. Dried egg. IV. Addition of carbohydrates to egg pulp before drying-method of retarding the effects of storage a t high temperatures and of improving the aerating power of spray-dried egg. J . SOC. Chem. Znd. 02, 165-167. Brooks, J., and Hawthorne, J. R. 1943b. Dried egg. V. pH of reconstituted dried egg. J. SOC.Chem. Znd. 62, 181-185. Brooks, J., and Hawthorne, J. R. 1944. Dried egg. IX. The lipids of fresh and spray-dried whole egg. J . SOC.Chem. Znd. 03, 310-312. Calvery, H. 0.) and Titus, H. W. 1934. The composition of the proteins of eggs from hens on different diets. J. Biol. Chem. 106, 683-689. Chargaff, E., Ziff, N., and Rittenberg, D. 1942. A study of the nitrogenous constituents of tissue phosphatides. J. Biol. Chem. 144, 343-352.
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Conquest, V., and Turner, A. W. 1943. Amour & Co., private communication. Conrad, R. M. 1946. Uniu. of Denver, private communication. CNckahank, E. M. 1934. 8tudiw in fat metabolism in the fowl. I. The composition of the egg fat and depot fat of the fowl aa dected by the ingestion of large amounts of different fats. Biochem. J. 48,966-977. Cruicbhax& E. M. 1940. The chemicd composition of the egg. C h i s t r y & I* dwtry 18, 416-419. Cruickshank, E. M., Kodicek, E., and Wang, Y. L. 1946. Vitamins in spray-dried egga. J . SOC.C h . Ind. 64,1617. Dawson, E. H., Shank, D. E., Lynn, J. M., and Wood, E. A. 1945. Effect of storage on flavor and cooking quality of spraydried whole egg. U. S. Poultry & Egg Mag. 61, 164-161. Denton, C. A., Cabell, C. A., Baatron, H., and Davis, R. 1944. The effect of spraydrying and the subsequent storage of the dried product on vitamin A, D, and riboflavin content of egga. J. Nutrition 48,421-426. DeVaney, C. M., Titus, H. W.,and Nestler, R. B. 1936. Vitamin A content of eggs produced by chickena fed vioeterol and various percentagea of cod liver oil. J. Agr. Research 50,853-860. Dingemam, J. J. J. 1931. Identification of preserved eggs. Chem. Veekblad 48 (23) 360-361. Dingemass, J. J. J. 1932. Results of investigation on eggs. Chem. W'eekblad 49 (9) 138-140. Dutton, H. J., and Edwards, B. G. 1946. Changes in stored dried eggs: Spectrophotometric and fluorometaic measurement of changes in lipides. Ind. Eng. Chem. 37, 1123-1 126. Dutton, H. J., and Edwards, B. G. 1946. Determination of carotenoids and lipid amine-aldehyde products in dehydrated egg. Ind. Eng. Chem., Anal. Ed. 18, 38-41. Edwards, B. G., Dimick, A. L.,and Fevold, H. L. 1946. Western Regioml Reesrch Laboratory, private communication. Edwards, B. G., and Dutton, H. J. 1946. Changes in stored dried eggs: Role of phospholipid- and aldehydes in discoloration. I d . Eng. Chem. 37, 1121-1122. Ellis, N. 1933. The relation of egg yolk color to some nutritive properties of eggs. U.S. Egg & Poultry Mag. 39 (lo), 47-48. Fevold, H. L., Edwards, B. G., Dimick, A. L., and Boggs, M. M. 1948. Dehydrated egg powders. Sources of off-flavors developed during storage. Ind. Eng. Chem. 38, 1079-1082. Fevold, H. L., and Lausten, A. 19468. Isolation of a new lipoprotein, lipovitellenin, from egg yolk. Arch. Biochem. l1,l-7. Fevold, H. L., and Lausten, A. 1940b. Western Regional Research Laboratory, private communication. Fevold, H. L., and Shapiro, E. 1946. Western Regional Research Laboratory, private communication. Folger, B. B., and K l e b h m i d t , R. V. 1938. Spray drying. Ind. Eng. Chem. SO, 1372-1384. Frankel, M., and Katchalsky, A. 1937. The interaction of a-amino-acids and peptides in aqueous solution. Biochent. J. 31, 1696-1604. Fryd, C. F. M., and Hanson, S. W. F. 1944. Spray-dried egg. The relation between flavour and physical and chemical charwteristics. J. SOC.Chem. Ind. 63, 2-6.
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Factors Affecting the Palatability of Poultry with Emphasis on Histological Post-mortem Changes BY BELLE LOWE Iowa State College. A m ? .Iowa CONTENT8
Page I. Introduction . . . . . . . . . . . . . . . . . . 204 I1. Composition of Edible Portion of Chicken . . . . . . . . . . 206 I11. The Proteins of Muscle and the Structure of Muscle aa Related to Poultry Cookery . . . . . . . . . . . . . . . . . . . 207 1 Structure of Proteins . . . . . . . . . . . . . . 208 2 The Fibrils and the Contractile Elements of Muscle . . . . . 208 3. Effect of Heatupon the Intracellular Proteins . . . . . . . 208 4. The Structure of Muscle . . . . . . . . . . . . . 209 5 The Connective Tissues . . . . . . . . . . . . . 210 a . Effect of Enzymes on Connective Tissue and Muscle Fibers . 210 b . The Effect of Cooking upon Connective Tissue . . . . . 211 c Fatty Tissue . . . . . . . . . . . . . . . 211 IV . Poultry Fat and Palatability . . . . . . . . . . . . . 211 1 Component Acids and Glycerides . . . . . . . . . . . 211 2. Similarity of Iodine Values of Depot Fats . . . . . . . . 212 3. Effect of Ingested Fat upon Iodine Value of Body Fat . . . . 213 4. Changes in Acidity of Fat during Storage . . . . . . . . 213 5. Changes in Other Fat Constants during Holding of Poultry . . . 213 a . Determination of Rancidity and Stability . . . . . . . 214 b . Peroxide Values . . . . . . . . . . . . . . 214 6. Stability of Poultry Fat . . . . . . . . . . . . . 214 a Storage Temperature and Relative Humidity . . . . . . 215 b . Effect of Preliminary Holding on Fat Stability . . . . . 215 c . Stability of Internal us. External Fat . . . . . . . . 217 d . Drawn os. Undrawn and Fat Stability . . . . . . . . 217 e. Ingested Food Fat and Stability . . . . . . . . . 218 f . Length of Storage . . . . . . . . . . . . . . 218 g. Starving Before Slaughter and Fat Stability . . . . . . 218 V . Factors Influencing Flavor . . . . . . . . . . . . . . 219 1. Production Factors and Palatability . . . . . . . . . . 219 a . Breed . . . . . . . . . . . . . . . . . 219 b . Ingested Food and Flavor of Poultry . . . . . . . . 219 c . Distribution of Fat and Flavor . . . . . . . . . . 220 2. Some Processing Factors Which Might Influence Flavor . . . . 220 a . Manner of Killing . . . . . . . . . . . . . . 220 b Bleeding . . . . . . . . . . . . . . . . 221 c . “Dry-Picking” us Scalding . . . . . . . . . . . 221
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3 Drawnu8 Undrawn . . . . . . . . . . . . . . . a. Time Undrawn Birda Can Be Refrigerated . . . . . . b. Rate of Freezing Undrawn Birds . . . . . . . . . c. Time Held before Drawing and Freezing . . . . . . . 4 Aging Drawn Birde . . . . . . . . . . . . . . . 6 FrozenPoultry . . . . . . . . . . . . . . . . 8 Aging Drawn Birde before Free~ing . . . . . . . . b Rate of Freezing Drawn Birda . . . . . . . . . . c Temperature for Frozen Storage . . . . . . . . . d. Length of Frozen Storage . . . . . . . . . . . e Type of Package . . . . . . . . . . . . . . VI. Factors Influencing Juiciness . . . . . . . . . . . . . 1 Feed. Finish. and Fat Distribution . . . . . . . . . . 2 Extent of Cooking . . . . . . . . . . . . . . . 3 The Processing Treatment . . . . . . . . . . . . . a. Freezingperse . . . . . . . . . . . . . . . b Time of Frozen Storage . . . . . . . . . . . . c. The Temperature of Frozen Storage . . . . . . . . 4. Bound Water and Juiciness . . . . . . . . . . . . VII Factors Influencing Tenderneea . . . . . . . . . . . . . 1 Aging after Slaughter . . . . . . . . . . . . . . a Aging before Freezing . . . . . . . . . . . . b.Age. . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Cutting the Muscle Soon after Slaughter VIII Postmortem Changes and Rigorin Poultry Muscle . . . . . . . Onset and Resolution of Rigor in Chicken . . . . . . . . a . Onset of Rigor . . . . . . . . . . . . . . . b . Resolution of Rigor . . . . . . . . . . . . . c Effect of Heat on Onset and Rerrolution of Rigor . . . . . IX . The Relation of Microscopic Appearance of the Muscle Fibers to Palat ability . . . . . . . . . . . . . . . . . . . . 1. Early Studies . . . . . . . . . . . . . . . . 2 . Rate of Freezing and Location of Ice Formations . . . . . . 3. Histological Changes in Fibers with Aging . . . . . . . . a . The Connective Tissue . . . . . . . . . . . . b Fat in Muscles . . . . . . . . . . . . . . c. Fiber Striations . . . . . . . . . . . . . . d . Turbulence . . . . . . . . . . . . . . . . e. Rigor Nodes . . . . . . . . . . . . . . . f . Disintegration . . . . . . . . . . . . . . . . . . . 4 . Tendernees cmd Histological Changes in Muscle Fibers X Information Lacking in the Literature . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .
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I. INTRODUCTION High quality in poultry. from the consumer’s standpoint. implies that it is very palatable; it makes excellent eating. Another inference of the term quality is that the edible portion be high in relation to the waste .
FACTORS AFFECTING PALATABILITY OF POULTRY
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For the convenience of discussing factors influencing quality, the type of bird, the breed, the feed, the degree of finish, the distribution of fat in the carcass, the percentage edible meat, and the age or maturity in relation to quality or palatability are discussed under production. Under handling or processing are discussed the practices which have for their aim the retention (or possible improvement), during subsequent distribution and storage, of the quality obtained in production. This includes the manner of killing, whether starved or full-fed prior to killing, thoroughness of bleeding, whether the carcass is drawn or left undrawn for storage, chilled rapidly or slowly, the aging of the carcass, the temperature of storage, the freezing, canning, or smoking, the packaging for processing, and the time and temperature of storage after processing. Quality obtained by good production procedures and good handling technics is ineffective, however, if the time, temperature, and method of cooking are inadequate. Obviously, all the literature relating to the different factors concerned with quality of poultry cannot be adequately reviewed in this paper. In general, the studies which are reviewed herein are those in which quality comparisons are made from subjective ratings or objective tests for one or more of the factors or sensations which determine good eating quality. The palatability factors most often used for comparison of quality are aroma, flavor (lean and fat combined or separately) , texture, tenderness, and juiciness. Less frequently aroma and flavor may be broken down into intensity and desirability, a procedure which complicates the scoring and hence is seldom desirable. There are no adequate objective tests that can replace subjective ratings for aroma and flavor. An objective test when used to determine a quality factor should adequately measure the factor for which it is used. Some chemical tests, such as those which indicate rancidity, imply that unpleasant aroma and flavor will be encountered in the food. Subjective ratings may make efficient tools for evaluating palatability factors if certain precautions are follswed. Among these are: (1) Ratings should be made by trained observers who can keep degrees of quality in memory over long periods of time and consistently rate these degrees of palatability; (2) only one or two production, processing, or cooking procedures should be compared at one time; and (3) the data obtained should be analyzed statistically. In this way not only the variations caused by treatment can be determined, but inconsistencies of members of the scoring panel can be measured. There are also essentials to be followed in collecting subjective data. Fresh controls should be used whenever processed birds are cooked. This aids in keeping scoring standards consistent. The portions of meat given
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the judges should be coded. The material should be scored as nearly as possible at the same comparative times, ie., while still warm or after having been cooled to a certain temperature. The same anatomical section of a muscle or muscles should always be given to a specific judge. The panel should not be distracted or interrupted during scoring. Replications should be made, the number depending on the divergence of the variations produced by the treatments. And, last but extremely important, the person or persons doing the tests should have repeated them a sufficient number of times before the study begins, so that all procedures are standardized as much as possible. During this preliminary period the panel can gain experience in scoring. This preliminary part of the study is seldom repeated an adequate number of times. The following is an example of how lack of standardization of one small point may complicate the analysis of the data. If thigh muscles are to be scored, i t makes little difference in what position the legs are during cooling and cooking. If thigh muscles are to be dissected and sheared, however, the legs should always be cooled and cooked in one position. If the muscle from one bird is stretched, less shear force is required than in a contracted muscle of equal tenderness. Studies involving subjective ratings may be good or poor. One study, in which the differences obtained are statistically significant, is worth several studies in which insignificant differences are found, particularly if long use and (or) practical experience indicate that differences should exist. 11. COMPOSITION OF EDIBLE PORTION OF CHICKEN
The chemical composition of the edible portion of the carcass of chicken varies greatly with the fat content (Holcomb and Maw, 1934;Harshaw, 1938;and others). Different results are obtained depending on whether only muscle tissue or muscle tissue plus the skin and visible fat are used in the analyses. Holcomb and Maw (1934)used the skin, fat, and muscle of the entire carcass to obtain the percentages which follow: Average (32birds) Maximum Minimum
Protein, % 19.79 22.33 16.08
Fat, % 13.99 28.92 6.35
Ash, % Moisture, % 0.89 65.33 0.98 71.81 0.79 54.23
Holcomb and Maw found that the fat content is inversely proportional to moisture content. Maw (1935b)observed that, in fattening cockerels for 21 days on 5 rations differing in animal protein level, the protein content of the carcass remained practically constant, the fat increasing as the moisture decreased. The gains in fat for the 5 different rations were
FACTORS AFFECTINQ PALATABILITY OF POULTRY
207
2.07, 2.31, 3.85, 3.62, and 1.73%, respectively, whereas the decrease in moisture in the same order was 2.20, 2.61, 3.76, 4.14, and 2.40%. Harshaw (1938) determined the chemical composition of the edible portions (skin and visible fat removed) of the breast, the leg, and the remaining edible portion of groups oicockerels (10 per group) before and after fattening. The composition was determined on the unfattened groups at ages of 8,12,16, and 20 weeks. At each of these ages, an additional group of similar cockerels were fattened for 2 weeks, after which time the composition of the fattened birds was determined. After fattening, the amount of fat increased in all the edible portions; breast over 120%, leg 95%, and the remaining edible portion 250%. In the breast with a very low initial and final fat content, the quantity of protein, ash, and water remained practically constant throughout the study. With increase of fat, however, the other constituents decreased slightly in the leg, and to a larger extent in the remainder of the edible portion. Only the ash decreased with increase of age. The minimum and maximum percentages of constituents for the breast, leg, and remaining edible portion (without consideration of age or extent of fattening) follow:
Breast Leg Remaining Portion
Protein, % Fat, % 23.6-24.4 0.31-0.84 20.0-21.2 1.554.16 15.1-21.9 5.90-29.6
Ash, % 1.11-1.25 1.03-1.14 0.76-1.16
Water, % 72.8-75.1 74.0-76.0 54.2-71.0
Pennington (1908) reported the chemical composition of the light and dark meat from 5 birds of varying ages; broilers, fryers, and roasters. The lowest and highest percentage of the different constituents follow: Light meat Dark meat
Protein, % 21.84-23.30 19.77-23.13
Fat, % 0.17-1.33 1.38-2.99
Ash, % 1.17-1.33 1.13-1.49
Water, % 73.30-75.73 71.75-75.94
111. THE PROTEINS OF MUSCLEAND THE STRUCTURE OF MUSCLEAS RELATEDTO POULTRY COOKERY If the enqmea are disregarded, muscle is composed largely of 2 types of proteins: (1) the intercellular or structural proteins, consisting mostly of collagen and elastin; and (2) the intracellular or protoplasmic proteins. For a comprehensive review of structural proteins in general, the reader is referred to a paper by Schmitt (1944). The intracellular protein is composed of a myosin and a nonmyosin fraction. The proportion of the intercellular proteins varies with the age of the animal and with the species. A late tabulation by Bailey (1944) from various sources, gives as little as 3% of the total protein nitrogen as
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intercellular in haddock, whereas in the same fkh the myosin content is 67%. The percentages of intercellular proteins are much higher for mammalian muscle. The percentages tabdated by Bailey vary from 16-27% for white and red rabbit muscles, respectively. The myosin content in the same order varies from 57-39%. Thus the proportions of the intracelIular proteins aho vary in muscle from different sources. The nonmyosin fraction contains globulin X,myogen, and an albumin (Bate-Smith, 1937-38).
1 . Structure oj Proteins The properties of proteins are closely related to their structure. Hence, the structure of proteins is of interest in relation to the changes obtained in proteins during processing, ie., frozen storage, cooking, canning, and curing. A discussion of the structure of proteins is omitted here, but the reader is referred to articles by Astbury (1941, 1943), Astbury and Dickinson (1940), Bergmann (1938), Fankuchen (1945), and Neurath (1940). 9. The Fibrils and the Contractile Elements of Muscle Szent-Gyorgyi (1945, 1946) states that the muscle fibril is constructed of 2 proteins, neither of which is contractiIe in itself. Together, they form a complex, which has the property of reversible contractility. Contraction or relaxation can be brought about by the constituents of the muscle, salts, and adenosine-triphosphate. One of the contractile proteins is obtained in the crystalline form. The name “myosin” is retained for this portion, although its properties differ from those of the previously defined myosin. The other protein is “actin.” The complex of the 2 proteins is called “actomyosin.” The previously defined myosin is actomyosin of indefinite composition. a. Myosin. Myosin is fibrous, Szent-Gyorgyi (1946). It is a hydrophilic colloid, soluble in water, and has a fairly high viscosity, which shows its particles are elongated. Its isoelectric point is pH 5.3. Myosin, although a hydrophilic colloid, can be precipitated by small concentrations of neutral salts but with high concentrations this precipitate redissolves. B. Actin. Actin (Smnt-Gyijrgyi, 1946) is also a hydrophilic colloid with isoelectric point at pH 4.7. It is unique, in that it can exist in both the globular and fibrous forms and can be reversibly transformed from one form to the other.
3. E@t of Heat upon the Intrmllular Protdns During the heating of red muscle, after a temperature of 50°C. (122’F.) is reached, the color gradually changes to a lighter color and after a suffi-
FACTORS AFFECTING PALATABILITY OF POULTRY
209
ciently high temperature is reached, to brown or gray. The red pigment, oxyhemoglobin, is decomposed by the heat to hematin. In addition to the color change, when muscle is cooked, part or all of the intracellular fiber proteins is denatured. The denaturation temperature varies somewhat for different proteins, but for any protein the rate of coagulation increases with elevation of the temperature. The heat coagulation of isoelectric protqin takes place 600 times faster for each 10°C. (18°F.) elevation in temperature (Anson, 1945). The reaction is endothermic and absorbs heat during the denaturation. This absorption of heat during coagulation explains why the temperature of the muscles, as indicated Idy a thermometer, when poultry is roasted a t very low temperatures, 100'120°C. (212"-248"F.), remains stationary, sometimes for quite long periods of time, The temperature a t which this occurs is usually around 76'78°C. (168.8"-172.4"F.). After a period of time, which varies with different birds, the temperature again increases. This effect is not noticeabIe at higher oven temperatures. Also of interest in poultry cookery is the fact that, with overcooking, the muscle fibers become tougher and dryer. Toughening of the muscle fibers also occurs during frozen storage of fryers, particularly at high storage temperatures, - 6.7%. and - 12.2"C. (20°F. and 10"F.), as shown by Wills (1946). Studies on the heat denaturation temperatures of proteins of muscle fibers, i.e., on the proteins as determined by Szent-Gyorgyi (1946) are needed in food preparation work. For a bibliography and discussion of denaturation of proteins the reader is referred to reviews by Anson (1945) and Neurath et al. (1944).
4. The Structure of Muscle Skeletal muscle is made up of bundles of fibers (the fasciculi) held together by connective tissue surrounded by a muscle sheath, the epimysium. The connective tissue entering the fasciculi is the endomysium, that surrounding the fasciculi, the perimysium. Muscle, depending upon its content of connective tissue, is classified as tender or as less tender. The breast and back muscles of poultry are tender ones, the others less tender. The length and diameter of muscle fibers varies with the species, the particular muscle, and the age of the animal. The length varies from 1 4 1 mm., the diameter from 10-100 p (Maximow and Bloom, 1939). The fibers are composed of fibrils, roughly 1 p in diameter, which run parallel to each other and to the fiber axis. The fibrils form the longitudinal striations. Skeletal muscle, optically, is characterized by cross striations, hence is often called striated muscle. In ordinary light the anisotropic, A or Q striations or bands appear dark, the alternate isotropic T or J bands light. The microscopic appearance of the cross striations is
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often changed in certain sections of the fiber in rigor, with the application of heat in cooking, and with disintegration of the cross striations following rigor. (See later discussions.)
5. The Connective Tissues The collagenous fibers are slender, often curly, and are birefringent, i.e., reflect the light readily so that the unstained fibers have a glistening appearance. Collagenous fibers have little elasticity] although they may be stretched until the wavy fibers are straight. The elastic fibers are more slender than the collagenous ones, are highly birefringent, and have great elasticity. a. Effect of Enzymes on Connective Tissue and Muscle Fibers. It has been assumed for years, and practical experience bears out the assumption, that as meat ripens autolysis occurs and the connective tissues are broken down thus increasing the tenderness of the meat. Enzymes act on the muscle fibers simultaneously with the action on connective tissue. Although much has been published concerning the proteinases and peptidases and their substrates, little is known about the mechanism by which meat is tenderised with aging. Yet to the poultry processor and food technologist, autolysis is important. Balls and Kies (1946) classify 4 types of proteinases: (1) those active in neutral or slightly alkaline medium, the trypsinases or tryptases; (2) those active in acid media, pepsin or the pepsinases; (3) the papainases (of plant origin, inactivated by oxidants and activated by reducing agents such as sulfhydryl and cyanide); and (4) the proteinases of cellular origin, the cathepsins with hydrogen-ion optima in a weak acid medium. Maximow and Bloom (1939) state that the collagenous fibers are acted upon by pepsin in acid solution but not by trypsin in a weak alkaline solution; the elastic fibers are digested slowly by pepsin and more rapidly by trypsin. After an animal is killed the pH of the tissue drops from 7.4 to about 5.5 to 5.7, occasionally it may be lower or higher. This increasing acidity would favor the breakdown of connective tissue by the pepsinases, provided autolysis in the muscle tissues proceeds in the same manner as i n uitro. Smorodintsev and Nikolaeva (1942) have reported results of experiments which indicate that autolysis in the tissues does not follow the expected pattern. These workers found that as beef ripens, the cathepsin (the main proteinase of muscle tissue) activity decreases in spite of the fact that the pH of the meat is optimal for its action. In an earlier report (1936) these same workers state that the activity of cathepsin is reduced 4045% in the first 24 hours of aging the carcass and an additional 20% during the next 5 days. On the other hand the activity of trypsin increases for the first 24 hours, then drops sharply thereafter.
FACTORS AFFECTING PALATABILITY OF POULTRY
21 1
Pepsinase activity increases steadily reaching 2.5 times its initial value after the first 10 days. That considerable autolysis occurs in aging poultry is shown by the increase in tenderness of old birds by the common practice of holding them 2 or 3 days. b. The Efect of Cooking upon Connective Tissue. Collagen is converted to gelatin when heated in the presence of water and normal muscle contains adequate water for this purpose. The rate of conversion is dependent upon the temperature reached, the time held a t a given temperature, the pH of the solution, the salts present, and the size and density of the tissue. Conversion of collagen to gelatin is not rapid at the pH of muscle. In addition, during ordinary cooking the interior temperature of muscle never reaches the boiling point, hence the conversion of thicker collagen tissues within the muscle to gelatin is probably relatively slow. The elastic tissues are not changed readily by heating. c. Fatty Tissue. Within the muscle, fat is usually laid down first in the connective tissue along the blood vessels between the bundles of fibers. (See photomicrographs accompanying later sections.) Gradually, this connective tissue may be filled with fat cells. However, only in very fat birds and then only in certain muscles are the fat cells found between individual muscle fibers. Fat is never found in large amounts in some poultry muscles, such as the breast muscles. Fat has an important role in the palatability of meat. When heated the fat in the muscle is melted. When the connective tissue surrounding the fat cell is broken or converted to gelatin the fat escapes from the cell. The major portion of the fat in poultry is found in the fat depots, i.e., beneath the skin, around the muscles, in the mesentery, and in the abdominal wall.
IV. POULTRY FATAND PALATABILITY 1. Component Acids and Glycerides
The chemical and physical characteristics of fats are largely dependent upon their component fatty acids and glycerides. Although other factors, such as the protection of the fat from contact with oxygen by the skin, extent of emulsification, other substances present such as antioxidants, synergists, etc., exert an influence upon the development of rancidity, the component fatty acids and glycerides also play important roles. The component fatty acids and glycerides are similar for fat from around the gizzard, the abdominal fat, and for the neck fat of hens (Hilditch et al., 1934). Further the component acids were about the same for a group of 12 hens (2 years old) receiving 7 4 % of fishmeal in their diet as for a
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group of 24 birds (7months of age) which did not receive fishmeal. (The diets of all birds contained less than 4a/,of fat.) The slight differences in the component acids could have been attributed to age as well as t o the diet. The 3 depot fats contained 60-65 mols yo of unsaturated fatty acids. The comment was made that in an animal with as high a body temperature as the hen another example is given showing that body temperature is by no means the sole determinant of the relative saturation of the depot fats. The major portion of the unsaturated acid content belonged to the Cle series of which oleic contributed about 35-3870 and linoleic 20-2201,. No evidence of linolenic acid was found. The body fat contained some unsaturated C 8 0 -acids. ~ One of the outstanding characteristics of chicken fat is its content of the unsaturated Cl6 hexadecenoic acid (sometimes known as palmitoleic or zoomaric acid). It comprises f3-801, of the total acid components. Although A9:10-hexadecenoicacid is common to all fats and is a major component acid of fats of aquatic animals and lower forms of life, it composes onIy about 2-3% of the-acid content of fats from animals such as swine, cattle, and sheep. Hilditch el a2. (1934) found that the component acids of chicken fat contain about 30-35y0 of saturated acids. Palmitic is present in about the same proportions as for all land animals, 25-3001,. Myristic is found in traces, whereas stearic acid forms 5-757, of the total acid components. Hilditch (1941) states that the component glycerides of hen body fats are differentiated from the body fats of pigs, sheep, and cattle by their unusually large proportions of tri-Cls (unsaturated) glycerides, and, particularly of the mixed semi-saturated di-Cla-mono-C1a glycerides. The result is a more heterogeneous mixture of mixed triglycerides than in fats of the lard type which is reflected in the physical consistency of the fats. Although a lard of similar unsaturation is fairly plastic, the “triedout” chicken fat separates into 2 phases, a clear liquid and a layer of solid glycerides. 2. Similuritv of Iodine Values of Depot Fats
The iodine value of the mixed fatty acids is similar for depot fats from the neck, leg muscle, abdomen, and from around the gizzard of a given bird (Cruickshank, 1934). The range in iodine value for the fatty acids of 4 depot fats from 4 hens was 90-93, 78-79, 64-67 and 88-90. This result was unexpected and differs from that obtained in studies by other investigators on pork and beef fats. Hence, the work was repeated using fats from 8 locations in the hen’s body. The same results were obtained, i.e., the iodine values were very similar, regardless of the location in the body from which the fat waa obtained.
FACTOR8 AFFECTING PALATABILITY OF POULTRY
213
3. E$ect of Ingested Fat upon Iodine Value of Body Fat The constitution of the fat in the hen's body is at least partially dependent upon the composition of the diet (Cruickshank, 1934). Since there is wide variation in the iodine value of body fats from bird to bird, Cruickshank studied progressive changes in the fat reserves of the same bird. Samples of fat (0.5-0.8 g.) were readily removed from the abdomen without injury to the bird. The iodine values of the 3 fats, which composed 28% of the experimental diets fed to the birds were: mutton tallow 45, palm kernel oil 15, and hempseed oil 164. The iodine value of fatty acids from the body fat of fowl' fed mutton tallow dropped from 85-88 to 59-66 in about 5 months, whereas that of body fat from fowl on the palm kernel oil diet dropped from 81-83 to 51-55 in 2 months, and that from fowl on the hempseed diet increased from 81-83 to 139-145 in 6 weeks. The rapidity of return of the iodine values to normal when the hens were put on the low-fat control diet was not in the same order as the response on the experimental diets. About 4 weeks were required for the hens which had been fed mutton tallow and palm kernel oil, but 6 months for those fed hempseed oil.
4. Changes in Acidity of Fat during Storage Early investigators, Pennington (1908) and Houghton (1911)) noted that acidity of fresh chicken fat varied considerably from bird t o bird, a fact substantiated by all subsequent investigators. Pennington (1908) found that of the various fat constants measured, only the free fatty acid content increased in the fat from chicken held in frozen storage. Fitzgerald and Nickerson (1939) held broilers near the freezing point and found that the free fatty acid value of the fat showed a tendency to increase with the holding period. Likewise Harshaw et al. (1941) found that the acidity of the fat increased during frozen storage at -17.8"C. (OOF.) and at -28.9OC. (-20°F.). However, they found more variation in the individual birds than in the different treatments. Cook and White (1939) showed that the free fatty acid content of poultry fat after storage varies somewhat among birds, is usually low, and is not related to the storage conditions a t freezing temperatures. 6. Changes i n Other Fat Constants during Holding of Poultry Pennington (1908) found that in fat of chickens stored for 4 months at 10°C. (14°F.) there was a general lowering of the iodine value, the saponi-
-
1
In this review the term fowl is used to indicate a sexually mature, female chicken
(hen)
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fication number, the ester value, and the Hehner number (amount of insoluble acids). These changes were small and not as great as the changes in the acid value. Houghton (1911)reported a gradual increase in the iodine value in the monthly examination of fat from ground chicken meat held at -21.1" to -16.7"C. (-6" to 2°F.)for 5 months. Fat aldehyde values and refractive index vary little in birds held near the freezing point according to Fitzgerald and Nickerson (1939). a. Determination of Rancidity and Stability. Beadle (1946)defines stability as a "measure of the extent to which the fatty substance resists the development of rancidity." It is usually expressed as the time elapsed under specified conditions before the fat becomes rancid. Beadle discusses the problems involved in the evaluation of fat stability and states that "just as there is no completely reliable chemical test for rancidity, there is no completely reliable laboratory test for stability. Rancidity is ultimately determined by taste and smell, and stability is ultimately determined by placing the fat in storage and allowing it to become rancid. Even then, the food products prepared from the fat may not possess a stability even remotely like that of the fat from which they were prepared." The last statement is applicable in reverse, i.e., the stability of the extracted or rendered fat does not always give a reliable indication of the stability of the fat in the tissues from which it is extracted. This is shown by the work of Schrieber et al. (1947) on development of rancidity in poultry. b. Peroxide Values. Although not entirely satisfactory the increase in peroxide oxygen is the chemical test most frequently used in following the development of rancidity in a fat. The peroxide value increases with progress of the induction period, but the results are difficult to apply practically, for the peroxide value at which a fat is judged rancid varies with different fats. The fat from different chickens, with its wide variation in iodine value from one bird to another, might be expected to vary considerably in its susceptibility to oxidation. Investigations show this is true. 6. Stability of Poultry Fat
Nickerson and Fitzgerald (1939), using an accelerated stability test, found that the "tried-out" abdominal fat of chickens became rancid in about 28 hours, at which time the peroxide value was about 20 meq./kg. of fat. The average lard usually becomes rancid also before 20 meq. is reached. Lea (1934) had suggested that the peroxide value for chicken fat to indicate rancidity would need to be higher than that for the average fat from hogs. Nickerson and Fitzgerald found that the keeping quality of the fat varied with the individual bird. The active oxygen, refractive index, and free fatty acids did not increase until the fat became rancid.
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215
The fat aldehyde value increased to a maximum value before the fat became rancid, then decreased to a comparatively low value as rancidity was reached. The data for the peroxide and aldehyde values obtained by Nickerson and Fitzgerald are shown in Fig. 1. a. Storage Temperature and Relative Humidity. The amount of peroxide oxygen increased with elevation of the storage temperature and with lower humidities (Cook and White, 1939). However, even after prolonged storage (25 months) at -13.3"C. (8.1"F.)the maximum peroxide
Fig. 1. Chemical determinations on chicken abdominal fats during heating at 97.8"C. (208°F.) while aerating (Nickerson and Fitzgerald, 1939).
oxygen recorded was not large. The fat from different birds varied in its susceptibility to oxidation. These investigators thought that the incipient changes indicated by the higher peroxide oxygen value might nave caused a loss in stability. b. Efect of Preliminary Holding on Fat Stability. Cook and White (1940) found that if delay in cooling and freezing occurs, serious decomposition of the fat may develop. The longer the poultry was stored before freezing the higher the peroxide value became and the greater the deterioration during subsequent frozen storage. The perioxide oxygen and free fatty acid values obtained under the various conditions are given in Table I. Cook and White (1940)state it is evident that poultry fat may become slightly or definitely rancid under storage conditions that prevent surface drying. Rancidity development is, therefore, one of the factors limiting the storage life of podtry stored at -112°C. (10.4"F.)even when surface
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drying is prevented. Cook and White imply, however, that in the birds stored an excessive period under commercial conditions the surface drying is the principle factor in limiting the storage life of the birds. Wagoner et al. (1947b) determined the effect of preliminary holding conditions on the stability of frozen poultry during storage. After chilling the birds were divided into 4 groups: (1) Control group-the birds were TAB= I
The Peroxide Oxygen and Free Fatty Acid Content of Poultq Fat Follmuing V a W Storage Treatmenlsa
Storage conditions Precooled commercially, stored 1 week at 0°C. (32°F.) followed by 32 weeks at -12°C. (10.4”F.)
Average Precooled commercially, stored 6 to 6 weeh at 0°C.(32°F.)and 27 weeks at - 12°C.(10.4”F.)
Average Precooled commercially, froien and stored in gastight tank containing ice for 87 weeks at -12°C. to -16°C. (10.4”F.to 6°F.) Average
Peroxide oxygen (as ml. 0.002 N NadjsOa per 9.1
Free fstty acid as yo oleic
4.0 2.7 3.0 0.8 4.9 1.7
0.67 0.69 0.67
0.66 0.63
2.86
0.69
6.3 8.8 11.1 6.1 18.4 9.92 9.3 6.3 0.4 11.7 8.42
0.54
6.3 19.9 16.2 31.4 12.9 16.92
0.95 0.60
0.88 0.89
0.83
held in the chill room for 24 hours, then eviscerated by splitting down the back, wrapped in cellophane, placed in poultry boxes with waxed liners, and frozen in the storage room at -13°C.(8.6OF.); (2) “Short hold”-the birds were eviscerated, wrapped, and placed in the freezer within 2 hours after slaughter; (3) “Long hold”-the birds were held in the chill room for 6 days before evisceration and freehg; (4) ‘Troaen and thawed,’-
217
FACTORS AFFECTINQ PALATABILITY OF POULTRY
the birds were held in the chill room 24 hours, then packed in poultry boxes and frozen. After 2 weeks in frozen storage, the birds were tempered in the boxes for 24 hours at 5°C. (41°F.), defrosted in tap water, eviscerated, then wrapped, and frozen. The thawing period was only 2-3 hours and the birds still contained ice crystals when drawn. The birds were returned to the freezer within 1 hour after drawing. The data for the effect of the preliminary holding on quality of the poultry after storage are given in Table 11. TABLE I1 Effect of Preliminary Holding on Quality of Poultry after Stmageo ~
I Holding treatment Control Short hold Long hold Frozen and thawed
ArOllM
score * 6.1 6.9’ 2.1 ‘ 4.4’
~
Internal fat Acid value aa % oleic 2.26 2.66 17.10a
11.37”
a
Wagoner et a2. (1947b).
c
Difference statistically significant at the 1% level.
Aldehyde units 57 M)
69 69
Peroxide meq./kg. 55 68 161‘ 195’
* Highest possible score equals 7.
Wagoner, Vail, and Conrad determined the changes in the skin fat as well as the internal fat. Except for the aldehyde values which were higher for the “long hold” and the “frozen-thawed” groups (the differences being highly significant),the results were in the same order as those shown for the internal fat in Table 11. c. Stability of Internal us. External Fat. The internal fatty tissues of poultry are less stable than those of the skin (Cook and White, 1939; Dubois et aE., 1942; and Schrieber et al. 1947). Evisceration increases the fat surface exposed to oxidation. In addition, after drawing, the internal fat surfaces are not a8 well protected as the skin surface. Wagoner et at. (1947~)found that the reduction in exposed surface by “cutting-up” the poultry and wrapping it in a compact package improves the stability of chicken during storage. d. Drawn us. Undrawn and Fat Stability. Since the internal fat is less stable than the external fat of poultry, it would be expected that the drawing would increase the rate of oxidation and the instability of the fatty tissues. This has been shown to be true by Dubois et al. (1942) and Wagoner et al. (1947b, 1947~).
218
BELLE LOWE
e. Ingested Food Fat and Stability. Schrieber et al. (1947), reasoning that diets which tended to increase the iodine number of the body fat of chickens would also increase the susceptibility to oxidation, fed diets to birds to produce body fat which would show differences in stability during storage. Since the fat from different birds showed great variation, they decided that all tests had to be applied to fat from each individual. Although the fat extracted from the birds showed greater differences in accelerated tests, there were no significant differencesdue to diet observed in the storage stability of the frozen carcasses. Since some rancidity was observed in these tests, it was concluded that factors other than the stability of the extracted fat itself play an important part in determining the stability of the fat in situ. Although the fat extracted from the birds fed alfalfa was the most stable of any of the groups of birds, the carcass fat of these birds was the least stable. Fish oil fed to chickens for 1 or 2 weeks before slaughter also decreased the stability of the carcass fat during frozen storage and thus decreased the storage life of the poultry. f. Length of Storage. Dubois et at. (1942) concluded that the interior chicken fat becomes rancid in: 5 months storage at -9.4OC. (15°F.) 5 months storage at - 12.2"C. (10OF.) 10 months storage at - 17.8"C. (0°F.) 18 months storage at -23.3OC. (- 1O0F.)
g. Starwing before Slaughter and Fat Stability. Wagoner et al. (1947a) found that starving poultry for 16 hours before slaughter tends to'increase the storage stability slightly, but significantly. No difference was found in the extent of fat oxidation. The fat of the starved birds had a greater acidity than that from the birds full-fed to time of slaughter. In the palatability tests the aroma of the starved birds (with higher fat acidity) scored higher than that of the full-fed birds. Because of this latter result and the high correlation between fat acidity and lipoid phosphorus in previous work with turkeys, it was suggested that the difference in fat acidity of the 2 groups was due t o phospholipids rather than to free fatty acids. It would be interesting to full-feed birds to the time of slaughter in order to build a high glycogen reserve. The high glycogen reserve with its consequent lower pH in the tissues, post-mortem, might be advantageous from some standpoints. Bate-Smith (1937) and Callow (1937) suggest advantages of a high carbohydrate supply in the carcass. Madsen (1943) found larger amounts of glycogen in the meat and liver of hogs which were fed 1-3 kg. of sugar in the feed the day before slaughter. This practice resulted in very paIatable meat and a Iard which kept twice as long as ordinary lard.
FACTORS AFFECTINQ PALATABILITY OF POULTRY
219
V. FACTORS INFLUENCING FLAVOR Flavor is defined as follows by Crocker (1945). “Flavor is the complex sensations through which the presence and identity of foods and beverages in the mouth are determined.” The 3 senses which may be engaged in the perception of a particular flavor are taste, smell, and feeling. Flavor as used in its broadest or over-all sense as related to poultry could also include tenderness and juiciness. In the following discussion the results of palatability tests for aroma and flavor are combined. Certain production and processing treatments may be so interrelated with other factors that they may or may not be detrimental to poultry quality, depending on previous and following treatments. Hence, it is not surprising to find some contradictory results in different studies which have been reported. Most of the reported palatability studies of poultry as influenced by production factom have been concerned with the effect of feed upon flavor and the distribution of the fat within the carcass. 1 . Production Factors and Palatability
a. Breed. Differences in flavor of the meat may occur among breeds of poultry. If they do occur, probably the ordinary scoring panel could not detect them. These differences would not be as great as those among various kinds of birds such as chickens, turkeys, geese, and ducks. The proportion of meat t o bone among breeds would probably vary to a greater extent than flavor. The amount of fat deposited within the muscle might also vary with breed. b. Ingested Food and Flavor of Poultry. Carrick and Hauge (1926) were the first to report that the flavor of chicken flesh can be adversely affected by feeding cod liver oil. They also point out individual variation in response to the effect of the diet in producing off flavor, and that the length of time upon the diet, the method of cooking, and the temperature of the chicken when served also influence the results. The kind of bird, the quality of cod liver oil, and fishmeal have considerable effect on the results produced. Offflavor is found in turkeys more commonly than in chickens. Only 1% of a poultry grade cod liver oil in the diet produced an off flavor in turkeys (Marble et a,?.,1938, and Murphy, et al., 1939). Cruickahank (1939) fed chickens 2% of cod liver oil without detrimental effect when fed with a high grade fishmeal at a 15% level. When a low grade fishmeal was substituted for the high grade, and when fed 4 weeks to 6 months previous to killing, a fishy flavor was observed in some birds. Marble et al. (1938) found that feeding 10% of vacuum dried white fishmeal to turkeys during the entire growth period produced a fishy flavor.
220
BELLE LOWE
The effect on the flavor of the poultry meat was more pronounced if both fishmeal and 1% of a poultry grade cod liver oil were fed. Likewise, Murphy et al. (1939) obtained an off flavor in turkeys with 10% menhaden (dark) fishmeal or 10%of vacuum dried white fishmeal; the flavor was more pronounced if 1% of cod liver oil was fed with the fishmeal. In addition to the diet, the processing practices may intensify the off flavor. Asmundson et al. (1938) fed turkeys rations containing 25% sardine or tuna fishmeals, with or without sardine or cod liver oil for 6 weeks or longer prior to time of killing. Both high grade and inferior grade fishmeals were fed. If the birds were not starved before killing and were kept in a warm room overnight after being killed the flavor of the flesh was usually adversely affected. Birds fed inferior fishmeals had poorer flavor than those fed high grade fishmeals. From 2-5% of fish oil also produced an off flavor. Marble et al. (1938) recommend removing the cod liver oil or fishmeals from the diet of turkeys 8 weeks prior to slaughter. Maw (1939) states that the characteristic flavors may be imparted to poultry meat by corn, wheat, oats, and barley when fed 21 days before slaughter. He adds that cod liver oil, mutton fat, soybean oil, peanut oil, and corn oil also impart characteristic flavors to the carcasses. c. Distribution of Fat and Flavor. Fat within the muscles has long been regarded as adding to the quality of poultry meat. The grading of poultry takes the amount of fat and its distribution into consideration in determining quality. Maw (1935a,b) showed that although the percentage of total fat in the carcass was nearly the same for birds fed different cereals for 21 days previous to slaughter (corn 13.4%, barley 12.175, oats 12.175, and wheat 12.8%) the percentage of fat in the muscle varied as follows: Corn 5.8oJ,, barley 4.20/,, oats 3.60/,, and wheat 3.40/00. Results of palatability tests rated the flavor of the birds in the order given, that of the cornfed being superior and that of the wheat-fed birds the poorest. 8. Some Processing Factors Which Might In$uence Flavor
a. Manner of Killing. The manner of killing may affect the flavor of poultry owing to its effect upon other factors. Birds killed by penetrating the brain to relax the muscles holding the feathers, flutter but do not usually flap the wings vigorously. Birds killed by disjointing the neck (hung head down for the blood to collect in the neck and head), by cutting off the head, or by cutting the blood vessels in the neck, usually struggle vigorously for a variable period of time. Ordinarily the struggling lasts about 1 minute; occasionally this time is trebled or quadrupled. Sometimes the bird scarcely flutters, but may stiffen. Of several hundred birds one may go into rigor during fluttering or scalding. What causes the dif-
FACTORS AFFECTING) PALATABILITY OF POULTRY
221
ference in the extent of this activity; nerve impulses; the release of hormones? From the physiological standpoint extreme muscular activity at the time of killing consumes a variable amount of the carbohydrate reserve of the muscles. In turn this may influence other reactions which eventually may have a decided effect upon flavor, particularly with a long storage period. Birds stunned by an electrocuting device (Stewart and Drews, 1938), then bled by cutting the blood vessels of the neck do not struggle. A study of the manner of killing on the rate of post-mortem changes should be interesting. b. Bleeding. During roasting, the skins of birds that are not well bled, turn very dark as compared with those that are well bled. No studies on the effect of poor bleeding on flavor have been reported. c. “Dry-Picking” vs. Scalding. “Dry-picking” involves almost perfect timing of several operations (Stewart and Drews, 1938). By piercing a certain portion of the brain with a sharp knife and cutting the arteries for bleeding, the muscle feathers relax for a short period of time. If the feathers are removed rapidly, before the feather muscles contract, well dressed poultry is obtained. Commercially, “dry-picking” has been superseded by the “semi-scald.” By this method the relaxing effect on the feather muscles is nearly permanent. Stewart and Drews suggest immersion for 15-20 seconds in a large quantity of vigorously agitated water at 52.2’-55.5’C. (126’-132’F.). In scalding, water of much higher temperature, 78’-88’C. (172’-187’F.) , with immersion of 3-6 seconds, is often advocated. If the temperature of the muscle is raised during scalding, the rate of its post-mortem changes might be influenced, since heat rigor can be induced in muscle by high temperature. Obviously, the extent to which the temperature of the muscle is raised, depends not only on the temperature of the water, but also on the time of immersion in the water. Other factors involved are the amount of water in relation to the size of the bird, the thickness of the feather cover, and the extent of agitation while the bird is immersed. 3. Drawn M. Undrawn The objection to drawing before storage is that the surface for fat oxidation and the action of bacteria is increased. Studies on this point have been considered under fat stability. If not drawn, undesirable odors and flavors may diffuse into the flesh from the intestinal tract. The extent to which the latter occurs depends largely upon 2 procedures: (1) whether the bird was starved prior to killing, which would lessen the material in the tract for decomposition and putrefaction; and (2) the rate of cooling the carcass. Rapid cooling of the carcass lessens the bacterial action and the amount of putrefaction in the intestinal tract. Under (l), since some
222
BELLE LOWE
foods are acted upon more readily by certain types of bacteria, and since the end products from bacterial action upon different foods vary, the type of food must also be considered. Pennington el al. (1911) found that undrawn poultry decomposes more slowly than does poultry which has been either wholly or partially eviscerated. a. Time Undrawn Birds Can Be Refrigerated. The time that undrawn birds can be held without off flavor developing, if cooled rapidly after killing, depends upon the holding temperature, Fitzgerald and Nickerson (1939). Hanson et at. (1942) found that the first decrease in aroma and flavor scores of spray-cooled undrawn or New York dressed broilers held a t 1.7"C. (35°F.) occurred after 40 hours. I n general, this decrease continued thereafter with increase of storage time to 120 hours (the longest period the broilers were held.) b. Rate of Freezing Undrawn Birds. Sair and Cook (1938) conclude that the rate of freezing has no effect on the flavor of undrawn birds. De: velopment of off flavors from the viscera depends primarily on the length of time the product is held at temperatures above freezing. I n addition, even if frozen soon after killing, the off flavors can develop during the thawing period. c. Time Held before Drawing and Freezing. Any treatment which minimizes the length of time undrawn poultry is held above freezing aids in preventing off odors and flavors in the carcass (Stewart et al., 1943). This conclusion is substantiated in other studies. Wills (1946) found that the aroma and flavor scores of fryers eviscerated soon after killing, chilled in ice water and frozen promptly, were higher than those of birds held 24-48 hours before evisceration and freezing. Similar results were obtained by Wagoner et al. (1947b). The aroma score, for birds held in the chill room 24 hours before evisceration and freezing was 5.1; that of the birds held under the same conditions, but for 6 days before evisceration was 2.1; and, that of the frozen and thawed birds (held in the chill room 24 hours, frozen, thawed, and eviscerated after 2 weeks of frozen storage, and refrozen) was 4.4. Birds were starved for 16 hours before slaughter.
4. Aging Drawn Birds The aroma scores of fowl, drawn and chilled immediately after killing, and then held a t refrigerator temperature, showed little variation until after 2 days' storage. Then the aroma scores dropped regularly and consistently with lengthening storage. Flavor scores followed the same pattern as aroma scores (Stewart, Lowe, et al., 1945). 6. Frozen Poultry a. Aging Drawn Birds before Freezing. The length of the aging or ripening period before off odors and flavors develop is closely interrelated with
223
FACTORS AFFECTING PALATABILITY OF POULTRY
other factors. In general, the shorter the aging before freezing, the better the initial flavor is retained. In broilers eviscerated and chilled rapidly after killing, however, no difference between flavor scores for broilers held 2 or 18 hours before freezing was obtained. See Table 111. TABLE III Analysie of Variance. T h effect of aging, freezing temperature, and storage tims on average palalability amea of broilers (Range of acme8 0 to 10, with 10 high).o
Breast Juiciness
Comparison
Av. -
Treatment Fresh controls Frozen
7.2 5.6
Aging time, hns. 2 18
5.4 5.7
Diff,
-
Flavor
Thigh
Liver
I
Flavor
Juiciness
Flavor
--Av. DifI. Av. D 8 . Av. - - - --
-
Diff. Av. D8.
---
9.3 1.6 7.8
8.2 1.5 7.4
9.0 0.80 7.2
8.8 1.8 6.6
7.6
7.1
7.3 0.50 7.2
6.6 0.1 5.5 0.1
7.3 0.4" 7.1 0.2 7.1
6.6 0.2 6.4 0.2 6.6
- 0.3 8.1 -0.5 7.6
3.3"
Freezing temperature,
"C.
-20.5 -46.6 -67.8
Storage period, days Fresh control 9 23 37 51 65 79
5.8 5.4 5.6
0.4 8.0 0.1 7.6
0.a 0.4
7.6 7.2 7.4
7.2 7.1 6.6 5.4 5.3 5.3 5.1
0.1 0.6 1.8 1.9 2.0 2.1
9.3 9.0 8.5 8.5 7.6 7.6 7.4
0.3 0.8 0.8 1,7 1.8 1.9
8.8 8.2 9.0 8.5 k0.3 9.4 k0.4 7.9 0.9 8.1 0.1 8.3 .0.7 6.7 2.1 * 7.4 0.8 7.5 1.5 6.1 2.70 7.1 1.lC 6.8 2.2 6.9 2.9" 6.7 1.5c 6.6 2.4 4.0 4.80 7.0 1.2° 6.3 2.7 5.3 3.5'
8.0
- - -- -
0.2 0.0
-
Stewart,Hamon el d. (1946). b Significant. 0 Highly significant.
a
b. Rate of Freezing Drawn Birds. N o significant differences were found in the flavor scores of broilers frozen at 3 temperatures, -20.5", -45.6', and -67.8OC. (-5", -50' and -90°F.). See Table 111. c. Temperaturefor Frozen Storage. It ia generally conceded in the frozen food industry that temperatures below -17.8OC. (OOF.) are preferable to those above for preserving the quality of frosen poultry and meat. Tem-
224
BELLE LOWE
perature of storage, however, cannot be considered apart from length of storage, the type of packaging, and the previous productinn and processing treatment.& Wills (1946) found that the aroma and flavor scores of roasters stored at -12.2"C. ( 1 0 O F . ) were lower than for roasters stored at -23.3"C. (-10"F.), the differences being highly significant for all storage periods (3,6, or 9 months). Wills also held fryers at 5 storage temperat>ures;6.7", -12.2", -17.8", -23.3", and -35°C. (20", lo", Oo, -lo", and -30°F.). After 3 months' storage the fryers at -6.7OC. (20"F.), especially those wrapped in paper, showed some desiccation and were less desirable than those stored at the other temperatures. After 6 months' storage at -6.7"C. (20°F.) the fryers were very undesirable. The skeletal muscles as well as the giblets had toughened and definite off odors and flavors had developed. The fryers wrapped in waxed paper cartons had developed mold growth on the surface of the skin and flesh. The Iower the storage temperature used, the better the condition of the birds when removed from storage. No mold growth occurred, except at -6.7"C. (20"F.), and desiccation was progressively less as the temperature was lowered. Flavor scores tended to be lower than aroma scores. Liver always scored the lowest, followed by gizzard, thigh muscle, and the breast in the order given. The giblets stored in waxed containers a t -6.7"C. (20°F.) for 6 months or longer were considered inedible and the panel was not asked to score them. The results of Wills' study and other studies indicate that the storage of livers and gizzards with the carcass at ordinary storage temperatures is unsatisfactory. The giblets deteriorate far more rapidly than the skeletal muscles. d . Length of Frozen Storage. Deterioration of flavor with lengthening storage has been shown by several studies. In general, the extent of deterioration is greater at higher storage temperatures. Harshaw et al. (1941) found that the flavor of roasters decreased after 1, 2, and 3 years of storage at - 17.8" and -28.9"C. (0' and -2OOF.). Wills found that the aroma and flavor scores of roasters were significantly lower after 9 months than after 6 months of storage. In turn, the flavor and aroma scores of all frozen birds were consistently lower than for the fresh controls. The storage period need not be long before the deteriorative changes become evident, even when the birds are stored at fairly low temperatures. For broilers stored at -23.3"C. (-10"F.), the differences in the scores between the fresh control and the stored birds became highly significant for the liver after 23 days of storage, for the thigh after 37 days, and for the breast muscles after 51 days. See Table 111. e. T y p e of Package. The aroma and flavor scores of fryers, other conditions being the same;were higher for fryers sealed in tin cans than for those stored in the regular commercial waxed paper cartons (Willsj 1946).
FACTORB AFFECTING PALATABILITY O F POULTRY
225
VI. FACTORS INFLUENCING JUICINESS Juiciness has been determined in 2 ways in studies with meats and poultry, subjectively by the feeling of moistness during eating, and objectively by determining the amount of fluid that can be pressed from a sample of the muscle being tested. In studies that have been reported on beef, pork, and lamb the results by the subjective and objective methods have not always been significantly correlated. The correlations are good, however, when different stages of “doneness” (rare and well-done) are compared. The lack of agreement between the subjective and objective methods has led to the conclusion that juiciness in meat as determined subjectively is influenced by other factors in addition to the amount and state (free or bound) of water present in the muscle. For example, it has been suggested that melted fat in the cooked samples gives an impression of moistness. Very few studies have been reported on poultry in which both subjective and objective methods have been used to determine juiciness. Stewart, Lowe el al. (1945) found that the correlation between juiciness scores and the amount of press fluid obtained was highly significant for the pectoralis major muscle, significant for the pectoralis secundus, and nonsignificant for the thigh muscles of fowl. The correlations were not significant for these same muscles in the study with roasters (Lowe et al., 1946). The relation of juiciness scores and press fluid values for the roasters are shown in Fig. 2. Many factors may affect the juiciness of cooked meat. Some are easily determined, others are more difficult to demonstrate. Among the factors which may influence the juiciness are: (1) feed; (2) the finish and fat distribution; (3) the extent of cooking; (4) the processing treatment (canning, smoking, and freezing); and ( 5 ) conditions of storage. 1. Feed, Finish, and Fat Distrcibution
Maw (1935a) found that although the percentage of total fat in the carcass was nearly the same for birds fed corn, barley, oats, and wheat, palatability tests showed increasing dryness of the cooked meat for the birds fed the different cereals in the order given. Since some fat aids in preventing an impression of dryness, the extent of the finish (fattening) should have some influence on the apparent juiciness of poultry flesh, but no study on this point has been reported. 1. Extent of Cooking
The weight loss during cooking, unless the bird is extremely fat, consists largely of water. Since any procedure that dehydrates the flesh tends to increase its dryness, the extent of the moisture loss during cooking is impor-
Fig. 2. The percentage of press fluid and the juicineea scores of cooked muscles from roasters aged varying periods of time before starting cooking. (The juiciness scores for the thigh are for cross sections of all thigh muscles; 10 represents B perfect score.) (Lowe et aZ., 1946).
227
FACTORS AFFECTING PALATABILITY OF POULTRY
I30
I80
a
3
rlo
'00
g
k
W
to
Fig. 8. (a) The Ioas in weight during cooking and the cooking time of roastm sged varying period^ before starting cooking. (Each point is an average of data from 10 roautem.) (b) The influence of the initial temperature upon the cooking time of roastera. (Each point is an average of data from 10 roasters.) (Lowe d al., 1946).
228
BELLE LOWE
tant. The cooking time, in combination with the cooking temperature and method of cooking, largely determines the weight loss during cooking. See Fig. 3. Higher cooking temperatures tend to increase the weight losses during cooking, but of the 3 factors mentioned, the cooking time usually exerts the greatest influence. Cooking time is dependent upon the cooking temperature, the method of cooking the size and shape of the bird, the composition, and the initial temperature of the carcass at the start of cooking. Higher temperatures and covered utensils, other conditions being standardized, shorten the cooking time. It is commonly known in work with meats, however, that covering the container in oven cooking increases the weight losses in cooking. Short, thick, wide muscles require a longer time for heat penetration than long, slender, narrow ones. The amount of fat in the carcass may have some role in rate of heat penetration during cooking. Heat penetrates fatty tissues more slowly than lean tissues. Even after the fat is melted, heat conduction may be retarded, for the specific heat of oil is 0.41-0.43as compared with 1 for water. The effect of initial carcass temperature (above freesing) upon the cooking time is depicted clearly in the lower graph of Fig. 3. When there was insufficient time to cool the carcass before cooking was begun, a shorter time was required to heat the interior of the thigh to 90.5"C. (195'F.). If the chicken is frozen when cooking is begun, a proportionally longer time is required for cooking. Hoffert (1947)split roasters into halves by sawing through the keel and backbone and found that the halves frozen at start of cooking required an average cooking time of 137.3 minutes and sustained a weight loss of 23.8%, whereas the thawed halves of the same roasters required 95.7 minutes for cooking and showed a weight loss of 19.4% during cooking. A defrosting weight loss of 2.8%) however, brought the total weight loss of the thawed halves to 22.2%. Lowe el al. (1946)noted a tendency, although the differences were not significant, for the meat to be more juicy with longer aging before cooking (Fig. 2). This would be expected if only the weight loss during cooking is considered (Fig. 3). 9. The Processing Treatment
a. Freeziw per se. PouItry may be froEen, and with proper packaging, stored for long periods of time with negligible loss of weight or moisture. If not properly wrapped, poultry will dehydrate during freezing and storage. In cases when actual dehydration does not occur, factors other than the freezing per se must cause the decrease in moistness. Possible causes are the changes brought about by enzymes, but the more probable explanation is that denaturation of the protein and/or binding of water occurs during freexing or frozen storage. Since denaturation or binding of water
FACTORS AFFECTIN‘Q PALATABILITY OF POULTRY
229
at temperatures of freezing or frozen storage is a gradual process, it is possible that the freezing itself does not decrease the moistness of the poultry meat, but that changes occur during storage. Evidence in support of the supposition that the greater dryness of flesh of frozen poultry is brought about during storage rather than by the freezing process is given by Wiley et al. (1908) and by Stewart, Hanson et al. (1945). Wiley et al, were chiefly concerned with the flavor changes of “coldstored” drawn and undrawn chickens. The descriptive term “dry‘, appears once in describing a frozen bird cooked after 3.5 months of storage. The term was not used again until comparisons were made of the 15.6month stored birds, when 4 out of the 6 “jurors” described the flesh of frozen birds as “dry.” Two out of 4 “jurors” used the term “dry” describing the meat of frozen birds of the 18.5-month storage group. The comments show that the flesh of the frozen birds was less juicy than that of the control ones and give some indication of increasing dryness with longer storage of the frozen birds. In the studies of Stewart, Hanson et al. (1945) the juiciness scores of breast and thigh muscles of broilers held in frozen storage a t -23.3”C. (- 10°F.) from 9-79 days became increasingly lower with lengthening storage. The differences in the scores from those for the fresh controls became highly significant for the breast muscle in 37 days and for the thigh muscles in 51 days. See Table 111. b. Time of Frozen Storage. The fryers used by Stewart et al. (1943) were stored for 6-12 months. The juiciness scores of the thigh for the 3 methods of freezing of the birds stored 12 months were approximately a whole point lower than for the birds stored 6 months. However, the significance of the differences was not analyzed. The time of storage affected the juiciness scores for the breast to a lesser extent than the thigh muscles. Wills (1946) reported that the juiciness scores of both breast and thigh muscles of the fresh control birds were higher than those of roasters from a frozen pack stored at -12.2” and -23.3”C. (10” and -10°F.). In addition, the juiciness scores indicated the breast and thigh muscles of the frozen pack were significantly less juicy after 9 months’ than after 6 months’ storage. c. The Temperature of Frozen Storage. The temperature at which a froaen pack is held, as well as the time of storage, has some effect upon the juiciness scores. Wills (1946) found that the scores of the breast and thigh muscles of roasters stored at -23.3”C. (- 10’F.) were juicier than those of birds stored at - 12.2”C. (10°F.)
4. Bound water and JU~&Tl.e88 Johnson (1946) determined by vapor pressure methods the amount of bound water in the pectoralis major breast muscles of the same roasters
230
BELLE LOWE
used by Wills (1946). Johnson found that there was greater binding of water with longer storage time and at the higher storage temperature. The quantity of bound water was also noticeably increased by cooking. Cooking masked the differences in amount of bound water caused by different storage temperatures, however, so that these differences were not evident after cooking.
VII. FACTORS INFLUENCINQ TENDERNESS Tenderness of muscle is affected by several factors. Of major importance are the extent of aging the carcass after slaughter and the age of the bird. In the first instance the lack of tenderness is connected primarily with the muscle fiber. It is of concern when the bird is cooked before the owet and resolution of rigor in the muscles, hence involves only a short period of time after slaughter of the bird. The amount of connective tissue found in older birds necessitates a much longer aging period and often different cooking methods to produce tenderness of the meat. Other factors which may affect the tenderness are whether the muscles are cut soon after slaughter, and the degree and distribution of fat in the c a r c & ~ ~ . Freezing may also increase the tenderness of poultry meat but no studies pertaining to it have been reported. 1. Aging after Slaughter Coincident with the development and resolution of rigor in poultry, other changes are occurring in muscle fibers. Increased tenderness is one result of these changes. The effect of aging upon tenderness of poultry meat has been shown in 3 studies. Hanson et al. (1942)found that even the muscles of very young birds are extremely tough if cooking is begun within a few minutes after slaughter. This was shown by a tenderness score of 1.8 (of a possible 10) for the breast muscle of broilers in the oven within 6 minutes after slaughter. In addition to the muscles being tough, they were also stringy and rubbery. However, holding the broilers only 3 hours after killing resulted in an average tenderness score of 8 for the breast muscle. Tenderness of the thigh muscles increased leas rapidly than that of the breast, probably because of the higher connective tissue content. The data from the work of Lowe et al. (1946)showing the effect upon tenderness of aging roasters various periods of time before cooking, is presented graphically in Fig. 4. In another study Stewart, Lowe, et al. (1945) obtained results with hens similar to those obtained with roasters, except the rate of tenderizing was slower. The comparative time for tenderizing muscle of birds of different ages is shown by the average scores. An average score of 8 was reached for the pectoralis major muscle of broilers in 3 hours, of roasters in 12 hours and of fowl in 48 hours of aging of the carcasses.
231
FACTORS AFFECTING PALATABILITY OF POULTRY
3
-a
-4
8
4
Bil --
-'I
R
-8
--
-m
-c
-44
tl
1
-4 K
Fig. 4. Average tenderneea scores and shear force (in pounds) of cooked muscles of roasters aged varying periods of time before cooking. The tenderness scores for the thigh are for cross sections of all muscles. (Each point is an average of data from 10 roasters.) (Lowe et al., 1946).
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Fitzgerald and Nickerson (1939) found that maximum tenderness of broilers developed in 2 to 3 days at temperatures near freezing. a. Aging before Freezing. If birds are processed too soon after slaughter, tenderndss may be adversely affected. Hoffman (as reported by Lowe, 1939) found that roasters frozen within 2 hours after killing were less tender than those aged 24 hours before freezing. Likewise, Wills (1946) found that fryers eviscerated and frozen within 2 hours after killing were less tender than similar fryers held 24 to 48 hours before evisceration and freezing. b. Age. Meat from young birds, if aged for the same period of time, is tenderer than that from older birds. I n general, older birds, as shown by histological sections, have more connective tissue within a given muscle than younger birds. 2. Cutting the Muscle Soon after Slaughter
Lowe and Stewart (1946) noted that if breast muscles of roasters or fowl are cut soon after slaughter, the shock of the cutting may induce a turgidity and roughness of the cut surface which persists even after 24 hours’ aging of the carcass and subsequent cooking. If cuts are made in the muscle beneath the skin on one side of the breast, whereas those on the other side are used as controls (not cut), the whole bird can be roasted, and the toughness of muscles on each side of the breast measured. The differences in tenderness of the muscles on the 2 sides is usually very striking. The extent of the toughness developed in the muscles by cutting appears to depend on several factors. These include the length of time after slaughter before the cut is made (the effect has been observed when cutting had been delayed as long as 60 minutes after killing on fowl), whether rigor has set in, the extent of the cutting, and other unknown factors. In general, the effect is greater the sooner after slaughter the muscle is cut, i.e., a larger area is affected and the muscle is tougher. If rigor has developed before the muscle is cut, however, the turgidity does not develop. VIII. POST-MORTEM CHANGES AND RIGOR IN POULTRY MUSCLE The post-mortem changes in the muscles of poultry are important in that they aid in tenderizing the muscles. Since BateSmith (1947) has discussed the physiology of rigor in this volume, the present discussion of the post-mortem changes will be limited to the development and resolution of rigor in poultry and the histological changes. After slaughter, changes occur in the muscles of animals. The fat becomes firmer as the carcass cools. The pliant, gel-like, yet viscous muscle fibers of the living animal pass into a state of turgidity known as rigor mortis. With resolution of rigor the muscles become pliant again and
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aging changes proceed. Finally with sufficient bacterial action incipient putrefaction occurs. There is no definite dividing line among the post-mortem changes. But, coincident with the development and resolution of rigor other changes are occurring in the muscles. Among these changes are the increasing acidity, lowering of the glycogen content, changes in elasticity, changes in the rate of conducting an electric current across the fibers, changes in the tenderness of the muscles, and changes in the microscopic appearance of the muscle fibers. Changes in tenderness have already been considered. Microscopic changes will be discussed in a later section.
Onset and Resolution of Rigor in Chicken The observations on onset and resolution of rigor as well as the major microscopic changes are based largely upon the results of 2 studies. One upon fowl by Stewart, Lowe et al. (1945) and one upon roasters by Lowe et al. (1946). Observations were made when the birds were prepared for the oven. u. Onset of Rigor. The extent of rigor can be determined subjectively by the firmness of the flesh and the ease with which the thigh joints can be flexed. The temperature of the carcass affects the stiffness to some extent, as the fat is not firm in an unchilled carcass. Although the time of onset of rigor varies considerably from bird to bird, it is usually gradual and for most birds the greatest rigidity of the muscles occurs between 6 and 12 hours after death. The time of onset of rigor, as shown by perceptible stiffening,is between 1 and 2 hours. Sometimes less time is required. The shortest observed time by the author for the onset of rigor in a roaster was less than 5 minutes. The degree of fluttering was not unusual, slightly over 1 minute, so that no explanation can be offered for this unusual happening. Hanson et al. (1942) observed that onset of rigor was more rapid in the breast than in the thigh of broilers. Work with fowl and roasters confirms this observation, provided a difference in time of onset of rigor in the different muscles was noted. b. Resolution of Rigor. The rate of resolution of rigor also varies in different birds. Hanson et al. (1942) found that some New York dressed broilers which were refrigerated 15 hours were still in rigor. I n these birds, however, resolution of rigor was usually complete after the birds had been drawn and prepared for the oven. This would indicate that handling in drawing and preparation for the oven may have hastened the passing of rigor. In the uncooked fowl, rigor was being resolved a t the end of 24 hours and no signs of rigor were evident in fowl held 2 days or more. All the roaster carcasses, aged 18 hours when placed in the oven were in rigor, but in all but one bird the stiffness was not considered maximum. After
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24 hours' aging, in 4 of the 10 roasters, rigor had been resolved, whereas resolution was in varying stages of the remaining 6 birds. c. Efect of Heat on Onset and Resolution of Rigor. Bate-Smith (1939) found that, compared with physiological factors, the external temperature had little effect on the rate of onset of rigor. The temperatures a t which measurements were made varied from 0"-25"C. (32O-77"F.). This observation, for the temperatures used by Bate-Smith, was roughly confirmed with fowl (Stewart, Lowe, et al., 1945). Since the short-time-aged fowl were not chilled, it was thought that this procedure might have some effect on the histological changes. Hence, 12 additional fowl were killed, half chilled in ice, half chilled at room temperature. All were cooked 6 hours after killing. Aside from individual variation, there were no detectable differences in onset of rigor, in the palatability ratings, shear force, or in the microscopic appearance of the muscle fibers. It is evident, however, that temperatures of 120"and 150°C. (about 250" and 300°F.) speed the onset and resolution of rigor, Lowe et al. (1946). None of the fowl or roaster carcasses, aged 10, 30, or 60 minutes, were in rigor as they went into the oven. All were definitely in rigor when removed from the oven, as shown by misshapen carcasses. The legs and wings were twisted and drawn, particularly for the birds aged the shorter time. The muscles were firm to the touch and when chewed were resilient, rubbery, and tough. (See Fig. 4 for average scores and shear force for roasters.) The average cooking time (see Fig. 3) for the roasters in the oven within 10 minutes after killing, was less than 90 minutes. If the oven temperature had not hastened the onset of rigor, and stiffening had proceeded at the same rate as at room temperature, the carcasses would have been only slightly stiff when removed from the oven. The oven temperatures used in cooking also speeded the resolution of rigor. No signs of rigor were observed in the cooked carcasses of fowl aged for 3 hours or longer before cooking, yet with normal onset of rigor the carcasses would have reached maximum turgidity in 6-12 hours. Note the decided increase in tenderness with 3 hours' aging (see Fig. 4).
IX. THERELATION OF MICROSCOPIC APPEARANCE OF MUSCLEFIBERSTO PALATABILITY
THE
1. Early Studies
Pennington (1908) made a microscopic study of the pectoralis major of freshly killed chicken and noted that the most striking appearance of the normal muscle is its wavy smoothness and the closeness of its individual fibers. The cross striations, although visible, were by no means as distinct as was expected. Other observations by this author on the pectoralis
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major of chicken frozen and stored at -10°C. (14°F.) were as acute and accurate as those of the fresh tissue. All the changes in the frozen tissues, however, were attributed to the freezing and frozen storage. Some of the changes could have occurred with aging of the carcass, which continues, even in frozen storage. Of the frozen tissue examined after 1 month’s storage at - 10°C. (14°F.) Pennington observed that the cross striations were more distinct and brighter than in tissues from freshly killed fowl, some of the fibers had begun to pull apart, and the small bundles were more noticeable. There was just a suspicion of breaking in the fiber which suggests the beginning of a degeneration. Sectioning became more difficult as the fiber became more brittle with long storage. A homogeneous material streamed from the ends of the fibers or from the broken sarcolemma along the edge of the fibers. This exuding material, with no form and no boundary, could be traced in some areas back through the sarcolemma to the cross striations within the fiber. In some areas the material was granular in appearance. When the sarcolemma is broken, the striations are lost at that point, but the striations in the neighborhood, even at the opposite edge of the fiber may remain intact. “Such changes as these seem to be due to enzymes, which experiment has shown, are still able to function at - 10°C. (14°F.) though their action is greatly retarded.” Richardson (1908) thought that the changes brought about in muscle fibers by freezing and storage are largely physical. 9. Rate of Freezing and Location of Ice Formations
Koonz and Ramsbottom (1939) studied the effect of freezing temperatures on the location of ice crystals in poultry tissues. They found that the rate of freezing affects the size, number, and location of ice formations. Nearly instantaneous freezing produced minute, evenly distributed ice columns within the fibers. With a somewhat slower rate of freezing the ice columns within the fibers were larger in diameter and fewer in number. With a further rise in the freezing temperature the water was frozen as a centrally located ice column in the center of the fiber. Finally, when the freezing process was sufficiently prolonged, the water was frozen externally to the fibers. Stewart, Hanson et al. (1945) noted that numerous vacuoles appeared in sections prepared from chicken tissue which had been frozen, thawed, and cooked. No vacuoles were found in the fibers of broilers held 18 hours before freezing. Vacuoles were found only in those broilers frozen within 2 hours after killing. All birds frozen at -67.8”C. (-90°F.) within 2 hours after killing had vacuoles within the fibers of both breast and thigh muscles. The same was true forbirds frozen at -45.5”C. (-50°F.), except that about half the thigh muscles of this group had no vacuoles. No
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vacuoles were found in the fibers of broilers frozen a t -20.5"C. (-5°F.) regardless of extent of ripening before freezing. 3. Histological Changes in Fibers with Aging
The general statements concerning the microscopic appearance of tissues are made with consideration of the following factors. Any microscopic section under scrutiny represents a very small area of the entire muscles. Different parts of a muscle contain varying amounts of connective tissue. Contraction of fibers or connective tissue affects adjacent fibers to a greater degree than more distant ones. If sections are made from different areas of a sample of muscle they may vary greatly. This was emphasized when a technician in the author's laboratory, unknowingly made 2 sets of slides of muscles from some birds. Further, in the same section, different areas may vary widely; some may have many nodes, others few; some areas have turbulent striae, others well defined cross striations; some have much disintegration, others very little or none. Photomicrographs show only a very small area of a section, yet they aid in portraying the post-mortem changes to the reader. However, with low magnification (which shows larger areas) more than half of the details of the original photomicrograph may be lost in the production. With these considerations, the photomicrographs of longitudinal sections which accompany this article were selected. Many points may be found in a. single photomicrograph. These were selected primarily to show connective tissue (Fig. 5 ) , fat and fat cells (Fig. S ) , longitudinal and cross striations of the fibers (Fig. 7), passive contractions in fibers (Figs. 8 and 9), turbulence in muscle fibers (Figs. 10 and ll),nodes (Figs. 12 and 13), and disintegration in fibers (E'igs.'14, 15, and 16). The microscopic changes in muscle fibers, like the onset of rigor, vary considerably in individual birds. However, the following generalizations occur with a majority of the observed birds. The work of Lowe et al. (1946) and Stewart, Lowe et al. (1945) showed that sections from the muscle of freshly killed chickens are characterized by poorly differentiated fibers with fairly distinct longitudinal striations. The fibers are usually wavy, but may be straight. With onset of rigor, rigor nodes are found. I-n general, nodes begin to appear in 1-2 hours after slaughter of the bird and increase in frequency with aging 4-6 hours. Soon after, or pmalleling the appearance of rigor nodes, disintegration of the cross striations starts. In general, disintegration is found in nearly all birds in 3 4 hours in the pectoralis major of the breast, about the same time in some thigh muscles, and later in others. The extent of disintegration of the cross striations increases with longer aging. a, The Connective Tissue. The connective tissue, holding the muscle
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fibers t,ogether, depending on its thickness and the magnification is sometimes discernible in the histological sections (Figs. 5 and 6), frequently it is not noticeable. In general, the connective tissue, for a particular muscle is thicker in the older birds (fowl) than in the roasters. The perimysium is shown in only 2 photomicrographs (lower half of Figs. 5 and 6).
Fig. 6. Upper: Cooked pectoralis major, fowl M), aged 10 minutes before cooking. Note the connective tissue (arrows) on each side of the middle fiber. X430. Lower: Cooked gluteus primus, fowl 21, aged 4 hours. The tiny black spheres in the perimysium around the fiber bundles are fat. Note also nodes (probably heat induaed) and internodes over entire surface of fibers. X100. (Stewart et d.,1946).
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During cooking the collagenous tissues first acquire a granular appearance (upper part of Fig. 5) and finally with longer cooking the fibers fall apart. Sometimes frayed pieces of collagen are found over the surface of the section. The pectoralis major and the secundus contain little connective tissue. Of the 5 muscles observed, the adductor iongus had the most
Fig. 6. Cooked adductor longus. Upper: Fowl 15, aged 6 hours. Note fat in connective tissues on each side of fiber and node in fiber. X430. Lower: Fowl 37, aged 4 days. The gray spheres in the perimysium are fat. Some fat cells (white spheres indicated by arrow) are empty, X100. The irregular black spots are crystals of dye. (Stewart, Lowe et at., 1945).
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connective tissue (Fig. 6). In some birds and some areas of the muscle, the adductor muscle has considerable connective tissue between nearly every fiber. b. Fat in Muscles. The photomicrographs (Figs. 5 and 6) showing the most fat are all of thigh muscles. Although more fat is found in the heavier
Fig. 7. Uncooked pectoralis major, fowl 78. X650. Upper: Note the longitudinal strise are more distinct (wction removed 10 minutes after killing the bird) than those shown in lower photograph. Lower: Same fowl, section taken 24 hours after slaughter. (Lowe et al., 1946).
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connective tissue of the breast muscles, in general, the small black spheres (fat) towards the upper left part of Figs. 8 and 13 are indicative of the amount of fat in the pectoralis major. The secundus usually contains more fat than the pectoralis major. Muscle fibers. Straight muscle fibers are shown in Fig. 7. However, waves, “zig-zags,” kinks, and twists (Figs. 8 and 9) are characteristic of uncooked muscle fibers and are often found in quite rhythmic design. The passive contractions (the waves and z-1’s) are the result of active contractions of adjoining fibers or the contraction of connective tissue. Passive contractions are shown in the lower photomicrograph of Fig. 8. Heat, if applied too long, intensifies the waves and 51formations. See the photomicrographs, Figs. 7 and 9. Although the magnification is too high to give more than an idea of the extent of the gyrations of the fibers (Fig. 9) the waviness was increased by holding the sample in water at 65°C. (149°F.) for 5 seconds. From this it might appear that more wavy, kinked fibers would be found in the cooked than in the uncooked muscles. The reverse is true, however, but in cooking, heat is applied for a long time and the muscles are anchored to the bones in birds dressed roaster style. The waves in the uncooked tissues persist over longer aging periods than those in the cooked muscle, some being found in muscle from birds aged 5 days. One possible reason for more wavy fibers occurring in the uncooked tissues is that the cutting across the fibers in removing the sample, might have stimulated contractions. When white connective tissues are heated, they contract, and part of the intensification of waves and kinks brought about by holding the small sample of muscle for 5 seconds in water at 65°C. (149”F.), Fig. 9 was caused by contraction of connective tissue. It is also possible that passive contraction in some fibers may have been caused by contraction of the unheated connective tissue. This is suggested on the basis of qualitative observations only. Carey (1940b) in discussing passive contraction of muscle fibers of the frog indicates that it is caused by the actively contracted fibers. In chicken the smaller breast muscle is anchored near the wing joint by a tendon that continues in approximately the center of the muscle. It was interesting in the shorttime-aged birds, in removing the pectoralis secundus for scoring, to cut this tendon at the wing joint and watch the muscle shrink, often over an inch. Macrowaves always appeared with the shrinking of the muscle. This did not occur or was not noticeable in fowl aged 1-5 days before cooking. Szmt-Gyorgyi (1946) states that contraction in muscle is only an extreme degree of colloidal shrinkage. Carey (1940b) suggests that the “alternate regions of condenfiation and rarefaction structurally expresaed
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in muscle as variable striae during physiological contraction and expressed as nodes and internodes during pathoIogic rigors and spasms” are similar to micropreasure waves in capillary tubes. c. Fiber Striations. In general, the longitudinal striations are more distinct in muscles from freshly killed chickens (see Figs. 7, 8, and 9). The
Fig. 8. Uncooked pectoralis major. Upper: Fowl 4, section taken 30 minutes after killing. Note prominent longitudinal striae, poorly differentiated fibers, and slight waviness. X100. Lower: Roaster 101, aged 3 hours. Note rhythmic waves of psseive contraction. X100. (Stewart et al., 1945).
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longitudinal striations also appear more distinct when the fiber is under longitudinal stress, i.e., when under passive contraction, as in waves (Fig. 9). Usually both aging and cooking of the muscle tend to lessen the distinctness of the longitudinal striations. Sometimes during disintegration of the striations the fibrils separate to a greater or lesser extent from each
Fig. 9. Uncooked pectoralis major. Upper: Roaster 152, aged 10 minutes. (Passive z-z contraction in fiber. X650.) Lower: Fowl 78, section taken 11 minutes after killing; held in water at 05°C. (149°F.) for 5 seconds before putting in fixative Note extensive waves. X260. (Lowe et al., 1946).
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other, as shown in Fig. 13 (lower part). This last, however, has not been a common occurrence in the sections that have been studied. The cross striations “are not constant morphologic structures but are influenced in number and pattern by the degree and duration of variations of temperature” (Carey, 1940a). In the obliquely placed heat nodes (Fig. 12) the coarse striations of the internodes split into 2 cross striations
Fig. 10. Turbulence in the pectoralis major. Upper: Section from fowl 78 taken 12 minutes after killing; held in water at 65°C. (149’F.) for 25 seconds. X650. Lower: Turbulence in uncooked muscles, fowl 29, aged 1 hour (left) and in the cooked muscle (right). X400. (Lowe et al., 1946; Stewart, Lowe et al., 1945).
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in the nodes. Although the spacings of the cross striations vary in nodes, they also vary in other parts of the fibers. It is not unusual to see one fiber or patt of the fiber with rather coarse, widely spaced striations. Adjoining fibers may have fine, narrow cross striae. The distinctness of the cross striations varies in different muscles in pouItry and in the same muscle with aging and cooking (Lowe et nl., 1946). The cross striations are usually more distinct in the cooked than in the uncooked tissues, probably because the cooking partially dehydrates the tissues. d. Turbulence. Turbulence is a disorganization of the cross striae. It is caused by exposing the muscle fibers to heat and possibly by other factors. The extent of the disorganization of the cross striae depends upon the length of time the heat is applied. It is much more likely to develop if the muscles have been aged for only a short time. The destruction of the cross striations are shown in the upper photomicrograph of Fig. 10, which illustrates clearly Carey’s (1940b) description. “The turbulence due to thermal agitation of the molecules and colloidal particles may be so great as to destroy by an explosive effect the cross striated internal structure.” Practically 100% of the entire surface of the sample from fowl 78, shown in Fig. 10, was covered with the turbulent striations. Usually only a part of the surface is affected. When the turbulent area covers only a few fibers along the edge of the section and, if these fibers had lain just beneath the skin, the scalding may have produced the turbulence. If only a small amount of turbulence was present, it was usually at or near the end of the fibers (Fig. 11). The turbulence a t the end of the fiber, shown in Fig. 11, extended only a short distance, less than M inch, from the end of the fiber. Extreme turbulence is found at the end of the fiber, which gradates into the regular cross striations at the other end of the fiber. For this magnification, however, the regular cross striae are extremely small. Turbulence is found more often in cooked than in uncooked tissues. In the uncooked tissues it appears as if causes other than the scalding might bring about some of the turbulence. It is possible, though not proven, that the spasmodic quivers of some birds (instead of the usual type of death struggle) might cause turbulence. If turbulence is found in uncooked fibers it usually persists through cooking (Fig. 10). Turbulence is found more often in the adductor longus than in the other muscles. No samples were taken from the uncooked adductor so that it is not known whether there is turbulence in tissues before cooking. If there is it can not be caused by the heat from scalding, except indirectly, for the adductor is an interior muscle and thus too far from the surface to be affected by the short time in the scalding water.
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e. Rigor Nodea. The shape of the nodes in chicken muscles varies, sometimes they are very long, sometimes quite rounded so that if a number occur in a row in a fiber, they appear like a string of beads. Some nodes are perpendicular to the long axis fibers, some are obliquely placed across
Fig. 11. Pectoralis major. Upper: Turbulence at left end of fiber. Section taken from fowl 78, 30 minutes after killing; held in water at 65°C. (149°F.) for 15 seconds. Note gradations in turbulence to the cross striae at right. X650. Lower: Heat induced nodes, turbulent-background, cooked muscle, roaster 109, aged 1 hour. X260. (Lowe et al., 1946).
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the fiber, and some do not cover the entire diameter of the fiber. Nodes are shown in Figs. 5, 6,11, 12, 13, and 14. The riggr nodes are of 2 types, those occurring in the normal onset of rigor and those induced by heat, The nodes are the result of strong contraction waves in muscle (Carey, 1940a). The pressure nodes have the
Fig. 12. Cooked pectoralis secundus, roaster 118. Heat induced nodes. Upper: The coarse striae of the internode divide into two finer striae in the node (arrow). Note the Z band (narrow dark line in white band) at lower right. X1350. Lower: condensed striae of node (arrow) and coarse striae of internode. X025. (Lowe el al., 1946).
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cytoplasm and cross striations condensed (the amount of condensation varies in different nodes) so that the fiber diameter is increased, thus the node forms a bulge in the fiber. The nodes are strongly anisotropic and stain deeply. This differential staining of light and dark internodes and nodes appears to be related to difference in either chemical composition or the concentration of constituents in the muscle fiber. The nuclei are compressed and appear round or nearly round. There is an increase in mineraI ash in the node (Carey and Zeit, 1939). The tension internodes stain relatively less deeply and have narrow diameters, rarefied fibrillated cytoplasm, greatly elongated nuclei, coarse, large striae, and a decrease in anistropy and mineral ash content (Carey, 1940a). Heat induced nodes, often with concave edges, were found in the muscles from poultry carcasses aged short periods of time before cooking (Lowe et al., 1946). (SeeFigs. 5, 11, 12, and 13.) They were more extensive in the 10, 30, and 60 minute aged groups. However, some were found in muscles of a few birds aged as long as 4 hours before starting cooking. The heat-induced nodes were often spaced fairly regularly, giving the effect of an all-over pattern. The frequency of these heat-induced nodes was seldom as great as shown in the upper photomicrograph (Fig. 13). Occasionally there was turbulence of the cross striations of the internodes as shown in the photomicrograph in the lower half of Fig. 11. f. Disintegration. Disintegration is the term applied to the disappearance of the cross striations of the muscle fibers and its replacement with a granular-like substance. Sometimes the substance is amorphous instead of granular, sometimes nothing is apparent in the place formerly occupied by the cross striae. Sometimes the sarcolemma is broken and some granular material may ooze from the break. Often no break can be detected in the part of t,he section under observation. The appearance of the disintegration varies somewhat in different muscles and in the same muscle in different birds. For convenience of discussion, disintegration may be divided into that found in the internodes, in the breast muscles, in the thigh muscles, and in the nodes. The first disintegration in either breast or thigh muscles is usually in the internodes, possibly because in the stretched internode the muscle constituents are less concentrated and thus the cross striae can be converted more rapidly into the granular tissue. Disintegration in the breast muscle is characterized, in short-time-aged carcasses, by short strips in one or more fibers in which the cross striae have been replaced with the granular material. As aging of the carcass proceeds these strips become longer and more frequent. The appearance of the disintegration in the breast muscles is shown in the lower photomicrograph, Fig. 16. The disintegrated areas in the fibers in this illustra-
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tion are very short, in order to include the entire area in the photomicrograph. It is common, after 24 hours’ aging, to find strips too long to be shown by low magnification. Quite regular spacing of disintegrated areas in fibers, as shown in the upper part of Fig. 16, is not as common as the irregularly spaced longer strips.
Fig. 13. Upper: Heat induced nodes in pectoralis major, fowl 36, aged 30 minutes before cooking. Note the frequency of the nodes. X100. (Stewart, Lowe el al., 1945). Lower: Separation of fibrils (arrow) in gluteus primus muscle, frozen fryer (Wills, 1946). X W .
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Long disintegrated areas in a fiber are seldom found in the thigh muscles. Small fissures, which involve only a few striae in the fiber or general disintegration are characteristic of the thigh muscles. The fissures (Fig. 14) may appear over most or small areas of a section. Again, the whole section or certain areas may appear fragile, somewhat resembling a worn
Fig. 14. Upper: Disintegration fissures in gluteus primus, frozen fryer 59 (Wills, 1946). Node at center top, with disintegration in internode at left. X430. Lower: Cooked semitendinosus, roaster 103, aged 6 hours. Higher magnification of fissures. X650. (Lowe et a!.,1946).
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textile (Fig. 15). This type of disintegration may look like turbulence with low magnification. With higher magnification the cross striae of fibers over wide areas may appear as if they were falling apart (Figs. 15 and 16). In other areas only a few cross striae may be involved. Disintegration in the condensed areas of the nodes seldom occurs. How-
Fig. 15. Cooked semitendinosus of thigh. Upper: Disintegration of crosa striae in muscle fibers, roaster 123, aged 6 hours before cooking. XB50. Lower: Fragile appearance due to disintegration of croB striae, roaster 133, aged 24 hours before cooking. X6W. (Lowe et al., 1946).
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25 1
ever, the extent, of condensation sometimes varies in different parts of a node, see lower photomicrograph, Fig. 11. In the slightly less condensed areas of the node, disintegration may occur. It has been seldom observed in the broilers (Hanson et al., 1942), roasters, or fowl. This type of disintegration has been found more frequently in the frozen fryers of Wills (1946). There is wide variation in the time a t which disintegration of the cross striae is first observed in different birds. In general, it is earlier in the roasters than in the hen and earlier in the breast than in the thigh muscles. Between the 2 breast muscles, it is earlier in the secundus than in the pectoralis major. Disintegration appears as early (perhaps earlier) in the broilers (Hanson et al., 1942) as in the roasters. It is difficult to compare the time of appearance of disintegration in the broilers with that in the roasters and fowl, for the time intervals for aging were not the same, and detailed records were not kept of the histologic appearance of each muscle for the broilers. In the pectoralis secundus disintegration is more extensive in a shorter aging period for broilers than for roasters. All carcasses in a particular aged group do not exhibit the same extent of disintegration. However, the first disintegrated areas in a muscle are few in number, but their increase in a particular muscle parallels the increase in tenderness of that muscle. Unlike the onset of and the resolution of rigor, disintegration does not appear to be appreciably hastened by cooking. Disintegrated areas are found in the uncooked and cooked muscles at the same time. However, cooking seems t o sharpen the outline of the disintegrated areas and to increase the amount of granular material within them. Onset of disintegration parallels or closely follows the onset of rigor. Whether disintegration is independent of or dependent upon onset of rigor, is not known. It appears logical, however, that the increasing acidity would have more influence upon the rate of disintegration than the rigor. The cause of disintegration is probably autolysis brought about by the action of the proteolytic enzymes during the aging period. The increasing acidity brings about a more nearly optimum pH for the action of pepsin. Carey (1940a) states that when frog's muscle is held 3 days a t 6"-10"C. (41"-60°F.), irregular replacement of the cross striae is manifested. In a personal communication to the author, Carey stated that he thought the replacement of the cross striae was brought about by autolysis.
4. Tenderness and Histological Changes in Muscle Fibers Heat-induced rigor, with its accompanying heat nodes and turbulence, is associated with extremely tough muscles. With the beginning of disintegration tenderness of the muscle increases. The time to reach maxi-
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mum tenderness varies with different muscles in a given bird. Since the characteristics of each muscle, ie., the spacing of the cross striations, the amount and density of the connective tissue, and the fatty tissue vary in different muscles. this result might be expected. Paul et al. (1944) found
Fig. 16. Upper: Uncooked pectoralis major, roaster 144, aged 6 hours before cooking. Note numerous short disintegrated areas (short white areas in fibers). X 100. Lower: Note areas of disintegration (strips) in cooked pectoralis major, roaster 133, aged 24 hours before cooking. (Compare with thigh muscle from same bird, Fig. 15.) X650. (Lowe el al.. 1946).
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that beef muscles from the same carcass tenderized at different rates during aging.
X. INFORMATION LACKING IN THE LITERATURE Perhaps a review of this type is of greater value in indicating what remains to be done than in emphasizing what has been accomplished. Under production factors little or no work has appeared in the literature on the effect of breed, the degree of finish, and the distribution of fat in the muscle upon the palatability of cooked poultry. The work of Maw (1935a, 1935b) on the effect of feeds upon finish and fat in the muscle of poultry should be extended. Under processing practices information is lacking for many factors that may affect the palatability of poultry, particularly for a long frozen storage life. These include the manner of killing, thoroughness of bleeding, and the glycogen content of the muscles. The last factor is closely related with the post-mortem changes in poultry. Studies upon the effect of a high glycogen content in the muscles as related to rate of acidification, rate of disintegration of the cross striae of muscle fibers, and the keeping quality in frozen storage should yield valuable information. Callow (1937) states that if cooling of the carcass is rapid, the electrical resistance of muscle remains high for a longer period. How is this related to length of frozen storage life of poultry? The work of Wagoner et al. (1947a) on the effect of different feeds upon fat stability in storage life should be extended. In addition to freezing, many processing problems in connection with canning and smoking of poultry are still unsolved. The dearth of literature upon cooking temperatures and methods suggests a fruitful field for exploration. Perhaps results of work that has been completed should be extended and published. Improvement of and standardization of methods for obtaining the data for palatability qualities are needed. Methods of cooking test birds should also be considered. A wide field, in which little has been done, is the precooked products containing poultry meat and the frozen storage life of these products. In short studies the author has observed that sometimes the poultry meat in precooked frozen foods tends to become less tender with lengthening storage. Fundamental studies on fats and proteins of poultry meat are needed. REFERENCES Anson, M. L. 1945. Protein denaturation and the properties of protein groups. Advances in Protein Chem. 2, 361. Asmundson, V. S.,Jukes, T.H., Fyler, H. M., and Maxwell, M. L. 1938. The effect of certain fish meals and fish oils in the ration on the flavor of turkey. Poultry Sci. 17, 147.
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Astbury, W. T. 1941. Proteins. Chemistry & Industry 60, 491. Astbury, W. T. 1943. X-rays and the stoichiometry of the proteins. Advances i n Enzymol. 3, 63. Astbury, W. T., and Dickinson, S. 1940. X-ray studies of the molecular structure of myosin. Proc. Roy. SOC.London Bl29, 307. Bailey, K. 1944. The proteins of skeletal muscles. Advances in Protein Chem. 1, 289. Balls, A. K.,and Kies, M. W. 1946. Enzymes and their role in wheat technology. Interscience, New York, pp. 231-273. Bate-Smith, E. C. 1937. The physiology of rigor mortis. Gt. Brit., Dept. Sci. Ind. Res., Rept. Food Investigation Board, p. 15. Bate-Smith, E. C. 1937-38. Native and denatured muscle proteins. PTOC.Roy. SOC. London BU4, 136. Bate-Smith, E.C. 1939. Changes in elasticity of mammalian muscle undergoing rigor. J. Physiol. 96, 176. Bate-Smith, E. C. 1947. The physiology and chemistry of rigor mortis, with special reference to the aging of beef. Advances in Food Res. 1, 1 (1948). Beadle, B. W. 1946. Problems in the evaluation of fat stability. Oil & Soap 23, 33. Bergmann, M. 1938. The structure of proteins in relation to biological problems. Chem. Revs. 22, 423. Callow, E. H. 1937. The electrical resistance and microstructure of muscular tissue. Gt. Brit., Dept. Sci. Ind. Rea., Rept. Food Investigation Board, p. 46. Carey, E. J. 19400. Microscopic structure of striated muscle in heat rigor. Arch. Pathol. SO, 881. Carey, E.J. 1940b. Wave mechanics in striated muscle. Arch. Pathol. 30, 1041. Carey, E.J., and Zeit, W. 1939. Microincineration of active smooth, transitional and skeletal muscles, Proc. Ezptl. Biol. Med. 41, 31. Carrick, C. W., and Hauge, M. S. 1926. The effect of cod liver oil upon flavor in poultry meat. Poultry Sci. 6, 213. Cook, W. H., and White, W. H. 1939. Frozen storage of poultry. 111. Peroxide oxygen and free fatty acid formation. Food Research 4, 433. Cook, W. H., and White, W. H. 1940. Frozen storage of poultry, IV. Further observations on surface drying and peroxide oxygen formation, Can. J. Research D18, 363. Crocker, E. C. 1945. Flavor. McGraw-Hill, New York. Cruickshank, E. M. 1934. Studies in fat metabolism of the fowl, I. The composition of the egg fat and depot fat of the fowl aa affectedby the ingestion of hrge amounts of different fats. Biochem. J . 28, 965. Gruickshank, E. M. 1939. The effect of cod liver oil and fishmeal on the flavor of poultry products. Proc. World's Poultry Congr. 7th Congr. Cleveland, Ohio, p. 539. Dubois, C. W., Tressler, D. K., and Fenton, F. 1942. The effect of the rate of freezing and temperature of storage on the quality of frozen poultry. Refrig. Eng. 44, 93. Fankuchen, I. 1945. X-ray diffraction and protein structure. Advances in Protein Chem. 2, 387. Fitzgerald, G. A., and Nickerson, J. T. R. 1939. Effect of time and temperature of holding undrawn poultry upon its quality. Proc. World's Poultry Congr. 7th Congr. Cleveland, Ohio, p. 509. Hanson, H. L., Stewart, G. F., and Lowe, B. 1942. Palatability and histological changes occurring in New York dressed broilers held a t 1.7"C. (35°F.). Food Research 7, 148.
FACTORS AFFECTING PALATABILITY OF POULTRY
255
Harshaw, H. M. 1938. The effect of fattening a t different ages on the composition of cockerels. Poultry Sci. 17, 163. Harshaw, H. M., Hale, W. S., Swenson, T. L., and Alexander, L. M. 1941. Quality of frozen poultry as affected by storage and other conditions, U. S. Dept. Agr. Tech. Bull. 768. Hilditch, T. P. 1941. The chemical constitution of natural fats. Wiley, New York, p. 245. Hilditch, T. P., Jones, E. C., and Rhead, A. J. 1934. The body fats of the hen. Biochem. J . 28, 784. Hoffert, E.1941. Palatability studies on poultry. V. Infiuence of defrosting method. Thesis (M.S.) , unpublished, Iowa State College Library. Holcomb, R., and Maw, W. A. 1934. The analysis and composition of the flesh of domestic fowl. Can. J . Research 11, 613. Houghton, H. W. 1911. The effect of low temperature on ground chicken meat. Ind. Eng. Chem. 3, 497. Johnson, H. V. 1946. Water content changes of poultry held in frozen storage as related t o palatability. Thesis (Ph.D.),unpublished, Iowa State College Library. Koonz, C. H., and Ramsbottom, J. H. 1939. A method for studying the histological structure of frozen products, I. Poultry. Food Research 4, 117. Lea, C. H. 1934. The cold storage of poultry, 11. Chemical changes in the fat of gas-stored chickens. J . SOC.Chem. Ind. 63, 347T. Lowe, B. 1939. Effect of drawing before freezing on the palatability of poultry. Proc. World'e Poultry Congr. 7th C o w . Cleveland, Ohio, p. 500. Lowe, B., and Stewart, G. F. 1946. The cutting of the breast muscles of poultry soon after killing and its effect on tenderness after subsequent storage and cooking, unpublished data, Iowa State College. Lowe, B., Stewart, G. F., Harrison, D. L., and McKeegan, M. 1946. Palatability studies on poultry. The organoleptic rigor and histologic changes in roasters after slaughter, Special Rept. (unpublished), Iowa State College Library. Madsen, J. 1943. Investigations on the keeping quality of pork from animals which have been fed feed containing sugar, Nord Jorbrugsforskning, p. 340 (Original not seen, C. A . 33, 3598, 1939). Marble, D. R., Hunter, J. E., Knandel, H. C., and Dutcher, R. A. 1938. Fish flavor and odor in turkey meat. Poultry Sci. 17, 49. Maw, W. A. 1935a. How quality in poultry meat is affected by the distribution of fat in the carcass. U.S . Egg and Poultry Mag. 41, No. 5, 32. Maw, W.A. 1935b. How quality in poultry meat is affected by the distribution of fat in the carcass. U . S.Egg and Poultry Mag. 41, No. 7, 16. Maw, W. A. 1939. How breeding and feeding control poultry character and meat quality. U. S. Egg and Poultry Mag. 46, 608. Meximow, A. A., and Bloom, W. 1939. A textbook of histology. Saunders, Philaclelphia, p. 49. Murphy, R. R., Boucher, R. V., and Knandel, H. C. 1939. Flavor of turkey meat as affected by feeding fishmeal and fish oil. Proc. World's Poultry Cmgr. 7th Congr. Cleveland, Ohio, p. 542. Neurath, H. 1940. Intramolecular folding of polypeptide chain in relation to protein structure. J . Phys. C b m . 44, 296. Neurath, H.,Greenstein, J. P., Putnam, F. W., and Erickson, J. 0. 1944. The chemistry of protein denaturation. Chem. Revs. 34, 156.
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Nickerson, J. T. R., and Fitzgerald, G. A. 1939. Problems arising during holding of poultry prior to evisceration and freezing. Proc. World's Poultry Congress, 7th Congr. Cleveland, Ohio, p. 605. Paul, P., Lowe, B., and McClurg, B. R. 1944. Changes in histological structure and palatability of beef during storage. Food Research 9, 221. Pennington, M. E. 1908. A chemical, bacteriological and histological study of coldstored poultry. First Intern. Congr. Refrig. II, 216. Pennington, M. E., Witmer, E., and Pierce, H. C. 1911. The comparative rate of decomposition in drawn and undrawn market poultry. U. S. Dept. Agr. Bur. Chem. Cz'rc. 10. Richardson, W. D. 1908. The cold storage of beef and poultry. First Zanlern. Congr. Refrig. U, 261. Sair, L., and Cook, W. H. 1938. Effect of precooling and rate of freezing on the quality of dressed poultry. Can. J . Research D16, 139. Schmitt, F. 0. 1944. Structural proteins of cells and tissues. Advances in Protein Chem. 1, 25. Schrieber, M. L., Vail, G. E., Conrad, R. M., and Payne, L. F. 1947. The effect of tissue fat stability on deterioration of frozen poultry. Poultry Sci. 26, 14. Smorodintscv, I. A., and Nikolaeva, N. V. 1936. Modification de la cathepsine en caa d'autolysis du tissue musculaire. Compt. rend. mad. an'. U.R.S.S. I11 (12), 375. Smorodintsev, I. A., and Nikolaeva, N. V. 1942. Change in activity of peptidase on autolysis of muscular tissue, Compt. rend. acud. an'. U.R.S.S. 34, 233. Stewart, G. F., and Drews, H. E. 1938. Poultry packers put quality under control. Food Znds. 10, 489. Stewart, G. F., Hanson, H. L., and Lowe, B. 1943. Palatability studies on poultry; a comparison of three methods for handling poultry prior to evisceration. Food Research 8, 202. Stewart, G. F., Hanson, H. L., Lowe, B., and Austin, J. J. 1945. Effects of aging, freezing rate, and storage period on palatability of broilers. Food Research 10, 16. Stewart, G. F., Lowe, B., Harrison, D. L., and McKeegan, M. 1945. Palatability studies on fowl. The organoleptic, rigor, and histological changes in fowl after slaughter. Special Rept. (unpublished). Iowa State College Library. Szent-Gyorgyi, A., 1945. Studies on muscle. Acta Physiologiea Sand. 9, Suppl. XXV. SzenbGyorgyi, A., 1946. Contraction and the chemical structure of muscle fibril. J . Colloid Sci. 1, 1. Wagoner, C. E., Vail, G. E., and Conrad, R. M. 1947a. The effect of premortal fast on deterioration of frozen poultry. Poultry Sci. 26, 167. Wagoner, C. E., Vail, G. E., and Conrad, R. M. 194713. The influence of preliminary holding conditions on deterioration of frozen poultry. Poultry Sci. 26, 170. Wagoner, C. E., Vail, G. E., and Conrad, R. M. 1947c. The effect of degree of surface exposure on deterioration of frozen poultry. Poultry Sn'. 26, 173. Wiley, W. H., Pennington, M. E., Stiles, G. W., Jr., Howard, B. J., and Cook, F. C. 1908. A preliminary study on the effects of cold storage on eggs, quail, and chickens. U.S. Dept. Agr. Bur. Chem. Bull. 116. Wills, R. F. 1946. Organoleptic and histological changes in eviscerated frozen poultry stored under varying conditions of temperature and time. Thesis (M.S.) unpublished, Iowa State College Library.
Deterioration of Processed Potatoes
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BY A FRANK ROSS1
Cornell University. Ithaca. New York CONTENTB
I. Introduction . . . . . . . . . I1. Common Types of Deteriorative Changes
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1 Dehydrated Potatoes . . . . . . . . . . . . . . . 2 Other Products . . . . . . . . . . . . . . . . 111 Factors Influencing Rate and Extent of Deterioration . . . . . . . 1 . Dehydrated Potatoes: Browning . . . . . . . . . . . a Temperature and Moisture during Drying . . . . . . . b Inhibition of Browning during Processing by Suliiting . . . . c Raw Material Factors in Relation to Browning during Drying d Temperature and Moisture Content during Storage e The Effect of Suliiting on Browning in Storage f Raw Material Factors Affecting Deterioration of the Dehydrated Product in Storage . . . . . . . . . . . . . . 268 g. Packaging and Other Factors . . . . . . . . . . . 271 2 Dehydrated Potatoes: Graying and Development of “Off” Flavors in Storage . . . . . . . . . . . . . . . . . . . 272 3 Other Products 272 I V Chemical Changes during Storage of Dehydrated Potatoes 273 1 Changes Associated with Browning . . . . . . . . . . . 273 2 . Sulfite Retention during Storage . . . . . . . . . . . 278 V Control of Browning 279 1 Methods of Suliiting . . . . . . . . . . . . . . . 279 2 Attainment of Low Moisture . . . . . . . . . . . . 280 281 3 Use of Potatoes Low in Reducing Sugars a Availability . . . . . . . . . . . . . . . . 281 b Factors Idluencing the Reducing Sugar Content of Potatoes 282 c General Considerations . . . . . . . . . . . . . 283 4 Combined Treatments . . . . . . . . . . . . . . 284 V I Summary 285 References 286
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About four hundred million bushels of potatoes are produced annually
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Formerly Biochedt, Maine Agr. Expt. Sta All of the author’s unpublished data cited in this review are from a joint project of the Quartermaster Corps, U.8.Army, and the University of Maine M . T . Hilborn, L C. Jenness, and Emily M Bartlett also participated in this project.
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in the United States. Of this amount, between 5 and 10% is utilized in the manufacture of potato food products. The potato industry is of comparable importance in many other countries. The production of dehydrated potatoes increased rapidly during the war years but this has declined to a much lower, peacetime level. On the other hand, the manufacture of certain other products, such as potato chips and frozen French fried potatoes is rapidly increasing. Although the production of processed potato products utilizes a comparatively small proportion of the potato crop, it is nevertheless an important stabilizing factor in the potato industry. Unless highly acceptable products are prepared and these products retain their quality until they reach the consumer, the potato processing industry will not prosper. Hence, the deterioration of potato products during and after processing is of interest not only to military authorities and to the consumer, but also to those concerned with the production, marketing and conversion of potatoes. Because of the special requirements of the Armed Forces, emphasis during the past several years has been placed on dehydrated potatoes. A comparatively small number of published papers dealing with other types of processed potatoes indicate the existence of certain phenomena common to all. Hence, certain of the data obtained with the'dehydrated product will be applicable to other products more in demand in the civilian market. While this review is concerned primarily with deterioration during storage of potato products, some attention will be given to changes during processing, or in other words, to deterioration during drying, canning, frying, etc. It is becoming increasingly clear that certain deteriorative changes that occur during storage are identical in nature with undesirable changes that may occur during processing. In the first case, the changes are brought about by mild conditions over a long period of time, whereas in the second, identical changes are the result of extreme conditions for a short period. Thus the potential storage life of a given sample may be materially shortened by processing under adverse conditions. Only those data on dehydrated potatoes obtained with a fully blanched product are reviewed here since it has been shown that blanching is essential to the production of a good quality product (Cruess and Mrak, 1940, 1942a, 194213; Cruess and Joslyn, 1942; Davis et al., 1942; Beckley and Notley, 1941; Chace et al., 1941) but nothing is to be gained by a blanch longer than that required t o inactivate enzymes (Cruess, Smith, and Balog, 1943; Campbell et al., 1945). Consequently, a discussion of enzymatic changes is not included.
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11. COMMON TYPESOF DETERIORATIVE CHANGES 1. Dehvdrated Potatoes
One of the most important types of deterioration in storage of dried potatoes which have been properly blanched is the development of 8 reddish brown to dark brown discoloration, commonly referred to as browning. Parallel to the development of color there is formed a bitter scorchlike (caramel) taste and an equally undesirable odor. The badly discolored pieces do not reconstitute properly. In strips or dice, the color development is most intense in the center of the piece. Browning may occur also during the drying process. This so-called “scorch” or heat damage has been a frequent cause of trouble in dehydration plants. It now appears that heat damage and browning in storage are very similar if not identical in nature, differing chiefly in the conditions under which they occur. Other types of deterioration have been described by workers in England. Tomkins el al. (1944) described a gray discoloration in potato strips stored at 15°C. (59°F.) or below, and Burton (1945b), the development of an “off-flavor’’ in low moisture potato powder stored a t high temperatures. 2. Other Products
Canned potatoes have been described as having a long storage life (Rendle, 1945). However, certain deteriorative changes have been recorded (Rogers, 1945; Rhodes and Davies, 1945). The former worker described the development of an amber-pink discoloration during heat processing and its intensification a t a storage temperature of 373°C. (100°F.). The discolored potatoes had a burnt flavor. Rhodes and Davies described a breakdown or crumbling of canned potatoes during storage with a resultant poor texture. Browning is also a problem in potato chip manufacture and in the preparation of French fried potatoes.
111. FACTORS INFLUENCING RATEAND EXTENT OF DETERIORATION I . Dehydrated Potatoes: Browning
a. Temperature and Mdisture during Drying. The early work of Mangels and Gore (1921) indicated that potatoes are rather sensitive to heat damage and that the extent of injury is a function of time, temperature, and humidity. More recent investigations have confirmed these results and have provided additional information on the conditions necessary to avoid this type of diacoloration. Nichols et al. (1925) recommended a finishing
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temperature of 62.So-68.3"C. (145"-155"F.) for potatoes. In a summary of dehydration work in Canada, Davis et al. (1942) reported that one of the greatest difficulties in dehydrating potatoes is the danger of discoloration near the end of the drying period. He recommended a reduction in temperature to 62.8"-65.5"C. (145"-150°F.) after an approximate moisture content of 10% has been reached. In their tabulated data, a greater sensitivity to heat damage of long stored potato tubers was recogniaed. A finishing temperature of 65.5"C. (150°F.) was recommended for early season material and one of 62.8"C. (145°F.) for that stored for a long period of time. Davis et al. pointed out that most material can be exposed to a high temperature a t the start of the operation while rapid evaporation is taking place and the temperature of the material itself is not much above that of the wet bulb, but that progressively lower dry bulb temperatures are required as drying takes place. Cruess and Friar (1943) observed discoloration when potatoes were dried a t high humidities, held too long at 60°C. (140"F.), and when they were dried at high finishing temperatures. They considered a 65.5"C. (150°F.) finishing temperature as above the "scorch line" or dangerously near it. These observations establish a relationship between heat reddening and moisture-temperature conditions during drying but data G Z ~the specific effect of a given temperature as the material reaches different moisture levels are lacking. It would appear that with a given temperature schedule during drying, browning can be minimized by a low relative humidity and a high rate of air flow. Reports are in disagreement however, on the effect of these factors. Caldwell e.! al. (1945) have pointed out that if drying is too rapid, the rate of evaporation may exceed the rate of transport of water from the interior and drying out of a surface layer or case hardening occurs, resulting in a rise of temperature in the interior of the piece. b. Inhibition of Browning during Processing by Suljiting. Nichols and Gross (1921) compared blanching in 0.1% sodium bisulfite with other methods and in general rated the sulfited samples above the others. Cruess et at. (1944a,b) reported data showing that dipping blanched potatoes in a 0.5% bisuifite solution prevented reddening or browning during finishing at 7323°C. (165°F.). It was concluded that sulfiting would permit finishing temperatures 11.1"C. (20°F.) above those in commercial use. Caldwell et at. (1945) found that sulfiting, either with SO2 gas or by dipping in sulfite solutions prior to blanching, effectively reduced or prevented heat damage. In none of the above reports were data given on the amount of sulfite retained in the dry product. Tressler (1944) treated blanched potatoes in 0.1% bisulfite, dried at temperatures of 98.9"C. (210°F.) and 85°C. (185OF.) in the dehydrator and one of 623°C. (145°F.) in the finishing bin. The dry product contained
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153 p.p.m. SOa, was of good color, and was free of scorched pieces. The untreated product was of a dull color and contained scorched pieces. Friar and Van Holten (1945) found that about 300 p.p.m. SO1 prevented discoloration during dehydration at 73.8OC. (165OF.) whereas control samples darkened. The sulfited sample was only very slightly damaged even after two additional hours of drying at 73.8"C. (165°F.) Green el al. (1946) made a study of the effect of different sulfiting practices on the color of the dried product. Since the samples were not examined until after 4% months' storage no distinction can be made between the discoloration which occurred during processing and that which occurred during storage. Samples sulfited after blanching, were of better color than those sulfited prior to blanching. Lots dipped in HSSOa and in "SO2 retained color equally well. The reports cited above show conclusively that sulfiting is an aid in the prevention of heat damage, but most of the data are only qualitative in nature. Little consideration has been given to the variability of the raw material. The indiscriminate raising of the dehydration Wishing temperatures could cause trouble with some lots and might nullify the beneficial effect of sulfite. It should be kept in mind also that lots quite sensitive to heat damage deteriorate rapidly in storage and that merely preventing discoloration during drying is no assurance that the finished product will store satisfactorily. Sulfiting destroys most of the thiamine (Morgan, 1935; Davis et al., 1942; Tressler, 1944) but decreases the loss of ascorbic acid (Davis et al., 1942; Tressler, 1944). c. Raw Material Fact#rs in Relation to Browning during Drying. Until recently, little attention was paid to the effect of variations in raw material on sensitivity to heat damage. Davis et al. (1942) recommended a lower finishing temperature for stored potatoes than for early season ones. Black (1943) reported that potatoes stored at 1.6'4.4"C. (35'40°F.) were more sensitive to heat than were new potatoes but no explanation was given. Friar (1943) encountered some lots of new potatoes of the White Rose variety that discolored seriously during dehydration while Cruess and Friar (1943) observed that potatoes which were immature or that had been stored a t low temperatures often became yellow during drying. It seems probable that at least some of the discoloration they noted was heat damage, yet no distinction ww made between the yellow color due to the presence of carotinoid pigments in such potatoes (Caldwell et al. 1943; 1945) and that due to heat damage. Caldwell et al. (1945) reported extreme sensitivity to heat damage with physiologically immature stock, with potatoes that had been in cold storage for long periods of time, and with tubers in which dormancy was "broken" and sprouting had begun. They
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suggested that the great sensitivity of such tubers was due to their high content of sugars and (or) amino acids. No analytical data, however, were reported in the foregoing papers. The behavior of potatoes from cold storage and possibly of those forming sprouts makes it appear likely that the presence of sugars was an important factor. The possible cause of the rapid discoloration of dehydrated potatoes from immature tubers is more obscure. The data of Appleman and Miller (1926) show that the sugar content of developing tubers decreases as maturity is approached, yet the sugar content of the most immature lots examined was about 570 total sugars on a dry weight basis and about 1% reducing sugars. Experience has shown that these levels of sugar would not cause undue sensitivity to heat damage during dehydration and storage (Campbell and Kilpatrick, 1945). On the other hand, the amount of amino nitrogen tends t o increase as the tubers approach maturity. Wright et al. (1945) found a definite correlation between the color of dehydrated potatoes and the storage temperature of the raw stock. The amount of color in the dried strips of various lots increased as the temperature at which the raw stock was stored approached 0°C. (32°F.). Those lots stored at 10°C. (50°F.) and 16.4"C. (60°F.) were lightest in color. These differences were correlated with changes in the total sugar content of the tubers, and the discoloration during processing was attributed to caramelisation of the sugar. Experiments with Maine potatoes showed that lots high in reducing sugars were quite sensitive to heat damage (Ross et al., 1945). Several commercial lots of dehydrated potatoes, rejected because of heat damage, were found to be high in reducing sugars. When discolored pieces were separated from those having good color, the former were found to be considerably higher in reducing sugars than the latter. These data point to high reducing sugar content as a primary cause of sensitivity to heat damage, but, in the absence of data relating to other types of sugars, they cannot be considered conclusive. Campbell and Kilpatrick (1945)obtained data correlating sensitivity to heat damage with the reducing sugar content rather than with that of total sugars. The raw stock (White Rose) was stored at 21.lo-23.9"C. (70"75"F.), 4.4"C. (40°F.), and 0°C. (32°F.) immediately after digging. Analyses for total sugars and for reducing sugars were made at regular intervals; at the same time samples were dehydrated under standardized conditions. A t the end of 8 weeks, the potatoes held at the lower temperatures were shifted to storage at 21.lo-23.9"C. (70"-75"F.). Samples for analysis and for dehydrating were taken at the end of 1,2,and 4 weeks of the additional storage. Both two stage and single stage RYS~~IIISof dehydration were
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263
used. The degree of heat damage was estimated by counting the discolored pieces and by measurement of the light absorbed by a clarified water extract of the dried potato. No heat damaged pieces were found in those prepared from the potatoes stored initially at 21.l0-23.1"C. (70"75OF.1, and, with the exception mentioned below, the extent of heat damage increased as the total and reducing sugars increased; i.e., as the storage temperature decreased. Subsequent storage of the potatoes high in sugar (first stored at low temperatures), at warm temperatures, reduced the extent of heat damage but did not completely prevent it. The exception referred to above is of particular interest. Potatoes stored at 4.4"C. (40°F.) for 4-6 weeks were more sensitive to damage by heat than those stored at 0°C. (32°F.) for the same length of time. The analytical data showed that there was a greater accumulation of reducing sugars in those samples stored at 4.4"C. (40°F.) than in those at 0°C. (32°F.) but just the reverse was true for total sugars. There was a highly significant correlrlr tion (coefficient, +0.89) between the percentage of light absorbed by the water extracts of the dehydrated potatoes and the reducing sugar content in the raw material. Campbell and Kilpatrick advise against the dehydration of potatoes containing over 2.5-3% reducing sugar (on the moisturefree basis). Doty et al. (1946) stated that within a given variety of potato there appears to be a rather close correlation between the degree of browning during dehydration and the amino nitrogen and reducing sugar contents. This relationship was not always observed when different varieties were compared. d. Temperature and Moisture Content during Storage. It has been known since 1921 (Gore and Rutledge, 1921) that the browning of dehydrated potatoes in storage occurs at high temperatures and that the rate of browning is influenced by the moisture level. Gore and Rutledge worked with slices and riced samples steamed 40 minutes before drying. When stored at 1.7"C. (35°F.) the slices did not discolor in 700 days. Storage life at 23.9"C. (75°F.)was dependent on the moisture content of the samples. Those containing 5.442% moisture discolored slightly in 700 days whereas those with 7.8-8.0% moisture turned brown in 222 days. When stored at 37.8"40.0"C. (100°-105"F.) , all samples became brown in 222 days; those highest in moisture were the most severely discolored. Tomkins et al. (1944), who worked with dried potato strips containing 50-100 p.p.m. SO, found that browning generally occurs at temperatures above 28°C. (82.4'F.) and not at lower temperatures. At 37°C. (98.7"F.) potatoes with 10% moisture browned much faster than those with 5%, which in turn discolored slightly faster than those containing 2.7% moisture.
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The workers at Continental Can Co. (1944) conducted storage tests on commercially dehydrated potatoes. In most instances, the samples were acceptable after 1 year at room temperature, but considerably darkened and inedible after 6 months at 36.7"C. (98'F.) and quite dark after 1 month at 54.4"C. (130°F.). The rate of darkening of a potato flour prepared from dried potato slices was found to be accelerated by an increase in moisture, particularly above 10.5%, and by high temperature (Burton, 1945a). Similar relationships were found with mashed potato powder (Burton, 1945b). The extent of discoloration was measured by means of a Lovibond tintometer and the limit of acceptability was stated to be between 1.0-1.5 Lovibond units. The storage life of samples held at 57°C. (134.6"F.) varied with the moisture content aa follows: Moisture content,% 4.9 6.5 7.1 11.5
Storage life, days 9 3 2 1
Samples with still higher moisture content became unacceptable in less than a day. Power containing 12% moisture retained color for 10 months at 20°C. (68'F.) but showed a slight discoloration a t 25°C. (77°F.). Further increase in storage temperature decreased the storage life as follows: Storage temperature, "C. ("F.) 28 (82.4) 37 (98.6) 47 (116.6) 57 (134.6)
Storage life, days 180 4249 14 3
Howard (1945a, b) determined the effect of absolute moisture content on the storage life of diced potatoes containing about 300 p.p.m. of SO2 and packed in nitrogen. The relation of absolute moisture content2 to storage life, as determined organoleptically, is shown below: Absolute moisture content, yo 4.5 6.7 8.0
Storage life at 48.9'C. (120"F.), daya 56 26 18
* Determined by the method of Mdtower et d. (1946). This method cliffera from that described in the tentative apeoificationa of the Quarterrnester Corps (1946) in that the entire sample is ground to &mesh, and a portion of the ground sample is held in a vacuum oven at 70'C. (158'F.) for a longer period of time (40hrs.).
DETERIORATION OF PROCESSED POTATOES
265
He also found that the higher the moisture content, the more rapid was the development of the brown color. Caldwell et al. (1945) noted that potato strips discolored more rapidly at high temperatures than at low ones and that samples stored in air with a high relative humidity discolored more rapidly than those stored in dry air. They claimed that browning practically ceases when the moisture content of the material is reduced below 5%.
Legault et al. (1946) reported that the time required for a given color development varies exponentially with the absolute temperature if the moisture content is constant (between the limits of 4 4 % moisture), or with the moisture content at constant temperature. Thus storage life was doubled by a 3.4"C. (6.2"F.) drop in temperature or by a 2% (absolute) drop in moisture. The present author obtained data on the effect of temperature on the storage life of dehydrated potatoes varying greatly in reducing sugar, sulfite, and moisture content. Several varieties were studied, including samples dehydrated in an experimental drier and in commercial dehydrators. Storage life was defined in terms of color development. The latter was followed by determining the transmission a t 390 mp, of an aqueous alcohol extract. It was found that, in general, the storage life of a given sample was 8.5 times as great at 37.8OC. (100°F.) as at 48.9OC. (120°F.); 3.1 times as great at 48.9"C. (120°F.) as at 54.4"C. (130°F.) and 27 times as great at 373°C. (100°F.) as at 54.4"C. (130°F.). Over the range studied, storage life was approximately tripled for each decrease in temperature of 5.6"C. (10°F.)or approximately doubled by a decrease of 3.3"C. (6°F.). The same relationship held regardless of the nature of the raw material used, the sulfite or moisture content, or the method of dehydration. These results appear to be in fair agreement with those of Legault et a2. (1946). Data were obtained also on the relation of moisture content to the storage of dried potatoes. Moisture levels of approximately 1% (2.5oj, absolute) were obtained by packaging with CaO in accordance with the method described by Howard (1945a,b,c). Storage life was about doubled by a decrease in moisture of 2.5% (2.2% absolute). This is in close agreement with the data of Legault et al. (1946). It was found that in practically all cases a straight line was obtained when the data were plotted on semi-log coordinates, using percentage moisture as abscissa and storage life in days as ordinates. The slope of the line was nearly always the same, apparently being independent of variety, and of reducing sugar and sulfite content. An equation was derived for calculating the anticipated storage life (L2)of provided the storage life (L1)at ana sample at any moisture level (M2) other moisture level (MI) is known. The storage temperature must, of
266
A. FRANK ROSE
course, be the same in both cmes. The equation formulated for the calculations of storage life is:
log L2 = m (MS
- MI) + log d
(1)
In this equation L1and Lz represent change in storage life in days, MI and
M zmoisture content in yo,and m is a constant, the value of which depends only upon the method used for determining the moisture content. Where the method described in the tentative specifications of the Quartermaster Corps (1945) is used, m = -0,119, whereas when the method of Makower et al. (1946) for absolute moisture is used, m = -0.131. It is not known whether or not these relationships hold outside of the moisture range studied (1.2% to 7.5%). e. The E$ect of Suljiting on Browning in Storage. The chronology of commercial sulfiting in England, Canada and the United States has recently been reviewed by Green et al. (1946) and need not be repeated here.s The beneficial effect of sulfiting on the storage life of dehydrated potatoes has been recognized by several investigators (Davis et al., 1942; Cruess and Mackinney, 1943; U. S. Dept. Agr., 1944; Tomkins et al., 1944; Mackinney, 1945a,b; Caldwell et al., 1945). In most cases, no data are given relative to the actual increase in storage life that could be attributed to sulfite. Statements range from “sulfite helps slightly to preserve quality during storage a t 37°C.” (Tomkins et al., 1944) to “sulfite affords a means of very considerably prolonging” storage life (Caldwell et al., 1945). It is probable that this difference is due in part to variations in the amount of SOZin the product. Howard (1945a) compared unsulfited and sulfited samples at the same moisture level and found that the former became unacceptable in 10 days at 48.9”C. (120°F.) while the latter, containing 300 p.p.m. SO2 was acceptable for 25 days. When the samples were packed in containers with CaO, the storage life of the unsulfited sample was 60 days and that of the sulfited one over 80 days. Unpublished data of the present author show that storage life is increased considerably by increasing the sulfite content of the finished product. The beneficial effect of sulfite, however, is not so apparent when the sugar content of the dried product is high (Table I). In the paper by Green et al. (1946) reference is made to a report by English investigators (Barker et al., 1943b) on the effect of a number of antioxidants on browning, none of which waa aa effective or as satisfactory aa sul6te. A second report is cited (Barker et aZ., 1943a) in which data are given on the beneficial effect of sulfite on ascorbic acid retention and on retardation of deterioration in respect to color and flavor. UnfortuaatslJr these reports are not available to the author.
267
DETERIORATION OF PROCESSED POTATOES
Irish Cobbler potatoes containing about 625 p.p.m. 501 7% moisture
Katahdin potatoes containing 7% moisture Reducing swam, %
p.p.m.
1.9 1.9 1.9 6.6 6.6 6.6
170 454 1200 323 813 1654
+
Increased
Reducing sugars, %
Increased storage lifea
1.7 2.2 5.2 1.9 2.1 3.1
0.5 2.7
6 1.6
* Ratio of storage life of suKted sample to that of the unsulfited sample.
These data show that progressively increasing amounts of sulfite generally cause progressively greater increments in storage life. It also appears that the effect of a given amount of SO2 depends in part upon the reducing sugar content of the sample; a given amount of sulfite being more effective at lower reducing sugar levels than at higher levels. All samples, however, did not behave in this manner. When the Green Mountain variety was sulfited, the reducing sugar content of the product had no consistent effect on the effectivenessof the sulfite added (up to 928 p.p.m.). It wa& concluded that sulfite in concentrations ranging from 200 to 500 p.p.m. would result in a 50-100% increase in the storage life of dried potatoes relatively high in reducing sugars. In cases when the reducing sugar contents are lower much greater increases in storage life can be expected. Legault et al. (1946) stated that the magnitude of the sulfite effect is a function of concentration and that it persists in accord with the factors that govern the rate of disappearance of sulfite (principally moisture and temperature). The combined effect of low moisture and suEte is greater than either alone (Howard, 1945a; Mackinney, 1945a,b). In the present author’s laboratory it was found that if a given amount of sulfite doubled the storage life of a sample at high moisture, the same amount of sulfite also would double storage life at a low moisture content. The amount of SO2 that can be used is limited by the quantity tolerable to the consumer. As losses during storage and during rehydration cannot
268
A. FRANK ROSS
be predicted, tolerances are established for the dry product at time of packaging. In this country the maximum acceptable limit is considered to be 500 p.p.m. (U. S. Dept. Agr., 1944;Tressler, 1944) while in England the limit is 300 p.p.m. (Lovern, 1945). Barker and Burton (1944)state that more than 150 p.p.m. in mashed potato powder causes the product to have an unpleasant taste. The fact that larger amounts cannot be tolerated in this type of product probably is due to smaller losses during rehydration. In a recent paper (Green et al., 1946), it was reported that the pH of the sulfiting solution affects not only the rate of loss of SO2 from dehydrated potatoes in storage, but also the amount lost during reconstitution. Potatoes dipped in an SO2 solution (with or without orthophosphoric acid) lost more SOs during storage and rehydration than those dipped in a sodium sulfite solution. A given amount of sulfite, however, in the acid dipped potatoes was much more objectionable to taste than was the same amount of SOa in potatoes dipped in the alkaline sodium bisulfite solution. These data help to explain some differences of opinion on the tolerable limits for SO,. They also offer the possibility of developing methods whereby a high SO2 content could be maintained in storage, without impairing the taste of the product. There is a belief in the potato drying industry, that sulfite imparted to the product by combustion fumes in direct-oil-fired tunnel dehydration is less objectionable (from the standpoint of taste), than that imparted to the product by use of a dip. f. Raw Material Factors Aflecting Deterioration of the Dehydrated Product in Storage. Although it has been known for many years that potatoes stored under different conditions vary greatly in sugar content (MiillerThurgau, 1882;Appleman, 1912), and that this factor is an important one influencing the browning of potato chips (Sweetmm, 1930, 1931;Rogers et al., 1937; Wright et al., 1936; Denny and Thornton, 1940, 1941b, 1942a,b), no report prior to 1945 has been found relating reducing sugars to the browning of dehydrated potatoes in storage. Several reports appeared in 1945 showing that the sugar content of dehydrated potatoes is definitely related to this type of deterioration and that it is reducing sugar, not sucrose, that is primarily concerned. Ross et al. (1946)reported a positive correlation between the amount of reducing sugars in the raw material and the relative amounts of color developed by the dehydrated product when stored at 54.4"C. (130'F.). At a constant moisture level, almost a straighbline relationship was obtained between these two factors. The data presented cannot be translated into terms of storage life; however, they show comparative effects of moisture and of reducing sugars. In another experiment by the author, samples varying in moisture and sugar content were compared after 14 days at 54.4%. (130'F.). One sample contained 8.2% moisture and was prepared from tubers containing 7 mg.
269
DETERIORATION OF PROCESSED POTATOES
reducing sugars per ml. juice. The figures for a second sample were 6% and 15.5 mg., respectively. The samples were identical in color after storage and were superior to a third containing less moisture and considerably more sugar (25.5 mg./ml. juice). Caldwell et al. (1945) state that the dehydrated potatoes produced from immature tubers, from sprouting tubers, and from tubers held in cold storage for long periods of time do not keep well in storage. They attributed this to high levels of sugars and amino acids but gave no data in support of this contention. Burton (1945b) found a good correlation between the TABLEI1 Effect of Initial Sugar Content upon the Degree of Browning of Mashed Potato Powder (13% Moisture) after 4 Days at 67OC. (13.4.6°F.)4 ‘rota1 sugars,
% 0.92 0.96 1.06 1.16 2.22 5.64 7.80 14.39 15.72 17.87 19.95 20.76
Sucrose,
Total reducing su8&w
Glucose,
Fructose,
%
%
%
%
Color after storage, Lovibond units
0.00 0.50 0.00 0.45 0.71 4.37 3.72 6.00 14.32 14.34 17.98 13.68
0.32 0.46 0.46 0.71 1.51 1.27 4.08 7.79 1.40 3.53 1.97 7.08
0.15 0.22 0.21 0.30 0.86 0.56 1.64 3.67 0.79 1.62 0.83 4.08
0.17 0.24 0.25 0.41 0.65 0.71 2.44 4.12 0.61 1.91 1.14 3.00
1.8 1.6 1.7 2.8 2.9 3.8 8.1 12.4 5.5 6.9 4.8 11.5
Compiled from data of Burton (194513).
initial reducing sugar content of mashed potato powder and the degree of browning after 4 days a t 57°C. (134.6’F.) (Table 11). Samples at a given reducing sugar level browned a little more if their sucrose content was high than if it was low. Burton concluded from these and other data on sugar changes during storage, that the rate of browning in the early stages is determined by the initial reducing sugar (hexose) content but that the intensity of color developed reaches a maximum which is determined by the total amount of sugar present. Data relative to the effect of different levels of reducing sugar on the storage life of diced potatoes have been obtained in the author’s laboratory. The methods used differed from those of Burton in that diced potatoes
270
A. FRANK ROSS
with a lower moisture content (7%) and the lower storage temperature of 48.9"C. (120'F.) were used. Furthermore, samples were judged by the time required to reach a certain color level, instead of measuring the color after a fixed time interval. The storage life of dehydrated potatoes was found to be roughly inversely proportional to the reducing sugar content. The graphical presentation of the data (Fig. 1) shows considerable scatter,
2-
A KATAHDIN ASEBAGO 0 I R I S H GOBBLER IDAHO RUSSET
L
Fig. 1. Storage life at 48.9"C. (120°F.) of dehydrated potatma containing 7% moisture and various amounts of reducing sugars. (Ross, 1947.)
but the line drawn on log-log coordinates appears to be a fairly good expression of the relationship between these two variables. The scatter could not be accounted for by the potato variety used, or by the relative amounts of sucrose, glucose and fructose. The line drawn in Fig. 1can be expressed by the equation log y = 1.38 - log 2
(2)
DETERIORATION OF PROCESSED POTATOES
271
where y = storage life (in days) a t 48.9"C. (12OOF.) of samples at 7% moisture (determined by the method described in the tentative specifications of the Quartermaster Corps, 1945) and 2 = reducing sugar content in %. Equation 2 was generalized into the form log ( y / a ) = b - log z
(3)
where a is a function of the temperature and b a function of the moisture content. Data previously discussed were used for deriving the following empirical equations for calculating a and b:
120-T log a = -log 3 10 b = 1.38
+ (7-M) log 1.315
(4) (5)
where T is the temperat'ure under observation in O F . and M is the moisture content in %. Equation 3 can be expected to give no more than a fair approximation but may prove useful in further study of these and other variables. Further data showing the marked effect of reducing sugars on browning were obtained by Wiegand et al. (1946). Color development was followed by measuring the amount of light absorbed by an alcohol-water extract by means of a colorimeter. I n a typical experiment, samples prepared from Netted Gems and containing 0.79, 0.99, 1.53 and 6.20% reducing sugars, respectively, gave readings of 98, 128, 167 and 888, respectively, after 15 days at 54.4"C. (130OF.). They also reported that samples prepared from Netted Gems from one area developed less color in proportion t o their reducing sugar content than did samples prepared from the same variety grown in other areas. They did not report moisture contents, however, and the differences noted may have been due in part, a t least, to variations in moisture. Doty et al. (1946) reported browning in low-sugar potatoes during dehydration, the extent of which was correlated with the amino acid content of the raw potatoes. g . Packaging and Other Factors. According to most investigators, packaging in inert atmospheres, such as nitrogen or carbon dioxide, has little or no effect on the rate or extent of the development of browning in dehydrated potatoes (Tomkins et al., 1944; Continental Can Co., 1944; Burton, 1945b). This does not necessarily mean that oxygen has no effect on the browning reaction, for small amounts of oxygen are usually present in gas packed cans. Consequently, the report of Legault et al. (1946) that the influence of oxygen on the rate of browning is positive, though relatively small, is not necessarily in disagreement with the earlier reports. Balog and Cruess (1946) noted that debydrated potatoes compressed
272
A. FRANK ROSS
and then sealed in cans were of much better color and flavor after storage for 2% years at 28.3O-29.4"C. (83"-85"F.) than were loosely packed samples. They attributed the superior keeping qualities of the compressed pack to a 95% reduction in the volume of air in the cans. In view of the previously discussed reports on gas packing, where the oxygen content was reduced to about 1%,the basis for this explanation is not apparent. It seems unlikely that the ratio of oxygen to product in air-packed compressed potatoes would be less than that in nitrogen-packed mashed potato powder (Burton, 1945b). Treatment of the potatoes with calcium chloride or sodium chloride solutions before or during blanching has been reported to decrease sensitivity to heat damage (Campbell and Kilpatrick, 1945). The same authors, as well as others (Legault et al., 1946), also report that extensive leaching results in decreased browning. The browning reaction in mashed potato powder is accelerated by an increase in pH, at least over the range pH 5.6 to pH 10 (Burton, 1945b). When the powder was subjected to pH 1.2, however, hydrolysis of sucrose occurred and the rate of browning was increased. In the same paper, data were given to show that the browning reaction is inhibited by hydroxylamine and by cyanide. 3. Dehydrated Potatoes: Graying and Development of '(O$" F ~ U O T inS Storage Tomkins et a2. (1944) found that sulfiting (50-100 p.p.m. SO,) appreciably delays the tendency of potato strips to turn gray at 15°C. (59°F.) or less. They observed that this type of deterioration was much the same in all samples, whether they varied in moisture content or were packed in air, nitrogen, or carbon dioxide. Their taste panel data, however, show progressively lower color and flavor ratings for low moisture samples (2.7%) as the storage period increased. This tendency became less apparent as the moisture content of the sample was increased. The estimated average storage life of samples stored at 15°C. (59°F.) was more than 15 months when judged on culinary quality. Individual lots varied from 7 to over 17 months. These samples contained 7y0 moisture and presumably were sulfited. The development of an "off" flavor in mashed potato powder at low moisture levels has been reported by Burton (1945b). The reaction is considered to be retarded by high moisture contents. 8. Other ~ T O d U C t S
Ruschmann (1932) has reported tBat silage made from potatoes high in sugar was brownish in color and inferior in quality. Rogers (1945) found
DETERIORATION OF PROCESSED POTATOES
273
that the reducing sugar content of potatoes used for canning affected flavor and color. Those high in such sugars, when canned by a variety of accepted methods, developed a pinkish-amber color at the centers of the tubers and were of poor flavor. At high temperature storage 37.8"C. (100'F.) there was further deterioration in both color and flavor. Several investigators have related the browning of potato chips to the sugar content of the potatoes used for their manufacture (Sweetman, 1930, 1931; Rogers el al., 1937; Wright el al., 1936). More recently, Denny and Thornton (1940, 1941b, 1942a) showed that this browning is correlated with the reducing sugar content rather than with that of sucrose. They recommended the use of potatoes for chip making containing not over 3 mg. of reducing sugars/ml. of juice. The color intensity of French fried potatoes also appears to be a function of the sugar content of the tubers used (Wright et al., 1936) although no attempt has been made to distinguish between the effect of reducing sugars and that of sucrose.
Iv.
CHEMICAL CHANGES DURING STORAGE OF DEHYDRATED POTATOES
1. Changes Associated with Browning
Several reports have been made showing that decreases in sugars and in amino acids accompany the development of the brown color. Analysis of a browned sample of potato flour showed a small decrease in sucrose, hexose sugars, and amino nitrogen (Burton, 1945a). In another report on the browning of potato powder induced by storage at high temperatures, Burton (1945b) gave the results of several analyses of browned samples. The figures given in Table I11 were calculated from the data of Burton to illustrate the relationship between the degree of browning and the decrease in total sugars. With few exceptions the degree of browning correlated well with the decrease in sugars but not with the decrease in amino nitrogen. Other of the data showed that in any particular lot of potatoes low in sugar a correlation existed between the degree of browning during the early stages of discoloration and the loss of hexose sugars. A loss of sucrose occurred in many but not all samples. A decrease in glucose content occuyed in all samples and this loss most nearly paralleled the increase in brown color. Analyses of potatoes containing considerably more sucrose than hexoae sugars indicated marked losses of the former and smaller losses of hexose sugars. Burton assumed that hydrolysis of sucrose had occurred in such samples at the high temperature. From these and other of the data showing a marked correlation between the initial hexose content and browning, he concluded that in the early stages of the reaction the rate of
274
A. FRANK ROSS
browning is determined by the initial hexose content but that the maximum amount of color developed may be determined by the total amount of sugar present. His surmise would appear to be justified in part but there is nothing in the results to show that sucrose itself does not participate in the later stages of the reaction. Unpublished data of the present author concerning changes in dried, diced potatoes during storage at 48.9"C. (120°F.)showed a consistent reduction in fructose, but the values of glucose were quite variable, sometimes showing increases of such magnitude that the total figure for reducing sugars in the browning product was TABLEI11 The Relation oj Color I n t m * t g to Loss of Total Sugars and of Amino Nitrogen i n Mashed Potato Powder Samples Containing 18% Moisture and Stored at 67°C. (134.6"F.)for VariozcS Periods of Timea Color of water extracta, Lovibond units 1.7 1.8 2.7 4.8 6.5 6.9 11.6 13.3 14.1 28 (approx.)
Total sugar Low g./1@Jg. 0.26 0.31 1.00 2.19 0.62 2.81 4.64
2.57 2.06 6.60
Amino-Nitrogen Loss" g./100 g.
0.03 0.04 0.01 0.04 0.05 0.10 0.07 0.03 0.12 0.22
larger than in the control sample. It was concluded that intermediate compounds were formed which reacted as reducing sugar during the quantitative determination for glucose by reduction of dinitrophenol, or that the increase in glucose may have come about as a result of a hydrolysis of sucrose (analyses for sucrose were not made). A loss of amino nitrogen also occurred during browning and for any given variety the reduction was greatest in the most severely discolored samples. In this respect, however, there was considerable variation between varieties. Some samples became severely discolored with only negligible losses in amino nitrogen while others lost up to half of t h t originally present in reaching the m e stage of discoloration. While it seems probable that losses in re-
275
DETERIORATION OF PROCESSED POTATOES
ducing sugars are associated directly with the development of the brown color, the relation of amino acid changes to browning is not as clear cut. The possibility of these being independent reactions has not been excluded. Absorption of oxygen and an evolution of carbon dioxide during storage of dried potatoes have been observed. Tomkins et al. (1944) noted these gaseous changes in sealed cans of potato strips and reported that the changes were greater at 37°C. (98.7"F.) than at 28°C. (82.4"F.) and were less at 15°C. (57°F.) than at 37°C. (98.7"F.). There was a greater change when samples were high in moisture than where they were low. Data obTABLE IV Changes in Gas Composition Occurring during Browning of Mashed Potato Powder Containing 1.2% Moisture and Stored at 67OC. ( 134.6'F.)
I
Length of atorage, days
Color
1 2 6 10 16 30 41 62
Lovibond units 0.3 0.4 1.0 6.1 7.5 11.8 12.9 13.9
Nitrogen pack ~
~~
CO,
absorbedo
COl evolvedo
Color
m1./100 g. 1.0 2.0 7.1 12.5 13.5 13.5 13.5 13.5
ml./lOO g. 1.2 1.6 6.2 8.0 13.7 20.7 25.7 25.9
Lovibond units 0.1 0.6 1.6 4.3 7.1 11.3 13.2 13.8
01
evolved ml./l00 g. 0.9
1.2d 2.3 4.2 8.0 14.8 17.4 17.4
Compiled from data of Burton (1945b). 13.5 ml. 01per 100 g. powder. 8 Normal temperature and pressure. d These results are presumably high because of traces of oxygen.
0
b Atmosphere contained
tained at the Continental Can Co. (1944) also indicate similar changes and agree with data referred to the above with respect to the effect of temperature. Burton (1945b) found that when mashed potato powder of low moisture content was stored at a high temperature there was an absorption of oxygen accompanied by the development of an "off" flavor, but very little browning Qr COZevolution occurred. With powder of high moisture content there was a more rapid intake of oxygen, evolution of C02, and development of brown color (Table IV). After the disappearance of all of the oxygen, evolution of carbon dioxide continued at a slower rate. This latter rate w1t9 about the same as that of samples packed in nitrogen. In
276
A. FRANK Roaa
both cases the rate of browning was the same, the color developed to the same maximum intensity, and the rate of browning fell as this was approached. Parallel with this decrease in rate of browning was a decrease in the rate of carbon dioxide evolution. These data were taken as an indication of the occurrence of two reactions in samples at a high moisture content : (1) Anaerobic development of a brown color accompanied by CO,evolution. (2) Absorption of oxygen accompanied by evolution of COI. A third possible reaction, occurring at low moisture levels, was stated to be an absorption of oxygen unaccompanied by evolution of carbon dioxide but resulting in the development of an “off” flavor. This type of flavor deterioration is reduced when the moisture content of the product is relatively high. It is quite possible that in potatoes relatively high in moisture, the substance causing off fiavor changes chemically, rather than accumulates as it forms. Reactions 1 and 2 are substantiated by data and their existence in dehydrated potato strips and dice appear to be consistent with the available data. There are fewer data relative to the third reaction and very little or no data are available on this type of change in potato strips, dice, or shreds. The data of Tomkins et al. (1944) show greater deterioration of flavor in diced potato strips containing 2.7% moisture than in strips containing 5%, when stored at 37°C. (98.7”F.). While this point may seem a minor one now, the development of this type of “off” flavor may become more apparent and of increasing importance as progress is made in the control of browning. Browning of dehydrated potatoes is accompanied by the development of ultraviolet fluorescence of extracts (Doty et al., 1946; Pyke et al., 1946; Patton and Pyke, 1946). The fluorescence of the extracts varies directly with color. The fluorescence varies with pH (Pyke et al., 1946; Patton and Pyke, 1946). The pH-fluorescence curve of the brown pigment extracted from dehydrated potatoes has been shown to be similar to that of the extract of potato chips. The browning of potato flour is accompanied by a decrease in pH and by an increase in buffering capacity (Burton, 1945a). A few data have been accumulated on the nature of the brown pigment or pigments produced in potatoes. Gore and Rutledge (1921) reported it to be water soluble, an observation that has been confirmed repeatedly since 1921. The pigment is insoluble in ether (Burton, 1945b), soluble in methyl alcohol, and somewhat soluble in ethyl alcohol (Doty et al., 1946). Dawson (1945) isolated a brown pigment from browned potatoes by a process involving clarification with amylase, dialysis, and precipitation with acetone. The latter treatment rendered the pigment imoluble in
DE1TERIORATION OF PROCESSED POTATOES
277
water. Both the dialyzed and nondialyred pigments reduced Fehling's solution and iodine. The former gave iodine titrations equivalent to 3540% glucose and about a third of the reducing value was retained after prolonged dialysis. The pigment reduced methylene blue and absorbed oxygen directly at 37°C. (pH 8.3 and 9.7). After prolonged dialysis the of which was amino nitrogen. The product contained 1.5% nitrogen, latter value was doubled by acid hydrolysis. Doty et al. (1946) stated that a purified preparation of the pigment gave certain carbohydrate and protein tests. As no statement was given as to the specific protein tests made, one cannot conclude that protein itself is a part of the pigment, since some mcalled protein tests give positive reactions with amino acids. A crystalline derivative of the pigment has been obtained by Caldwell el al. (1945) but no analytical data concerning it are available. Pyke and associates (Pyke et al., 1946; Patton and Pyke, 1946) have reported results indicating that reducing sugars and amino acids are both concerned in the browning reaction in potatoes. They worked chiefly with potato chips but were able to show that the browning reaction in this product was quite similar to that in dehydrated potatoes. Extraction of potato slices with hot water yielded an extract containing considerable amounts of reducing sugars and amino acids. When dried in vacuo the extract yielded a dry powder that browned if moistened and heated. The extracted potato chips did not brown when fried in hot fat but did so if first impregnated with the concentrated extract. Impregnation of the potentially white slices with glucose or other reducing sugars failed to produce browning during subsequent frying; similar results were obtained by impregnation with glycine or other amino acids. When the slices were impregnated simultaneously with both types of compounds, browning occurred during subsequent frying. These results appear a t variance with those of Denny and Thornton (1940) who obtained browning of filter paper discs impregnated with glucose, dipped into potato starch, dried and then fried in hot fat. Moisture relationships were probably different in the two cases and the possibility of the presence of impurities in the potato starch was not eliminated. Unpublished results of the present author indicate that some constituent other than reducing sugars is concerned in the browning of dehydrated potatoes. Samples of low-sugar potatoes were rehydrated in glucose and in fructose solutions and in a solution containing both. Efforts to avoid leaching of other constituents were unsuccessful, for appreciable quantities of liquid drained from the potatoes. The samples were redried and when stored at 48.9OC. (120OF.)failed to brown at rates anticipated from their reducing sugar contents. It is concluded that sufficient quantities of other constituents essential for browning were leached from the samples, to appreciably affect the rate of browning.
278
A. FRANK ROSE
The available data constitute strong evidence that browning in potatoes is due to a reaction between reducing sugars and amino compounds, usually referred to as the Maillard reaction. The disappearance of both reducing sugars and amino nitTogen during the browning of potatoes has been demonstrated. For browning to occur in potatoes, both types of compounds that have been leached must be replaced. The high temperature coefficient of the reaction, changes in pH during browning, development of fluorescence, evolution of carbon dioxide, effects of pH and of moisture on the reaction, and the reducing properties and nitrogen content of the pigment are all consistent with this view. Caldwell et al. (1945) have pointed out that reducing sugars and amino acids are always present in potatoes and undergo an enormous concentration during dehydration. 2. Sulfite Retention during Storage
Variables that effect the rate of browning also appear to affect the rate of sulflte loss. Howard (1945a) measured the rate of sulfite loss in potatoes stored in nitrogen at 48.9"C. (120°F.). These samples were dehydrated to 8.0, 6.7, and 4.6y0 moisture (absolute), respectively. The original SO2 content of about 300 p.p.m. decreased considerably during storage. This is shown below: Storage life', days 18 26 66
Moisture content, % 8 6.7 4.6
SOs after storage, p.p.m. 60 25 50
Burton (1945a) noted that sulfite loss from dried potatoes is dependent on both moisture content and temperature. This is indicated by data obtained with a sample of potato flour, containing 340 p.p.m. of SO2, held in storage for 4 months. Storage temperature
Moisture content, %
SO2 after storage, p.p.m.
8.6
300
"C.
OF.
1 1 37 37
(33.8') (33.8O)
16
(98.6") (98.6')
8.6 15
200
110 60
The present author (unpublished data) found that sulfite loss was 5 to 10 times as great at 37.8"C. (100°F.) as a t room temperature. With samples containing 2.5y0or more reducing sugars, the rate of loss was about 7 times aa great at 48.9"C.(120°F.) as a t 37.8"C. (100°F.); but where the
' The time intervah indicated were those at which the respective samples were considered to have become organoleptically undesirable.
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279
reducing sugar content was less than 2.5%, sulfite loss wm only 3 to 4 times as great at the higher temperature as a t 37.8OC. (100OF.). Other data were obtained indicating an effect of reducing sugar content on rate of sulfite low. Two samples containing approximately 650 p.p.m. of SO2 and 6.19% moisture but differing widely in sugar content were stored at 48.9"C. (120°F.). The losses in SO2 observed during storage are shown below: Potatoes containing 0.46% reducing sugars Storage period, days SOBloat, % ' 13 22 23 30 39 47
Potatoes containing 4.7% reducing sugara Storage period, days SO*lost, yo 13 w 23 80 39 80
Green et al. (1946) noted better retention of SO, in storage in potatoes given alkaline dips than in those dipped in acid solutions prior to drying. Very little information on the nature of the inhibiting action of sulfite can be gained from the work on potatoes, Mackinney (1945a), speaking of vegetables in general, stated that sulfite effects an inhibiting rather than a masking action in the prevention of darkening. It is apparent from the data given by different authors that the formation of the brown color and sulfite loss occur simultaneously, that browning is not completely inhibited by the presence of sulfite, and that appreciable amounts of sulfite may be present in the product after it has become unacceptable. The present author (unpublished data) found a fairly good correlation between rate of sulfite loss and rate of color development at a given temperature. Estimates of the proportion of the initial sulfite lost a t the point where samples became unacceptable were all very close to 60% in 48.9%. (120°F.)storage and to 70% in 37.8OC. (100'F.) storage. This held true regardless of reducing sugar content. This does not necessarily indicate a stoichiometric relationship between the two. It may be that both are affected independently by the same factors at approximately the same degree. Burton (1945b) pointed out that the substances known to inhibit the browning reaction are reducing agents as well as compounds capable of reacting with aldehydic or ketonic groups, hence it is possible that this might be the property (of SO2) effective in preventing browning.
V. CONTROL OF BROWNING 1. Methods of SulJiting There is considerable variation in the methods used for sulfiting. In England, where hot water blanching is practiced, sulfite salts are usually
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A. FFlANH ROSS
added to the blanching water. In the United States, most plants blanch the potatoes with steam. For tray blanching, Mackinney (1945) recommends application of sulfite in the form of a spray. Subject only to the stipulation that the tray should emerge without dripping, Mackinney found that the later the spray is applied in the blancher, the more efficient will be the penetration and absorption of sulfite. Where potatoes are blanched on continuous belts, a similar procedure is satisfactory provided the belt is made of stainless steel. If corrosion of the belt is a factor, Mackinney (1945b) recommends the application of the sulfite solution as a drip from a perforated pipe while the potatoes are on the vibrating screen (Syntron). The technological problems involved in obtaining the desired concentration of SO2in the dry product have been discussed by Mackinney (1945b), Beavens and Bourne (1945), Wager et al. (1945), and by Green et al. (1946). Blanched potatoes absorb SO2 much more readily than raw ones whereas the pH of the sulfite solution has little or no effect on rate of absorption (Green et al., 1946). Approximately neutral sulfite solutions generally are recommended, and for a given plant, the desired SOa content in the dried product is obtained by proper adjustment of the concentration of the sulfite solution. Sulfite solutions apparently do not cause serious corrosion if trays or belts are made of stainless steel, galvanized iron or iron with Bakelite-type finish (Mackinney and Howard, 1944; Western Regional Research Laboratory, 1945). 8. Attainment of
Low Moisture
It is agreed generally that moisture levels less than 6% cannot be obtained by the usual dehydration methods without damage to the product. Howard (1945a,b,c) has developed what appears to be a satisfactory method for reducing the moisture contents of potatoes well below 6% moisture. This method, termed “in-can desiccation,” involves placing calcium oxide (in a porous package) in the container at the time of packaging. This entails a sacrifice of about 14% of the-container space. This worker clearly demonstrated that this method greatly extends the storage life of dehydrated potatoes. Diced potatoes containing 300 p.p.m. SO2 packed in nitrogen together with CaO were of good quality after 80 days a t 50°C. (120°F.) while a similar sample packed without CaO was unacceptable in about 25 days. Nonsulfited potatoes lasted 6 times as long when CaO-packed as did those containing 6% moisture. These data have been substantiated by the present author. It was found, however, that the effectiveness of in-can desiccation depends in part upon the reducing sugar content of the sample. When the latter was quite high (10.7%) storage life a t 37.8”C. (100°F.) was a little more than doubled, at a medium sugar level (3.5%) storage life was increased 3 to 4 times, and
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281
at a low sugar level (0.8%) storage life was increased 6 times or more. The high sugar samples dried at a slower rate than the others. The storage life of suliited samples (2.3 to 3.6% reducing sugars, 216 to 504 p.p.m. SO2) waa increased about 5 fold by in-can desiccation. Potatoes subjected to in-can desiccation and then held a t a moderate temperature for 3 months before storing at 37.8"C. (100°F.)were somewhat more stable although the storage life was not always prolonged by following this procedure. During storage at moderate temperature the moisture content dropped to slightly less than half of the original value during the first 42 days. An additional 42-day period caused the moisture content to drop by about 0.6%. Drying was much faster and more complete at higher temperatures. Certain dehydrated vegetables that are easily compressed may be dried to low moisture levels by high frequency radiation, preferably at reduced pressures (Sherman, 1944; Rushton et al., 1945). No satisfactory process, however, for compressing the usual forms of dehydrated potatoes has been developed (Proctor and Sluder, 1943; Magoon and associates, 1946). It has been found that dehydrated potatoes are too brittle and that breakage releases free starch, causing gumminess in the reconstituted product. Whether or not mashed potato powder or other powdered or granulated potato products can be satisfactorily compressed is yet to be demonstrated. Drying of this type of product to low moisture levels by the usual methods has not been fully investigated. 9. Use of Potatoes Low in Reducing Sugars
a. Availability. Few data have been published on the reducing sugar content of fresh potatoes at the time of removal from common storage. In Maine, appreciable quantities of potatoes reasonably low in reducing sugars were found during the first half of the winter (Ross, unpublished data). Considerable variation was found in the Pacific Northwest by Bedford and Lusk (1946) but their results indicate that it is possible and practical to obtain potatoes fairly low in reducing sugars during the winter. In Idaho, the average sugar content for potatoes from 15 cellars had definitely increased by November and continued to increase through March (Stamberg and McKinnon, 1946). Large quantities of low sugar potatoes should be available for drying during the winter and spring, provided they can be conditioned for 2-3 weeks at warm temperature. On the other hand, it is a common experience to find occasional lots of potatoes extremely high in sugar content. The present writer has analyzed commercially dehydrated samples containing over 12% reducing sugars. More southernly areas are more fortunate in this respect. Southern grown potatoes are, however, as a rule, lower in specific gravity than northern sown ones aud hence are less suitable for dehydration. Potatoes of low
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A. FRANK ROBS
specific g r a i t y give high dehydration ratios and may result in a product of inferior quality (Caldwell et al., 1943a,b). b. Factors Influencing the Reducing Sugar Content of Potatoes. The most important factors affecting the reducing sugar content of potatoes are storage temperature and variety. That potatoes in cold storage accumulate sugars has been known for many years (Miiller-Thurgau, 1882); numerous papers have been published on the effect of different temperatures on the rate and extent of sugar accumulation (Appleman, 1912; Wright, 1932; Wright et al., 1936; Denny and Thornton, 1941b, 1942a; Ross et al., 1946; Campbell and Kilpatrick, 1945). I n general, it has been found that both sucrose and reducing sugars accumulate a t low temperatures and that the lower the temperature, the faster is the rate of accumulation and the greater is the amount accumulated. This, however, is not always the case since with a t least one variety it has been found that reducing sugars are formed a t a faster rate a t 4°C. (40°F.) than a t 0°C. (32°F.) (Campbell and Kilpatrick, 1945). Reducing sugars and sucrose, generally, but not always, show parallel changes. Varieties differ greatly in the rapidity and extent of the accumulation of reducing sugars a t low temperature (Denny and Thornton, 1941b, 1942a; Ross et al., 1946). Fertilizer practices apparently have little or no effect on sugar-forming characteristics of potatoes (Denny and Thornton, 1941b; Ross, unpublished data). Some investigators report that tubers of the same variety grown in different locations show no important differences in the amounts of reducing sugars developed in cold storage (Denny and Thornton, 1941b) while others report significant differences (Wiegand et al., 1946). Potatoes held in an atmosphere containing 4.9% carbon dioxide while in cold storage form reducing sugars at a lower rate than when stored in air (Denny and Thornton, 1941a, 1942b, 1943b). Prestorage of tubers a t warmer temperatures before they are placed in cold storage causes a small reduction in the rate of reducing sugar formation during subsequent low temperature storage (Barker, 1939; Denny and Thornton, 1943a). The temperature range at which there is little or no change in reducing sugar content has been variously placed between 4.4" and 10°C. (40"and 50°F.), (Wright et al., 1936; Butler, 1919; Barker, 1938). Denny and Thornton (1942a) found a variation from 7"-8"C. (44.6"46.4"F.) to be critical for reducing sugars as well as for sprouting. Reducing sugar values were about twice as high at 7°C. (44.6"F.) as at 8°C. (46.4"F.) and a t 7°C. (44.6"F.) none of the varieties tested showed an excessive amount of Bprouting in about 7 months. The reducing sugar content of potatoes from low temperature storage can be lowered by subsequent storage a t higher temperatures (Wright,
DETERIORATION OF PROCESSED POTATOES
283
1932; Wright et al., 1936; Denny and Thornton, 1941a; 1942b; Ross et al., 1946). The rate of loss is much faster at 21.1”C. (70°F.) than at 15.5”C. (60°F.) but only slightly more rapid at 26.7”C. (80°F.) than at 21.1”C. (70°F.) (Ross, unpublished data). The rate at which reducing sugars disappear under such conditions depends upon the variety and upon the length and temperature of previous storage (Denny and Thornton, 1943a; Ross, unpublished data). Excessive sprouting may cause an increase in reducing sugars (Stamberg and McKinnon, 1946). Data from a number of potato producing areas show that conditioning in a warm room can be depended upon to appreciably lower the reducing sugar content of a11 varieties (Bedford and Lusk, 1946; Wiegand et al., 1946; Stamberg and McKinnon, 1946). In a test conducted on a pilot-plant scale the present author demonstrated that conditioning of the raw material for 2% weeks at 21.1OC. (70°F.) increased the storage life of the dry product from 2% to 3 times. c. General Considerations. The use of potatoes low in reducing sugars has advantages other than those cited. Wright et al. (1936) have shown that the culinary quality of potatoes in general is inversely correlated with their sugar content. They found that as sugar accumulates, the texture becomes soggy or watery and the flavor unpleasantly sweet. Later it was demonstrated that dehydrated potatoes prepared from potatoes of high sugar content are also of poor culinary quality, for in addition to being discolored, they are soggy and sweet (Wright et al., 1945). Frequently an undesirable yellow color develops in potatoes held in cold storage (Caldwell et al., 1943a, 1943b, 1945). Finally losses during the processing of potatoes high in soluble sugars may be high because of the removal of soluble materials by leaching. Conditioning of raw stock at warm temperatures is a common practice among manufacturers of potato chips. It wa8 not possible, however, for dehydrators to rely on this procedure alone during the war years because of large volume of production, In a set of recommendations drawn up for dehydrators, Ross et al. (1945) pointed out the advantages of obtaining desirable potatoes by selective buying as well as the use of warm storage conditioning when necessary. Rapid sugar tests have been developed (Peacock and Brunstetter, 1931; Ross et al., 1946) that make possible the procurement of potatoes on the basis of their reducing sugar content. In addition, the results of Denny and Thornton (1940) suggest the possibility of using a “chipping test” to estimate the reducing sugar content of potatoes. This “chipping test” involves preparing potato chips under standardized conditions and comparing their color with similar ones prepared from potatoes of known reducing sugar content. Sprouting is the chief difficulty encountered in storing potatoes so that
284
A. FRANK ROSS
reducing sugars do not accumulate, during conditioning. While it is possible to avoid sprouting by proper choice of time and temperatures, the use of sprout inhibitors is also promising. The methyl ester of a-naphthaleneacetic acid appears to be the most useful and satisfactory (Guthrie, 1939; Denny, 1942; Denny et al., 1942). These authors have shown that potatoes treated with this chemical can be stored from autumn to spring at temperatures ranging from 10"-22"C. (50°-71.6"F.) without appreciable sprouting. The most suitable storage temperature (for treated tubers) to avoid shrinkage and accumulation of reducing sugars is in the range 1Oo-15"C. (15O-59"F.). The sugar forming characteristics of the potatoea are not altered by the treatment with the sprout-inhibiting chemical. More recently Denny (1945) has found that lots of potatoes treated with sufficient chemical to prevent sprouting when stored for 5 months at 12.5"C. (54.5"F.) will remain firm and contain only traces of reducing sugars after storage, and when made into potato chips the product has a good color. Potatoes treated with the methyl ester of a-naphthaleneacetic acid do not appear to be toxic. Finch and Harteell (1945) fed mice with treated potatoes and with diets containing up to 90 times the amount of the chemical in treated tubers. No harmful effects attributable to the chemical were found.
4. Combined Treatments The method used to prevent browning depends upon the degree of stability that is required. From a standpoint of military requirements, it generally is considered that a product should have a storage life of 6 months at 37.6"C. (100°F.) (Wodicka, 1945) and needless to say, a longer storage life would be even more desirable. This degree of stability may not be essential for the civilian market. According to unpublished data of the present author, if dehydrated potatoes are to have a storage life of 6 months at 37.6"C. (100°F.) the reducing sugar content cannot be ignored, even when sulfiting and in-can desiccation are utilized. It was found that for a sample of dried potatoes containing 7% moisture to be able to withstand storage for 6 months at 37.6"C. (lOO°F.), the reducing sugar content should not be over 1.2% if unsulfited or over 2% if sulfited. For similar storage requirements, dried potatoes containing 6% moisture must not contain over 1.6% of reducing sugars if unsulfited or 2.7% if sulfited. CaO-packed (in-can desiccation), unsulfited samples containing over 3.5% reducing sugars will not tolerate 6 months of storage at 37.6"C. (100°F.). Samples receiving both sdfiting and in-can desiccation are not considered sufficiently stable if containing as much as 6% of reducing sugars. In aotual practice, the storage life might be greater because dried potatoes would rarely if ever be removed from the dehydrator, packed, and then subjected
DETERIORATION OF PROCESSED POTATOES
285
to continuous storage at 37.6"C. (100°F.). If the combined treatment is used, it would then be necessary to avoid only those potatoes which are excessively high in reducing sugars. Gas packing does not appear to be justifiable for the purpose of retarding discoloration. Additional data of the effects of this treatment on flavor at low moisture levels are needed. VI. SUMMARY Browning is frequently encountered in most types of processed potatoes.
It may occur during processing or during subsequent storage. The available data indicate that browning of dehydrated potatoes is due to a reaction between reducing sugars and amino acids. It is most severe with potatoes of high reducing sugar content and is very slight if the reducing sugar content is low. The rate of formation of the brown color is directly proportional to the reducing sugar content. Thus the storage life of a sample containing 2% of reducing sugars will be twice that of one containing 4%, provided other conditions are the same. The rate of discoloration also is affected by moisture content and temperature. The time required for a given color development varies exponentially with the absolute temperature if the moisture content is constant, or with the moisture content at constant temperature. Thus storage life of the product is doubled by a 3.4"C. (62°F.)drop in temperature or by a 2% (absolute) drop in moisture. Sulfiting inhibits the browning reaction in dehydrated potatoes during processing and also during storage. Sulfite at concentrations ranging from 200-500 p.p.m. results in a 50-100010 increase in the storage life of dried potatoes relatively high in reducing sugars. In cases where the reducing sugar contents are lower, much greater increases in storage life can be expected. Packaging in inert atmospheres has little or no effect on the rate of browning. The effects of sulfiting, of the use of potatoes with low reducing sugar contents, and of attainment of low moisture contents are additive in the sense that a combination of any two methods is more effective than either alone and a triple combination is the most effective. Low moisture levels can be reached satisfactorily by packaging the product together with a porous container containing calcium oxide (termed in-can desiccation). The reducing sugar content of potatoes is affected principally by storage temperature and by variety. Raw stocks suitable for processing can be obtained by selective buying and by conditioning lots with high reducing sugar content at warm temperature. Dehydrated potatoes occasionally turn gray during low temperature storage. This type of discoloration is inhibited by sulfite. Mashed potato powder sometimes develops an "off" flavor other than that asso-
286
A. FRANK
Roaa
ciafed with browning. The reaction is retarded by high moisture content. Potatoes high in reducing sugars generally give products with inferior texture and color. If canned, such potatoes may develop a pinkish-amber color in their centers and be of poor flavor. The browning of potato chips and of French fried potatoes is correlated with the sugar content of the tubers used for their manufacture.
REFERENCES* Appleman, C. 0. 1912. Change in potatoes during storage. Md. Agr. Exp. Sta. B d l . 167. Appleman, C. O., and Miller, E. V. 1926. A chemical and physiological study of maturity in potatoes. J. Agr. Research 33, 569-577. Balog, E. G., and Cruess, W. V. 1946. A note on compressed dehydrated potatoes. Fruit Products J. 16, 38, 54. Barker, J. 1938. Changes in sugar content and respiration in potatoes stored a t different temperatures. Gr. Brit. Dept. Scd. Znd. Res. Food Invest. Bd. Rept. 1937, 176-117. Barker, J. 1939. The effect of temperature-history on the sensitivity of the sugar/starch balancing system in potatoes. Gr. Brit. Dept. Sn‘. Ind. Rea. Food h v e e t . Bd. Rept. 1938, 193-195. Barker, J., and Burton, W. G. 1944. Mashed potato powder, I. General characteristics and the “brush sieve” method of preparation. J . SOC. Chem. Znd.63,169-172. Barker, J., Tomkins, R. G., Allen, R. J., and Mapson, L. W. 1943a. The packing and storage of dried vegetables-January, 1943. Dehydration, United Kingdom Prog. Rept. Sect. VI, Part 2, 21 pp. Barker, J., Tomkins, R. G. L., and Wager, H. G. 1943b. Summary account of investigations a t present in progress. Dehydration, United Kingdom Prog. Rept. Sect. VI, Part 1, 19 pp. Beavens, E. A., and Bbume, J. A. 1945. Commercial sulfiting practices. Food I d . 17, 1044-1046. Beckley, V. A., and Notley, V. E. 1941. The ascorbic acid content of dried vegetables. Biochem. J. 86,1396-1403. Bedford, C. L., and Lusk, J. L. 1946. Personal communication, State College of Washington. Black, H. G. 1943. The effect of storage on Irish Potatoee used for dehydration. Fruit. Products J. 28, 370, 377. Burton, W. G. 1945a. The storage life of a sample of potato flour produced from potato slices dried in a sugar beet factory. J. SOC. Chem. Znd. 64,85-86. Burton, W. G. 1945b. Mashed potato powder, 111. High temperature browning of meshed-potato powder. J . Soe. C h . I d . 64,216-218. Butler, 0.1919. Storage of potatoes. New Hampahire Agr. Ezpt. Sta. Circ. 10. Caldwell, J. S., Brunstetter, B. C., Culpepper, C. W., and Eaell, B. D. 1945. Causes and control of discoloration in dehydration of white potatoes, Parta 1 and 2.
* Many of the data were taken from reports of joint projects of the Quartermaster Corps, U. 5. Army, and various institutions, and from reports preeented at various QMC conferences. Since few, if any, of these reporta are avdable for general distribution, they me listed aa personal communications.
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The Cunner 100 (13), 35-39, 112, 114, 318, 120, 122; 100 (14) 15-16, 18, 30-32, 34;100 (is), 14, 16,24,26-27. Caldwell, J. S., Lombard, P. M., and Culpepper, C. W. 1943. Variety and place of production aa factors in determining suitability of dehydration in white potatoes. The Cunner 97 (3),30,32,34-35,42,44; 97 (4),14-17,24;97 (5),15-16,18-19,28. Campbell, H., and Kilpatrick, P. W. 1945. Effect of storage temperatures in sensitivity of white rose potatoes to processing heat. Fruit Products J . 26, 1Oe108, 120-122. Campbell, H., Lineweaver, H., and Morris, H. J. 1945. Severe blanch doesn’t improve dehydrated potato quality. Food Inds. 17, 384-386, 478, 480, 482, 484,486. Chace, E. M., Noel, W. A., and Pease, V. A. 1941. Preservation of fruits and vegetables by commercial dehydration. U.S. Dept. Agr. Circ. 019. Continental Can Co. Research Staff. 1944. New facts about packaging and storing dehydrated foods. Food Inds. 10, 991-993. Cruess, W.V., Ea!og, E. G., Friar, H. F., and IRw, M. 1944a. SuMting to improve vegetables for dehydration. Food Pucker 26 (1). 31, 62. Cmess, W.V., Balog, E. G., Friar, H. F., and Lew, M. 1944b. Effect of sulfiting on dehydration temperature. The Cunner 98 (5),18. Cruess, W. V., and Friar, H. F. 1943. Notes on dehydration of potatoes. The Canner 97 (14),14-15. Cruess, W.V,,and Joslyn, M. A. 1942. Significance of enzyme reaction to dehydretion of vegetables. Prac. Inat. Food TechmZ. pp. 84.110. Cruess, W. V., and M a c k i e y , G. 1943. The dehydration of vegetables. CaZij. Agr. Expt. Stu. BuEt. 000. Cruess, W. V., and Mrak, E. M. 1940. The dehydration of vegetables. Fruit Products J . 20, 100-103. Cruess, W.V., and Mrak, E. M. 1942a. The dehydration of vegetables. Fruit Products J . 21, 201-204, 241-242, 269-272, 302-307, 337-340. Cruess, W. V., and Mrak, E. M. 1942b. What’s known today about dehydrating vegetables. Food Inds. 14 (l),67-80;14 (2)41-43, 96-97. Cruess, W.V., Smith, M., and Balog, E. G. 1943. Enzyme reactions in dehydrated potat3es. Fruit Products J . 23, 135, 155. Davis, M. B., Eidt, C. C., MacArthur, M., and Strachan, C. C. 1942. Factors affecb ing the quality of dehydrated vegetables. Proc. Inat. Food Technol. pp. 90-98. Dawson, C. R. 1945. Personal communication, Columbia UniverSity. Denny, F. E. 1942. The use of methyl ester of alpha-naphthaleneacetic acid for inhibiting sprouting of potato tubers, and an estimate of the amount of chemical retained by tubers. Contrib. Boyce Thompson Znat. 12, 387-403. of the methyl ester of alpha-naphthaleneDenny, F. E. 1945. Further tests of the acetic acid for inhibiting the sprouting of potato tubers. Contrib. Boyce Thompson Zwt. 14, 1620. Denny, F. E., Guthrie, J. D., and Thornton, N. C. 1942. Effect of the vapor of the methyl ester of alpha-naphthaleneacetic acid on the sprouting and the sugar content of potato tubers. Contrib. Boyce Thompson Znet. 19, 253-268. Denny, F. E., and Thornton, N. C. 1940. Factors for color in the production of potato chips. Co:ontrz%.Boyce Thmp8on Inat. 11, 291-303. Denny, F. E., and Thornton, N. C. 1941a. Carbon dioxide preventa the rapid increase in the reducing sugar content of potato tubers stored at low temperatures. Contra%. Boyce Thompson Zmt. 19, 79-84. Denny, F. E.,and Thornton, N. C. 1941b. Potato varieties: sugar-forming charac-
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teristics of tubers in cold storage, and suitability for production of potato chips. Contrib. Boyce Thompson Inst. 12, 217-252. Denny, F. E., and Thornton, N. C. 1942a. The third year’s results on storage of potato tubers in relation to sugar content and color of potato chips. Contrib. Buyce Thompson Inst. 12, 405430. Denny, F. E., and Thornton, N. C. 1942b. Interrelationship of storage temperature, concentration, and time in the effect of carbon dioxide upon the sugar content of potato tubers. Contrib. Boyce Thompson Inst. 12, 361-374. Denny, F. E., and Thornton, N. C. 1943a. Effect of post-harvest pre-storage conditions on the rate of development of sugar in potato tubers during subsequent cold storage. Contrib. Boyce Thompson Inst. 13, 65-72. Denny, F. E., and Thornton, N. C. 1943b. The effect of low concentrations of carbon dioxide upon the sugar content of potato tubers in cold storage. Contrib. Boyce Thompson Inst. 13, 73-78. Doty, D. M., Bergdoll, M. S., Greene, L., Lewis, W. R., and Ellis, N. K. 1946. Personal communication, Purdue University. Finch, N., and Hartzell, A. 1945. Effects on mice of a diet containing methyl ester of alpha-naphthaleneacetic acid. Cantrib. Boyce Thompson Inst. 14, 69078. Friar, H. F. 1943. A problem in dehydration of new potatoes. Fruit Products J. 22,339. Friar, H. F., and Van Holten, P. 1945. Effect of sulfiting on maximum drying temperature of vegetables. Fruit Products J . 24, 337-339. Gore, H. C., and Rutledge, L. F. 1921. Control of the darkening of dehydrated potato. Chem. Age 2Q, 457-458. Green, E. L., Culpepper, C. W., Caldwell, J. S., and Hutchins, M. C. 1946. The use of SO, in dehydration of eastern potatoes and other vegetables. Fruit Products J. 26, 15-20, 26, 39-44, 81-86. Guthrie, J. D. 1939. Inhibition of the growth of buds of potato tubers with the vapor of the methyl ester of naphthaleneacetic acid. Contrib. Boyce Thompson Inst. 10, 325-328. Howard, L. B. 1945a. Personal communication, U. S. Dept. Agr. Howard, L. B. 194513. Factors of processing and storage that affect quality. The Canner 100 (13), 46, 48, 50. Howard, L. B. 1945c. Desiccants improve dry packs. Food Packer 26 (3) 31. Legault, R. R., Talburt, W. F., Mylne, A. M., and Bryan, L. A. 1946. The browning of dehydrated vegetables during storage. Abstr. Papers 110th Meeting Am. Chem. SOC.pp. 7a-8a. Lovern, J. A. 1945. Personal communication, British Food Mission. Mackinney, G. 1945a. Personal communication (University of California). Mackinney, G. 1945b. The sulfiting of vegetables. Fruit Products J. 24, 300-301. Mackinney, G., and Howard, L. B. 1944. Sulfite retards deterioration of dehydrated cabbage shreds. Food I d s . 16, 355-356. Magoon, C. A., and associates, 1946. Peacetime food compression. Food I d . 18, 362-364 Makower, B., Chastain, S. M., and Nielsen, E. 1946. Moisture determination on dehydrated vegetables; vacuum oven method. Ind. Eng. Chem. 38, 725-731. Mangels, C. E., and Gore, H. C. 1921. Effect of heat on different dehydrated vegetables. Ind. Eng. Chem. 13, 525-526. Morgan, A. F. 1935, Nutritive value of dried fruits. Am. J . Public Health 26, 328. Muller-Thurgau, H. 1882. Ueber Zuckeranhaufungin Pflanzentheilenin Folge niederer Temperaturen. Landzu. Jahrb. Bd. 11, 751-828.
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Nichols, P. F., and Grow, C. R. 1921. Methods of preparing vegetables for dehydration. Chem. Age 29, 139-141. Nichols, P. F.,Powers, R., Gross, C. R., and Noel, W. A. 1925. Commercial dehydration of fruits and vegetables. U. S. Dept.' Agr. Dept. Bull. 1336. Patton, A. R., and Pyke, W. E. 1946. The role of amino acids and glucose in the browning of potato chips and dehydrated potatoes. Abstr. 110th Meeting Am. Chem. SOC.pp. 9a-10a. Peacock, W. M., and Brunstetter, B. C. 1931. A simple chemical test for predetermining the culinary quality of potatoes aa affected by the accumulation of soluble sugars. U . S.Dept. Agr. Circ. 168. Proctor, B. E., and Sluder, J. C. 1943. The compression of dehydrated foods. Proc. Znst. Food Technol. pp. 132-142. Pyke, W. E.,Patton, A. R., Croye, E. B., Durham, H. A., and Allison, W. W. 1946. Personal communication, Colorado A. & M. College. Rendle, T. 1945. The preservation of potatoes for human consumption. Chemistry & Industry 1946, 354-359. Rhodes, W.E., and Davies, A. F. 1945. The selection and pre-processing of potatoes for canning with special reference to control of texture by calcium chloride. Chemistry & IndWtTY 23, 162-163. Rogers, H. B., Jr. 1945. Personal communication, QM Food and Container Inst. for the Armed Forces. Rogers, M. C., Rogers, C. F., and Child, A. M. 1937. The making of potato chips in relation to some chemical properties of potatoes. Am. Potato J . 14, 269-290. &om, A. F. 1947. Unpublished data, Univ. of Maine. Ross, A. F., Hilborn, M. T., and Jenness, L. C. 1945. Discoloration can be avoided. Food Packer 26 (lo),38, 40,42, 78. Ross, A. F., Hilborn, M. T., Jenness, L. C., and Bartlett, E. M. 1946. Selecting and storing potatoes to avoid darkening. Food Znds. 18, 1011-1013, 1144, 1146, 1148, 1150, 1152. Ruschmann, G. 1932. Daa Braunwerden der Kartoffeln beim Dampfen und ihre schlechte Sauxung (Kartoffelsilage). Arch. Tierernahr. Tierzucht 8, 1-30. Rushton, E., Stanley, E. C., and Scott, A. W. 1945. Compressed dehydrated vegetable blocks. Chemistry and Industry 23, 274-276. Sherman, V. W. 1944. Electronics in food industry. Proc. Inst.. Food Technol. pp. 87-101. Stamberg, 0.E., and McKinnon, M. I. 1946. Personal communication, Univ. of Idaho. Sweetman, M. D. 1930. Color of potato chips as influenced by storage temperature of the tubers and other factors. J . Agr. Research 41, 479-490. Sweetman, M. D. 1931. The relation of storage temperature of potatoes and their culinary quality. Am. Potato J . 8, 174-176. Tomkins, R. G., Mapson, L. W., Allen, R. J. L., Wager, H. G., and Barker, J. 1944. The drying of vegetables, 111. The storage of dried vegetables. J . SOC.Chem. Znd. 68, 225-231. Tressler, C. J., Jr. 1944. The use of sulfite solutions in potato dehydration. The Canner 99 (26),12-13. U. S. Dept. Agr. 1944. Vegetable and fruit dehydration. A manual for plant operators. U . S . Dept. Agr. Misc. Publ. 640. Wager, H. G., Tomkins, R. G., Brightwell, S. T., Allen, R. J. L., and Mapson, L. W. 1945. Drying of potatoes, I, 11, 111. Food Manuf. 20, 289-293, 321-325, 367371, 375.
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Western Regional Research Laboratory, 1945. Effects from sulfiting; studies m de on tray materials used for vegetable dehydration. Food Packer 26, 50, 52, 54. Wiegand, E. H.,Litwiller, E. M., and Hatch, M. B. 1946. Personal communication, Oregon State College. Wodicka, V. 0. 1945. Personal communication, QMC. Wright, R. C. 1932. Some physiological studies of potatoes in storage. J. Agr. Research 46, 543-555. Wright, R. C., Caldwell, J. S., Whiteman, T. M., and Culpepper, C. W. 1945. The effect of previous storage temperatures on the quality of dehydrated potatoes. Am. Potato J. 22, 311-323. Wright, R. C., Peacock, W. M., Whiteman, T. M., and Whiteman,E. F. 1936. The cooking quality, palatability and carbohydrate composition of potatoes as influenced by storage temperaturas. U. S . Dept. Agr. Tech. Bull. SOT.
The Influence of Climate and Fertilizer Practices upon the Vitamin and Mineral Content of Vegetables BY G. FRED SOMERS AND KENNETH C. BEESON
U.S. Plant, SoiE and Nutrition Laboratory, Itham, N. Y. CONTENT0
. . . . . . . . . . . 1. Analytical Methods . . . . . . . 2. Sampling Methods . . . . . . . 11. Influence of Climate on Vitamin Content . . 1. Ascorbic Acid . . . . . . . . . a. General Effects of Climate . . . . b. Influenceof Light . . . . . . c. Influence of Temperature . . . . d. Iduence of Rainfall . . . . . 2. Carotene . . . . . . . . . . 3. Other Vitamins . . . . . . . . 111. Influence of Fertilizers on Vitamin Content . . IV. Influence of Fertilizers on Mineral Content . . V. Discussion . . . . . . . . . . . VI. Summary. . . . . . . . . . . . 1; Introduction
References
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291 292 294 295 295 295 299 .303 .304 306 306 307 310 315 318 318
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I. INTRODUCTION Any detailed compilation of the nutritive values of foods reveals the wide ranges that have been reported. A main purpose of many of the investigations of factors affecting nutritive value has been to determine the reasons for these variations. It is clear that faulty techniques are responsible for some differences, but in general, it is recognized that environmental and soil factors contribute a profound influence on the composition of plants. It is a consideration of such factors that is the subject of this review. In the preparation of this review we have restricted our consideration primarily to vegetables. We have felt free, however, to include data obtained with other plants when these would aid in clarifying or amplifying the results obtained in the more limited field. In discussing the mineral contents of plants we have limited the discussion to those minerals of particular importance in human nutrition. It is recogniied, of course, that climate and fertilizer practices are not the only forces acting to determine the vitamin and mineral content of vegetables. There are other factors which may be of equal or greater importance. Some of these are processing, handling, and genetics. Thus. the vitamin or mineral content of canned vegetables at the time they are consumed is determined by a large 291
292
G. FRED SOMERS AND KENNETH C. BEESON
number of factors. In the first place, the vitamin content of the vegetables as they are produced a t the farm is important, and this is determined by such fmtors as variety, climate, fertilizer practices, etc. What proportion of the vitamins initially present in the fresh vegetables reaches the consumer depends upon such factors as processing, handling, cooking, etc. In this review, then, we are dealing with only a limited portion of the over-all picture. Most of the material presented in this review represents the work published during the past few years; since the subject has been reviewed previously (Giroud, 1938; Hamner and Maynard, 1942; Maynard and Beeson, 1943-44; Hamner, 1945; Beeson, 1946). The amount of material to be considered is restricted further by the nature of the work that has been done. It would be extremely interesting, for example, to be able to give an account of the influence of climate upon all of the well-known vitamins found in plants. This is not possible because only a few vitamins have been studied in this regard. For example, a great deal of research of all degrees of quality has been done concerning the ascorbic acid content of foods; many aspects of that problem have been investigated. Fewer investigations have been made of the influence of climatological variables upon the carotene and thiamine content of plants, and the other vitamins have received virtually no attention in this respect. This probably is largely the result of relative facility of experimentation, e.g., ascorbic acid is comparatively easy to determine quantitatively, and hence it has been widely studied. Nevertheless, it seems obvious that more emphasis should be placed upon studying the influence of various factors upon vitamin content of plants with respect to vitamins of the B-complex and carotene. While it is not the purpose of this review to deal at length with methods, it seems worthwhile to point out some of the difficulties that are involved, particularly in reference to analytical and sampling methods, since they are essential to a critical evaluation of the published data. These will be illustrated by using ascorbic acid studies as an example. Similar crit,icisms apply to other studies, but they will not be discussed in detail in this review. 1. Analytical Methods First of all, it is essential that a satisfactory analytical method be used. Some of the essential points to be checked in an analytical method are: (1) If a chemical method is used, it should be compared with bioassay methods. (2) Sources of variability in the results obtained with duplicate aliquots should be located and placed under control. (3) Precautions must be taken to avoid destruction of unstable constituents for which an
INFLUENCES ON V I T ~ X I N AND MINERAL
CONTENT OF VEGETABLES
293
analysis is desired. (4) Unnecessary complications should be avoided siace they only waste time and materials. In general, the evidence (King, 1941; Harris and Olliver, 1942; Hartzler, 1945) indicates that the titration of ascorbic acid by means of 2,6-dichlorophenolindophenol gives results which agree with bioassays for fresh vegetables and fruits. Recent experience has shown that, under suitable conditions, various photometric methods likewise give valid results (Bessey, 1938; King, 1941; Loeffler and Ponting, 1942; Hochberg et al., 1943; Nelson and Somers, 1945; Robinson and Stotz, 1945). These photometric methods have the advantage of being more objective than the titration methods. Both the titrimetric and photometric methods considerably facilitate experimentation, but bioassays are essential to an evaluation of the chemical methods. There are some pitfalls in the chemical methods which should be pointed out. Some of these have been recognized for a long time, but still they occasionally cause trouble. For example, ascorbic acid appears to be largely in the reduced form in most fresh vegetables (see Lampitt et al., 1943 and 1945; Giral and Torre, 1947; and McMillan and Todhunter, 1946, for some recent values). This and the fact that dehydroascorbic acid is more difficult to determine has led a number of investigators to analyze only for the reduced form of this vitamin. Since ascorbic acid is very readily oxidized as soon as the tissue is damaged by mincing or grinding, it is difficult to evaluate the analytical data obtained, unless suitable precautions are taken to avoid oxidation during the preparation and manipulation of extracts for analysis. Oxidation not only invalidates the results obtained when analysis is made solely for the reduced form, but also gives erroneous values for the relative amount of dehydroascorbic acid when this is included in the analysis. Some workers consider that the use of an inert atmosphere is necessary (Hochberg et al., 1943), but if sufficiently concentrated metaphosphoric acid is used during grinding, similar results are obtained in air and in an inert at.mosphere (Vavich et al., 1945). Other workers have used metaphosphoric acid in conjunction with a relatively strong acid such as sulfuric acid because they thought that a strong acid was necessary to inactivate the oxidizing ensymes (Mack and Tressler, 1937). The use of a strong acid does not appear to be essential. In fact, some workers have found such a practice to be undesirable for one reason or another (Bessey, 1938; King, 1941; Nelson and Somers, 1945). It should be pointed out that metaphosphoric scid is a good protein precipitant; hence a strong acid generally is not necessary to inactivate the enzymes. If a sufficiently large amount of metaphosphoric acid is used in proportion to the amount of material being extracted, it will satisfactorily protect ascorbic acid in plant extracts (Loeffler and Ponting, 1942; Nelson and Somers, 1945; Vavich et al., 1945).
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FRED 80MERS AND KENNETH C. BEESON
The presence of a strong acid catalyzes the hydrolysis of metaphosphoric acid and thus reduces its efficiency as a protectant for ascorbic acid (Nelson and Somers, 1945). It is important, however, to observe two precautions in using metaphosphoric acid: (1) The acid used for extracting should be sufficiently concentrated. Metaphosphoric acid solutions of less than 301, frequently yield low results (Reder, Speirs et al., 1943l; Vavich et al., 1945). (2) Too much tissue should not be used at one time. Our experience indicates that, using the Waring blendor, up to about 25 g. of some fresh tissues per 100 ml. of 3% metaphosphoric acid is satisfactory. We have found that the amount of material which can be used safely vanes from plant to plant, and each time a new type of plant material is to be studied, the stability of the extracts should always be tested. These comments are not intended to represent a complete review of ascorbic acid determination methods; they simply illustrate some difficulties which are encountered. A consideration of such difficulties is essential in evaluating the results which have been published. There are other factors which should be considered, such as magnitude of interfering substances, the normal amount of dehydroascorbic acid, etc. All plant material with which a worker experiments should be tested with these in mind. 8. Sampling Methods
Another extremely important point in connection with all studies of this kind is that of sampling techniques. It would seem that this is self-evident, yet data of doubtful value are frequently encountered because of faulty sampling techniques. The problem of sampling is different for nearly every plant, and hence detailed criticisms cannot be made here. An illustration of the type of problem encountered, and not always considered, can be given using studies dealing with the ascorbic acid content of potato tubers. In this case, at least the following must be considered in selecting samples for analysis: variety, replication in the field, locality a t which grown, fertilizer treatments, time of harvest, whether the vines are dead or alive at harvest time, and temperature and length of storage. Some of these variables are more important than others. One of the most important case is storage (Karikka et al., 1944;Murphy et al., 1945);yet even this is not always controlled. It has been reported, for example, that potatoes grown in Maine show seasonal differences in ascorbic acid content (Murphy et a?., 1945). In this case the same varieties were analyzed in each of three years, but in each case the potatoes had been stored for different lengths of time and the temperature of storage WBS not controlled. It seems possible that changes during storage very largely obscured whatever 1 See
footnote on page 19 of their paper.
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AND MINERAL CONTENT OF VEGETABLES
295
effects may have been produced by the different seasons of growth. In any case it is not possible from the published data to separate the effects of season and storage. This serves as one example of the type of difficulty which can be encountered. It is not always possible to control such variables experimentally, but if such is not feasible, it is important to place such factors under st.atistica1control.
11. INFLUENCE OF CLIMATE ON VITAMINCONTENT 1 . Ascorbic Acid a. General E$eck of Climate. In considering the influence of climate upon the composition of plants a number of variables must be taken into account. Some of them act indirectly, e.g., over long periods of time climate influences the soil type and produces concomitant changes in the amount and kind of minerals supplied to the plant. Also, during a single season, climate may influence the amount and kind of minerals supplied to the plant without changing appreciably the soil type. These changes in mineral supply may in turn influence the composition of the plant, Such indirect effects as these will not be considered in this section; instead, the emphasis will be placed upon factors that act more directly upon the plants. The factors, sunshine, temperature, and rainfall, will be considered primarily. In field experiments, however, it is usually not possible to separate the effects of these three variables. All of them, along with other factors, are included under the effects produced by season, location of experimental plot, and date of harvest. Table I gives some selected recent data showing effects of season and location upon the ascorbic acid content of a few vegetables. In the case of snap beans, Heinfie et al. (1944)found that pods from the spring harvest of both the pole and bush varieties were significantly higher in ascorbic acid content than those of the fall harvest. Wade and Kanap a w (1943)report similar differences (about 15y0)between fall and spring crops of snap beans. In this case, however, it is pointed out that the low mean value for the fall harvest results largely from the fact that one picking in the fall was very low in ascorbic acid content. If this picking is disregarded, the results are the same for fall and spring. Janes (1944),who grew various varieties of snap beans and cabbage in different locations in Florida, reported that the location influenced the ascorbic acid content of the crop. In this case the beans were harvested a t different times, depending upon the location, and therefore, time of harvest is confounded with location. Harvesting dates ranged from January to May. Hansen (1945a) also observed seasonal differences in the ascorbic acid content of vegetables. His results, however, are not convincing since the experi-
296
a.
FRED SOMERS AND KENNETH C. BEESON
ment was not designed in such a way that the validity of the results could be tested statistically. Poole et al. (1944), in a well-designed experiment, observed seasonal differences in the ascorbic acid content of some cabbage lines, but not others. TABLEI Selected Data on the Ascorbic Acid Cmtenta of Some Crops Grown at Various Locations
Location
Season
1
Ascorbic acid content" (mg./lWg.)
Crop
Variety
Fall Fall Fall Fall Fall
Potatoes Potatoes Potatoes Potatoes Potatoes
Earlaine Irish Cobbler Chippewa Katahdin Green Mtn.
45.3 40.3 29.6 28.1 19.9
Genesee County, N. Y.
Fall Fall Fall Fall Fall
Potatoes Potatoes Potatoes Potatoes Potatoes
Katahdin Earlaine Irish Cobbler Chippewa Green Mtn.
36.6 33.4 30.3 22.3 22.1
Knoxville, Tenn. Holgate, Ohio Wenatchee, Wash
Summer Summer Summer
Tomatoes Marglobe Tomatoee Marglobe Tomatoes Marglobe
Experiment, Ga.
Fall
Stillwater, Okla.
Fall
Norfolk, Va.
Fall
Turnip greens Turnip greens Turnip greens Turnip greens Turnip greens
Tompkins County, N. Y.
Experiment, Ga.
Spring
Stillwater, Okla.
Spring
-
14.4 22.3 30.6
Seven Top
100.0
Seven Top
122.0
Seven Top
393.0
Seven Top
144.0
Seven Top
151.0
Reference
Karikka et al., 1944
Harmer et al., 1945
Reder, Speirs et at., 1943
Expressed as mg./100 g. fresh weight.
They grew 25 breeding lines both in the fall and in the spring. Of these, only 6 lines differed significantly in ascorbic acid content from seaaon to season. In only one line was the ascorbic acid content higher in the fall than in the spring. They conclude that such seasonal effects are of less importance than hereditary factors. Smith and Walker (1946) likewise
INFLUENCES ON VITAMIN AND MINERAL CONTENT OF VEGETABLES
297
found seasonal differences in the ascorbic acid content of cabbage. In a series of transplantings and harvests there was a tendency for heads harvested in August to contain more ascorbic acid than those harvested in either July or September. They used statistical methods in an attempt to evaluate different components of the seasonal effect, but were not able to find a simple relationship between any one factor and the ascorbic acid content . Karikka et al. (1944) reported that potatoes grown in various locations in New York State differed in their ascorbic acid content. On the other hand, Murphy et al. (1945) failed to find such a difference between potatoes grown on two soils in Maine. In such a comparison as this it is essential that storage be considered. Murphy et al. took storage into account in this case since they state that. all of the tubers used in this study of the effect of location were harvested and analyzed a t approximately the same time; however, they apparently paid no attention to the date at which the tubers reached maturity. This may be important, since after the tubers are mature and the vines dead, the tubers are essentially in storage even though they are still in the ground. Both storage and maturity were considered in the comparisons which Karikka et al. made for the year 1939 between potatoes grown in Lordstown stony silt loam in Tompkins County, New York, and in the muck soil of Genesee County. They state that the varieties were harvested soon after they matured, and were analyzed within a few days after harvest. Under such circumstances, comparisons between locations would probably be valid. Some of their data are shown in Table I. They found that the ascorbic acid content of tubers of the variety Katahdin grown in the organic soil was about 30% greater than that grown in the inorganic soil. On the other hand, the ascorbic acid content of the tubers from the inorganic soil was about 30% more than that from the muck in the case of the varieties Irish Cobbler, Chippewa, and Earlaine, and about 60% more in the case of the variety Houma. For the varieties Green Mountain and Sebago, the differences between locations were about loyo,or less. Presumably the soils used by Murphy et al. were both inorganic. This may be a factor in explaining the difference between the results in Maine and New York, but the data of Lampitt et al. (1945) indicate that such a difference in soil type produces no differences in the ascorbic acid content of potato tubers. In all of these studies, soil type is only one of the many variables associated with a difference in location. Earlier studies similarly are not in agreement as to the effect of locat,ion upon the ascorbic acid content of potato tubers (see literature reviews by Karikka et al., 1944, and Murphy et al., 1945). Hamner et al. (1945) obtained tomato fruits of several varieties grown in different parts of the United States. Some of their data are shown in
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Table I. They found, as had been indicated earlier, that tomatoes from different localities differed widely in ascorbic acid content. Lo COCO (1945) has reported similar results for tomatoes grown in Northern California. Another crop which has been studied extensively in recent years with respect to its ascorbic acid content is turnip greens. Reder, Ascham and Eheart (1943) grew turnips in well-replicated fertilizer experiments a t four locations in the southern United States. Wide variations were obtained in the ascorbic acid content of the greens produced a t the four places (see Table 11). The influence of location was about 14 times aa TAB^ I1
Selected Data on Aambic Acid Content of Turnip Greens (Seven Top) Grown in 16 Duplicate Fertilim- Tredmente at 6 Locationsa Treatment
I
Ascorbic acid content' Norfolk,
None N K Average a
243 249 230 240
Blacksburg, Va. 128 128 129 128
Stillwater,
Okla. 163
157 162 161
Experiment, Ga. 193 200 216 203
Average 178 179 173
-
Reder, Ascham and Eheart (1943). Mg./100 g. fresh weight.
great as the most important average effect produced by fertilizer treatment. These variations did not appear to be directly related to differences in soil composition or to differences in temperature. The results indicated that the formation of ascorbic acid may have been influenced by the amount of sunlight or the amount of rainfall, since a higher ascorbic acid content was obtained at locations which had more sunshine and less rainfall. It is not possible in this case to separate the effects of sunlight and rainfall since there is, in general, a reciprocal relationship between the two. However, in view of the data obtained by other workers (see InJluence of Light, p. 299), it may be that the amount of sunlight was the factor of primary importance. A later experiment which was even more extensive in scope was conducted also in the South by Reder, Speirs et al. (1943). Some of their data are given in Table I. This experiment was very well designed so that the effects of the various variables could be evaluated statistically. Seasonal and location effects were observed. This experiment will be discussed further elflewhere (see InJluence of Rainjull, p. 304). Bernstein, Hamner and Parks (1945) studied the influence of mineral
INFLUENCES ON VITAMIN AND MINERAL CONTENT OF VEGETABLES
299
nutrition, soil fertility, and climate on the ascorbic acid content of turnip greens. They made a careful study of variables involved in sampling and conducted two complete experiments, one in the early summer and one in the fall. These workers concluded that the date of harvest ( i e . , season) and location were the factors primarily responsible for determining the ascorbic acid content of turnip greens. In the experiment conducted in the early summer, the average ascorbic acid content was much greater than in the fall experiments. There were also large differences between the averages for the ascorbic acid content of turnip greens produced at different locations, even though all of the locations were within a few miles of each other. Kostenko (1943) has published the ascorbic acid content of a number of vegetables and other plants grown at high altitudes in Pamir. No details, however, are given concerning analytical method, sampling technique, the part of the plant analyzed or whether the data are expressed on a fresh or dry weight basis. b. Injluence of Light. Previous reviews have indicated that light is a factor of primary importance in determining the vitamin C content of some crops (Hamner and Maynard, 1942; Maynard and Beeson, 194344). Light apparently is much more important in this respect than the mineral nutrition of the plant. In general, recent work substantiates this view. However, Platenius (1945) found that a single day of cloudy, rainy weather did not decrease the ascorbic acid content of spinach. This is a rather short interval. On the other hand Koizumi and Kakukawa (1940) found that the ascorbic acid content of the leaves of bean plants placed in total darkness decreased about 20% in 24 hours. When the plants were placed again in the light (Mazda) the ascorbic acid increased. Wberg (1945) similarly found significant (about 25%) decreases in the ascorbic acid content of tomato leaflets within 24 hours after the plants were placed in the darkness. This decrease in the dark was influenced by the age of the leaf and other factors. Lantz (1945) obtained some evidence of a diurnal variation (about 20%) in the ascorbic acid content of peppers, but his results are presented on a fresh weight basis which is rather unsatisfactory for comparisons of this kind (cf. Platenius, 1945). In a study of diurnal variation, comparisons based either on unit fresh weight or unit dry weight are unsatisfactory because b0t.h of these units are themselves subject to diurnal variation. One must select a basis for reference which is as stable as possible. This is not always easy to do, but plant physiologists have used such measures as per unit plant, unit of area, per leaf, half-leaf, or leaflet, etc., for studies of this kind. A number of other experiments have indicated an association between the ascorbic acid content of plants and the amount of sunshine. Platenius
300
0. FRED SOMERS AND KENNETH C. BEEBON
(1945) found that the ascorbic acid content of kale and the amount of solar radiation and the average daily temperature decreased during the growing season. The relative decrease in the latter two factors was much greater than the change in the vitamin content. Pepkowitz (1944) found that when peas were planted close together the plants contained less ascorbic acid than when they were planted farther apart. The effect of sunlight upon the ascorbic acid content of tomato fruits has received considerable attention recently. Wokes and Organ (1943) found that tomat.0 fruits ripened (off the vine) in sunshine at “room” temperature contained on an average nearly 50% more vitamin C than similar TABLEI11
Ascorbic Acid Content of Tomato Fruits from Plank Transferred from Shade to Sunlight and yice versa at Various Stages of Fruit Developmenta
Treatment of Plants Grown continuously in sunshine Grown continuously in shade Transferred from sun to shade: At time of first blossom When first fruits were “mature green” Transferred from shade to sun: At time of first blossom When first fruits were’“mature green” a
Ascorbic acid content
No. of fruits analyzed
25.8 f 0.63 15.5 f 0.43
56 48
17.0 0.88 16.8 f 0.72
17 23
26.1 f 0.83 23.4 f 1.08
27 17
Hamner el al. (1945). Mg./100 g. fresh weight.
fruit ripened at “room” temperature in the dark, and twice as much as similar fruit ripened a t 37°C. (98.6’F.) in the dark. Kaski el al. (1944) collected tomato fruits from the field at various times. They obtained simultaneous data on the “per cent possible sunshine” and rainfall. Their data indicate that a low ascorbic acid content is associated with cloudy or rainy days. More convincing evidence of the influence of light was obtained by McCollum (1944) and by Hamner, Bernstein and Maynard (1945). The latter grew tomatoes in sand culture with controlled temperature, humidity, light intensity, and length of photoperiod. In one experiment the design was such that all of these factors except light intensity were varied. I n another experiment plants were grown in sand culture outdoors, some in full sunshine and some under a cloth shade which reduced the light intensity by 75% during bright days. Some of these data are given in Table 111. When plants were shifted from shade to sun,
INFLUENCES ON VITAMIN
AND MINERAL CONTENT OF VEGETABLES
301
and vice versa, the fruits which ripened under the new conditions had an ascorbic acid content characteristic of the conditions under which they ripened. The conclusion drawn by Hamner et al. from these experiments was that the amount of light was the principal factor in determining the ascorbic acid content of tomato fruits. McCollum (1944), using field-grown tomato plants, found that fruits which ripened while exposed to the sun on normal plants, or were exposed as a result of defoliation of the plants, were higher in ascorbic acid than those which ripened in the shade of the leaves. This indicates that the direct illumination of tomato fruits is of importance in determining their ascorbic acid content. This conclusion has been substantiated by unpublished experiments in our laboratory (Somers, Kelly and Hamner). In this case tomatoes were grown in sand culture in the sun and in the shade. Some fruits from both shade- and sun-grown plants were ripened in the sun, and others were ripened inside cloth bags. Some fruits were ripened on plants exposed to full sunshine and others on plants with all of the leaves shaded. In all cases the ascorbic acid content of the fruits was determined by the degree of illumination of the fruits rather than by the amount of illumination of the foliage. Field experiments by Somers, Hamner and Nelson (1945), in which a continuous record was made of the amount of sunshine prior to harvest, show that under some conditions there is a correlation between the amount of sunshine and the ascorbic acid content of tomato fruits. I n this case three fields in New York State were used, the greatest distance between fields being approximately thirty miles. The fruits in all cases were relatively exposed. A similar relationship might not be expected in cases where the fruits were relatively shaded, since the amount of direct illumination of the fruits determines their ascorbic acid content. In such a case the amount of sunshine on the field as a whole would be expected to bear little or no relationship to the ascorbic acid content of the tomato fruits produced. Unpublished experiments conducted in several states simultaneously (Somers, Kelly and Hamner) support this view. Other experiments indicate that the ascorbic acid content of other plants is influenced by light. Hansen and Waldo (1944) reported that strawberries and raspberries which ripened in the shade contained much leas ascorbic acid than those which ripened in the sun. In this case it seems desirable to add a word of caution concerning the analytical method used. The extracts for ascorbic acid determination were buffered at pH 3.6 and made up to convenient volume before being analyzed. Ascorbic acid is less stable at this pH than in the initial extract. In fact, in some cases appreciable losses of ascorbic acid can occur in a short time (Nelson and Somers, 1945).
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Q. FRED SOMERS AND KENNETH C. BEESON
Smith et al. (1943) found that growing cantaloupes under a cloth shade resulted in a lower ascorbic acid content of the edible portion of the fruits. Finch et aE. (1945) found similar results with cantaloupes and with both leaves and tubers of potatoes. Hiberg (1945) has studied the influence of light upon the ascorbic acid content of tomato leaves. In this case the ascorbic acid content was determined by the amount of illumination. The magnitude of the response was influenced by the age of the leaves. It was found that ascorbic acid formation did not require the blue end of the spectrum which confirms the earlier results of Sugawara (1939).
0 m
a 0 0
1 100 Fig. 1. Ascorbic acid values (per unit fresh weight) of leaves from three large turnip planta which were grown in the greenhouse for about 4 months and then shifted (at 0 days) to control rooms illuminated at various light intensities. Plants A and C were both placed at about 800 foot candles (fluorescent lights). Plant B was placed at about 6000 foot candles (Masda lights). After 1 week plants A and B were interchanged. (Hamner & Parks, 1944.)
Hamner and Parks (1944) showed that the ascorbic acid content of turnip greens could be varied by about 800% over a period of a week with light (Mazda) intensities ranging from 200-5000 foot candles. The changes produced by changes in light intensity were found to be reversible (see Fig. 1). These observations have been extended by Somers, Kelly and Hamner (1948) who used discs cut from turnip leaves. By this means it is possible to study more easily the influence of various factors upon ascorbic acid accumulation. So far the results with discs have confirmed studies with intact plants with respect to the essentiality of light for ascorbic acid accumulation. It has been found, in addition, that carbon dioxide is required for ascorbic acid accumulation in these discs. When all this evidence is considered together, it appears conclusive that
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303
light is an important factor in determining the ascorbic acid content of vegetables. Similar conclusions have been reached earlier (see Hamner and Maynard, 1942; Reid, 1942; and Maynard and Beeson, 194344). It must be pointed out, however, that ascorbic acid may be formed in the absence of light. This is clearly established by the fact that ascorbic acid is formed in germinating seeds in the dark. This fact has long been known (Heller, 1928; see Giroud, 1938 for other early references) and has been repeatedly confirmed (Johnson et al., 1945; Moldtmann, 1939; Reid, 1941a and 1941b; Shen et al., 1945; Sugawara, 1939; Weissenbock and Weissenback, 1940). In addition it should be mentioned that Hamner and Nightingale (1946) found that pineapple juice from plants which were shaded for about 6 weeks prior to harvest contained about 20% more ascorbic acid than juice from unshaded plants. c. Influence of Temperature. There have been comparatively few investigations in which the influence of temperature upon the ascorbic acid content of plants has been studied. Temperature effects are difficult to sort out from other variables associated with locational differences. Reder et al. (1943), in their field experiments, observed no direct relationship between temperature and the ascorbic acid content of turnip greens. Moldtmann (1939) obtained variable results. In some species of plants the ascorbic acid content was greater a t higher temperatures, in some cases it was lower, and in still other cases temperature had little influence. Hamner et al. (1945) grew tomatoes in sand culture under controlled conditions. Some plants were grown continuously a t 17°C. (63°F.) and others at 25°C. (78°F.). The lower temperature is just about as low a temperature as tomato plants will stand continuously and still produce a crop, whereas the higher temperature is approaching the upper temperature limit. The fruits from the low temperature plants contained significantly less ascorbic acid than those from the high temperature plants. The difference was about 16% of the higher value, which is not a very great difference in comparison with differences which can be produced by varying the amount of light. Aberg (1945) studied the influence of different temperatures upon the ascorbic acid content of tomato leaves. One lot of 6-week old plants was subjected to 155°C. (60°F.) for 2 weeks, and another lot to 25OC. (73°F.) for a similar period. The leaves from the plants at the lower temperature contained about 30% more ascorbic acid than those from plants at the higher temperature. Hamner and Nightingale (1946) similarly observed an inverse correlation (coefficient = -0.767) between the air temperature at which pineapple plants were grown and the ascorbic acid content of processed juice from these same plants. In this experiment, an even more striking correlation was obtained between the total acidity of the pineapple juice and its ascorbic acid content (correlation coefficient
304
G . FRED SOMERS AND KENNETH C. BEEEON
= 0.956). (Ascorbic acid accounts for only about 1% of the total acidity of pineapple juice.) d. Influence of Rainfall. This factor is likewise difficult to separate from other variables associated with location. Some observations have been reported. Thiessen (1936) found that potatoes grown on dry land in Wyoming rated about 15y0 higher in vitamin C potency (by the bioassay method) than potatoes grown on irrigated land. These assays necessarily lasted over a period of several weeks following harvest, during which time considerable changes in the ascorbic acid content occurred as a result of storage. The data of Reder, Ascham and Eheart (1943) suggest a possible connection between rainfall and the ascorbic acid content of turnip greens. In summarizing these data they state, “The highest ascorbic acid content of the three spring crops (1.9065 mg. per gm.) was found in greens which were produced a t the place having the lowest average daily rainfall and where 49 per cent of the days in the growing season was clear; the lowest ascorbic acid content (1.2842 mg. per gm.) was found in greens which received the greatest average daily rainfall and the least amount of sunshine.” As these workers realize, it is not possible to separate the effects of sunshine and rainfall in this case. Nevertheless these results do not indicate a positive correlation between the amount of rainfall and the ascorbic acid content of turnip greens as was reported for later results obtained by Reder, Speirs et al. (1943). An inspection of the data published for this latter experiment raises some questions. The value for the ascorbic acid content of the fall crop a t State College, Mississippi, seems rather low. As these workers themselves suggest in a footnote on page 19 of their paper, there is some indication that a t least some of the low results may have been caused by using 1% mataphosphoric acid instead of 3% in the analyses. To what extent would the correlation between ascorbic acid content and rainfall still hold if values obtained using the dilute extractant were omitted in the statistical analysis? By a comparison of their tables 2 and 9, it is seen that, in the case of the spring harvest (where 3% metaphosphoric acid was used in all of the analyses), the highest ascorbic acid values were obtained a t the locality with the least rainfall. Hunter and coworkers (unpublished data) in our laboratory have conducted some preliminary studies in which turnip greens were grown in the greenhouse with three levels of moisture supply. The moisture content of the soil was permitted to fluctuate between saturation and the desired tension. The maximum tensions in the three treatments were 0.25, 0.85, and 15 atmospheres. The plants grown in the driest soil, i.e., which was allowed to reach the highest tension, contained about 15 to 3Oy0 more ascorbic acid, on a fresh weight basis, than those grown in the two wetter soils. These results indicate that when turnip greens are grown in dry
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305
soils they contain more ascorbic acid per unit fresh weight than when grown in soils in which the water supply is more nearly optimal. It would be interesting to repeat this investigation under field conditions. The studies in the greenhouse indicate that the greater ascorbic acid content of the plants from the driest soil may not be of practical significance since the yield of plant material is markedly reduced. It may be that the greater ascorbic acid content of these plants results simply from their small size and relatively low moisture content. In summarizing the influence of climate upon the ascorbic acid content of vegetables, it may be said that light seems to be the only climatic variable which has been shown clearly to have a significant effect. The evidence concerning other climatic variables is either limited in amount or is controversial, or is both. Even in the case of light it is still not clear just how illumination produces an increase in ascorbic acid content and why the studies with pineapple should indicate an inverse relationship between ascorbic acid content and the amount of light. The fact that the amount of light can influence markedly the ascorbic acid content of plant materials is not one that can be used immediately in commercial production. In most cases it is not feasible to do anything about the amount of light under commercial growing conditions. Even in greenhouses where it might be possible to change the light intensity the cost would probably prove prohibitive. It does seem important, however, to recognize the importance of light as a factor in influencing the vitamin C content of plants and to take this fact into consideration in planning and interpreting experiments in which an attempt is being made to evaluate the effect of other variables. I n other words, whereas the fact of the influence of light may not be of commercial importance, it certainly is of fundamental scientific interest. 2. Carotene
Previous reviews (Hamner and Maynard, 1942; Maynard and Beeson, 194344) have indicated that there is little or no clear-cut evidence concerning the influence of specific elements of the climate on the carotene content of plants. There is some indication that either the quality or quantity of light, or both, has an effect, since greenhouse-ripened tomatoes contain less carotene than sun-ripened tomatoes (Smith, 1936; Ellis and Hamner, 1943). There is very little of a specific nature that can be added at the present time. Bernstein el al. (1945) grew turnip greens in sand cultures supplied with varying amounts of macronutrient elements to make a total of 87 treatments in soil-pot cultures, and in soil-field plots with 26 fertilizer treatments. Two complete experiments were conducted; one in late spring
306
0. FRED SOMERS AND KENNETH C. BEESON
and early summer, and the other in the early fall; in both experiments, three locations were used. The carotene content of the plants grown in the separate experiments varied greatly, indicating a marked influence of season. Those grown in the late spring and early summer contained more carotene than those grown in the fall. The location at which the plants were grown also influenced their carotene content. In this case the locations were all within a few miles of Ithaca, New York. Hathaway et al. (1945) found that the carotene content of most of the Nebraska grasses they tested declined during the growing season. Porter el al. (1946) studied the carotene content of corn plants during their growth and concluded: “Significant differences in carotene between stocks, both inbred and hybrid, exist, but are small relative to those associated with stage of development of the plant and with seasonal factors.” Hansen (1945b) reports that winter-grown carrots are much lower in carotene content than those matured during the summer and fall. This may be related at least in part to temperature, for Barnes (1936) found that the temperature at which carrots were grown influenced their carotene content. Bondi and Meyer (1946a) analyzed a number of plants grown in Palestine at various times of the year. These data are interesting since plants are cultivated all year round in Palestine, and the summer and winter climates are very different from each other. Rain falls only in the winter months, and at the place where this work was done the annual average rainfall is 577 mm. (22.7 inches). They found that the carotene content of wintergrown plants was higher, in some cases twice as high, as that of summergrown plants. Unpublished data obtained by Hunter and associates indicate that the moisture tension of the soil may influence the carotene content of turnip greens. (Pertinent details of the experimental method are given above in the discussion of ascorbic acid.) The carotene content of the plants from the driest soil was about 65% greater (on a fresh weight basis) than that of the plants from the two wetter soils. It appears from these results that one or more factors associated with climate influence the carotene content of plants, but as yet it is not possible to separate the effect of the individual factors.
3. Other Vitamins There are very few studies with other vitamins which deal with cliiatological variables. Whiteside and Jackson (1943) report that the environment influences the thiamine content of Canadian hard red spring wheat. Hamner and coworkers (unpublished data) have analyzed a number of wheat varieties and selections grown in many parts of the United States.
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307
It was found that the location a t which the wheat was grown produced differences in thiamine content which were greater than those resulting from genetic differences among the varieties observed. Some of their data are given in Table IV. TABLSI IV Selected Data on the Thiamine Content” of Varieties of Southern Wheat Grown at Various Locationsb
Location l\;lanhattan, Kans. Amarillo, Texas Ames, Iowa Alliona, Nebraska Fort Collins, Colo. Fort Lewis, Colo. Akron, Colo.
Kharkof
Blackhull
8.3 5.9 4.4 4.4 5.1 6.8 4.6
6.6 5.5 4.9 5.4 6.3 5.0
-
Comanche
Average of thirty varieties
6.6 7.2 5.4 5.5 4.7 7.0 5.4
7.2 6.9 4.9 5.4 5.6 6.2 5.2
Mg./g. From Hamner et al. (unpublished).
Bondi and Meyer (1946b) found that plants grown in Palestine during the summer months contain more riboflavin than similar plants grown during the rainy winter months. 111. INFLUENCE OF FERTILIZERB ON VITAMINCONTENT The effects of soils and fertilizers on the vitamin content of plants have been the subjects of reviews by Maynard and Beeson (1943-44) and Hamner (1945). The general conclusion reached by Hamner follows: “It seems probable that variations in the ascorbic acid content of plants such as might be encountered under field conditions are influenced so markedly by differences between varieties and by climatic conditions that the possible influence of soil conditions and fertilizer practices will be found to have little practical importance. It seems probable also that any fertilizer treatment (or lack of treatment) which causes the development of chlorosis in the plants will likely decrease the carotene content of the leaves.’’ Recent contributions in this field tend to support these views. The effect of manganese on the ascorbic acid content of tomatoes and other foods of plant origin is still controversial. Gum et al. (1945) found no effect of manganese on the ascorbic acid content of tomatoes grown in culture solutions, although large Werences occurred in the manganese
308
0. FRED SOMERS AND KENNETH C. BEESON
content of the foliage which was definitely chlorotic in the manganese deficient cultures. A boron deficiency in these experiments resulted in strikingly lower levels of carotene and riboflavin in the tomato fruit, although no effect on the foliage was reported. Harmer and Sherman (1943) have reported on the effect of manganese on the total and reduced ascorbic acid content in spinach, oat plants, and sudan grass. Manganese apparently had no effect on the reduced ascorbic acid content of spinach. Some variations were observed in oats and sudan grass, but they are probably nonsignificant. In a footnote the authors state, at least for one set of data, that their analysis of variance shows significant differences only on a 17% level, which is generally considered to be nonsignificant. Sideris and Young (1944) in agreement with earlier work (see Hamner, 1945) found that the ascorbic acid content of the pineapple plant was higher than normal when the iron supply was deficient. The effects of nitrogen on the ascorbic acid content of plants seems still to be in doubt as far as any generalizations are concerned. Reder, Ascham and Eheart (1943) have reported that although some significant effects of nitrogen and potassium were obtained in their comprehensive experiments using turnip greens, the over-all effect of location or place was 13.75 times as great as the most important average effect of potassium. This is illustrated in Table 11, where a part of their data from a factorial experiment is presented. Variations of nearly 100% occur between locations, whereas variations of less than 10% occur between treatments. Potassium fertilization resulted in the most consistent effects (decreased ascorbic acid content) of any fertilizer treatment. In later work Reder, Speirs et al. (1943) and Bernstein, Hamner and Parks (1945) found no significant effect of nitrogen on ascorbic acid. Others (Holmes et al., 1945; Karikka et al., 1944; Sideris and Young, 1945b; Smith and Walker, 1946) have reported that they observed no effect of fertilization or fertility level of the soil on the concentration of ascorbic acid in plants. Jones et al. (1945) have reported an inverse relationship between nitrogen supply and the ascorbic acid content of grapefruit juice: Their conclusions are based on average values for the entire season, September 16 to April 24, 1944; and they noted that the fruit produced on the different plots grew and matured under quite different conditions as regards the nitrogen nutrition of the tree. At none of the 12 sampling dates, however, was there any deviation from this inverse relationship. Petrosini (1945) has also reported an inverse relationship between nitrogen supply and the ascorbic acid content of pepper pods (Capsicumfrutescens). Lo and Chen (1944) reported that zinc applied to soils increased the ascorbic acid content of tomatoes. Their experimental design seems to be satisfactory. Wynd and Noggle (1945a, 1945b) have reported on experiments deal-
INFLUENCES ON VITAMIN AND MINERAL CONTENT OF VEGETABLES
309
ing with the effect of various soil properties on the ascorbic acid and carotene content of young oat plants harvested for their content of vitamins. According to these investigators the amount of ascorbic acid in the plants is related to such factors as the amount of nitrogen in the soil and the b a s e exchange capacity. The samples used in this investigation were harvested over a period of 23 days, however, and no consideration was given to this important fact. An examination of their data reveals that there is as close a correlation between ascorbic acid in the plant and the date of harvest as between ascorbic acid and any soil factor studied. In a later paper (Wynd and Noggle, 194613) these authors reported on their results with rye which showed an opposite trend to the earlier experiments with oats. The rye was harvested in the spring and the oats in the fall. The experimental design is inadequate, however, and the cultural, harvesting, and other essential information presented is meager. Pollard (1941), working with carrots, reported a direct relationship between the content of carotene and the supply of nitrogen in the soil. The differences are small, however. Smith and Wang (1941) found a correlation coefficient of +0.85 for total nitrogen and carotene in 63 samples of orchard grass (Dactylis glomerata). In this experiment a late dressing of ammonium sulfate increased the carotene content of rye grass leaves and heads by 55% and 17% respectively. Bernsteili et a?. (1945) found that deficiencies of sulfur, nitrogen, and potassium in sand cultures resulted in lower levels than normal of carotene in turnips. Well-replicated soil experiments, however, did not show this relationship when these elements were omitted in the fertilization program. This indicates, possibly, an adequate supply in the soil. They state that any treatment that resulted in visible chlorosis also resulted in appreciable decreases in carotene. Wynd and Noggle (1946a) have reported certain relationships between the carotene content of rye plants and soil characters, such as nitrogen supply and loss on ignition. The samples, however, were not collected at the same time, and other factors were not held under, statistical control. Finch et al. (1945) have reported an inverse relationship between the nitrogen and ascorbic acid content of the edible portion of cantaloupes and potatoes, but no measure of variability is presented. The following papers were not examined in the original, but the abstracts indicate the importance of their contributions. Sapun (1940) has reported some effects of fertilizers on the ascorbic acid content of cabbage, tomatoes, cucumber, and onions. The direction of the change varied with the species and, in general, the changes are small. Rangnekar (1945) reported that 0.05-0.3 g. of Mn as MnS01.4H20 per 6 pounds of soil did not affect the carotene content of Amaranthus gangeticus ( A . tricolor), although the ascorbic acid was increased. Greater quantities of manganese
310
0. FRED SOMERS AND KENNETH C. BEESON
resulted in lower ascorbic acid values. He states that the effect of manganese on ascorbic acid paralleled its effect on plant growth. Ferres and Brown (1946) reported that additions of potassium, zinc, or molybdenum to soil pot cultures did not modify the ascorbic acid content of lettuce or peas. OF FERTILIZERS ON MINERALCONTENT IV. INFLUENCE Although it is well recognized that many factors act together in influencing the mineral concentration in plants, few experiments have been devised to supply information which is adequate for the evaluation of the relative effects of any one factor. Consequently, any review of even recent experimental results in this field must of necessity be restricted to a consideration of the summation of influences for which the plant is the end result. In general, there is more evidence of a basic nature concerning the effect of variation in mineral supply than of the influence of climate or other factors on the concentration of minerals in plants. Many experiments, for example, particularly in solution culture, have confirmed earlier observations made under practical conditions about the pronounced interrelationships between the different nutrient elements. There still is, however, much confusion and uncertainty with respect to any general principles concerning the effect of fertilizers on plant composition. A critical discussion of this problem has been given elsewhere (Beeson, 1946). The probability of a calcium deficiency in the human diet is greater than that for most of the other mineral elements. Consequently, production methods for the protective foods of vegetable origin should stress optimum quantities of this element. Recent experiments in which calcium-supplying materials were added to the soil are not in agreement as to the magnitude of any effect they have on the concentration of calcium in the plant. In Massachusetts, Holmes et al. (1945) obtained very marked increases in the calcium concentration of kale when limestone was added to a Merrimac fine sandy loam. Hansen (1945a), working,with broccoli, collards, and kale on a Chehalis clay loam in Washington obtained less consistent increases in calcium concentration, particularly in kale. Sheets et al. (1944), in very comprehensively designed experiments, found that applications of calcium sulfate to soils in the southeast significantly increased the calcium concentration in turnip greens in only 4 of 30 experiments carried out in 12 localities. It is clear that in all of these field experiments factors other than calcium supply influenced the calcium content of the plant. An evaluation of climate is needed in considering these results. Thus, Tremblay and Vandecaveye (1944) have shown that liming several Washington soils did not greatly affect the calcium content of spinach under conditions of normal growth when the experiments were carried out under
311
INFLUENCES ON VITAMIN AND MINERAL CONTENT OF VEGETABLES
uniform conditions in pots. Sheets el al. (1944) have clearly shown in their work that of the three variables that had significant effects, e.g., location, treatment, and replication, location had much the greatest effect (refer to Table V). Location, in these experiments, includes, of course, soil and all climatic factors. Many recent experiments have emphasized the importance of the interrelationships of the mineral elements in the nutrient supply. Those elements that seem to influence the absorption of calcium by plants to the greatest degree are nitrogen and potassium. Addition of either of these TABLE V Selected Data Showing the Efect of Fertilization on the Calcium Content of Turnip Greens, MoistureFree Basis, Spring of 1940" Location in Mississippi ~~
~
Treatment
Crystal Springs g. Ca/100 g.
Poplarville g. Ca/100 g.
Stoneville g. Ca/100 g.
None Gypsum Nitrogen
2.080 2.610 1.740
2.195 2.246 1.755b
3.085 3.070 2.4000
~
Sheets el al. (1944). Significantly lower at the 6% level. Significantly lower at the 1% level. The test of significance is based on the entire study of 10 duplicate fertilizer treatments in 30 experiments of which the results of the 3 treatments and 3 experiments given here are representative. 0
to the soil is normally associated with a lower calcium concentration in the plant (Wittwer and Goff, 1946). The mechanism of this relationship is not clear from present evidence, but that the magnitude may be of importance in some cases has been demonstrated in the work of Sheets et al. (1944), where nitrogen had the greatest effect of any of 4 fertilizer factors studied. The depression of calcium content by nitrogen was significant in 24 of the 30 experiments and averaged 0.36% of calcium in the moisturefree turnip greens. This depression was 6 times greater than the average increase obtained when gypsum was added to the soil. Some representative data illustrating this effect are given in Table V. An important contribution to the problem of nitrogen-calcium relationships has been made by Sideris and Young (1945a, 1946) working with the pineapple. Their data (See Table VI) indicate that in solution culture work, the calcium content of fresh tissue is greater for the high-nitrogen culture, where nitrates supplied the nitrogen, and in the low-nitrogen cul-
312
Q. FRED SOMERS AND KENNETH C. BEESON
ture, where ammonium salts supplied the nitrogen, than for either of the other extremes in nitrogen supply. I n these experiments it is assumed that the effect of the form of nitrogen added predominated a t all times, since the culture solutions were renewed every 2 weeks. Rose and McCalla (1944) also found a direct relationship in solution culture tests between nitrogen supply and calcium concentration in wheat plants. I n the soil system, of course, both forms of nitrogen would normally be present, and interpretations are more difficult. The effect of potassium supply on the calcium concentration in plants is less striking and in some cases not significant (Hampton and Albrecht, 1944; Sheets et al., 1944; Vandecaveye and Baker, 1944; Finch and McGeorge, 1945; Rose and McCalla, 1944). Sideris and Young (1915a) TABLE^
VI
Eflect of the Amount and Source of Nitrogen on the Calcium Content of the Leaves of the Pineupple PIQnt. Fresh-Weight Basis, SoLutwn Cultwee.
g. Ca/100 g.
Nitrate series g. Ca/100 g.
Ammonium series g. Ca/lOO g.
140 mg. N/1. 2.8 mg. N/1.
0.158 0.037
0.018 0.030
Treatment High N Low N 0
Sideris and Young (1946).
have reported that the calcium concentration in tissues of the pineapple plant was definitely lower where the potassium supply was high. Rose and McCalla (1944) observed a significantly lower concentration of calcium in wheat plants at all levels of nitrogen supply as the potassium supply was increased. Fried and Peech (1946) have shown recently that calcium absorption by plants was inhibited in the presence of large quantities of manganese. Leaves of the alfalfa plant grown in pots receiving 200 pounds of manganese per 2 million pounds of soil contained about half as much calcium as did the control. The concentration of phosphorus in plants is not as critical to the human diet as is calcium, but a few recent observations may be worth attention. Whether or not applications of phosphorus to soils will result in a change in the phosphorus content of the plant depends upon the supply of available phosphorus in the soil (Sheets et aZ., 1944; Beeson, 1946). In the presence of minimum quantities of phosphorus, all that is available will be utilized in growth. Not until growth is satisfied or until other elements become limiting in turn will the phosphorus concentration in the plant be materially increased. Consequently, any attempt to summarize the re-
INFLUENCES ON VITAMIN AND MINERAL CONTENT OF VEGETABLES
313
sults in the literature is likely to lead to confusion. Thus, several workers have found that an increase in nitrogen supply will result in a decrease in phosphorus concentration in wheat (Rose and McCalla, 1944), barley (Hamy, 1943), New Zealand spinach (Wittwer and Goff, 1946), and turnip greens (Sheets et al., 1944). Others working with the grapefruit (Finch and McGeorge, 1945), rice (Aiyar, 1946), and potatoes, wheat, barley, and alfalfa (Greaves and Pittman, 1946) have reported no significant changes in phosphorus when nitrogen supply was increased. Sheets et al. (1944), working with soil plots, and Sideris and Young (1946), working with culture solutions, have observed increases in tissue phosphorus of turnip greens and the pineapple plant, respectively, when the nitrogen supply was increased. Most of these variations, it is believed, could be ascribed to the relative quantities of phosphorus and nitrogen supply in either the solution cultures or the soil. Similar variations in the effect of potassium supply on tissue concentration of phosphorus (Sheets et al., 1944; Hampton and Albrecht, 1944; Vandecaveye and Baker, 1944; Finch and McGeorge, 1945; Sideris and Young, 1945a; Aiyar, 1946; Arnon and Hoagland, 1943) and of the effect of phosphorus supply (Sheets et al., 1944; Tremblay and Vandecaveye, 1944; Aiyar, 1946; Greaves and Pittman, 1946; Amon and HoagIand, 1943; Vandecaveye and Baker, 1944; Finch and McGeorge, 1945) and liming materials (Holmes et aE., 1945; Tremblay and Vandecaveye, 1944; Rose and McCalla, 1944) could probably be explained in the same way. Iron, like calcium, is of special significance in the protective foods of plant origin. Consequently, any procedure that will result in an improvement in the iron content of the plant may be ultimately beneficial in human nutrition. Iron compounds added to the soil have had little effect in increasing the iron content of the plant (Beeson, 1946). In solution culture work Sideris and Young (1945a) 1946) observed a small reduction in the concentration of iron in the pineapple plant when nitrates Rerved as the source of nitrogen. Large quantities of ammonium salts, however, increased the concentration of iron as compared to minimum quantities of nitrogen supply from this source. They found that iron precipitation on the epidermal root cells was greatest for the high-nitrogen cultures in the nitrate series and smallest for the high-nitrogen culture in the ammonium series. Iron concentrations in the combined leaf and stem sections were inversely proportional to the amounts of iron precipitated on the roots. They point out that such precipitation on the epidermal root layer is greatly influenced by changes in the hydrogen-ion concentration at the interfacial layer, resulting from an unequal absorption of anions and cations. Under either condition of nitrogen supply and hydrogen-ion concentration, there was a low translocation rate of iron from the roots to the other plant organs.
314
G. FRED SOMERS AND KENNETH C. BEESON
Variations in potassium supply in these experiments had little effect upon iron concentration in the pineapple plant (Sideris and Young, 1945a). In field experiments, Speirs et al. (1944) reported that the fertilizer treatment that had the greatest influence on iron concentration in turnip greens was nitrogen. A reduction in iron with increased nitrogen was found in 26 of the 29 locations studied, and was significant at 14 places. Sodium nitrate was the source of nitrogen in these experiments, but it would not necessarily follow from the work of Sideris and Young (1945a, 1946), previously referred to, that the source of nitrogen in soil cultures would always have the same relative importance as in solution cultures. Holmes et aZ. (1945) noted, in agreement with earlier work (Beeson, 1946), that limestone added to the soil was followed by a marked reduction in the iron content of kale. Their data are given in Table VII. This obTABLEVII InfEuence of Calcium and Magnesium Applied to the Merrimac Fine Sandy Loam on the Iron Content of Kale, Fresh-Weight Basisa
I None, mg./100 g.
Item Fe in fresh tissue
Treatment
1
3.3
+
MgSOi limestone, mg./100 g.
MgSO4, mg./100 g.
I
4.7
I
4.5
Limestone, mg./100 g.
I
2.4
Holmes et al. (1945).
servation is also in harmony with the results from solution culture work reported by Sideris and Young (1945a, 1946). With the exception of iron, and possibly copper, recent investigations have emphasized that the concentration of important micronutrient elements such as zinc, manganese, cobalt, and iodine in plants is directly proportional to the supply in a nutrient medium of normal composition (Beeson, 1946). Excesses of an element like boron will modify the uptake by and concentration of other elements in the plant (Parks et al., 1944). Riou and Delorme (1941) have reported that the copper concentration in turnips in the presence of boron fertilizers is lower than normal. They feel that the absence of boron resulted in an abnormal accumulation of mineral matter in the plant. This observation is in agreement with Parks et aZ. (1944) for, the deficient-to-normal range of boron supply in their solution culture work. In the normal-to-toxic range, however, the copper concentration of the tomato plants increased with increase in the boron supply.
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V. DISCUSSION The results presented in this review can be discussed and evaluated conveniently in terms of the reviewer’s opinion as to what constitutes an adequate experimental approach to this problem. In the first place, it seems that the primary purpose for undertaking studies of this kind is to improve the nutritional quality of the basic food supply. Any improvement which is made should be of sufficient magnitude to justify the effort and expense involved, but first of all, the possibilities for improvement must be explored. In these exploratory studies, a number of points must be considered : 1. What nutritional factors are of interest, and which of them can be studied? Any selection of the nutritional factors to be studied depends upon both the relative importance of the factors and the ease with which they can be measured. Reliable analytical methods must be available and these must be adaptable to routine determinations, preferably on a large scale in different laboratories. 2. What precautions are known, or believed to be known, to be essential in measuring the nutritional factors to be studied? Here careful consideration must be given to such factors as the stage of development of the plant and the parts or organs to be selected, sampling method, and the variation between samples. Only by statistical methods can such variables be adequately controlled. In addition, consideration must be given to harvesting techniques and the changes which occur between harvesting and analysis. In some cases, e.g., ascorbic acid in ripe tomato fruits, changes that follow harvesting are not a serious matter, but in other cases, e.g., ascorbic acid in potato tubers, they can be serious. The significance of these changes, if any, should be considered in relation to the nutritive value of the crop as it is consumed. 3. What soil and climatic influences are to be studied? Here there are a qumber of possibilities such as mineral supply, moisture supply, amount of bght, temperature, etc. Some can be studied more easily than others. 4. What general methods are available for a study of the influence of soil and climatic factors? In the first place, it should be realized that, under natural conditions, it is not possible to separate the influence of soil and climatic factors. Various soil factors are determined by climate, but climate apparently also has a direct effect upon the plant. This interdependence very greatly complicates the evaluation of individual variables. The tendency is to attempt avoidance of this complication by using controlled conditions-conditions under which only one, or a t most a few, variables are introduced into the experiment a t any one time. This is not an entirely satisfactory expedient, although such techniques have produced
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valuable results. One of the chief drawbacks of this approach is that natural forces and conditions can seldom be duplicated under controlled conditions. This is particularly true of studies involving light. So far as the authors are aware, there are nowhere existent any facilities which permit studies under controlled conditions with light quality and light intensities approaching those of full sunlight on a clear day in the temperate zones. There are a limited number of facilities available for studies a t from about 5 2 0 % of full sunlight intensities, but these facilities are by no means adequate for a study of a problem of this scope. In the final analysis, a measure of the effects of environmental variables -both those of climate and those of the soil-are obtained when plants are grown a t different locations and a t different seasons of the year. When a sufficient number of analyses have been made of samples obtained from various locations or during different seasons, some measure of the relative magnitude of the influence of environment on plant composition is obtained. In interpreting such data, one is naturally interested in the relative contribution to the differences made by soil variations as compared to climatic variations. By simply growing plants a t different locations or harvesting crops a t different seasons, some measure is obtained of the relative magnitude of the influence of environment, but no measure is given of the relative importance of the two groups of variables, climatic us. soil. Since the natural variations in climate and soil cannot be produced at present under controlled conditions, the only way t o carry out experiments of this type is to grow plants under exposure to natural conditions. By properly designing the experiments, it seems possible to make some progress toward the measurement of the relative influence of individual environmental vrlriations or groups of variables. For example, plants may be grown at different locations with exposure to the natural variations in climate and soil and a measure obtained of the influence of location on plant composition. If at these same locations, comparable plants are grown in a homogeneous medium that provides a similar root environment a t each location, then a measure of the relative influence of climate could be obtained by comparing the composition of the plants produced in such a medium. Several methods are available whereby standardization of the root environment is reasonably accurate. Plants may be grown in solution cultures or in sand or gravel cultures using exactly the same techniques a t each location. Plants may also be grown in a fertile, welldrained soil which has been obtained a t a single location and shipped to the other locations for use under as nearly identical conditions of culture as possible. Under such circumstances, the sand or solution cultures, or the soil cultures, provide a nearly uniform basis for the comparison of results between locations. The differences between plants grown under field conditions in
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various locations and those of plants grown in the homogeneous medium at the same locations would serve as a means of comparison of the field soil effects. If such an experiment is conducted at a sufficient number of locations and the experiment is designed in such a way that statistical analysis of the results is justified, then the inclusion of a sufficient number of records as to the environmental conditions at each location should provide a means of obtaining some information as to the specific influence of each environ; mental variable. This approach to the problem is open to criticism, but it seems to be about the only feasible one at the present time. It is obvious, of course, that this use of a single soil at several locations is not for the purpose of studying the characteristics of that soil. It serves only as a uniform medium for plant growth at different locations. In this discussion so far, no mention has been made of the influence of genetic variables. These are likewise of importance, but their proper evaluation is somewhat easier, provided one does not attempt to evaluate interactions between climate and soil factors and genetic factors. Such interactions may be important, but it is not desirable to discuss them further here. In any case, however, genetic variables must be recognized and taken into consideration in the design of the experiment. A critical examination of the experiments described in this review, as well as in similar reviews published previously in light of the above discussion, leads to the conclusion that only a limited number of them have been so designed as to evaluate adequately the effect of soil and climatic factors upon the vitamin and minerd content of plants. Most experiments have been too limited in their scope. There are a number of obvious reasons for this lack of a comprehensive approach, Two of the more important of them are: (1) Insufficient knowledge of the factors which are operating and how they can be controlled; (2) A lack of sufficient time, labor, and facilities to undertake a project of the magnitude that is required. The problem is so complex that no one investigator has the ability, funds, or time to handle at one time all of its aspects. We feel that it would be helpful to illustrate our point of view by reference to specific examples. Several of the experiments reported are certainly of considerable merit and could be used for illustrative purposes, but the cooperative experiment on the ascorbic acid cotltent of turnip greens carried out by 6 of the southeastern experiment stations (Reder, Speirs et al., 1943)will be used because it is outstanding in many respects. This experiment meets most of the criteria enumerated above. The component being studied, ascorbic acid, is easily determined and is of nutritional significance. These workers have considered not only the ascorbic acid content of the fresh material, but also the effects of cooking and storage. The statistical design of the experiment is particularly commendable. The
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plans laid down for the experiment seem to have been well carried out, except that in one part of the fall harvest, one of the cooperators used a different analytical method. Such a change as this should be avoided since it introduces uncontrolled variables. Perhaps the data obtained by this method should have been disregarded in the statistical analysis of the results. Despite this criticistn of this experiment, we feel that these cooperators have made an excellent start toward the proper study of problems of this kind. They have gained experience which should prove invaluable in planning further experiments. It is our feeling that work of this kind should be expanded and that the cooperative approach is the only one feasible for future expansion. VI. SUMMARY Recent advances in studies on the influence of climate and fertilizer practices upon the vitamin and mineral content of vegetables have been summarized in this review. It is pointed out that, among the vitamins, ascorbic acid has received much more attention in this regard than other vitamins. A number of experiments have shown that the location and season in which plants are grown can have a profound effect upon their ascorbic acid content. Investigations of the individual variables which are associated with season and location have shown that light is one of the most important of these factors in determining the ascorbic acid content of plant tissues. Other environmental factors appear to be of less importance in this regard, although under experimental conditions, temperature, carbon dioxide concentration, soil moisture, and possibly rainfall have been found to affect the amount of ascorbic acid in plants either directly or indirectly. Under field conditions the amount of rainfall and the amount of sunlight are so closely interdependent that it is difficult to separate the effects of the two. With respect to other vitamins, the studies to date have shown that location and/or season influence the amount of carotene, riboflavin, and thiamine in plant materials. Variations in light and soil moisture apparently influence the carotene content of at least some plant tissues. Recent studies on the influence of fertilizers on the vitamin content of plants support earlier findings which indicated that the differences in vitamin content of plants produced by such variables as variety and climate were so marked that the smaller differences produced by fertilizers were likely to be of little practical importance. Climate is a variable which has not always been adequately evaluated in studies on the influence of fertilizers on the mineral content of plants. This possibly accounts for some of the apparent contradictions which are reported in the literature; e.g., recent experiments in which calcium-sup-
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plying materials were added to the soil are not in agreement as to the magnitude of the effect (if any) they may have on the concentration of calcium in the plant. More consistent results have been obtained in studies of the interrelationships of the mineral elements in the nutrient supply and their effect upon the mineral content of plant tissues. A number of experiments dealing with this problem are summarized in this review.
REFERENCES Aberg, B. 1945. Effects of light and temperature on the ascorbic acid content of green plants. Ann. Roy. Agr. College Sweden 13, 239-273. Aiyar, S. P. 1946. Effects of phosphate deficiency on rice. Proc. Indian Acad. Sci. 23B, 165-193. Amon, D.I., and Hoagland, D. R. 1943. Composition of the tomato plant as influenced by nutrient supply in relation to fruiting. Botan. Qaz. 104, 576-690. Barnes, W. C. 1936. Effects of some environmental factors on growth and color of carrots. Cornell Univ. Agr. Expt. Stu., Mem. 186. Beeson, K. C. 1946. The effect of mineral supply on the mineral concentration and nutritional quality of plants. Botan. Rev. 12, 424455. Bernstein, L., Hamner, K. C., and Parks, R. Q. 1945. The influence of mineral nutrition, soil fertility, and climate on carotene and ascorbic acid content of turnip greens. Plant Physiol. 20, 540-572. Bessey, 0. A. 1938. A method for the determination of small quantitie; of ascorbic acid and dehydroascorbic acid in turbid and colored solutioq in the presence of other reducing substances. J. Biol. Chem. 126, 771-784. Bondi, A,, and Meyer, H. 1946a. Carotene in Palestinian crops. f. Agr. Sci. 36, 1-5. Bondi, A., and Meyer, H. 1946b. The riboflavin content of poultry feeding stuffs. J. Agr. Sci. 36, 6-9. Ellis, G. H., aud Hamner, K. C. 1943. The carotene content of tomatoes as influenced by various factors. J Nutrition 26, 539-553. Ferres, H. M., and Brown, W. D. 1946. The effects of mineral nutrients on the concentration of ascorbic acid in legumes and two leaf vegetables. Exptl. Biol. Med. Sci. 24, 111-119. Original not seen. C. A . 40,6573. Finch, A. H., Jones, W. W., and Van Horn, C. W. 1945. The influence of nitrogen nutrition upon the ascorbic acid content of several vegetable crops. Proc. Am. SOC.Hort. Sn'. 46, 314-318. Finch, A. H., and McGeorge, W. T. 1945. Fruiting and physiological responses of Marsh grapefruit trees to fertilization. Ariwnu Agr. Expl. Sta. Tech. Bull. 106, 42-54. Fried, M., and Yeech, M. 1946. The comparative effects of lime and gypsum upon plants grown on acid soils. J. Am. SOC.Agron. 38, 614-623. Giral, F., and Torre, L. M. 1947. Vitamin C content of Mexican ornamental plants. Science 106,65-66. Giroud, A. 1938. Ascorbic acid in cells and tissues. Protoplasma Monographien 16, 1-187. Greaves, J . E., and Pittman, D. W. 1946. Influence of fertilizers on the yield and composition of certain crops and on the soil. Soil Sn'. 61, 239-246. Gum, 0.B., Brown, H. D., and Burrell, R. C. 1945. Some effects of boron and manganese on the quality of beets and tomatoes. Plant Physiol. 20, 267-275.
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Hamner, K. C. 1945. Minor elements and vitamin content of plants. Soil Sci. 60, 166-171. Hamner, K. C., Bernstein, L., and Maynard, L. A. 1945. Effects of light intensity, day length, temperature, and other environmental factors on the ascorbic acid content of tomatoes. J . Nutrition 2Q,85-97. Hamner, K. C., and Maynard, L. A. 1942. Factors influencing the nutritive value of the tomato. U.S. Dept. Agr. M i x . Publ. 602, 23 pp. Hamner, K. C., and Nightingale, G. T. 1946. Ascorbic acid content of pineapples as correlated with environmental factors and plant composition. Food Research 11, 535-541. Hamner, K. C., and Parks, R. Q. 1944. Effect of light intensity on ascorbic acid content of turnip greens. J . Am. SOC.Agrcm. 86, 269-273. Hampton, H. E.,,and Albrecht, W. A. 1944. Nitrogen fixation, composition, and growth of soybeans in relation to variable amounts of potaasium and calcium. Mo. Agr. Expt. Sta. Res. Bull. 381, 36 pp. Hamy, A. 1943. The influence of fertilizers on the composition of vegetable juices. Ann. Agrcm. 13, 117-129. Hansen, E. 1945a. Seasonal variations in the mineral and vitamin content of certain green vegetable crops. Proc. Am. Soc. Hort. Sn'. 46, 299-304. Hansen, E. 1946b. Variations in the carotene content of carrots. Proc. Am. SOC. Hort. Sci. 46, 355-358. Hansen, E., and Waldo, G. F. 1944. Ascorbic acid content of small fruits in relation to genetic and environmental factors. Food Research 9, 463-461. Harmer, P. M., and Sherman, G. D. 1943. The influence of manganese deficiency on the synthesis of ascorbic acid (vitamin C) in foliage of plants. Soil Sn'. SOC.Am. Proc. 8, 346-349. Harris, L. J., and Olliver, M. 1942. The reliability of the method for estimating vitamin C by titration against 2,6dichlorophenolindophenol. I. Control testa with plant tissues. B i o c h . J . 86, 156-182. Hartzler, E. R. 1945. The availability of ascorbic acid in papayas and guavas. J . Nutrition 80, 356-366. Hathaway, I. L.,Davis, H. P., and Keim, F. D. 1945. Carotene content of native Nebraska grasses. Nebraaka Agr. Espt. Sta. Res. Bull. 140, 15 pp. Heinze, P. H., Kanapaux, M. S., Wade, B. L., Grimball, P. C., and Foster, R. L. 1944. Ascorbic acid content of 39 varieties of snap beans. Food Research 9, 19-26. Heller, V. G. 1928. Vitamin synthesis in plants as affected by light source. J . Bio2. Chenz. 76,499-511. Hochberg, M., Melnick, D., and Oser, B. L. 1943. Photometric determination of reduced and total ascorbic acid. Ind. Eng. Chem., Anal. Ed. 16, 182-188. Holmes, A. D.,Crowley, L. V., and Kuame ki, J. W. 1945. Influence of supplementary calcium and magnesium fertilizers on nutritive value of kale. Food Research 10, 401-407. Janes, B. E. 1944. Relative effects of variety and environment in determining the variations of per cent dry weight, ascorbic acid, and carotene content of cabbage and beans. Proc. Am. Soc. Hort. Sei. 46, 387-390. Johnson, L. P. V., Young, G. A., and Marshall, J. B. 1945. A note on the production of vitamin C by sprouting seeds. Sci. Agr. 26, 499-603. Jones, W.W.,Van Horn, C. W., and Finch, A: H. 1945. The influence of nitrogen
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nutrition of the tree upon the ascorbic acid content and other chemical and phyeical characteristics of grapefruit. A r i m Agr. Expt. Sta. Tech. Bull. 106. Karikka, K. J., Dudgeon, L. T., and Hauck, H. M. 1944. Influence of variety, location, fertilizer, and storage on the ascorbic acid content of potatow grown in New York State. J. Agr. Research 68, 49-03, Kaski, I. J., Webster, G. L., and Kirch, E. R.1944. Ascorbic acid content of tomatoes. Food Research 9,386-391. King, C. G. 1941. Chemical methods for determination of vitamin C. Znd. Eng. Chem.,Anal. Ed. 18,226-227. Koizumi, T., and KakukawtL, T. 1940. Vitamin C content of herbaceous plants and marine algae considering factors influencing it. Sci. Repls. Tohoku Imp. Univ., Fourth Ser. 15, 1OS-120. Kostenko, V. D. 1943. Content of vitamin C in cultivated and wild plants growing in high region8 of P a d . Compt. rend. acad. eci. U.R.S.S. 88, 42-43. Lampitt, L. H., Baker, L. C., and Parkinson, T. L. 1943. The vitamin C content of raw and cooked vegetables. J. SOC.Chem. Znd. 62, 61. Lampitt, L. H., Baker, L. C., and Parkinson, T.L. 1946. Vitamin C content of potatoes. I. Distribution in the potato plant. 11. The effect of variety, soil, and storage. J. SOC.Chem. Z n d . 64, 18-26. Lantz, E. M. 1945. Some factors affecting the ascorbic acid content of chile. New Mexica Agr. Expt. Sta. Bull. S24, 14 pp. Lo, T.-Y., and Chen, S.-M. 1944. The effect of chemical treatment on the carotene and ascorbic acid contents of tomatoes. J. Chinese Chem. SOC.11, 96-98. Lo COCO,G.1946. Composition of northern California tomatoes. Food Research 10, 114-121.
Loeffler, H. J., and Ponting, J. D. 1942. Ascorbic acid-rapid determination in fresh, frozen, or dehyd ated fruits and vegetables. Znd. Eng. Chem., Anal. Ed. 14, 846-849.
McCollum, J. P. 1944. Some factors affecting the ascorbic acid content of tomatoes. Proc. Am. SOC.Hort. Sci. 45, 382-386. Mack, G. L., and Treader, D. K. 1937. Vitamin C in vegetables. VI. A critical investigation of the Tillmans method for the determination of ascorbic acid. J. Biol.
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McMillan, T. J., and Todhunter, E. N. 1946. Dehydroascorbic acid in cabbage. Science 108, 196-197.
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a.
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Petrosini, G. 1945. The action of mineral fertilization on the vitamin content of plants: investigation of pepper pods. Ann. chim. applicuta 36, 81-93. Original not seen. C. A . 40, 7478. Platenius, H. 1945. Diurnal and seasonal changes in the ascorbic acid content of some vegetables. Plant Physiol. 20, 98-105. Pollard, A. 1941. Effect of fertilizer treatment on the carotene content of carrot roots. Ann. Rept. Agr. H o d . Res. Sta., Long Ashton, Bristol, 32. Poole, C. F.,Grimball, P. C., and Kanapaux, M. S. 1944. Factors affecting the ascorbic acid content of cabbage lines. J. Agr. Research 68, 325-329. Porter, J. W., Strong, F. M., Brink, R. A., and Neal, N. P. 1946. Carotene content of the corn plant. J. Agr. Research 72, 169-187. Rangnekar, Y. B. 1945. Manganese in the formation of vitamin C and carotene in plants. Current Sci. 14, 325. Original not seen. C . A . 40, 5472. Reder, R., Ascham, L., and Eheart, M. S. 1943. Effect of fertilizer and environment on the ascorbic acid content of turnip greens. J. Agr. Research 66, 375-388. Reder, R., Speirs, M., Cochran, H. L., Hollinger, M. E., Farish, M., Gieger, M., McWhirter, L., Sheets, 0. A., Eheert, J. F., Moore, R. C., and Carolus, R. L. 1943. The effects of maturity, nitrogen fertilization, storage, and cooking on the ascorbic acid content of two varieties of turnip greens. Southern Coop. Ser. Bull. 1, 30 PP. Reid, M. E. 1941a. Metabolism of ascorbic acid in cowpea plants. Bull. Torreg Botan. Club 68, 359-371. Reid, M. E. 1941b. Relation of temperature to the as-orbic acid content of cowpea plants. Bull. Torrey Botan. Club 68, 519-530. Reid, M. E. 1942. Effect of variations in light intensity, length of photo-period, and availability of nitrogen upon accumulation of mcorbic acid in cowpea plants. Bull. T m e y Botan. Club 69, 204-220. Riou, P., and Delorme, G. 1941. The influence of soil and fertilizer on the assimilation of copper by turnips. Ann. 1'Acfm. 7 , 74-5. Robinson, W. B.,and Stotz, E. 1945. The indophenol-xylene extraction method for ascorbic acid and modifications for interfering substances. J. Biol. Chem. 160, 217-225. Rose, D. and McCalla, A. G. 1944. Effects of limiting ions on the absorption of nutrients by wheat. Can. J. Research 22C, 87-104. Sapun, M. P. 1940. The effect of fertilizers and of liming on the content of vitamin C in vegetables. Doklady Vsesoyuz. Akad. Sel'skokhoz. Nauk. im. V . 1. Lenina, NO.17, 28-32. Original not seen. C. A . 37, 3548. Sheets, 0. McWhirter, L., Anderson, W. S., Gieger, M., Ascham, L., Cochran, H. L., Speirs, M., Reder, R., Edmond, J. B., Lease, E. J., Mitchell, J. H., Fraps, G. S., Whitacre, J., Yarnell, S. H., Ellet, W. B., Moore, R. C., and Zimmerley, H. H. 1944. Effect of fertilizer, soil composition, and certain climatological conditions on the calcium and phosphorus content of turnip greens. J. Agr. Research 68, 1945-1990. Shen, T., Hsieh, K. M., and Chen, T. M. 1945. Effects of magnesium, chloride, and manganous nitrate upon the content of ascorbic acid in soybean during germination, with observations on the activity of ascorbic acid oxidaae. Biochem. J . 39, 107-110. Sideris, C. P., and Young, H. Y. 1944. Effects of iron on chlorophyllous pigments, ascorbic acid, acidity, and carbohydrates of Ananas cornsus (L.)Merr., 8uppli.d with nitrate or ammonium salts. Plant Physiol. 19, 62-75.
A.,
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Wynd, F. L., and Noggle, G. R. 194613. Influence of chemical characteristics of soil on production of vitamin C in leaves of oats. Food Research 10, 637-646. Wynd, F. L., and Noggle, G. R. 1948e. Influence of chemical characteristics of soil on production of carotene in leava of rye. Food Research 11, 148-168. Wynd,F. L., and Noggle, G. R. 194613. Influence of chemical propertiea of soil on production of vitamin C in leaves of rye. Food Reseurch 11, 169-178.
Nonemymatic Browning in Fruit Products
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BY EARL R STADTMAN University of California. Berkeley. California CONTENTB
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I Introduction
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. . . . . . . . . . .. . . . . . . . . . . . . . I1. Methods of Color Measurement . . . . . . I11. The Effect of Storage Temperature on Browning . 1. Dried Fruit . . . . . . . . . . 2. Citrus Juice and Citrus Juice Concentrates . 3. Other Juices . . . . . . . . . . .
1 General 2 Experimentd Approach 3 Theories of Browning
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IV. The Effect of Processing and Drying Temperature on Browning V. The Infiuence of Moisture on the Rate of Browning . . . . 1. Fruit Concentrates . . . . . . . . . . . . 2 DriedFruit . . . . . . . . . . . . . . V I The Iduence of Oxygen on Deterioration . . . . . . . 1. CitrusJuices . . . . . . . . . . . . . . 2. Other Juices . . . . . . . . . . . . . . 3. Fruit Concentrates . . . . . . . . . . . . 4 Dried Fruita . . . . . . . . . . . . . . VII Changes in Chemical Composition Which Accompany Browning 1 Carbon Dioxide Production . . . . . . . . . 2. Ascorbic Acid Destruction . . . . . . . . . . . . . . . . . 3. Changes in Nitrogen Constituents 4. Changes in Sugar Concentration . . . . . . . . 5 . The Formation of Furfuraldehydes . . . . . . . 0. The Nature of the Brown Pigments Produced . . . . VIII " h e Use of Inhibitors to Delay Browning . . . . . . . 1. SulfurDioxide . . . . . . . . . . . . . 2 Sirup Treatment . . . . . . . . . . . . 3 Other Treatmenta . . . . . . . . . . . . 4. Formaldehyde . . . . . . . . . . . . . IX Statua of the Browning Problem in Fruit . . . . . . . X NeededReaearch References
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325 325 326 327 328 329 329 330 332 333 334 334 335 336 336 341 341 342 346 346 348 351 354 357 369 300 360 364 365 366 300 368 369
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1 General Most fruit products readily undergo changes in color during preparation. processing. or storage. The natural color of the product may be destroyed. and dark brown substances are formed causing the product to become 326
326
EARL R. BTADTMAN
brownish to black in appearance. Such “browning” is usually accompanied by undesirable changes in flavor, odor, and nutritive value. This type of deterioration has long been recognized as one of the most important problems of fruit preservation, and has been the subject of research for many years. Surprising, however, is the fact that our knowledge concerning the chemistry of food discoloration is still very limited. This is due primarily to the fact that color deterioration involves a large number of highly interrelated variables. Some of these can be controlled by methods of processing, manufacture, or handling of the fruit. Among these are temperature, moisture, oxygen, and the use of inhibitors such as sulfur dioxide. Variables which are not so readily subject to control are those having to do with the constitution of the natural product. Thus the rate of deterioration is influenced by the nature and relative concentration of carbohydrates, nitrogen compounds, acids, and metal ions present in the fruit. During storage, each of these is subject to changes which may be more or less independent of the others. The problem of browning is therefore complicated by the fact that it involves a characterization of specific reactions in a complex system where a multiplicity of unrelated changes are taking place simultaneously. The problem is further complicated by the fact that the end products of the reactions, i.e., the brown pigments produced, are huminlike substances which are difficult to characterize. Quite aside from the complexity of the problem itself, the slowness with which our understanding of the chemistry of the reactions has developed is due mainly to the fact that most of the research has been directed at efforts to find a cure without first having found the cause of browning. Indeed, relatively little effort has been directed to elucidate the fundamental nature of the reactions involved. 2. Experimental Approach
The experimental approach to the problem of browning has been of four general types: (1) The effects of various packaging and processing treatments have been studied in attempts t o discover means for retarding browning. Nearly all of the earlier investigations were confined to experiments of this type. Unfortunately, many of these experiments were conducted in a haphazard fashion without regard for the influence of even the more obvious variables such as temperature, oxygen, etc. I n many instances, therefore, the observaQionsmade are meaningless. (2) A more fundamental approach to the problem has been made by correlating color deterioration with chemical changes which occur in fruit during storage, (3) Other efforts have been made to isolate and characterize the dark, huminlike compounds formed, with the hope that this information would disclose the identity of the reactants. (4) An attempt to characteriae the
NONENZYMATIC BROWNING IN FRUIT PRODUCTS
327
reactions has been made by studying the inhibitory action of a wide variety of chemical substances when added to the fruit. 3. Theories of Browning As yet there is no very well-developed theory for the mechanism of browning. This is undoubtedly due to the lack of any really concrete information regarding reactions which have been postulated. Three theories will be mentioned, however, since they are the most generally accepted and will serve as a basis for further discussion: (1) The most popular explanation for browning is found in the Maillard OT mehnoidin condensation theory. The Maillard reaction involves a condensation of amino acids and reducing sugars and gives rise to the formation of dark colored substances which have properties not unlike those found in darkened fruit products. Actually, the chemical evidence to support this theory of darkening in fruit products is very meager and not altogether consistent. (2) Another theory for which there is considerable evidence so far as citrus products are concerned, is the ascorbic acid theory. According to this the most important precursors to browning are ascorbic acid and related compounds, which upon oxidation yield reactive products that may polymerize or react with nitrogenous constituents of the products to form brown pigments. (3) The most recent theory may be called the “aclive-aldehyde” theory. It is in reality but a modification of the above theories. It is postulated that browning involves the decomposition of sugars and sugar acids to furfuraldehydes or similar compounds characterized by having an active carbonyl group, and that these products condense with nitrogen compounds and/or polymerize to form brown, resinous materials. Actually all three of the above mechanisms may be involved in the browning of fruit products. Until such time as specific processes are positively identified] the common tendency to refer to discoloration as the “Maillard reaction” should be avoided. A more general term such as the “browning-reaction,” which carries with it no implication as to reaction mechanism, is to be preferred, and will be used, therefore] throughout this paper. It is the primary aim of this discussion to present a critical review of the literature on browning in fruit products, to emphasize the deficiencies in our current knowledge, and thereby clarify the status of the problem. Browning reactions mhich are enzyme-catalyzed are not considered here, and may be distinguished for present purposes from nonenzymatic reactions on the basis of their susceptibility to inactivation at high temperatures. A general review of the problem of enzymatic browning has been made by Joslyn (1941a).
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EARL R. STADTMAN
11. METHODS OF COLORMEASUREMENT In most experiments on darkening of fruit, little or no attempt has been made to measure the amount of color produced. Usually the time required for discoloration first to become detectable is noted; further darkening is described as slight, medium, great or light, brown, black, etc. Obviously such relative expressions are of little value when it is desired to relate browning quantitatively to other variables. Color changes have been roughly evaluated by direct visual inspection of the fruit, the color grades being determined by comparing the experimental samples with a series of standard color samples which were arbitrarily assigned numerical ratings (Nichols and Reed, 1931; Nichols el al., 1938). The use of Lovibond red, yellow, and blue or brown tintometers t o measure browning is general. In the case of orange juice (Joslyn et al., 1934; Joslyn and Marsh, 1935) there is a progressive increase in the number of units of red, while little if any change in the yellow occurs during browning. Tintometers have been particularly useful in following color changes in strawberry, currant, and raspberry juices (Beattie et al., 1943; Tressler et al., 1943; Pederson et al., 1941). The most obvious change in these products during storage appears to be a destruction of the natural fruit pigments, and accordingly a decrease in the red components relative to the yellow is observed. In these cases the use of tintometers can, therefore, not be used to measure the extent of browning that may occur concurrently with pigment decomposition. The color of dried apples, cranberries, and vegetables has been evaluated in numerical terms by use of Munsell color disks (Continental Can Company, 1944a). The results are reported in terms of the percentage of each color required to match the color of the sample tested. Moore et al. (1944), Curl et at?. (1946) measured the browning in orange juice by comparing it with charts of Maerz and Paul (1930) using a standard light source. An undesirable feature of using tintometers or color disks for color measurement lies in the fact that all components may vary simultaneously, thus making it impossible to express the degree of browning on a single value basis. Hamburger and Joslyn (1941) determined the absorption spectra (in the visahle region) of filtered lemon and orange juices at various stages of browning. Absorption was greatest in the ultraviolet portion of the spectrum. As browning increased, the absorption increased only slightly at 7000 A, but increased very rapidly at 4800 A. As a measure of browning, ttey used the absorption at wave lengths go00 A and 4800 A. A similar technique waa used by Esselen el al. (1946) in studying brow'ning of apple, cranberry, and grape juices.
NONENZYMATIC BROWNING I N FRUIT PRODUCTS
329
Loeffler (1941) and Moore el al. (1942b) measured the transmission of 50% acetone solutions of filtered orange juice by means of a photoelectric colorirneter (using a blue filter #420). This method was found to be quite sensitive; changes not apparent to the eye were claimed to be measurable. Browning in dried apricots was determined by using a photoelectric colorimeter to measure the light transmission through 50% alcoholic extracts of the fruit (Stadtman, Barker, Mrak, and Mackinney, 1945). This method was found to be satisfactory provided fruit of a given SO2 concentration was used. Spectrophotometric analysis of extracts of darkened apricots which had been sulfured to different SO2 levels prior to storage revealed a marked difference in the absorption characteristics. A high SO2 level resulted in increased absorption in the blue relative to the green as compared with a low SO2 level. As a result, when samples of different SO2 treatments are compared, photoelectric measurements do not give values consistent with the visual appearance of the fruit. A method for determining the “browning index” was finally developed which involves a visual comparison of 50% alcoholic extracts of dried fruit with standard reference solutions under standard conditions. By this method, a measure of browning was obtained which was consistent with the visual appearance of the fruit, irrespective of the SO2 treatment. A general review of the problem of measuring color in canned foods has been made by Kramer and Smith (1946). The relative merits of using disc colorimeters and spectrophotometer to measure color are considered in detail.
11. THEEFFECTOF STORAGE TEMPERATURE ON BROWNING By far the most important single factor influencing the rate of browning in fruit products is temperature. Yet very little reliable quantitative data has been obtained relating browning to temperature. 1. Dried Fruit
Nichols snd Reed (1931)vstudied the influence of temperature on the rate of browning in dried apricots, peaches, and pears. They concluded that the rate of darkening at 2324°C. (75°F.) was approximately double the rate at 0°C. (32”F.), while the rate at 373°C. (100°F.) was 3 or 4 times as severe as at 0°C. (32°F.). The experimental data do not seem to justify this conclusion which is apparently true only when slight initial changes in color are compared. It is noted for example, that only 2 4 months were required for dried fruit to darken to a point described as the “poorest acceptable commercially,” whereas the same degree of discoloration was not reached even after 23 months at 0°C. (32°F.). In any event the method of color measurement was highly subjective; it seems in-
330
EARL R. STADTMAN
advisable to place much emphasis on the quantitative data of these experiments. However, these are typical of 8 great deal of the work on browning. Too often generalizations are made without defining the conditions under which they apply. Cruess and Pancoast (1933) canned commercially dried apricots, containing 2700 p.p.m. SO2 in hermetically sealed jars and stored samples at 0°C. (32"F.), room temperature [approximately 21.1"C. (70"F.)] and 46.1"C. (115°F.). Within 3 weeks at 46.1"C. (115°F.) the fruit had darkened considerably; at room temperature, darkening did not occur until after 3 months; fruit a t 0°C. (32°F.) showed no detectable darkening after 6 months. In another study aprieots containing 26% moisture and 3560 p.p.m. SOa were stored at -18, 0, 21, and 373°C. (-0.4, 32, 69.8 and 100°F.) (Nichols, Mrak, and Bethel, 1938). After 19 months, the fruit at - 18°C. (-0.4'F.) and 0°C. (32°F.) had undergone no changes in color. There was, however, a loss of about 500 p.p.m. SO2. The samples at 21°C. (69.8"F.) had darkened appreciably a t the end of 18 months, and samples at 37.8OC. (100°F.) darkened after only 8 months' storage. Stadtman, Barker, Haas, and Mrak (1945), determined the rate of darkening, C02 production, SOa loss and oxygen uptake in dried apricots at temperatures of 22'49°C. (71.6"-120.2"F.). They found the logarithms of the rates for all these processes to be proportional to the reciprocal of the absolute temperature (Fig. 1). An apparent activation energy of about 26 Kg.Cal. was calculated for the browning reaction by means of the Arrhenius equation. The corresponding &lo value was 3.9. Almost identical temperature relations were observed for C02production, SO2 loss, and oxygen uptake. The temperature coefficient was found to be more or less independent of the SOz concentration over a range of 1800 to 9600 p.p.m. and of moisture over a range of 5 to 24%. These results may be of considerable practical importance since it was shown that data obtained from storage tests a t high temperatures where browning is very rapid (2-3 weeh) may be used to predict the storage life at lower temperatures where appreciable browning may take years to occur. Results on storage of apple nuggets (ground apples containing 0.8% moisture) packed in air, vacuum, C02, and nitrogen and stored at temperatures of 23.8O-54.4"C. (75"-130°F.) (Continental Can Company, 1944a; Heberlein and Clifcorn, 1944) show that after 2 months' storage at 54.4"C. (130°F.) this product had darkened appreciably. Significant changes occurred after 6 months at 36.7OC. (98"F.), but only a slight change in color was observed after 12 months at 23.8O-26.7"C. (75"-80"F.). 2. Citrus Juice and Citrus Juice Concentrates
The browning of orange juice is always associated with a decline in its ascorbic acid content, and, as pointed out in the Introduction, a number
331
NONENZYMATIC BROWNING IN FRUIT PRODUCTB
of investigators believe that browning involves a breakdown of ascorbic acid. Therefore, the effect of temperature on both browning and ascorbic acid content will be considered here. A further discussion of ascorbic acid losses in citrus products will be presented later. Loeffler (1941)stored samples of pasteurized bottled orange juice at 4.4", 15.6' and 32.2"C. (40",60", 95'F.), and at room temperature. Changes in ascorbic acid, turbidity, color, amino nitrogen, COz production, and I
i
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Fig. 1. Influence of temperature on rate of oxygen uptake, SO2 disappearance,
CO,production, and darkening of apricots (Stadtman, Barker, Haas, and Mrak, 1945.) Fruit contained 5350 p.p.m. SO, and 23.5% moisture. Darkening = reciprocal of storage life; COn production = initial rate in mg./100 g. dry fruit/day X 10; SO2 disappearance = reciprocal of half life X 10'; 0 1 uptake = reciprocal of half life X 10'. Temperature is in degrees absolute. Storage life is time in days required for the fruit to become so brown as to be considered inedible.
oxygen uptake were studied. Browning was slight after 4 months at room temperature, but was very noticeable after only 1-2 months at 35°C. (95°F.). Unfortunately the data presented are inadequate t o enable a quantitative comparison of browning rates to be made. Storage was too short; the changes observed were small. Indiscriminate sampling, and at an insufficient number of time intervals, make it impossible to deduce much about the rate-time relationships. In addition the results showed considerable variability. In Table V of his paper, Loeffler (1941) at-
332
EARL R. STADTMAN
tempted to summarize the data by averaging the results obtained for all samples at each temperature, irrespective of the storage time; such averages were assumed to represent the change which occurs in the average storage time of the samples. This type of statistical analysis is valid only when the changes taking place are linear functions of time. Such may be the case for orange juice, but until this is definitely established the data presented in this manner should be considered only as trends rather than actual quantitative relationships. Similar studies on orange and grapefruit juice were made by Moore et al. (1944). Here, too, the experiments were of too short duration to allow strictly quantitative comparisons of darkening rates to be made. The loss in ascorbic acid during 6 months at approximately 26.7"C. (80°F.) was 19-25% for oranges, and 18-26% for grapefruit juice. The losses at 4.4"C. (40°F.) were 4 1 2 % and 7-9%, respectively. With orange juice concentrates the effect of storage temperature has been determined more carefully. Curl et al. (1946) studied changes in orange concentrates (65" Brix) stored a t 4.4", 15.6', 32.2") 35.4" and 49°C. (40°, 60") 80")95") and 120°F.). The rates of COa production, ascorbic acid loss, and darkening (initial rate) increased approximately 4 times for a 10°C. (18OF.) rise in temperature. The findings are in agreement with data reported by Hall (1927) and Stephens et al. (1942). Thus the temperature coefficient of these processes is of the same order of magnitude as darkening and COn production in dried apricots (Stadtman et al., 1945). According to Curl et al. (1946) similar studies have been madeby Chaves (1945) who followed changes in vitamin C during storage of Brazilian orange juice concentrates containing 62% solids, 3-574 acidity (as citric), and 2 4 7 mg. vitamin C/g. A t 10°C. (50°F.) the loss of vitamin C was 18% in 30 days and 19% in 58 days; a t 27°C. (80.6'F.) the corresponding losses were 28% and 36%) and at 38°C. (100.4"F.) 82% and 94%) respectively. Dried orange juice powder containing 0.8% moisture was prepared (Harden and Robison, 1920, 1922) without any appreciable loss in antiscorbutic potency. After 14 months at 29°C. (84.4"F.) the powder had darkened slightly and about 85% of the antiscorbutic potency was destroyed, while at room temperature a loss of 50% was observed after 2 years. Darkening at room temperature was not mentioned. 3. Other Juices Berry juices do not offer a very satisfactory medium for studying browning. The intense color of the naturally occurring pigments tends to mask changes due to the brown pigmbnts formed from colorless precursors. Moreover, the decomposition of the naturally occurring pigments may also
NONENZYMATIC BROWNING IN FRUIT PRODUCTB
333
result in the formation of brown substances which cannot be differentiated from the usual type of browning. The effect of temperature on changes in bottled processed strawberry, currant, and raspberry juices has been investigated by several workers (Pederson ef al., 1941; Beattie et al., 1943; Tressler et ab., 1943). Lovibond tintometers were used to determine the change in color which was characterized by a decrease in the red as well as the yellow components due to a destruction of the natural pigments. The authors pointed out that changes in Lovibond readings failed to show the actual degree of browning at the higher temperature. Losses in ascorbic acid were related to the decrease in red tintometer units. The ascorbic acid loss during one month at 32°C. (89.6"F.)was the same as that lost in 17 months at 1°C. (33.8"F.)
IV. THEEFFECTOF PROCESSING AND DRYINQTEMPERATURE ON BROWNING From the above discussion it is apparent that the browning reaction in fruit has a relatively high temperature coefficient. Accordingly, it might be predicted that the rate of browning is effected by the time and temperature of processing or dehydration. There is, in fact, considerable evidence to support this conclusion. In the concentration of orange juice (Hall, 1927) the importance of mild heat treatment during pasteurization and concentration is emphasized. A 30-minute pasteurization a t 65°C. (149°F.)was sufficient to cause noticeable darkening in concentrated orange juice. In another study (Tressler and Pederson, 1936) a comparison of grape juice pasteurized at 71.1", 82.2", and 97.1"C. (160°, 180" and 207°F.) for 20 minutes and stored in partly filled bottles revealed that color changes due to oxidation were more rapid when high pasteurization temperatures were used. Completely filled bottles, however, showed no great differences as a result of pasteurization, even after 9 months' storage. The pasteurization of strawberry, raspberry, and currant juices at temperatures varying from 73.9O-96.9"C. (165"-206"F.) resulted in slight differences in stability (Pederson et al., 1941). Lovibond color measurements showed a greater loss in red and yellow units a t the lower pasteurization temperatures; however, by vimal examination the juice pasteurized at higher temperatures appeared to have browned more. Flavor changes were less severe when lower temperatures were used. Other data (Beattie et al., 1943) showed that differences in pasteurization temperatures [over a range of 74"-93"C. (165.2"-199.4"F.)] had no apparent effect on the rate of deterioration of bottled currant and strawberry juice. The effect of drying temperatures on the deterioration of apples was investigated by Schrader et al. (1943). The temperatures of the first half
334
EARL R. STADTMAh'
of the drying period were maintained at 62.8", 79.4", 90.6") 93.3"C. (145"' 175") 190") and 200°F.). Drying was finished at 73.9"C. (165°F.) in all cases. Only those samples dried a t 93.3"C. (200°F.) showed any apparent browning during dehydration. Ascorbic acid losses and browning were determined on subsequent storage a t 2l.l0-32.2"C. (70°-900F.). After 5 months' storage, browning was greatest for the fruit dried at high temperature. The effect of drying at both high and low humidities was tested also and it was found that drying at a high humidity favored browning during storage. The effects of drying time and temperature on the rate of darkening of apricots and peaches have been investigated (Stadtman, Barker, Haas, and Mrak, 1945). Fruit was dried at 60" and 71°C. (140" and 159.8OF.) to various moisture levels. Drying temperature appeared to be of no great consequence provided the fruit was not dried to a moisture content less than 25%. Drying below this level, however, caused incipient damage in the fruit a t both temperatures, e.g., drying apricots from 25%-11% moisture at 60°C. (140°F.) decreased the storage life 10%; whereas drying a t 71°C. (159.8"F.) decreased the storage life 24%. These differences in darkening were not detectable immediately after drying but were manifested only on subsequent storage.
V. THEINFLUENCE OF MOISTURE ON
THE
RATEOF BROWNING
1 . Fruit Concentrates
It is generally agreed that the rate of browning in fruit juices and concentrates is increased as the concentration of solids is increased. Hall (1927) reported that the rate of darkening of orange concentrate increases with an increase in solids; however, no data were presented to substantiate this conclusion and the experimental conditions were not described. Cruess and Glenn (1930) observed an increase in the rate of darkening of grape sirup with increasing concentration (over a range of 67"-78" Balling). It is not clear from a description of the experiments under precisely what conditions the concentrates were prepared. It is apparent that, in studies of this kind, special precautions must be taken to prevent differences due to heat treatment; otherwise the effects of temperature and concentration cannot be distinguished. Harden and Robison (1920) dried orange juice under vacuum a t 4.4"C. (40°F.) to a thick sirup, and then in a desiccator to a hydroscopic solid containing 0.8% moisture. This material retained about 50% of its antiscorbutic potency after 2 years' storage a t room temperature. Unfortunately samplesat other moisture contents were not prepared for comparison.
NONENZYMATIC BROWNING IN FRUIT PRODUCTB
335
2. Dried Fruit
Contrary to the general conclusion drawn regarding the effect of concentration on browning of fruit juices, it was generally concluded by the early investigators that browning in dried cut fruits is decreased by decreasing the moisture content. Jewel1 (1937) emphasized the importance of maintaining low moisture levels in the storage of dried apricots. Wiegand et UI!. (1943) recommend low moisture levels for the storage of all cut fruits. It has been a general observation in industry that dried fruits placed in bin storage darken more rapidly if the moisture content is high (20-25%). Nichols and Reed (1931) stated that "Drying to approximately 10% moisture improved the color permanence of apples; apricots were unchanged; pears were poorer for the treatment." It should be emphasized! however, that in spite of this general conclusion that high moisture content is detrimental, there is very little experimental evidence in support of it in the literature. Culpepper and Caldwell (1927) studied the influence of humidity on the storage quality of dried apples. They suspended the fruit in muslin bags in storage chambers (bell jars of 11,000 ml. capacity) a t relative humidities of 0, 8.5, 18.8, 37.1, 47.7, 58.3, 70.4, 88.8 and loo%, and stored them at 25°C. (77'F.). Higher humidities favored a rapid rate of darkening. Samples at 0% humidity remained in good condition for over 3 years a t which time they contained 0 . 4 4 7 % moisture. Recent work by Stadtman, Barker, Mrak, and Mackinney (1945) have shown that the influence of moisture on darkening of dried apricots is greatly modified by the presence or absence of oxygen. Under anaerobic conditions a maximum rate of darkening was found to occur between 5 and 10% moisture (Fig. 2). Increasing the moisture content from 10-25Oj, caused a 15-30% increase in the storage life of apricots a t 37" and 49°C. (98.6"and 120.2"F.), in an oxygen-free atmosphere. As the fruit was exposed to increasing quantities of oxygen, however, the beneficial effect of high moisture became progressively smaller, and in the presence of very large amounts of oxygen (200 mg. 02/100 g. of fruit) the rate of deterioration was nearly the same for fruit at all moisture levels. The detrimental effect of high moisture when fruit is stored in the presence of large amounts of oxygen was shown to be due to a greater rate of oxygen uptake (Stadtman, Barker, Haas et ul., 1945). These results emphasize the importance of carefully controlling oxygen in studies on the influence of moisture in relation to the browning of apricots. Differences in the tightness of pack (ratio of oxygen to fruit) must be avoided. Unfortunately, the interdependence of oxygen and moisture in the deterioration of dried fruit
336
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PERCENT MOISTURE Fig. 2. Influence of moisture and oxygen on storage life a t 49°C. of Blenheim apricots containing 2800 p.p.m. SO*. Dry weight. (Stadtman, Barker, Haas et a2. 1945). The quantity of fruit (dry weight) per No. 2 can (585-ml. capacity) was varied aa follows: half-closed circles, 200 g. in N1; crossed-circles, 300 g. in air; closed circles, 200 g. in air; open circles, 75 g. in air. Quantities of oxygen refer to the amount originally present per 100 g. of dry fruit. Storage life = time required for the fruit to become so brown aa to be considered inedible. A short storage life, therefore, means a rapid rate of browning.
other than apricots does not appear to have been studied. It may be that, as with apricots, the detrimental effect of moisture on browning, so often observed, is actually due to an increase in the rate of oxygen uptake.
VI. THEINFLUENCE OF OXYGENON DETERIORATION 1. Citrus Juices
It is a generaIly accepted fact that oxygen has a pronounced effect on the rate of browning in orange juice. In early studies by Gore (1915) and McDermott (1916) itt was observed that color was more stable in the absence of oxygen. Matthew (1928) conducted a series of carefully controlled experiments to study the effect of oxygen on orange juice. Samples of fresh, filtered juice (100 ml.) in 120 ml. bottles were evacuated and the
NONENZYMATIC BROWNING IN FRUIT PRODUCTS
337
vacuum released with oxygen-free Na, COZ and Ha; this operation was repeated 5 times before finally sealing. Other samples were bottled under vacuum and in air. Storage was at 0", 15.6"-21.1", 26.7O-32.2"C. (32", 60"-70" and 80"-90"F.). The samples stored in oxygen-free gas atmospheres did not darken during 1 year a t 15.6"-21.loC. (60"-70"F.), but at 26.7O-32.2"C. (80"-90°F.) slight darkening occurred by the end of 1year. All samples stored in air darkened more rapidly at both temperatures] the rate being greater a t the higher temperature. Since the exclusion of oxygen did not completely prevent darkening at 26.7O-32.2"C. (80"-SOOF.), atmospheric oxygen may not be absolutely essential to this process. It should be pointed out, however, that although very careful precautions were apparently taken to exclude oxygen from the samples immediately prior to storage, it is not evident that similar care was taken during the initial extraction of the juice and preparation of the samples. Oxidation during this operation may account for the darkening eventually observed at the higher temperature. Moore et al. (1942b) were able to show that the rate of browning in pasteurized] bottled orange juice was correlated with the volume of air-filled head space; as the volume of air was increased the rate of browning was also increased (Table I). This oxidative effect was associated with constituents in the juice not removed by filtration or extraction with petroleum ether. The addition of ascorbic acid seemed to enhance the rate of browning caused by oxidation, whereas the addition of sucro8e or citric acid had no effect. There is considerable evidence that one important consequence of exposure to oxygen is the oxidation of ascorbic acid which in turn is believed to be in some way responsible for browning. This will be discussed more fully later. Joslyn, Marsh, and Morgan (1934) followed changes in reducing value (by iodine and indophenol titrations), vitamin C (animal assay), and browning in orange juice exposed to air. Large quantities of filtered Valencia and navel orange and unfiltered navel juice were stored in cotton plugged 19-1. carboys at room temperature. Sodium benzoate (0.2%) was added as a preservative. Small aliquots of the juice were withdrawn from time to time over a &day period and analyzed. A decrease in vitamin C, iodine- and indophenol-reducing values accompanied increased exposure to oxygen at room temperature. Browning, as measured by an increase in red Lovibond tintometer units, was parallel to the loss in vitamin C suggesting a possible relationship between the two processes. Additional experiments by JoslyQ and Marsh (1935) showed that the iodine-reducing value of fresh, filtered orange juice could be almost completely destroyed by shaking with oxygen a t 25°C. (77°F.) for only 8 hours, during which time no change in color occurred. On subsequent storage of
EARL R. STADTMAN
338
the oxygenated sample the rate of browning was almost identical with that of fresh untreated juice exposed to air in a cotton-stoppered bottle (1 1. juice in a 4 liter flask) (Table 11). Furthermore, browning occurred in oxygenated juice even in the complete absence of molecular oxygen, to the TABLEI Darkening of Orange Juice as Influenced by DifferentParts of the Orange, Omgen, and Light"
I Color index before and after 1 week's storage' +
Start
Cold 4.4"C. (40'F.)
Heat 48.9"C. (120'F.)
Light Heat 48.9"C. (120'F.)
made up to 1000 ml.-with material Head space -----indicated. 10 cc. i0 cc. !O cc. 50 cc. 10 CC 50 cc LO cc 50 cc. .__
--
1. Whole juice 2. Filtrate 3. Filtrate pulp and color bodies 4. Filtrate ground peel 5. Filtrate 324 mg. 1-ascorbic acid 6. Filtrate citric acid and sucrose (25 ml. each of saturated sol.) 7. Filtrate split seeds 8. Filtrate extracted with petroleum ether 9. Filtrate pulp, color bodies, ground peel, and split seeds
+
+ + + + +
64.5 66.5 67.0 55.0 66.0
64.5 66.5 67.0 56.0 66.5
65.0 64.0 66.0 53.5 64.5
63.5 59.5 63.5 55.5 60.0
51.0 46.0 51.0 39.5 50.0
-
32.0 28.0 36.5 29.0 20.5
58.5 49.0 51.O 40.5 50.0
28.5 29.5 27.8 23.5 19.0
69.6 68.5 67.5 65.5 50.5 26.0 54.0 40.5 66.5 66.5 63.5 61.0 48.0 31.O 45.5 27.0 66.5 64.5 65.0 63.5 54.0 26.5 52.5 28.5 59.5 60.0 57.5 56.0 51.5 29.0 49.5 31.5
--
Moore et al. (1942b). Color index = transmission of juice to which 50% by volume of acetone has been added, as measured by Evelyn colorimeter, using filter #420. Highest values correspond to least browning. a
b
same extent as in samples exposed to air. It was concluded that "primary products of oxidation, probably peroxides, are involved in browning." In this connection, it might be well to point out that darkening in dried fruit occurs even when they are stored under completely anaerobic conditions (Stadtman, Barker, Haas et al., 1945). This does not preclude the possibility that molecular oxygen may be required. As with orange juice a preliminary absorption of oxygen may result in the formation of compounds (possibly peroxides) which can undergo secondary reactions even
339
NONENZYMATIC BROWNING IN FRUIT PRODUCTS
in the absence of oxygen. To the present author's knowledge, no experiments have been made with dried fruit in which there has been a rigid exclusion of oxygen during drying and processing and packing for storage. It is possible that oxidation may occur during these preparatory procedures. A general discussion of the role of autooxidation in browning has been made by Joslyn (1936, 1941a, 1941bl.
Storage period, Days 0 2 3 6 6 10 12 19
'
TABLE I1 Browning of Oxidized and U n o d k d Orange Juice0
Units of color' Iodine titration of juice exposed to air 23.3 11.6 6.0 0.7
-
-
Juice exposed to air
I
Oxidized juice.
Yellow
Red
Yellow
Red
12 12 12 16 17 20 26 25
1.3 1.3 1.5 2.0 2.8 4.5 6.0 5.5
12 12 12 15 20 20 26 26
1.3 1.3 1.8 2.5 3.1 4.8 6.0 6.5
Joalyn and Marsh (1935). Lovibond color units. Juice waa shaken with oxygen for 15 hours prior to storage, during which time the iodine reducing value decreased to about 1.0. 4
b
A manometric technique was used by Eddy (1936) to measure the rate of oxygen uptake by fresh, filtered Valencia orange juice. A loss in indophenol-reducing value was correlated with oxygen consumption. After 18 hours of thorough aeration with oxygen, at 3OOC. (86OF.) the rate of oxidation of the juice had become almost nil, and the indophenol titration had dropped to almost zero (Table 111). At this time, 6.1 ml. of oxygen had been consumed/100 ml. of juice. The initial indophenol titration equivalent was only 4.4 ml. of oxygen/100 ml. of juice. Therefore, about 30% of the oxygen consumed was used for purposes other than the primary oxidation of indophenol-reducing substances. The, effect of metallic catalysts on the rate of oxidation was also investigated. Cupric ion increased the rate very greatly; zinc ion increased the rate slightly; and stannic and stannous ions had a slight inhibitory effect. These results are summariced in Table 111. Qualitative results obtained by Joslyn and Marsh (1934) are in agreement with these.
340
EARL R. STADTMAN
Pully and von Loesecke (1939) found that the rate of oxygen disappearance was greatly accelerated a t high temperatures. Of the oxygen initially present, 27% was lost from fresh orange juice in 2 hours a t room temperature while 95% was lost during 30 minutes a t 180°F. Results of experiments by Loeffler (1941) cast some doubt on the idea TABLEI11
Effectof Catalysts and Inhibitors on Rate of Absorption of Ozygen by Orange Juice=
Total Time Hours
0 2
+ 20
p.p.m. Cupric Ion
1
+
On 20 p.p.m. Zinc Ion
- -- --Oxygen
A'
-0 0.5 1 2 3 4 5 6 7 8 9 11 12 14 21 22 23 26 28
I
0
-
0.53 0.90 1.60 2.68 3.30 4.20 4.77 5.43
-
6.97 6.13
-
Bc
4.4 4.3 3.9 3.5 2.9 2.2 1.65 1.01 0.73
-
0.18
-
0.09
-
I
A'
Be
Ab
Bc
3.7 3.1 1.8 0.18
0 0.07 0.52 1.45 2.59 3.40 3.84 3.91
3.9 3.6 3.1 2.3 1.2 0.65 0.18
0.09
4.25
-
-
5.3
-
-
-
-
0.04
-
-
-
-
-
4.46
-
-
0.09 -
6.41
-
-
-
-
-
-
+ 20
p.p.m. Stannous Xon
+
0% Ascorbic acid preparation
-Be A' -
---
0 0.63 1.63 4.03 4.25 4.25
-
01
Bc
0
4.0
0
6.1
0 0.69 1.08 1.66 2.10 2.60 3.16 3.66
4.0 3.2 2.75 2.02 1.37 0.83 0.46 0.28
0.24 0.72 1.33
4.5
-
-
4.23 -
0.07 -
5.24 -
-
-
-
-
-
-
3.51 4.66 5.08
-
-
-
3.6
-
2.2
-
1.47
-
0.23
.0.47
0.07
.0.51 10.61
0.07
8.72
-
-
-
-
-
Eddy (1936). A = cc. oxygen (S.T.P.) absorbed per 100 ml. orange juice. e B = 2,6-dichlorophenolindophenol reducing value expressed as ml. oxygen per 100 ml. orange juice. 0
that oxygen is essential to darkening or ascorbic acid loss a t high storage temperatures. Orange juice obtained by hand reaming Valencia oranges was deaerated until it contained 0.1 ml. oxygen/100 ml. It was then flash pasteurized a t 105a-1100C. (221"-230°F.) and sealed into 12-or. bottles while in an atmosphere of steam. The bottles had 8-10 ml. head space and were evacuated to 4-8" of mercury. Samples were stored at 4", 15", and 35°C. (39.2", 59" and 95°F.) and a t room temperature. Little
NONENZYMATIC BROWNING IN FRUIT PRODUCTS
341
change in vitamin C or color occurred during 4 months’ storage at room temperature or below; however, darkening and a loss in vitamin C did occur after 1-2 months at 35°C. (95°F.). Calculations were made showing that the molltl loss of ascorbic acid during 5 months’ storage at 35°C. (95°F.)was 10 times as great as the molal loss of oxygen. On further storage, ascorbic acid continued to be lost even though no oxygen was present. Of interest in this connection are experiments carried out by Proctor (1943-1944) in which it was shown that browning of ascorbic acid-citric acid mixtures occurred just as rapidly when heated at 100°C. (212°F.)in the presence of oxygen aa when heated in tubes evacuated to 0.075 mm. of mercury and sealed. Thus there appears to be good evidence to indicate that ascorbic acid destruction may take place by either aerobic or anaerobic processes. Further consideration of the role of ascorbic acid in browning will be taken up later. 8. Other Juices
The influence of oxygen on the deterioration of other fruit juices has not been extensively investigated. Tressler and Pederson (1936)noted that deterioration of grape juice in bottles without head space was less rapid than with head space. Deterioration in the latter case was decreased by replacing the air with COS or steam before sealing. Juice stored at room temperature for 15 months in half-filled and completely filled bottles was analyaed; losses in protein, titratable acidity, sugar, tannin and “total astringency” were noted. The losses of protein and titratable acidity were greater in the half-filled bottles. The amount of sediment in the half-filled bottles was 2.5 times as great as in the completely filled bottles. Juice sealed in glaes tubes evacuated to 10 mm. mercury showed only slight changes after 20 months at room temperature whereas similar tubes filled half full and sealed without evacuation had deteriorated considerably after 3 months’ storage. Pederson et al. (1941)found that even after thorough deaeration, strawberry, raspberry, and currant juices deteriorated in color on storage. Beattie et al. (1943) found that raspberries, strawberries, and currants stored in partly filled bottles lost ascorbic acid and deteriorated in color more rapidly than samples stored in completely filled bottles. 3. Fruit Concentrates Cruess and Glenn (1930)observed that darkening of grape concentrates is more rapid in the presence than in the absence of oxygen but that it proceeds rapidly even when containers are evacuated to remove the dissolved oxygen. Similar observations are reported by Richert (1930a). In neither paper, however, are the conditions described under which the ob-
342
EARL R. STAD'l"
servations were made. Hall (1927) points out that oxidation is of minor importance in the darkening of orange concentrates.
4. Dried Fruits Many conflicting reports have been made concerning the effect of oxygen on the darkening of dried cut fruits. This lack of agreement is undoubtedly due to differences in the experimental conditions under which the observations were made. These will be discussed more fully later. Some of the first controlled experiments on cut fruits were made by Nichols and Reed (1931). They packed apples, apricots, and pears in vacuum-sealed glass jars and in air and stored them at 0", 21.1" and 37.8"C. (32", 70°, and 100°F.). The vacuum-packed samples darkened slightly less rapidly than the samples packed in air. In another experiment, they packed these same fruits in tin cans under vacuum, in hydrogen and in air, and in cardboard cartons. Samples were sent to Manila (P.I.) and to Singapore (F.M.S.) for storage. They were examined for changes in color and SO2 after various periods of time. These workers concluded that cartons protected the color of apricots as well as did cans, but that in the case of apples and pears SO2 was lost and the color deteriorated faster in the carton packs than in cans. Vacuum-packed cans gave the best protection and were better than cans filled with hydrogen and these in turn were better than air-packed cans. Cruess and Pancoast (1933) conducted experiments with apricots. They packed the fruit in 4-05. jars treated as follows: (a) jars containing dried fruit and air; (b) jars sealed under 25 inches of vacuum after 60 seconds at this vacuum; ( c ) jars vacuumized to 29 inches, then released with various gases (N2,COz,Hz, and 02).The fruit in jars filled with COz darkened much less rapidly than did fruit in other jars, while that in oxygen darkened most rapidly. Darkening was considerably retarded by nitrogen and hydrogen atmospheres. The temperature of storage was not stated; however, in an identical experiment at 46.1%. (115°F.) fruit was stored in air, COz, 0 2 , and vacuum. Again the fruit in 0 2 darkened most rapidly and that in COz least rapidly. An almost identical experiment was carried out by Bedford (1936b) who stored apricots at 40.6"C. (105'F.) in Nz,02,SO2,H2,He, air, and vacuum. A detrimental effect of packing in oxygen and a beneficial effect of packing in SO2 were observed, but otherwise the results were quite irregular. Nichols el al. (1936, 1938) using apricots containing 26.7% moisture and 3,560 p.p.m. SO2, stored samples packed in vacuum, air, N2, and C02 in 4-01;. "anchor-top" sealed jars, at - 17.8", Oo, 21.1", and 373°C. (0", 32", 70", and 100°F.) and observed changes in color and SOz content. Fruit was also packed in wax-paper cartons. They found the rate of darkening
NONENZYMATIC BROWNING IN FRUIT PRODUCT8
343
in the sealed jars to be nearly independent of the type of atmosphere in which the fruit was stored. Apricots in paper cups decreased in SO2 and deteriorated in color more rapidly than those packed in jars. A more extensive study of the effect of oxygen on darkening of dried apricots was carried out by Stadtman, Barker, Haas et al. (1945). Fundamentally, these experiments were not unlike those just described; however, the composition of the gas in the cans of dried fruit was determined by analysis after various periods of storage, and it, was thus possible to relate changes in darkening, COzproduction, and SOaloss to the quantity of oxygen reacting with the fruit. The influence of moisture, SO2 concentration, partial pressure of oxygen, and temperature on the rate of oxygen uptake by the fruit in relation to darkening was determined. A number of different experiments were made. The original papers should be con-
207
19
-
18
-
v)
> allP t
z -16w
k
J 15-
w t a a 14a 13-
12
-
10
0
20
40
60
80
100
120
140
OXYGEN UPTAKE Fig. 3. Relation between quantity of oxygen consumed and storage life in dried apricots (Stadtman, Barker, Haas et al., 1945). Oxygen uptake in mg. 0~/100 g. dry fruit. Upper line (closed circles) refers to apricots containing 5350 p.p.m, Son and 23.5% moisture. Values in % refer to initial partial pressure of oxygen in gas phase. Lower 2 lines (open circles) refer to apricots containing 2800 p.p.m. SO2. The quantity of oxygen consumed was varied by varying the ratio of 0 1 to fruit in the cans. Storage life is time in days required for the fruit to become so brown aa to be considered inedible.
344
EARL R. ETDTMAN
sulted for the experimental conditions as too much space would be required to discuss them all here. The results may be summarized as follows: (1) Oxygen increases the rate of darkening by an amount that is proportional to the quantity of oxygen taken up by the fruit. (Fig. 3.) Less than 15 mg. of oxygen/100 g. of dry fruit causes such a slight increase in darkening that it is hardly detectable, but larger amounts produce noticeable damage, and when an unlimited supply of oxygen is available, the storage life may be decreased by as much as 30% compared to vacuum pack (30mm. Hg). (2) The rate of oxygen consumption is about 10 times greater for fruit of 25% than for that at 10% moisture. Thus the detrimental effect of oxygen is much greater at the higher moisture level. (3) The initial rate of oxygen uptake is directly proportional to the partial pressure of oxygen. (4) Oxygen causes an increase in the rate of COz production but the increase is more than proport$nal to the increase in the rate of darkening. ( 5 ) With sulfured apricots there is no loss of SO2by conversion to sulfate under anaerobic storage conditions. The detrimental effect of oxygen at storage temperatures of 43.3"C. (llO°F.), or below, appears to be due to its action in decreasing the SOa level by oxidation to sulfate; however, only 30% or less of the oxygen consumed can be accounted for as sulfate. (The influence of oxygen on the rate of deterioration of unsulfured apricots has not been studied.) (6) The rate of oxygen uptake is independent of the SO2 concentration over a range of 0 to 8,000 p.p.m. (7) Temperature is the most important single factor controlling the rate of oxygen uptake; the rate is increased nearly 4 times for every 10°C. (18°F.) rise in temperature. From the above results, it is suggested that lack of agreement in observations on the influence of oxygen reported earlier (Nichols and Reed, 1931; Nichols et al., 1938) may be due to differences in one or both of the following experimental conditions: (1) The ratio of oxygen to fruit; if the fruit is tightly packed in a sealed container, the total quantity of oxygen present may be so small as to make it impossible to distinguish between aerobic and anaerobic packs. (2) Moisture content; the lower the moisture content, the smaller is the effect of oxygen (Fig. 2). Experiments in which the above factors are not adequately controlled obviously are incapable of strict interpretation. The Research Department of the Continental Can Company (1944a, 1944b) has compared the storage qualities of apple nuggets (0.8% moisture) and cranberries (2% moisture) when stored in air, Nz and C01. Changes in the composition of the gas were followed by analysis after
NONENZYMATIC BROWNING IN FRUIT PRODUCTS
345
various periods of storage. Gas-packed products were found to be superior in flavor and odor to those packed in air. However, so far as color, ascorbic acid, thiamine, and riboflavin content are concerned, there was little difference between air- and gas-packed samples. With apple nuggets sealed in air, only a slight decrease in the percentage of oxygen in the gas was observed even after 3 months' storage at 54.4"C. (130°F.). On the other hand, with cranberries nearly all of the oxygen originally present had been consumed. The rate of COz production was greatest in the cans containing oxygen. Unfortunately, these results are expressed only in terms of the percentages of COZand OZpresent in the gas, without any reference to the total volume of gas in the container. This mode of expressing results should be avoided because a strictly quantitative interpretation of the results is quite impossible. For example, if the container is very tightly packed with the fruit being studied, the gas volume-fruit ratio will be small, and a very small uptake of Oz/unit weight of product will produce very large changes in the percentages of the gas in the atmosphere. On the other hand, if the fruit is packed loosely so that the gas volume-fruit ratio is high, then a large unit uptake of O2may be reflected in a relatively small change in the percentage composition of the gas. Determinations of gas production or utilization should always be presented in terms of the absolute quantity of gas/unit weight of material studied. This is, of course, a very elementary rule, but unfortunately, often has been disregarded. On the basis of the results on apricots (StadtmaqBarker, Haas et at., 1945) a very low rate of oxygen uptake by apple nuggets would be expected because of the extremely low moisture content of the product. This may account for the absence of an appreciable oxygen effect in the experiments just described. It is of interest to note that Nichols and Reed (1931) working with sulfured dried apples at high moisture levels did observe a beneficial effect of packing in the absence of oxygen. The latter observation may, however, be due to the sulfur dioxide treatment rather than differencein moisture content, or perhaps, a combination of both factors. Such is the case with apricots where the chief effect of oxygen in aceelerating browning appears to be due to a lowering of the sulfur dioxide level by oxidation to sulfate (Stadtmen, Barker, Haas et al., 1945). All of this discussion brings UB to the conclusion that, for the most part, the investigations on the effects of oxygen are very inadequate. Several interrelated factors are obviously involved in the reaction, namely, moisture content, sulfur dioxide concentration, partial pressure of oxygen, temperature of storage, ascorbic acid content, and the total amount of oxygen available to react with the fruit. Most investigators have failed to exercise adequate controls over these variables. Consequently, a more satis-
346
EARL R. STADTMAN
factory understanding of the role of oxygen will have to await more careful experimentation.
VII. CHANGES IN CHEMICAL COMPOSITION WHICHACCOMPANY BROWNING 1. Carbon Dioxide Production
The browning of dried fruit and fruit concentrates is usually accompanied by a production of carbon dioxide. The development of "swells" in cans of sterile, darkened orange concentrate was shown to be caused by C02 formation (Hail, 1927). The rate of formation was nearly doubled by increasing the temperature 5.6"C. (10°F.). Studies by the Research Department of the Continental Can Company (1945) and of Curl el al. (1946) show that C02 from pasteurized orange concentrate is not of microTABLEIV
Gas Analysis of Swelled Cane of Concentrated Orange Juices.' of
of
of residual
0 The cans contained approximately 750 g. of orange concentrate (65" Brix) . The volume of gas measured at room temperature. b Curl el al. (1946).
biological origin. The latter investigators observed a temperature relationship similar to that found by Hall. They also noticed that browning and ascorbic acid losses paralleled C02 production. Greer (1944) states that "During storage there is a chemical breakdown of sugars which manifests itself by the evolution of C02 gas, darkening of the concentrate and a cooked or caramelized flavor in the reconstituted beverage." No evidence was offered to support the idea that a breakdown of sugars was actually responsible for the gas production. In the absence of such evidence, this conclusion should be regarded with suspicion. From data obtained by Curl et at. (1946) (Table IV) it can be calculated that only 37 ml. of C02 are produced/100 g. of orange concentrate (65" Brk) during atorage at 120°F. for 1 month. During this time the concentrate becomes very dark. The concentrate contains approximately 50% sugar, nearly 280 millimoles calculated as glucose. Considering the high degree
NONENZYMATIC BROWNING IN FRUIT PRODUCTS
347
of nonspecificity of the methods commonly used for the determination of sugar, the problem of proving that such small amounts of C02 are derived mainly from sugars is difficult. Experiments by Loeffler (1941) have demonstrated that the amount of CO, produced is increased by raising the storage temperature. The total quantity of COz produced during 5 months' storage at 35°C. was 10 times as great as the oxygen which had disappeared. The absolute amount of gas formed was rather small, 1.87 m1./100 ml. juice. Other investigators (Matthew, 1928; Eddy, 1936) were unable to detect the formation of COz in orange juice; however, this may be due to the fact that their experiments were of shorter duration, and COz production, being small, could probably not have been detected by the methods used. Nichols and Reed (1931) noticed that sufficient gas was produced in jars of dried apricots stored for 2 years a t 37.8"C. (100°F.) to cause the lids to be blown off. The composition of the gas was not determined. Stadtman, Barker, Haas et al. (1945) determined the quantity of C02produced by dried apricots under various conditions. The influence of temperature, sulfur dioxide concentration, oxygen pressure, and moisture content on the rate of carbon dioxide production was determined by sealing the fruit in hermetically sealed tin cans and following the gas changes during subsequent storage. The rate of COZproduction was found to increase about 4 times for every 10°C. (18°F.) rise in temperature over the range of 22'49°C. (71.6"-120.2"F.). (Fig. 1.) Carbon dioxide was produced almost as rapicUy under anaerobic conditions as in air; however, as the oxygen pressure was increased from 12-77% of an atmosphere there WM a gradual increase in the rate of COzformation. The rate at 77% was twice as great as the rate at 12%. The results indicate a general proportionality between anaerobically produced COZ and browning, whereas the increased production caused by aerobic oxidation was apparently unrelated to the browning. Sulfur dioxide retarded the rate of COZformation to about the same extent that it retarded discoloration. The total quantity of Cot produced during storage to the limit of edibility was found to be about 35-40 mg./ 100 g. dry fiuit. These relationships are illustrated in Table V. In the absence of oxygen a maximum rate of C02 production was observed at a moisture content of about 15%, but this decreased at higher or lower moisture levels (in the range 10-25%): Investigations by workers a t the Continental Can Company (1944a, 1944b) have revealed that apple nuggets produce small amounts of COz during storage while considerable amounts are produced in dried cranberries containing only 2% moisture. Carbon dioxide production in the cranberries was greater in samples canned in air than in samples stored in an atmosphere of nitrogen. Browning and losses in ascorbic acid occurred
l A R L R. BTADTMAN
348
simultaneously; however, the increased C02 production caused by storage in air as compared to storage in nitrogen was not associated with any significant effect on the rate of ascorbic acid loss or browning. These results are, therefore, qualitatively in agreement with those obtained on apricots. 8. Ascorbic Acid Destruction From the preceding discussion it is evident that browning in citrus fruit is always associated with a destruction of ascorbic acid (Joslyn and Marsh, 1935; Joslyn et al., 1934; Loeffler, 1941; Moore et al., 1942a; Stephens el d., TABInjeuence o j Suljur %xi&
Lot No.*
6
3
Initial SO1 level, p.p.m.
V
on Carbon f i d e Produclion in Bied Apricolsa
Rate of COI production, mg./100 g. dry fruit/day
Total COIduring storage life, mg./lOO g. dry fruit*
49°C.
36.7"C.
49°C.
36.7"C.
2180 4070 6700
3.6 3.0 2.7
-
34 36 36
-
6360
2.3 1.6
0.47 0.31
36 37
37 37
9600
-
-
1942; Joslyn, 1941a; Chaves, 1945; and Curl et al., 1946). Thia fact has led to the suggestion that ascorbic acid is involved in the browning reaction in one of two ways: (1) It may act as an antioxidant, being oxidiwd in preference to other substances present in the juice which upon oxidation yield dark compounds or precursors of dark compounds. (2) The oxidation products of ascorbic acid may themselves be the actual precursors of dark compounds. In recent years a number of investigations have been made to elucidate the role of ascorbic acid in browning. Studies on the oxidation of orange juice in the presence and absence of SO2 were made by Hamburger and Joslyn (1941). Reduced ascorbic acid was determined by means of a double iodine titration method. This method consists of an initial iodine titration to oxidize all free-reducing substances present, followed by HnS treatment, and Snally a second iodine titration to determine the total
NONENZYMATIC BROWNING I N FRUIT PRODUCTS
349
ascorbic acid. In fresh orange juice the second iodine titration gives a reducing value that is probably due mainly to ascorbic acid; however, in partially oxidized juice the H S treatment produces substances which are oxidized by iodine, but not by indophenol. Filtered orange juice was treated with 0, 250, and 500 p.p.m. SOa and stored in air at room temperature. There was a rapid decrease in reduced ascorbic acid, the rate being slower in the SO2 treated samples. The total ascorbic acid (second iodine reducing value) decreased to about half its original value and then became constant, at which time the reduced ascorbic acid content had become negligible. Thus SO2 caused a decided lag in the loss of dehydroascorbic acid; the Iag was greater with 500 p.p.m. SO2 than with 250 p.p.m. 502. Browning was determined in the above-mentioned work by spectrophotometric analysis. No browning occurred in juices containing 500 p.p.m. SOa during the 90-day storage period; browning was greatly retarded by 250 p.p.m. SO2. In juice con taining no SO2, browning occurred at an appreciable rate after a slight lag period, reaching a constant value after 20-30 days a t room temperature when all the reduced ascorbic acid had disappeared. The authors concluded : "Darkening did not occur until all the vitamin C was in the dehydro or some other form and when there were no more readily oxidizable substances, such as sulfur dioxide, present in the juice." This conclusion is not in agreement with their experimental data which show that appreciable darkening occurred in 5 days at which time the reduced ascorbic acid had decreased to only about one-half the initial value. The latter observation is more consistent with that made by other investigators (Joslyn and Marsh, 1935; Joslyn et al., 1934; Loeffler, 1941; and Curl et al., 1946). Moore et al. (1942b) obtained convincing evidence that ascorbic acid is involved in the browning of orange juice. Data from their paper are presented in Table VI which show that the addition of ascorbic acid to orange juice results in a marked increase in the rate of browning when the juice is stored in the presence of oxygen. Similar results were obtained by Beattie et al. (1943) when ascorbic acid (50 mg./100 ml.) was added to strawberry juice. These results would indicate that ascorbic acid is effective not as an antioxidant as implied by Hamburger and Joslyn (1941) but rather as an intermediate in the browning reaction; otherwise the addition of ascorbic acid should retard rather than accelerate browning. The addition of 50 mg. of ascorbic acid/100 ml. to apple, cranberry and grape juices has been reported to decrease the rate of browning when these juices are stored a t 21.lo-26.7"C. (70"-80"F.) (Esselen et al., 1946). Browning was determined by spectrophotometric analysis over a range of wave lengths (350-800 mp). The results indicated that a bleaching of the
350
EARL R. BTADl"'
juices occurred immtdiately following the addition of the ascorbic acid and it is not certain how much of the alleged decrease in rate of browning on storage was the result of this initial bleaching action. In any event these observations are not necessarily contradictory to the results obtained on orange juice since the former were made on juices which had been "vaporvacuum" packed and were, therefore, stored under relatively anaerobic conditions. As had been pointed out, browning in orange juice is apparently associated with a preliminary oxidation of the ascorbic acid with molecular oxygen (Tables I, 11,and 111). Stephens et al. (1942) studied the effect of temperature -20.6"-37.8"C. (-5"-100°F.) on the stability of ascorbic acid in concentrated citrus juice. TABLEI VI Egect of Ascorbic Acid on the Browning of Orange Juice. Filtered Valencia orange juice pasteurized at 71.1"C. for 30 minutes and bottled in 8-or. bottles with 100 ml. headspace containing air ~~
Color index after 1 week's storage at temperature indicatedb
Sample Juice Juice Juice
+ 64.8 mg. d-glucoascorbic acid per bottle + 64.8 mg. d-ieoascorbic acid per bottle
I
4.4"C. 77.5 72.0 67.0
I
48.9"C. 42.5 21 .o 12.0
Moore et al. (1942b), Color index = transmission of juice to which 50% by volume of acetone has been added, m measured by Evelyn colorimeter, using a filter #420. Highest values correspond to least browning.
The effect of storage temperature on the rate of darkening and stability of flavor was found to be roughly proportional to its effect on ascorbic acid stability. This observation was confirmed by Curl et al. (1946). These investigators also observed a parallel effect with respect to COaproduction and thereby contributed support to the suggestion that COz formation in these products is derived from a breakdown of ascorbic acid (Joslyn et al., 1934; Nelson et al., 1933). It should be pointed out, however, that the C02 production reported by Curl et al. (1946) was measured by following pressure changes in cans during storage, rather than by measuring absolute quantities of the gas. The molar ratio of COZproduced to ascorbic acid lost is, therefore, not obtainable. Loeffler (1941), found that the quantity of COz produced in canned orange juice during 5 months' storage a t 35°C. (95°F.) was almost equiv-
NONENZYMATIC BROWNING IN FRUIT PRODUCTS
351
alent to the quantity of ascorbic acid lost. The significance of these findings is questioned, however, on the basis that the quantities involved were very small (less than 0.1 millimole/100 ml. juice). Experiments to determine the effectof added ascorbic acid on the rate of C02 formation would probably yield useful information regarding the validity of the conclusion that ascorbic acid is the COz-forming substance. Of some interest in this connection are studies carried out by Proctor (1943-1944) in which the evolution of C02 from citric acid-ascorbic acid solutions (containing approximately 500 mg. of ascorbic acid in 5 ml. of a 50% citric acid solution) was determined by means of a Warburg respiration apparatus. The temperature was held at 60°C. (140°F.) and the determinations lasted from 4-7 hours. The molecular ratio of COz evolved to ascorbic acid lost was about 0.74. Noteworthy is the finding of Koppanyi et al. (1945) that dehydroascorbic acid reacts very rapidly with a-amino acids to produce strongly colored (reddish to brown) complexes. The color reaction was apparently specific for the oxidized form of ascorbic acid. In view of the fact that browning in citrus products is preceded by an oxidation of ascorbic acid, the above observation suggests a possible mechanism for the browning of these products. The need for investigations along this line is indicated. While considerable evidence favors the hypothesis that ascorbic acid is important in the browning of citrus products, there is little, if any, such evidence in the case of the dried fruits. In apricots, peaches, and dried apples the ascorbic acid is present usually in negligible amounts by the time the product is processed and ready for packaging (Haas and Mrak, 1945). It must not be concluded, however, that ascorbic acid is not of fundamental importance to the browning of these products also. The highly aerobic conditions under which dried fruits are prepared may leave them in a state which corresponds to the so-called “oxidized-juice” state described by Joslyn and Marsh (1935) (Table 11). This would account for the lack of any appreciable oxygen effect on subsequent storage of the fruit. A thorough investigation of the influence of ascorbic acid on the browning of fruit products other than citrus would be desirable, particularly with fruit which has been dried without the usual losses in ascorbic acid. 3. Changes in Nitrogen Constituents Hall (1927) and Wilson (1928) were probably the first to suggest the possibility that the Maillard reaction was responsible for the darkening in citrus products. According to Hall it waa definitely established that the amino nitrogen content of orange concentrates steadily decreases in storage and may drop to zero. The decrease in samples of varying N con-
352
EARL R. STADTMAN
centration is practically the same when gas bubbles (COJ first appear. He states that: “Roughly within a variety, the life of a concentrated juice, other things being equal, will be inversely proportional to its amino N concentration. This is true down to an amino N concentration of about 0.175% in Valencia juice or 0.12% in navel juice which corresponds to approximately a 3 to 1 concentration of these juices.” Apparently the amino compounds of navel orange juice are more reactive than those of Valencia orange juice when in similar concentrations. The concentration of amide nitrogen in orange juice remains practically constant in storage and, therefore, does not appear to be involved in darkening. The above observations are reported in a summary of work done by Hall and associates (1927), but none of the experimental details are described. These observations should be confirmed since the implications are of considerable importance to the problem under discussion. Katz and Mackinney (1943) determined the amino-N content of dried apricots after various periods of storage at 48.9”C. (120’F.). In the method used, 10 g. samples of ground fruit were extracted by refluxing for 3-4 hours with 200 ml. of 5oY0 acetone and also with water. Aliquots were taken for amino-N determination using the Van Slyke method. After 12 days at 48.9OC. (120°F.) the fruit had darkened considerably, the water extractable amino-N had decreased about 20Y0, and the 50% acetone extractable amino-N had decreased 43%. Weast and Mackinney (1941) found that nitrogen partition was quite different in darkened than in undarkened apricots. A lower percentage of amide, aspartic, and glutamic acid nitrogen, and a greater percentage of humin nitrogen was observed in darkened as compared to undarkened samples. There is some doubt as to the significance of these results, however, since t.he darkened apricots were from the 1932 fruit crop while the undarkened samples were from the 1940 crop. Contrary to the observations on orange concentrates and dried apricots, no changes in amino nitrogen have been observed during the darkening of orange juice. Joslyn and Marsh (1935) followed changes in Valencia and navel orange juice by means of the formol titration and also by the Van Slyke method. Practically no change in amino-N could be detected even after 126 days’ storage a t room temperature when the juice had become very dark brown in color. It was pointed out that hydrolysis of a soluble polypeptide might maintain a fairly constant amino-N content even though the substances containing amino groups were involved in darkening. It would seem rather unlikely, however, that the rate of amino-N formation by such a process would exactly parallel the loss of amino acids through darking. Loeffler (1941) determined the amino-N concentration in orange juice by
NONENZYMATIC B R O W I N Q IN FRUIT PRODUCTS
353
means of formol titration. No correlation between darkening and changes in amino-N was found in samples stored a t temperatures varying from - 18"C.-35"C. (-0.4O-95'F.). If anything, there appeared to be a slight increase in amino nitrogen during storage. It should be pointed out that the author made an error with respect to the factor used to calculate the amino-N content from the formol titration; accordingly, the absolute values for amino-N are incorrect. Nelson et aE. (1933) determined the distribution of nitrogen in various fractions of darkened and undarkened, filtered orange juice by the use of various precipitating agents. The agents used were Ba(OH)2, lead acetate, Neuberg's reagent, and phosphotungstic acid. Ammonia-N, amide-N, amino-N and total-N were determined for each fraction. Stachydrine, arginine, choline, asparagine, and aspartic acid were actually identified. Mixtures of amino acids were obtained from which no definite compound could be isolated. The darkened juice contained about twice as much soluble nitrogen as the fresh juice. No nitrogenous constituents were found in the darkened juice which had not been identified in the fresh juice. There was, however, a considerable increase in the arginine content with darkening of the juice; no change in the concentration of ammonia-N occurred. Their results do not rule out the possibility that nitrogen compounds are involved in the browning of orange juice, but they do indicate that browning can occur without the transformation of large amounts of these substances. Bedford (1936a, 1937) followed changes in various nitrogen fractions of concentrated aqueous extracts of apricots during storage at 40°C. (104'F.I. Total-N, ammonia-N, amino-N, and amide-N were determined. Two separate methods were used: (1) that of the Committee on Analysis of Plant Physiologists; and (2) that of Muth and Malsch. The data obtained by the f is t method showed slight, progressive decreases in amino and amide nitrogen on storage. After 8 weeks the amino-N had decreased 14y0 and the amide-N had decreased ISYO. The ammonia-N remained constant. Analysis by the method of Muth and Malsch revealed no significant changes in any of the nitrogen fractions. In view of the 1att.er results, it was concluded that the changes observed by the first procedure were probably within the experimental error in the method. There does not appear to be any justification for this reasoning because the magnitude of the experimental error was not actually determined in either procedure and, therefore, the results obtained by the first method may be as significant. Unfortunately, in the published report (1936a) only data obtained by the second method were given. In light of the above considerations these data should not be taken as conclusive evidence that no changes in active nitrogen groups occur.
354
EARL R. STADTMAN
The Eflect of Adding Nitrogen Compounds to Fruit. The effect of adding various nitrogen compounds to fruit juices and sirups has been studied. Matthew (1928) added asparagine in amounts varying from 0.014.1 g./ 100 ml. to orange juice with and without additions of 0.5 g. of glucose and citric acid. The effect of urea and casein was also studied. All samples were pasteurized a t 79.4"C. (175°F.) for 5 minutes and stored in air and in nitrogen at 26.7O-32.2"C. (80"-90°F.) for 1 year. Darkening in treated and untreated juice occurred at the same rate and to the same extent. Joslyn and Marsh (1935) added a wide variety of nitrogenous substances to Valencia and navel juice in amounts to give 0.1% nitrogen in the juice. The samples were stored at room temperature. For controls, similar amounts of the compounds were added to 10% invert sugar solutions containing 1% citric acid. Aniline and tryptophane caused immediate browning in the controls; butylamine caused a slight yellowing. Evacuation and vacuum packing had no effect on the reactions. The addition of all nitrogen compounds tested increased the rate of browning of the orange juice; the effect varied greatly with the different compounds. Moreover, the order of effectiveness of the various compounds in producing browning was different for the navel than for the Valencia juice. The results are, therefore, not subject to a simple interpretation. Richert (1930b) showed that the addition of ammonium salts was part.icularly effective in hastening darkening of grape concentrates. The addition of 0.2% ammonium tartrate caused the concentrates to darken about 10 times faster than normally.
4. Changes i n Sugar Concentration Following the suggestion that the Maillard reaction was responsible for browning (Wilson, 1928) a number of investigators have attempted to correlate browning with changes in reducing sugars. Hall (1927), in a summary of work done on darkening in orange concentrates, states that slight decreases in reducing sugar during storage have been observed. He states, "It has further been shown that the presence of reducing sugar is necessary for the darkening of concentrates." Unfortunately, the data from which these conclusions are drawn are not given and, therefore, cannot be evaluated. Obviously, the term "reducing sugar'' is meaningless unless the conditions of measurement are specified. Curl e2 al. (1946) have verified the conclusion that slight losses in reducing value (as determined by the Lane-Eynon method) do occur during the storage of orange concentrates. Data from their experiments are presented in Table VII. Other data (not given in the table) show that these losses in reducing value are roughly parallel to changes in color. Bedford (1936, 1937) reported that no measurable changes in sugar
TABLEVII Average Analysis of Stozed Concentrated Orange Juice Samplesa ~
~~
~
~
Storage temperature Duration of storage Initial 3 mo. Vacuum (in.) or pressure (Ibs.) llv llv Brix (Calc. to ZO'C.) 64.2 63.9 3.46 PH Ascorbic acid (mg./g.) 2.03 2.05 Total acid (as % anh. citric acid) 4.97 5.03 Reducing sugars (as % invert) 24.3 24.5 Total sugars (as % invert) 50.7 50.6 Nonreducing sugars (as yosucrose) 25.0 24.9 101 yo Retention ascorbic acid yo Retention total sugars 100.3 yo Retention sucrose 99.7 a
Curl et d.(1946).
~
~~
40''F. 80°F. 6 mo. 9 mo. 12 mo. 3 mo. 6 mo. 9 mo. 12 mo. 12V 1l v 5v llv 3P 6P 9P 64.0 64.0 64.1 64.0 64.2 64.3 64.5 2.02 2.04 1.97 1.69 0.91 0.44 0.16 5.06 5.05 5.03 5.01 5.00 5.00 4.88 21.4 24.7 30.4 24.5 35.5 38.1 40.5 50.5 50.7 60.6 50.6 50.1 50.0 50.0 24.8 24.7 19.1 14.0 24.8 11.9 9.1 100 97 101 83 45 21 9 99.9 100.0 100.2 100.0 99.1 98.8 98.9 98.9 99.4 99.2 76.5 55.8 45.4 36.3
95°F. 3 mo. 6 mo. 9P 14P 64.3 64.3 0.32 0.11 4.99 5.00 41.6 45.6 50.2- 48.8 3.1 8.2 15 5 99.2 96.6 32.8 12.4
120°F.
dass 16P 64.2 3.52 0.14 5.03 46.7 49.4 2.5 7 97.6 10.0
356
EARL R. BTADl"
content occurred during the browning of concentrated apricot extracts. Since important experimental details are not presented this conclusion is difficult to evaluate. The sugar content was determined by the method of Lathrop and Holmes but the method of preparing the samples prior to estimation of the reducing value is not described. It should be emphasized that nearly all methods available for the determination of reducing sugars are nonspecific. Therefore, the failure to observe a change in reducing value during storage cannot be interpreted as meaning that no changes in the sugar concentration have occurred. Decomposition of sugars need not be associated with a loss in reducing property. Moreover, there is always the possibility that losees in reducing value resulting from the decomposition of sugars may be compensated for by the formation of reducing substances derived from other constituents of the fruit. For example, Weast and Mackinney (1941) have isolated brown pigments from darkened apricots which were shown to have reducing values of the same order of magnitude as glucose. An important consideration also is the possibility that relatively small chemical changes are required to produce brown pigments of intense color; thus only small amounts of pigment could cause extensive discoloration. If this is the case, then the changes in reducing sugars, or amino nitrogen, necessary to produce large changes in color might be so small as not to be detectable by the methods ordinarily used. Noteworthy in this connection are the following facts: (1) Small amounts of ascorbic acid have a significant influence on the rate of browning of orange juice (Moore et al., 1942b);(2) COz production is apparently associated with browning yet the absolute quanties of COZ evolution corresponding to large changes in color are very small (Stadtman, Barker, Haas et al., 1945; Curl et al., 1946). (3) The addition of only very minute amounts of furfuraldehyde to apricot sirups will cause a great acceleration in the rate of browning (Stadtman et al., 1946). Evidence in support of the idea that extensive chemical changes are responsible for browning was obtained by Weast and Mackinney (1941) who found that brown pigments isolated from darkened apricots represented 5-7% of the total dry weight of the fruit. There is considerable doubt, however, that the material isolated was pure pigment. I n fact it was obtained as an amorphous precipitate; nearly all attempts to characterize it chemically were unsuccessful. The Effect 01Removing Sugars by Fermentation. I n order to see if reducing sugars were involved in browning of orange juice, Joslyn and Marsh (1935) studied the effect of their removal by fermentation. The treatments were as follows: unfermented juice plus 0.2% sodium benzoate; fermented juice; fermented juice dealcoholizedby vacuum distillation; fermented juice dealcoholized by atmospheric distillation; fermented juice refluxed for 30
NONENZYMATIC BROWNING IN FRUIT PRODUCTS
357
minutes; benzoated juice refluxed for 30 minutes; fermented juice plus 0.2% sodium fluoride; benzoated juice plus 5% alcohol; benzoated juice plus yeast juice. 1300 ml. portions of each sample were stored in cottonstoppered 4-1. bottles at room temperature. All samples darkened a t about the same rate. Fermentation did have a stabilizing influence on vitamin C as determined by iodine titration. Just how completely fermentation removed the sugars is not evident from the data. The juice after fermentation, however, contained 5% alcohol by volume, thus indicating that most, if not all, of the sugar was removed. Fermentation of apricot sirups resulted in almost complete removal of the reducing sugars (as determined by the Hassid ceric sulfate procedure) yet the rate of browning was decreased to only about one-half the rate in unfermented samples. The addition of fructose and glucose to fermented sirups in amounts equal to the sugar lost by fermentation, resulted in a restoration of the normal browning rate. (Stadtman et al., 1946). These results indicate that sugar may not be involved in the darkening of orange juice, and that probably only part of the browning in apricots involves sugar reactions. 6. The Formation of Furfuraldehydes Sugars and sugar acids decompose in acid solutions to form furfuraldehydes. These unsaturated aldehydes may polymerize and yield brown, resinous compounds. It has, therefore, been speculated that browning in fruit is due partly to this process (Matlack and Sando, 1933;Joslyn, 1941). Recent experiments with apricots have verified this contention (Stadtman et al., 1946). Spectrophotometric analysis of darkened apricot extracts showed the development of a characteristic absorption band, having an absorption maximum at 285 mp and a minimum at 245 mp. The substances responsible for this absorption were extracted with ether. Furfural, and hydroxymethylfurfural were both positively identified as constituents of the extract (Wahab, 1946). That furfuraldehydes are actually involved in browning was evidenced by the fact that apricot sirups continuously extracted with ethyl acetate (to remove the furfurals as they were formed) did not darken during storage at 57°C. (134.6"F.);whereaa darkening proceeded rapidly as soon aa such extraction was discontinued (Stadtman et d., 1946). The ethyl acetate extracts were found to contain a number of carbonyl compounds, among which furfural and hydroxymethylfurfural were identified. Further evidence that furfuraldehydes are involved in browning was obtained from the observation that the addition of furfural to apricot sirups in concentrations of O.l-l.O% caused a marked increase in the rate of browning. These results suggest the obvious conclusion that browning
358
EARL R. STADTMAN
is preceded by an acid decomposition of sugar to furfuraldehydes. Removal of the sugar by fermentation, however, decreased the rate of browning in apricot sirups to only about half the original rate (Stadtman et al., 1946) and had no effect on the browning of orange juice (Joslyn and Marsh, 1935). This means that compounds other than fermentable sugars are involved. These findings do not exclude the possibility that furfuraldehydes are intermediates in the fermented sirups since uronic acids and ascorbic acid and related compounds such as reductic acid readily decompose in acid solutions to form furfuraldehydes and COZ (Isbell, 1944). The latter reaction may account for the involvement of ascorbic acid in the browning of citrus products. It would also explain the production of COz usually associated with browning (see section under COZ production and ascorbic acid, p. 346). There is in fact some direct evidence that the latter is actually the case. Proctor (194344) found that browning in citric acid-ascorbic acid mixtures was accompanied by the production of furfural and COz. The production of COZwas 74% of the amount theoretically expected by a conversion of the ascorbic acid lost into furfural. The rate of browning was increased by increasing the concentration of ascorbic acid over a range of 0-500 mg./lO ml. of citric acid solution (50% by weight); it was also increased by increasing the concentration of citric acid over a range of 1-507- (pH range, 2.42-1.07). Malic, tartaric, and oxalic acids were found to catalyze darkening of ascorbic acid solutions. No attempt was made to correlate browning with pH. Browning under essentially anaerobic conditions (pressure, 0.074 mm. mercury) was as rapid as in air. The latter observation is of considerable interest in view of the fact that the polymerization of pure furfural to form dark compounds is an auto-oxidative process which requires the presence of molecular oxygen or peroxides (Dunlop et al., 1946). Spectrophotometric analysis of the pigments produced by browning of pure furfuraldehyde solutions and of the browning induced by the addition of furfuraldehyde to apricot sirups indicated that the two types of browning are not identical (Stadtman et al., 1946). It has been shown that furfural will react with amino acids to form huminlike substances (Dowel1 and Menaue, 1919). The latter reaction may be analogous to that occurring in fruit. It would account for the high nitrogen content of the black pigments formed. Whether oxygen is essential t o the condensation of furfural and amino acids has not been determined. The hypothesis that browning in orange juice is due to a breakdown of ascorbic acid to furfural is subject to criticism on the basis of the fact that the ascorbic acid is apparently oxidized prior to its utilization in a browning reaction (Joslyn and Marsh, 1935; Moore et al., 1942b; Hamburger and Joslyn, 1941). It has been reported (Roe, 1936) that furfural is produced
NONENZYMATIC BROWNING IN FRUIT PRODUCTS
359
from reduced ascorbic acid only. The oxidized compound, presumably dehydroascorbic acid, which can be reduced reversibly to ascorbic acid by treating with ZnCh in acid solution does not yield furfural. Results by Borsook et al. (1937) led them to believe that the ZnClzreducible compound described by Roe is not all dehydroascorbic acid. In any event, until such time as the production of furfural in orange juice has been definitely established, the above hypothesis should not be regarded very seriously. For that matter, it still remains to be shown to what extent furfural accounts for the browning in other products. Although furfurals were positively identified in apricots during storage, numerous other carbonyl compounds, as yet unidentified, were also formed (Stadtman and Stadtman, 1946; Wahab, 1946);these may play equally important roles in the browning of this product. 6 . The Nature of the Brown Pigments Produced
Comparatively little work has been done on the dark compound. Joslyn and Marsh (1935) found that an amorphous brown pigment could be isolated from darkened orange juice by precipitation with neutral lead acetate, followed by deleading, concentrating, and reprecipitating with alcohol and acetone. The material was insoluble in ethyl ether, petroleum ether, benzene, toluene, and practically insoluble in strong ethanol, methanol, or ethyl acetate. Tests for phenolic residues and aromatic nuclei were negative. The ash contents of pigments obtained from benzoated browned Valencia orange juice, benzoated browned navel juice and old Valencia concentrate, respectively, were as follows: 28.4, 18.1 and 8.0%. On an ash-free basis, the respective elementary compositions were as follows: (in yo)carbon 40.6, 41.8, 46.0; hydrogen 5.0, 4.15, 4.8; and nitrogen 2.44, 2.44,2.22. In view of the high ash contents, the purity of the precipitates is questionable. Weast and Mackinney (1941) also found that the dark compounds formed in dried apricots could be precipitated by neutral lead acetate, by high concentrations of alcohol or acetone and by increasing the acidity. These workers were able to obtain a dark brown precipitate which was ash-free and which, after repeated reprecipitation, did not change in composition. The dark material represented 5-7% of the total dry weight of the apricot. The following observations were made on it: (1) The elementary composition was carbon 53.2%; hydrogen, 5.0%; nitrogen, 3.26%; which corresponds to an empirical formula, ClrHlaOeN. (2) 98% of the nitrogen was recovered in the humin fraction. (3) Protein tests were negative. (4) Attempts a t acetylation were unsuccessful. (5) The ash-free material was virtually insoluble in water, but was solu-
360
EARL R. STMTMAN
ble in dilute alkali and in aqueous sodium acetate. I t was practically insoluble in concentrated hydrochloric acid, camphor, dioxane, and pyridine, and in all other organic solvents tried (not listed). (6) It was bleached by alkaline hypochlorite solution. The chlorine uptake was equivalent to 0.37 ml. of 0.1 N thiosulfate/mg. (7) It had a reducing value to ferricyanide 0.87-1 (glucose equivalent). (8) Glass electrode titrations indicated the absence of any buffer effect from pH 11-2; precipitation occurred at pH 2.6. (9) A weakly basic solution, when viewed through the ultramicroscope, showed considerable light scattering indicating the presence of patticles of colloidal size. VIII. TEE USE OF INHIBITORS TO DELAYBROWNING 1. Suvur Dioxide
The search for processes to prevent the deterioration of dried fruit and fruit juices is probably as old as the fruit industry itself. Sulfur dioxide has proven to be by far the most effective inhibitor tried and is widely used commercially. Numerous investigations have been made t o ascertain the influence of sulfur dioxide on browning in dried fruit (Quinn, 1926; Anderssen, 1929; Cameron et al., 1929; Jewell, 1927, 1937; Nichols and Reed, 1931; Nichols e l al., 1938; Fisher et al., 1942; Mrak et al., 1942; Stadtman et al., 1945). Most of the earlier investigations were qualitative and, except for the general conclusion that a8 the sulfur dioxide level is increased the rate of browning is decreased, the results are not particularly enlightening. It is also a commonly observed fact that the sulfur dioxide content of dried fruit steadily declines on storage (Nichols and Reed, 1931; Roleson and Nichols, 1933; Nichols et al., 1938; Mrak, 1943; Sorber, 1944; and Stadtman et al., 1945). Similar observations have been made on orange juice stored in air (Hamburger and Joslyn, 1941). The most extensive studies of the effect of SOt on the darkening of fruit have been made on dried apricots (Stadtman, Barker, Mrak, and Mackinney, 1945). The role of temperature, sulfur dioxide concentration, oxygen, and moisture, as factors affecting the influence of SOz on darkening, was thoroughly investigated. The important conclusions may be summarized as follows : (1) The rate of darkening is inversely proportional to the sulfur dioxide concentration over a range of 1000-8000 p.p.m. (Fig. 4). (2) The rate of Cot production is inversely proportional to the sulfur dioxide concentration (Table V). (3) The rate of SO2 loss increases with increasing SO2 concentration. Under anaerobic storage conditions, the logarithm of the rate is nearly a
361
NONINZYMATIC BROWNINQ IN FRUIT PRODUCTS
20
I
18-
18
-
v)
+l4-
a
0
z
-12 W
!k
A WIO (3
a a
-
$6-
64-
2' 0 0
1
2
3
RIM.
4
SOe x
5
6
7
6
IO+'
Fig. 4. Influence of initial 802 level on storage life of dehydrated Blenheim apricote at 49°C. (Stadtman, Barker, Mrak, and Mackinney, 1946).
linear function of storage time. Thus the loss of SO2 appears to be a first order reaction. (4) The rate of SOaloss is increased nearly 4 times for each 10°C. (18°F.) rise in temperature. The logarithm of the relative rate is directly proportional to the reciprocal of the absolute temperature (Fig. 1). ( 5 ) Sulfur dioxide is lost from fruit stored in the absence of oxygen. The rate of loss in air is only slightly greater than under the anaerobic conditions, but it is progressively increased by increasing the partial pressure of oxygen. (6) No sulfate is formed in the absence of oxygen; however, the increased
362
EARL R. B T A D W N
rate of loss of SO2from fruit stored in oxygen is entirely due to its oxidation to sulfate (Table VIII). (7) Although the loss of SO2is increased by storing fruit in air, the rate of oxygen uptake is independent of the SO2 concentration over a range of 0-8000 p.p.m. It was once thought that when the concentration of SO2was maintained above some critical level (500-1000 pap.m.for dried fruit) the fruit would not darken at all (Morgan el al., 1931;Nichols and Reed, 1931;Hamburger TABLE VIII Influence of Ozygm on Sulfur D i o d e Loss and Sulfate Formation i n Dehydrated Blenheim A pricotsa (Lot 3,2243% Hz0, SO1 initially 5350 p.p.m.) OPin Gas, %
Vacuum 11.8 20.6 38.8 57.5 77.5 a
Disappearance of SOZ,p.p.m. 2900
3400 3700 4000 4300 4700
Change due to 01, p.p.m.
500
800 1100
1400 1800
Sulfate, reptd., as SOZ,p.p.m. None 300 920 1200 1760 2220
Stadtman, Barker, Haas e l al. (1945).
and Joslyn, 1941;and Nichols et al., 1936). This is now known to be incorrect. Stadtman, Barker, Mrak, and Mackinney, (1945) have shown that dried apricots may darken to the point of inedibility when about 6070% of the SO2 originally present has disappeared. Thus, fruit initially containing 10,000p.p.m. may become quite dark even though it still contains 3,500p.p.m. SO2. The effect of SO2 on darkening of fruit juices and fruit concentrates has not been studied extensively. Matthew (1928)found that the addition of 10,50, and 90 p.p.m. SO2 prevented the darkening in orange juice stored at 15.6"-21.loC.(60-70°F.)for 1 year, but that 10 and 50 p.p.m. only retarded the rate when stored a t 32.2"C. (90'F.); 90 p.p.m. prevented darkening for 1 year at this temperature, Greer (1944)found that 250 p.p.m. of SO2 retarded the rate of darkening, ascorbic acid loss, and C02 production in orange concentrate. The effect of SO2 in concentrations varying from 0-100 p.p.m. on the darkening of grape concentrates was determined by Richert (1930a). He reported that SO2 appears to have a slight bleaching action but that the main effect is to retard darkening.
NONENZYMATIC BROWNINQ IN FRUIT PRODUCTS
363
Concentrations as low as 20 p.p.m. were beneficial; even very high SO2 concentrations did not bleach darkened sirups completely. The Mechanism of SO2 Inhibition. An understanding of the mechanism by which SOZ exerts its inhibitory action undoubtedly would help to elucidate the mechanism of browning. Three modes of action have been suggested: The first of these is the “antioxidant theory.” It has been suggested that because of its reducing property, SO2 acts mainly as an antioxidant and that, so long as it is present in a sufficient quantity, a low oxidation-reduction level is maintained, and darkening is inhibited. Evidence to support this theory has been obtained in the case of orange juice (Hamburger and Joslyn, 1941), where it was demonstrated that the inhibitory action of SO2 is due partly, a t least, to the fact that it is oxidized by molecular oxygen in preference to ascorbic acid (indophenol-reducing material). With apricots it has been shown that part of the SO2 added to fruit stored in the presence of oxygen is oxidiaed to sulfate (Stadtman, Barker, Haas et al., 1945). Under anaerobic conditions, however, none of the SO2 added is converted to sulfate (Table VIII). There is in fact no evidence to indicate that SO2 is oxidized in fruit stored under anaerobic conditions. A second explanation for the inhibitory action of SO2 might be called the “addition compound t.heory.” This is based upon the fact that SO, readily undergoes addition reactions with compounds possessing an active carbonyl group. Should the process of browning involve condensation reactions of the aldol or melanoidin type, then the formation of sulfite addition complexes would tend to prevent the reactions from taking place. There appears to be no direct evidence for or against this theory. The inhibitory effect of other compounds which form strong addition products with carbonyl groups, e.g., HCN, might be studied to test the validity of this argument. Finally there is the “bleaching theory” which postulates that SO2 acts, not by inhibiting the darkening reactions, but by reacting with the dark products formed in such a manner as t o bleach them. In other words, the compounds formed in the presence of SO2 are merely lighter in color than those formed in the absence of SO2, thus making it necessary for more material to be transformed before the fruit becomes dark. This explanation is in agreement with the observation that the absorption spectra of extracts, obtained from apricots darkened in the presence of high concentrations of SO2, are different from those for low sulfured fruit. Also, the color obtained by photoelectric measurement does not agree with that made by visual inspection (Stadtman, Barker, Mrak, and Mackinney, 1945). Consistent with the bleaching theory is the observation that dried apricots which have darkened only slightly can be almost completely re-
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EARL R. STADTMAN
stored to their original oolor by treating with SO2 (Jewell, 1937). On the other hand, if-the fruit is allowed to become very dark brown it is not possible to bleach it with SO*. It is possible that the darkening involves a series'of polymerization reactions in which SO2 may act, as a bleaching agent, on only the simpler polymers. Additional support to the bleaching hypothesis is given by Hall (1927) who reports that SO2 will greatly retard browning of lemon and orange concentrates. He suggests that this is merely a bleaching effect in as much as the decrease in amino-N concentration continues at the same rate in the presence, as in the absence, of SOZ. Evidence against the bleaching theory is found in the fact that SO2 retards COZ formation in apricots to about the same extent that it retards darkening (Table VIII). This suggests that the action of SOa on darkening may be intimately associated with the reactions giving rise to COZ. The sulfur dioxide which disappears during the storage of dried fruit in the absence of oxygen is irreversibly "bound" in combination with as yet unidentified organic compounds. (Stadtman, Barker, Mrak, and Mackinney, 1945.) If the SO2 is lost by virtue of a reaction with normal intermediates in browning, then the identification of the compound will contribute to our understanding of the mechanism involved. The compound has been isolated in a relatively pure form but has not yet been completely characterized (Stadtman et al., 1946). The substance is a relatively strong polyhydroxy acid (pKa approximately 2.2) containing about 10% sulfur. The free acid is nearly colorless but in concentrated solution it darkens very rapidly a t room temperature. On the other hand, its salts are quite stable. Stability to alkaline or acid hydrolysis indicates that the compound is a sulfonic acid. It is a well-known fact that SO2will react irreversibly with unsaturated compounds with the formation of sulfonic acids. The recent indications that darkening involves a desaturation of sugars and sugar acids to active aldehydes (Stadtman et al., 1946), provides basis for the speculation that the inhibitory action of SO2 results from its reaction with partly unsaturated sugars to form sulfonic acids. 2. sirup Treatment
Cruess et al. (1945) have demonstrated that darkening in dried apricots may be retarded by treating the fruit with sugar sirups prior to drying and sulfuring. Dried apricots were treated as follows: (1) sulfured; (2) steam blanched 2 minutes, then sulfured; (3) steam blanched 2 minutes and soaked in 40" Brix sirup (1:l cane sugar and corn sirup) 2 hours and sulfured; and (4) like (3), but soaked in sirup 11 hours. After drying, all samples were adjusted to 25% moisture and stored a t 42.8"C. (109°F.). The storage life of all samples, based on color comparisons, was 21, 23, 36,
NONENZYMATIC BROWNING IN FRUIT PRODUCTS
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and 37 days, respectively. Similar results have been reported elsewhere (Cruess, 1942b). Beneficial effects of adding sugar to fruit prior to drying have been observed by Mrak et al. (1942). It is not clear whether the beneficial effect of the sirup is due to some chemical property of the sugar or whether it is merely due to a dilution of constituents naturally present in the fruit. Stephens et al. (1942) studied the effect of adding cane sirup to orange and lemon concentrates. Sufficient quantities were added to increase the sirup concentration from 40°-70" Brix. The addition of sugar had no beneficial effect on vitamin C retention and, in some instances, appeared to increase both the rate of vitamin C loss and darkening. Similar results were obtained by Matthew (1928) who was unable to detect any difference between untreated orange juice and juice to which had been added 10, 20, 80, 40, and 50 g. of sucrose/100 ml. 3. Other Treatments The effects of a wide variety of chemical compounds and of aqueous extracts of plant materials on the darkening of ,apricot concentrates was studied by Bedford and Fisher (1935), and Bedford (1936b). Sulfur dioxide, thioglycolic acid, ethyl mercaptan, and HaOa were the only compounds found to inhibit browning. After two months' storage at 41°C. (105°F.) samples treated with 100 p.p.m. of SO2 darkened sligbtly, whereas thioglycolic acid and ethyl mercaptan in the same concentrations prevented darkening completely. Elion (1942) has obtained a patent on the use of thiosulfate to inhibit darkening of fruit. He reports that dipping cut fruit in 0.01-0.5% thiosulfate solutions for 1 minute prior to drying will prevent darkening. Contradictory evidence on this point has been obtained by C. D. Fisher (personal communication). Mrak et al. (1942) studied the effect of dipping apricots in various concentrations of sodium citrate, citric acid, tartaric acid, sodium tartrate, calcium carbonate, tri-sodium phosphate, calcium hydroxide, sodium bicarbonate, and sodium carbonate prior to sulfuring and drying. A slight beneficial effect of sodium citrate was observed. Part, but not all, of this effect was due to a higher retention of SO2 by the fruit during drying. Nichols and Reed (1931) treated dried apples, apricots, and pears with glycerin, ethylene glycol, and mineral oil prior to storage. The only generalization made was that dipping apricots and apples in 2% reiined mineral oil in carbon tetrachloride improved the permanence of color slightly a t high storage temperatures.
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4. Formaldehyde Hall (1927) found that the addition of formaldehyde in quantities of from 20-100% of that theoretically required to combine with all the amino-N was effective in preventing darkening in orange and lemon concentrates. Carbon dioxide production, however, was not stopped; the rate actually increased. The addition of formaldehyde to citrus concentrates is accompanied by a decrease in amino-N. Formaldehyde is apparently a specific inhibitor of this type since acetaldehyde accelerated darkening and cyclic aldehydes were without effect. Formaldehyde in concentrations of 0.15-0.30% (dry wt.) will also retard browning in apricot sirups (Stadtman and Stadtman, 1946). It is equally effective in retarding the browning induced by the addition of furfuraldehyde to apricot sirups as well as the browning of furfuraldehyde solutions when stored alone. IX. STATUSOF THE BROWNING PROBLEM IN FRUIT Although the dark pigments produced in various fruit products are indistinguishableby the methods thus far employed for their study, evidence is available to indicate that they are formed by several entirely different mechanisms. This conclusion can be deduced from the fact that the deterioration of different fruit products is not effected in an identical manner by variations in processing and storage conditions. Moreover, it has been demonstrated that the browning of a single product may be the manifestation of several different types of reactions. Using ionexchange resins (Duolites A-3 and C-3) undarkened apricot concentrate (35" Brix) was fractionated into 3 distinctly different groups of compounds: (1) a cationfraction containing all inorganic cations and 81% of the organic nitrogen; (2) an anion-fraction containing 88% of the fruit acids other than the acidic proteins and amino acids; (3) a neutral-fraction containing 98% of the sugars. (Haas and Stadtman, 1947). The pH of the fractions was adjusted to 3.6 (pH of the original fruit). When these 3 fractions were stored separately at 57°C. (134.4"F.), only slight browning occurred in the anion-fraction and little or no browning occurred in the other 2 fractions. When all 3 fractions were recombined in their original concentrations, however, the mixture browned at a rate identical with that of the unfractionated concentrate. But more important from the standpoint of browning is the fact that browning occurred when any two fractions were combined, though the rate was less than when all 3 fractions were present. It was further shown that the inorganic ions of the cation-fraction (obtained by ashing this fraction) were only slightly effective in deterioration. Therefore, it must be the organic nitrogenous constituents of this fraction
NONENZYMATIC BROWNING IN FRUIT PRODUCTB
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which are important in pigment formation. These results show that browning of apricot concentrate is the result of at least 3 general types of reactions : (1) reactions involving nitrogenous compounds and sugars, (2) reactions involving organic acids and sugars, (3) reactions involving nitrogenous compounds and organic acids. It is to be emphasized that these represent only general types of reactions. Undoubtedly each of the 3 groups of compounds contains several substances which are capable of participating in browning reactions. In the above study, for example, it was shown that the importance of the union-fractionwas due not only to acidity but to specific organic acids which varied in their ability to produce browning. Thus galacturonic acid, though present in relatively small amounts, was 12 times more effective in causing browning than citric acid and 14 times more effective than malic acid, when compared on an equimolecular basis. Phosphoric acid, on the other hand, was almost completely ineffective in causing browning. In all probability, the general types of reactions shown to produce browning in apricot concentrate are operative in most other fruit products. The variability in behavior between the various fruits undoubtedly is due to differences in the relative importance of some one or another of these possible reactions, depending upon the exact composition of the fruit products, In certain extreme cases one of these types can dominate the browning picture. For example, browning of unconcentrated orange juice apparently is limited to reactions between the anions and the cations, since the removal of sugar from this product by fermentation does not influence the rate of browning (Joslyn and Marsh, 1935). Moreover, it has been shown that the acid most important in this type of browning is ascorbic acid which after oxidation with molecular oxygen can give rise to dark pigments. With concentration of the orange juice the above type of reaction no longer dominates the browning picture as is evidenced by the fact that oxidation with molecular oxygen has a negligible effect on the browning of orange concentrate. Some evidence has been obtained (though not yet conclusive) that browning of the concentrate involves a sugar-amino acid reaction (Hall, 1927). In view of the fact that browning is the result of several reactions, it is interesting that the continuous extraction of apricot concentrate with ethyl acetate almost completely prevents the browning of this product. (Stadtman et uZ., 1946). This means that all types of the browning reactions in this case have, as critical intermediates, substances which are extractable from aqueous solution with ethyl acetate. These intermediates might have other properties in common also. Indeed, the observation that sulfur dioxide is inhibitory to all types of browning could be explained by its action on critical intermediates having common chemical character-
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istics. It hac3 been shown that among the ethyl acetate-extractable intermediates there are furfuraldehydes which have been derived from the sugars and presumably from sugar acids. It, therefore, seems logical that other extractable intermediates might be unsaturated aldehydes. In fact, it haa been shown that the continuous extraction of apricot sirups with ethyl acetate does remove a number of as yet unidentified aldehydes which may prove to be important in browning (Stadtman and Stadtman, 1946).
X. NEEDEDREBEARCH With the evidence that browning is produced by several mechanisms, it is obvious that future research must be directed toward the complete characterization of the individual reactions capable of producing browning. This means that the usual techniques employed for the study of this problem must be abandoned. We have seen that there is considerable evidence that browning is the result of small quantity changes (page 356). For this reason the attempt to determine the identity of the reactions by correlating the change in color with changes in the concentrations of suspected precursors is not satisfactory. The inadequacy of this approach has been demonstrated with studies on apricot concentrates where it was impossible to detect any change in the concentration of sugars or of any organic nitrogen fraction during the browning of this product (Bedford, 1936a); yet, with fractionation of the apricot concentrate by means of ion-exchange resins, it was possible to prove that both of these groups of compounds were involved in browning (Haas and Stadtman, 1947). The application of the ionexchange technique to the study of browning offers numerous possibilities. By this means, it is poEsible to isolate the various kinds of reactions in order that they can be studied separately. By a judicious use of this technique, in conjunction with other techniques such as the continuous extraction with suitable organic solvents, spectrophotometry, etc., it should be possible to determine the relative importance of the various general types of browning reactions in a given product, and furthermore, to identify the specific reactants. We have seen that in the deterioration of fruit products there are reactions in which COa is produced, oxygen is consumed, and furfuraldehydes are formed. But as yet, no information has been obtained concerning the specific nature of the reactions whereby these changes occur. Now that a method is available for the isolation of individual reactions, all of these processes should be reinvestigated with the object of identifying the particular reactions associated with each. In recent years, a great deal of research has been carried out on the browning of simple synthetic systems with the object of elucidating the mechanism of browning in food materials. In this review, the writer has purposely avoided considering any of the work on these well-defined syn-
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thetic Bystems, even though studies of this kind would seem to constitute a more fundamental approach to the problem. The difficulty of such research lies in the fact that one cannot bc sure that the simple chemical systems which have been studied are important in the natural product. In the absence of such information, it is unwise to place too much emphasis on these studies. Once specific reactions are identified in the natural product, however, we will be in a position to appraise the vast amount of work already done on the simple synthetic systems, and can proceed logically in further studies along the same lines. Certainly, the establishment of well-defined, simple chemical systems is the ultimate objective for a study of the actual mechanism of browning.
REFERENCES Andereen, F. G. 1929. Sulfur dioxide in dried fruit. Union S. Afrim, Dept. Agr. Bull. 84, 3-14. Barger, W . R. 1941s. Effect of cold storage conditions on the keeping of dried fruit. Western Canner and Packer 93, 47-50. Barger, W. R. 1941b. Experimenta with dried fruit in storage. Publication of Pacific States Cold Storage Warehousemen’s Assn., San Francisco. Beattie, H. G., Wheeler, K. A., and Pederson, C. S. 1943. Changes occurring in fruit juice during storage. Food Research 8, 395-404. Bedford, C. L. 1936a. A note on the nitrogenous constituents of dried apricota during storage. Food Reseurch 1,.337-339. Bedford, C. L. 1936b. Further obeervations on the darkening of apricots. Annual Meeting of the Dried Fruit Assn. of Calif., Tech. Dept. Bull. No. 6. Bedford, C. L. 1937. Changes in nitrogenous constituents of apricota on browning. Univ. Calif. Coll. Agr. Expt. Sta. Proj. Rept. 614, 248-258. Bedford, C. L.,and Fisher, C. D. 1935. Apricot darkening studies. Uniu. Calif. 1702. Agr. Ezpt. Sta. Proj. Rept. 614,230-239. Borsook, H., Davenport, H.W., Jefferys, E. P., and Warren, R. C. 1937. The oxidation of ascorbic acid and its reduction in vitro and in vivo. J . BioE. C h m . 117, 237-279. Cameron, 8.S., Quinn, G., Savage, C. G., Jewell, W. R., and Lyon, A. V. 1929. The sulfuring of dried fruits. J . Council Sci. Ind. R?aearch 2, 151-166. Chaves, J. M. 1945. Rev. quim. ind. Rio de J a m b 14, No. 154, 23. New facts about packaging and storing dehydrated foods. Continental Can Co. 1%. Food In&. 16, 171-176. Continental Can CO. 1944b. New facta about psckaging and storing dehydrated foods. Food I&. 16, 267-269. Continental Can Co. 1945. The problem of swells in orange juice concentrates. Food Packer 4, 32-33. Cruese, W. V. 1Wa. Fruit concentratea and their use. Fruit Produck J . 21, 165169; 187; 190. Cruese, W. V. 1 W b . Experimenta on drying unsulfmd apricots and peaches. Fruit Produ& J . 21, 136-137. Cruese, W. V., Friar, H. F., and Van Holten, P. 1945. Dried syrup treated fruit. Fruit Prodzcds J . 24, 241-248.
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Cruess, W. V., and Glenn, D. S. 1930. Table syrup from grapes. Fruit Prodz& J . 9, 366-368. Cruess, W. V., and Pancoast, H. M. 1933. Observations on the oxidase system of the apricot. Univ. Calif. CoU. Agr. Expt. Sta. Proj. Rept. 614, 180-201. Culpepper, C. W., and Caldwell, J. S. 1927. The relation of atmospheric humidity to the deterioration of evaporated apples in storage. J. Agr. Reseamh 36, 889-906. Curl, L. C., Moore, E. L., Wiederhold, E., andveldhuis, M. K. 1946. Concentrated orange juice storage studies with particular reference to the development of swells. Fruit P d u c t s J . 20, 101-109. Dowell, C. T.,and Menaue, P. 1919. The action of furfural and dextrose on amino acids and protein hydrolysates. J . Biol. C h . 40, 131-136. Dunlop, A. P., Stout, P. R., and Swandish, 6. 1946. Auto-oxidation of furfural. Znd. Eng. Chem. 38, 705-708. Eddy, C. W. 1936. Absorption rate of oxygen by orange juice. Znd. Eng. Chem. 28, 480-482. Elion, E. 1942. Method of treatment of plant tissue. U. S. Patent 2,298,933. Eeaelen, W. B., Jr., Powers, J. J., and Fellers, C. R. 1946. Thc fortication of fruit juices with ascorbic acid. Fruit Products J . 26, 11-14. Fisher, C. D.,Mrak, E. M., and Long, J. D. 1942. Effect of time and temperature of sulfuring on absorption and retention of sulfur dioxide in fruits. Fruit Products J . 21, 175-176;199-200;237-238. Gore, H. C. 1915. Studies on fruit juices. U.S. Dept. Agr. Bull. 241, 1-19. Greer, L. P. 1944. New techniques produce better war-time citrus concentrates. Food Znds. 10, 626-627. H w , V. A., and Mrak, E. M. 1945. Unpublished data, Univ. of Calif. Haas, 1’. A., and Stadtman, E. R. 1947. Unpublished data, Univ. of Cdif. H w , V. A., Stadtman, E. R., and Barker, H. A. 1945. Behavior of natural and golden bleach raisins during storage. Univ. Calif. Coll. Agr. Expt. Sta. Proj. Rept. 1266, 216-233. Hall, J. A., 1927. Summary of the orange juice problem. Unpublished .repart of Research Lab., Calif. Fruit Growers Exchange, Los AngeIes. Hamburger, J. J., and Joslyn, M. A. 1941. Auto-oxidation of filtered citrus juices. Food Research 0, 699-619. Harden, A., and Robison, R. 1920. The anti-scorbutic properties of concentrated fruit juices. Part 111. Biochem. J . 14, 171-177. Harden, A., and Robison, R. 1922. The anti-scorbutic properties of concentrated fruit juices. Part IV. Biochem. J. 16, 912-917. Heberlein, D.G., and Clifcorn, L. C. 1944. Vitamin content of dehydrated foods. Effect of packing and storage. Znd. Eng. Chem. 80, 912-917. Isbell, H.S. 1944. Synthesis of vitamin C from pectic substances. J . Research Natt. Bur. Siunohrds 33, 45-46. Jewell, W. R. 1927. Sulfuring dried fruit. J . Dept. Agr. VictWia 26, 457-462. Jewell, W. R. 1937. “Moist Pack” processing of dried apricots. J. Dept. Agr. Victoria 36, 498-600. Joslyn, M. A. 1936. The role of autooxidation in browning. Annual Meeting of the Dried Fruit Assn. of Calif., Tech. Dept. BuU. No. 5. Joslyn, M. A. 1941a. Color retention in fruit producta. Znd. Eng. Chem. 33,308-314. Joslyn, M. A. 1941b. Current developments in science likely ta affect the food industries. Fruit Products J . 20, 277-282. Jwlyn, M. A., and Marsh, G. L. 1934. Iodine reducing value of orange juice. I d . Eng. Chem. 40, 867-860.
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Joslyn, M. A., and Marsh, G. L. 1935. Browning of orange juice. Ind. Eng. Chem. 27, 186-189. Joslyn, M. A., Marsh, G. L., and Morgan, Agnes Faye, 1934. The relation of reducing value and extent of browning to the vitamin C content of orange juice exposed to air. J. Biol. Chem. 1011, 17-28. Katz, J., and Mackinney, G. 1943. Changes in amino nitrogen content of darkening dried apricots. Unpublished data, Univ. of Calif., Division of Food Technology. Koppanyi, T., Vivino, A. E., and Veitch, F. P., Jr. 1945. A reaction of ascorbic acid with a-amino acids. Science 101, 541-542. Kramer, A., and Smith, H. R. 1946. Preliminary investigations on measurement of color in canned foods. Food Research 11, 14-31. Loeffler, H.J. 1941. Processing of orange juice. Effect of storage temperature on quality factors of bottled juice. I d . Eng. Chem. 83, 1308-1314. McDermott, F. A. 1916. The utilization of cull Florida citrus fruit. Ind. Eng. Chem. 8,136. Maer5, A,, and Paul, M. R. 1930. A Dictionary of Color. 1st ed., McGraw-Hill, N. Y. Matbck, M. B., and Sando, C. E. 1933. Color in tomato Products. Fruit Products J. 13,81-82. Matthew, A. 1928. M.S.Thesis, Library, Univ. of Calif. Moore, E. L., Esselen, W. B., Jr., and Fellers, C. R. 1942a. Causes of darkening of packaged orange juice. The Cunner 96 (16) 13. Moore, E. L.,Esselen, W. B., Jr., and Fellers, C. R. 1942b. Factors responsible for the darkening of packaged orange juice. Fruit Products J . 22, 100-102. Moore, E. L.,Wiederhold, E., and Atkins, C. D. 1944. Changes occurring in oranges and grapefruit juices during commercial processing, etc. Fruit Products J. 23, 270-275. Morgan, A. F., Field, A., and Nichols, P. F. 1931. Effect of drying and ulphuring on vitamin content of prunes and apricots. J . Agr. Research&, 35-46. Mrak, E. M. 1941. Retention of vitamins by dried fruits and vegetables. Fruit Products J . 21, 13-15. Mrak, E. M., 1943. Developments in dehydration. J. Am. Dietet. Assoc. 10, 6-11. Mrak, E. M., and Mackinney, G. 1944. The dehydration of foods. Chemistry and Technology of Food and Food Products, Vol. 11. Edited by M. B. Jacobs. Interscience, New York. Mrak, E. M., Fisher, C. D., and Bornstein, B. 1942. The effect of certain substancee and pre-treatments on the retention of color and sulfur dioxide by cut fruits. Fruit Products J . 21, 297-299. Nelson, E. K., Mottern, H. H., and Eddy, C. W. 1933. Nitrogenous constituents of Florida Valencia orange juice. Fruit Products J . 12,231-235. Nichyls, P. F., Bethel, R., Mrak, E. M. 1936. Annual Meeting of the Dried Fruit Assoc. of Calif. Tech. Dept. Bull. No. 6 (unavailable). Nichols, P. F., Mrak, E. M., and Bethel, R. 1938. Effect of drying and storage conditions on color and SO2 retention of dried apricots. Food Research 4, 67-74. Nichols, P. F., and Reed, H. M. 1931. What happens in the tropics. Western Cunner and Pucker 28,ll-15. Pederson, C.S., Beattie, H. G., and Beavens, E. A. 1941. Proceasing and storage of fruit juice. Proc. Inst. Food Techml. 75-83. Proctor, B. E. 1943-1944. Factors in the browning of dried citrus juice powders during storage. Unpublished data, Maw. Inst. Tech. M y , G. N., and von Loesecke, H. W. 1939. Gases in the commercial handling of citrue juices. I d . Eng. C h . 31, 1276-1278.
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Quinn, G. 1926. Notes on sulfuring of fruits prior to drying. J. Dept. Agr. S . Awr-
tralia 30, 500-510. Richert, P. H.1930s. Darkening of grape syrup. Fruit Products J . 9, 149. Richert, P. H.1930b. Darkening and other grape products problems. Fruit Prodwta J.10,Oct., 36-37. Roe, J. H., 1936. The determination of ascorbic acid aa furfural and a comparison of the results obtained by this method and by indophenol titration. J. Biol. Chem. 116,609-619. Roleson, E. P. and Nichols, P. F. 1933. Pointers for sulfur dioxide. Western Canner and Packer 21, July. Schrader, A., Thompson, A. H., and Burkhardt, G. J. 1943. Considerations of Bmperature, humidity and storage in the dehydration of apples. Proc. Maryland Dehydration Coqfermce Mimeo 42-63. Sorber, D. G., 1944. The rclation of the sulfur dioxide and total sulfur contents of dried apricots to color changes during storage. Fruit Prodwta J. 23, 234-237. Stadtman, E.R., Barker, H. A., Haas, V. A,, and Mrak, E. M. 1945. Studies on the storage of dried fruit. 111. The influence of temperature on the deterioration of dried apricots. Ind. Eng. Chem. 58,541-543. Stadtman, E. R., Barker, H. A,, H w , V. A., Mrak, E. M., and Mackinney, G. 1946. Studies on the storage of dried fruit. 11. Gas changes during storage of dried apricots and the influence of oxygen on the rate of deterioration. Id. Eng. Chem. 88, 324-329. Stadtman, E. R., Barker, H. A., Mrak, E. M., and Mackinney, G. 1945. Studies on the storage of dried fruit. I. Experimental methods and the influence of moisture and sulfur dioxide on the rate of deterioration of dried apricots. I d . Eng. Chem. 38,-104. Stadtnum, E. R., Haas, V. A., Mackinney, G., and Temmer, 0. 1946. Darkening in dried fruit. Unpublished data, Univ. of Calif. Stadtman, F. H., and St9dtman, and E. R. 1946. Unpublished data, Univ. of Calif. Stephens, J. W.,Shipston, G. T., and Wilson, C. P. 1942. Value and u8e8 of concentrated citrus juices. Unpublished data, Calif. Orange Growers Exchange, Loe Angeles. Treaeler, D. K., and Pederson, C. S. 1936. 11. Factors controlling the rate of deteriore tion of bottled concord juice. Food Reaeurch 1, 87-97. Tressler, D. K.,Pederson, C . S., and Beattie, H. G. 1943. Fruit and vegetable juice preparation and preservation. Ind. Eng. Chem.86, 96-100. Wahab, A. 1946. M.S. Thesis, Library, Univ. of CaW. Weast, C.A., and Mackinney, G. 1941. N o n e w t i c darkening of fruita and fruit products. Ind. Enq. Chem. 33, 1408-1412. Wiegand, E.H.,Madaen, H. S., and Price, F. E. 1943. Commercial dehydration of fruit and vegetables. Food Packer 24, 604-806. Wilson, C. P. 1928. Relation of chemistry to the citrus products industry. Ind. Eng. Chem. 20,1302-1306.
Microbial Inhibition by Food Preservatives BY ORVILLE WYSf3 University of Tauu, Austin, Team CONTENTS
I. Introduction . . . . . . . . . 11. Interference with the Genetic Mechanism 111. Interference with the Cell Membrane .
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IV. Interference with Enzyme Activity . . . . . . . . . . . V. Applications
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VI. Summary and Conclusions References
Page 373 . 374 . 377 . 380 . 385 . 389 . 391
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I. INTRODUCTION Bacteria and related forms are essentially masses of protoplasm consisting largely of enzymes so organized as to permit the orderly progress of metabolic processes. This organization involves the following factors : (1) a controlling genetic mechanism regulating cell division and insuring that reproduction results in new cells essentially identical with the parent, (2) the cell contents separated from direct contact with the environment by means of a living membrane, and (3) the contents consisting of enzymes and enzyme systems with definite spacial orientation in regard both to each other and to the geography of the cell. Severe disturbances of any of these factors result in death of the cell and trivial disturbances may result in partial or complete inhibition of cellular activity. Disruptive influencefi may vary from profound effects such as the actual destruction of the cell by physical forces, e.g., supersonic waves, to the slowing of metabolic activity by the presence in excess of some necessary food constituent. A vast literature has accumulated on each detail of the subject. This review will be limited to a discussion of the mechanism of the antimicrobial action brought about by chemical substances and possible applications of this knowledge in the food industries. Most in uitro tests of the activity of chemical agents on microbial cells concentrate the attention of the investigator on the limiting cases. The actual measurements made and reported are either in terms of the lowest concentration of a chemical agent required to bring about an effect, or in terms of the shortest time of action required for a certain concentration of the chemical to exhibit its antimicrobial action under stated environmental conditions. By such methods one presumably studies the inhibition of that one mechanism in the cell most sensitive to the chemical under test. 373
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In actual practice, however, the concentration employed will be many times in excess of that minimal quantity required for inhibition; or the time permitted for action will be many times in excess of that measured in the latloratory experiment. It is likely that under such circumstances several cell-destroying mechanisms may be exhibited by a single chemical agent and the effectiveness of t,he chemical will be a summation of the several actions. Nevertheless, since the antimicrobial action will fail only when the inhibition of the most sensitive function in the most resistant organism is no longer operative, a study of such limiting mechanisms is essential to our understanding of the basic problem.
11. INTERFERENCE WITH THE GENETICMECHANISM Our knowledge of chemical interference with the genetic mechanism of the cell is based largely on indirect evidence and analogy. As pointed out by Rahn (1945) the microbial cell is generally regarded as dead when it has lost its power to reproduce. Under many practical conditions, if microorganisms are prevented from reproducing or are even slowed down in their reproductive capacity the objective of the chemical inhibitor is attained. This is due t o the fact that in most circumstances the metabolic activity of the organisms makes their presence undesirable only when large numbers of cells are present. Consequently, interference with reproduction is usually an adequate control measure. Strong evidence for the theory that chemical substances destroy bacteria by interference with the genetic mechanism can be inferred from the shape of curves obtained by plotting data from experiments on bactericidal agents. In many instances it will be observed that when the log of the surviving bacteria is plotted against the time of exposure to a chemical agent, a straight line results. Based on analogy with monomolecular chemical reactions Rahn offersa clear statement of the case for gene interference: “The conclusion is that a logarithmic order of death can be obtained only if the death of the bacterium is brought about by the reaction of one single molecule. This conclusion is absolute. The logarithmic order of death is entirely impossible if more than one molecule must be inactivated to produce death of the cell . . . an approximation of the logarithmic order is obtained if death is caused by inactivation of a very few molecules but if this number is higher than 4 or 5 the order is very definitely not logarithmic. This eliminates denaturation of enzymes or cytoplasm as the cause of death, wherever the logarithmic order prevails because it does not seem possibIe that the inactivation of a very few of numerous identical molecules can kill the organism. It would probably exclude also the disruption of the cell membrane as a cause of death for this is likely to
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require inactivation of a considerable number of molecules before damage becomes irreparable.” “Let us now consider the bacteriologists’ definition of death: a bacterium is dead when it has lost the power to reproduce. Cell division is linked with chromosome division, the chromosomes contain the genes, and the inactivation of certain genes is known to cause “lethal mutations”, i.e., to prevent the cell from multiplying. While chromosomes and genes have not been definitely found in bacteria, these organisms undoubtedly have some hereditary mechanism, and hereditary units. As a rule there are two genes of the same kind in a diploid cell and one in a haploid cell. According t o Fricke and Demerec (1937): “we may assume a gene contains about 2500 atoms . . . This would indicate an average gene diameter of about 25A.” This is the size of a small protein molecule. A gene, then, would consist of only one or two molecules. Each gene is different from the others and has its own rate of denaturation. If one vitally essential gene is denatured, the cell can no longer divide, the bacterium is sterile, that is, dead, according to the bacteriological definition.” Mutations induced by chemical agents do not necessarily result in death. They may result in the loss of some function of the cell which may definitely retard its growth or stop growth entirely under certain environmental conditions. For example, by ultraviolet and x-ray radiations it is possible to induce mutations involving the loss of ability to synthesize a vitamin or amino acid. This has been done with a number of bacterial species (see Ryan et al., 1946) and a tremendous amount of work has been done with fungi (Beadle, 1945). The recent work of Stone, Wyss, and Haas (1947) indicates that certain mutations may result from the irradiation of the substrate, which is evidence that mutations may be induced in the bacteria by definite chemical agents. It is a common experience among bacteriologists who work with antibacterial agents to observe “small colony” variants which result from experiments on chemical inhibition. These are undoubtedly biochemical mutants which exhibit a nutritional deficiency when growing on the medium which was satisfactory for the production of large normal colonies by their parent strain. Nucleotides interfere with the action of basic dyes in a manner suggesting that part of the inhibition by a dye may be a result of its union with acid groups in the nuclear apparatus. The inhibition of cell division by penicillin and the recent work by Krampitz and Werkman (1947) showing that penicillin inhibits the dissimilation of ribonucleic acid suggests that this inhibition involves the nuclear apparatus of the cell. It is true that in the few cases which have been carefully studied thus far, the rate of induced mutation to any one particular mutant has been
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extremely low and would not account for any degree of inhibition of activity of the cell population. It must be borne in mind, however, that when- mutations are produced the majority are lethal to the organism. This is the expected result since a mild chemical treatment may produce a few single gene transformations, but more drastic treatment would undoubtedly produce multiple genetic disturbances in the cell and these are extremely unlikely to be other than deleterious to the welfare of the organism in its accustomed environment.. This is emphasized in the review on mustard gas by Gilman and Philips (1946). They have summarized the evidence that high concentrations of mustard produce many chromosomal breaks which ultimately result in death. Mild treatment also results in chromosomal breaks and if these are not too numerous they may be transmitted to daughter cells in the subsequent mitosis as heritable chromosome abnormalities. Although these experiments were carried out with higher forms, they probably apply to microorganisms since exposure of yeast to mild doses of mustard reduces the growth rate in a manner which is inherited by several succeeding generations of daughter cells. It follows, therefore, that even if minor, specific genetic changes are brought about in a fraction of the organisms present in a population, the ultimate result may be an inhibition of the microbial growth. In addition to mustard gas other chemicals are known to bring about mutations. High concentrations of BaCla (Schnitzer et al., 1943), nitrite and a number of other agents (Steinberg and Thom, 1940) added to the medium may result in mutant forms. Some such mutations may arise by selection of pre-existing spontaneous mutants, but others appear to be the result of induced hereditary changes in the population. A mechanism for induced mutations might be developed based on Delbriick’s (1941) theory of autocatalytic synthesis of polypeptides. If the assumption is made that all primary gene actions involve one general mechanism, then since the reproduction of the gene is the only primary gene action of whose general occurrence we are certain, it would appear that the gene acts primarily because and while it is reproducing itself. The gene probably reproduces several times during each cell cycle and the extra replicas diffuse into the cytoplasm where they control the observable characters of the cell. Delbriick offers the following explanation as to how the autocatalytic reproduction of the gene proceeds. He assumes with LinderstrGm-Lang that proteins are synthesized not from amino acids but from the aldehydes uniting fist to form an imide bond which is then oxidized to a peptide bond. In the oxidation involving the removal of two hydrogen atoms, the first step requires the most energy because it leads to the formation of a radical with a free valency (Michaelis and
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Schubert, 1938). It is the function of the catalyst to reduce the size of the first step so that it may be accomplished by the available hydrogen acceptors in the cell and not require some much more powerful oxidant. The size of the first step can be reduced by lessening the energy of the radical and this can be done by having the radical form an intimate complex with a molecule that has already lost both hydrogens. The complex would be a structure that is stabilized by resonance between the two equivalent electronic configurations. The amount of resonance energy involved will depend on, first, the closeness of approach of the two molecules, and second, the near equality of the energy of the two resonating states. Both of these conditions are most easily fulfilled if the catalyzing molecule is identical in structure (including side chains) with the oxidized product of the substrate molecule, i e . , if it reproduces itself exactly. The introduction of a nonphysiological chemical into the system might easily disturb the progress of the autocatalytic reproduction. If the chemical does not prevent the process entirely it might, by uniting with the substrate or the catalyst, introduce distortions which would result in inexact replication. In this manner mutants may arise.
CELLMEMBRANE The cell membrane is the outermost layer of highly reactive protoplasmic material of the cell. Naturally, reactive chemical compounds diffusing into the cell will unite with the components of the membrane. Chlorine is an example of a highly reactive chemical which reacts indiscriminately with many substances. Therefore, it is unlikely, in its active state, to get beyond the cell membrane without satisfying the groups present there which can react with it. Chang (1944)reviews the evidence and presents data obtained with cysts of Entameba histolytica. Using the indicator, o-tolidine, which forms a yellow color with chlorine, he was able to determine that when the cysts were killed by free chlorine the halogen was present inside the cyst, a phenomenon that may have been a result rather than a cause of death. On the other hand, with low concentrations of some less active chloramines, the cysts survived because the chloramine did not, or was unable to, penetrate into the protoplast. Membrane damage can also be attributed to such agents as the fat solvents which, if present in sufficient concentration, dissolve out the lipoidal constituents and thus destroy the functioning membrane. This can be demonstrated readily by placing yeast cells under ether and observing the rapid and complete dissolution of the cells. High concentrations are required since the action depends on differential solubilities. The enzyme, lysozyme, attacks specifically the acetylaminopolysaccharide in the cell 111. INTERFERENCE WITH
THE
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envelope of certain susceptible microorganisms. A number of species are inhibited by the action of the enzyme but a very few species are completely lysed by it. The large group of compounds known as surface active agents produce their antibacterial effect, at least in part, by action on the cell membrane (Hotchkiss, 1946). Experiments with compounds of this type show that within a few minutes after application of a lethal dose there appears in the surrounding medium various phosphate esters and nitrogen compounds which were an integral part of the functioning protoplasm of the living ceI1. Such effects occur a t low temperature, indicating that they are not the result of a disturbed autolytic mechanism which often functions when death has been brought about under conditions favorable for enzyme action. The surface active agents effect the destruction of the semipermeable nature of the cell membrane. That these phenomena are the cause rather than the result of death is proved by experiments with a wide variety of nonsurface active killing agents which bring about death but do not result in the rapid diffusion of the cell contents into the medium. It is assumed that surface active substances not only may disturb the permeability of the cell membrane, but that they also may dest,roy the orientation of various constituents within the cell, an orientat,ion that is preserved only as the surface tension is maintained within definite’limits. When a disturbance is introduced, certain enzymes come in contact with substrates from which they are ordinarily restricted by means of cell geography or cell architecture, and the resulting action is disastrous to the cell. An example of a substance bringing about membrane damage is tyrocidine, a surface active polypeptide having a molecular weight of 2500, which can be extracted from culture fluids of Bacillus brevis. The action described for this compound in damaging the cytoplasmic membrane does not appear to differ greatly from other antibacterial substances with surface active properties. These include not only such simple compounds as fatty acids, alcohols, and aldehydes, but also most of the hundreds of synthetic wetting and emulsifying agents and detergents which have been so extensively studied over the past ten years. The activity of the phenols appears to belong to this group, and substitution on phenolic compounds to give a greater heteropolar character to the molecule results in stronger activity. I n keeping with all compounds of this type a hydrophobic group is attached to one or more hydrophilic groups. Increase in the hydrophobic group results in greater water insolubility and this is often accompanied by greater antibacterial activity when tested by conventional methods. The total activity depends on two factors: (1) the tendency to concentrate in or about the bacterial cell thus giving a higher concentra-
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tion than is present in the menstruum, a property governed by the water solubility and the surface activity and (2) the actual toxicity of the compound when at the site of action. Attempts to prepare the most active germicidal compound of a series of surface active agents are complicated by the fact that modifications in the molecular structure may result in an increase in absolute toxicity without this fact being evident from the antibacterial tests. The advantage gained by increased toxicity may be masked by a simultaneous decrease in the tendency of the modified molecules to concentrate on or in the bacterial cells. Surface active ions also have a strong affinity for proteins and combine readily with them. The results of Anson (1941) indicate, however, that the amounts required for denaturation of protein are generally far in excess of those required for killing bacteria. From this and other experimental evidence, Hotchkiss (1946) concludes that when limiting concentrations of surface active agents are considered, the effect on the membrane is the phenomenon which causes death or inhibition of the organism. Confirmation of this view is offered by the analytical work of Gale and Taylor (1947) and the electron micrographs of Mitchell and Crowe (1947). The former workers show that the glutamic acid and lysine from the internal environment of bacteria and yeast rapidly leaks out of the cells which are treated with detergent substances. This did not occur after the application of inhibitory concentrations of substances which were not surface-active. The substances employed included phenol, the antibiotic, tyrocidine, the cationic detergent, cetyltrimethylammonium bromide and the anionic agent, Aerosol 0. T. Gale and Taylor believed the lytic action of these compounds to be sufficient to account for their antibacterial effect. Mitchell and Crowe found the cell wall disrupted in streptococci treated with surface active agents. Presumably the cell wall is itself the amino acid-confining barrier, or it may be attached to the cell membrane or some other confining structure, which is disrupted with it. On gram positive bacteria there appears to be a definite layer of a specific chemical substance which absorbs basic dyes such as gentian violet (Henry and Stacey, 1946). The exact location of the material is in doubt, but in many organisms it appears to be a part of, or layered on, the cytoplasmic membrane. This dye absorbed in the surface layer is probably harmful to the cell, since organisms having the dye-absorbing layer are inhibited by dyes to a greater extent than are those in which the layer is absent. It seems likely, therefore, that at least part of the bacteriostatic effect of dyes is due to their interference with proper functioning of the membranes of the cell.
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IV. INTERFERENCE WITH ENZYME ACTIVITY Interference with the activity of enzymes results in microbial inhibition or death. Such interference is readily accomplished. Unfortunately, our studies on enzymes have been largely limited to those exerting katabolic activity since most anabolic enzymes can be studied only through measurements on the over-all life process. A beginning has been made in the elucidation of the enzymes involved in phosphorylation which link the energyyielding and the energy-consuming reactions in the cell. The enzymes involved in synthesis are undoubtedly similar in many respects to those involved in katabolic reactions and inhibitors will affect them in an analogous manner. Enzymes are protein colloids which contain reactive groups that unite with a substrate molecule. For activity, the colloidal nature of the protein must be maintained, the prosthetic group, if there be one, must be properly attached and functioning and various functional groups on the protein moiety must be maintained in their free and active state. Any interference with these properties results in inactivation. A wide variety of agents is known to be protein denaturant. They may act by rendering the protein molecules incapable of maintaining themselves as a colloidal suspension. For example, wetting agents may irreversibly denature enzymes by so altering the colloidal properties and the surface of the molecules as to interfere with the enzyme action. Enzymes, like other colloids, are sensitive to marked changes in the ionic content of the medium. This may be due partly to the effect on reactive groups, but more often is concerned with maintenance of the colloid. High concentrations of salts precipitate proteins or so alter the colloids as to drastically curtail their biological activity. The effect of some ions is more marked than others, e.g., NaCl inhibits some proteinases a t concentrations where most other sodium salts are not effective. From the data of Ingram (1947) it appears that the action of concentrated salt solutions on bacterial respiration may be partly explained in terms of “salting out” the enzymes. The limiting action is probably on a dehydrogenating enzyme. There are some factors of salt action not yet explained, for although the data on halophiles bear out this conception, the enzymes of nonhalophiles are more sensitive in the intact cell than when isolated. One of the most powerful chemical agents for inhibiting enzyme action is the hydrogen ion. When large departures in pH from the optimum for enzyme action are maintained, irreversible denaturation results, which is undoubtecjly due to disturbance of the colloidal nature of the enzyme. The bacteriostatic effect obtained by adjusting the pH slightly above or below that which permits growth is an example of bacteriostasis which is
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38 1
due to enzyme inhibition. The assumption is usually made that only uncharged or slightly charged particles of enzymes are active. The adjustment of the hydrogen ion concentration away from the isoelectric point reduces enzyme action. The pH activity curves of enzymes, however, are complicated by the effect of the pH on the substrate, and when living cells are involved there are other factors, such as cellular pH and the relative permeability of the membrane to ions vs. molecules, to be considered. The enzyme chemist studies the active groups on the enzyme proteins by noting the effectof mildly reactive inhibitors. For example, an enzyme which is inactivated by very mild oxidizing agents and which can be reactivated by reducing agents is thought to contain sulfhydryl groups which are essential for activity. The studies on the active groups of enzyme proteins by means of inhibitors have beeqreviewed by Barron and Singer (1945) and Singer (1946). From such studies we are able t o obtain a clearer picture of the mechanism of action of many inhibiting agents since the experimental data indicate the relative susceptibilities of enzymes and their active groups. Of the reactive groups attached to enzyme proteins, the -SH groups have received widest attention. The development of the concept of the active sulfhydryl group as an essential part of the functioning enzyme is summarized by the work of Hellerman (1937). In a series of experiments he brought about the inactivation and reactivation of enzymes by alternating the application of selected oxidizing and reducing agents. Hellerman suggested that the disulfide form of the enzyme (Enzyme-S-S-Enzyme) was inactive and that reduction of the disulfide group to active sulfhydryl groups activated the enzyme thus : Enzyme-S-S-Enzyme (inactive)
+ 2H
-
2 Enzyme-SH (active)
Of a list of about 40 enzymes tabulated by Singer (1946), it can be noted that for over of the enzymes studied, functioning sulfhydryl groups are essential for activity. These include respiratory enzymes as well as hydrolases. Obvious poisons for sulfhydryl enzymes are the oxidizing agents, e.g., the halogens, ozone, hydrogen peroxide, and permanganate which in high dilution may destroy cellular activity by oxidation of sulfhydryl enzymes. Many microorganisms do not maintain the enzymes in the active reduced state during inactive periods, and it is necessary to supply the proper reducing material in order to restore active -SH groups. The activity of the enzyme may be indefinitely postponed by maintaining a high oxidation-reduction potential and it is believed that the bacteriostatic action of certain dyes is due in part t o their ability t o poise the 0-R potential of the medium at such a level that the sulfhydryl enzymes
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of the inoculum have difficulty in getting under way. There are a great many inhibitory substances which are mild oxidizing agents and they may function in this manner. Another common poison of the sulfhydryl groups on the protein is of the heavy metal type. Copper, mercury and silver ions, compounds of bismuth and arsenic, and other mercaptide forming agents combine with the sulfhydryl groups on the protein and the result is “poisoning” of the enzyme. This inhibition of enzyme activity may require only trace quantities of the heavy metals. In some instances the amount required may be so small that the inactivation is regarded as the oxidation of the essential -SH catalyzed by a trace of metal, but in most cases the poisons form more or less tightly bound compounds with the -SH group in the enzyme. In such cases the inactivation can be reversed by removing the toxic agent with adsorbents or by selectively precipitating them with lower molecular weight thiols, but this must be done before the cell dies from lack of the functioning enzyme. Numerous other compounds will react with the -SH group and consequently destroy the activity of enzymes where the free group is necessary for functioning. Among those which have been useful tools in biological research are iodoacetate and bromoacetate which form alkyl derivatives with the enzyme a t the -SH linkage. Though their reaction is slow it is irreversible and at low concentrations they produce strong inhibition of certain enzymatic processes. It must be pointed out here that a number of agents which are powerful enzyme inhibitors when tested on isolated enzyme systems, fail to show great inhibition of the intact microbial cell since at normal pH values they pass only with great difficulty through the cell membrane. I n some cases this is the effect of the dissociation as described by Jacobs (1940). He points out that most undissociated molecules pass through the membrane and through protoplasm with great rapidity, limited only by the diffusion rate, but that the permeability of cells to ions is a more complex phenomenon and considerably more limited in its extent. The work of Anderson (1945) showing that pyruvic acid can pass into cells only in its undissociated form is good confirmation of this view. Reducing agents also inhibit enzyme action. There is a class of enzymes known as disulfide enzymes which require for functioning the group S-S. Such enzymes are inactivated by reducing agents such as SO2, HZS, HCN, dithionate or formaldehyde. The unit in the enzyme protein sufficiently labile to reduction to account for the effect is probably in the amino acid, cystine. Reducing agents act by splitting the S-S linkage into S-H groups. The inactivation is generally not reversible since the split S-S groups would, upon mild oxidation, probably not unite in the
MICROBIAL INHIBITION BY FOOD PREBERVATIVEB
383
original positions to restore the enzyme activity. A t least this was found to be the case in studies on the biological activity of insulin (du Vigneaud et al., 1931). Inhibition of enzymes by such reducing agents as H2S or HCN cannot be ascribed to the destruction of disulfide groups unless it has been ascertained that the enzyme in question does not contain a metallic prosthetic group which is sensitive to these reagents. The phenolic hydroxyl group, essential for the activity of certain enzymes, is also sufficiently reactive to be attacked by various chemical agents. Numerous possibilities exist including any esterifying reagent and acylating agents. Herriott and Northrop (1984) observed that pepsin could be inactivated by acetylation of both the amino and phenolic groups with ketene and that the subsequent liberation of the phenolic hydroxyls by gentle hydrolysis restored the activity of the enzyme. Of the inhibitions caused by iodine, at least part are dne to reaction with the hydroxyl in the tyrosine groups in the enzyme proteins. Herriott (1936) was able to isolate the iodinated tyrosine from pepsin inactivated by iodine. Similarly prolonged treatment with nitrite under acid conditions (Philpot and Small, 1938) attacks the OH groups and renders certain enzymes inactive although milder treatment with nitrite appears to bring about a reversible oxidation in addition to its well-known action on primary amino groups. The chemicals which attack the amine and amide groups in enzyme proteins are usually nonspecific. Formaldehyde owes at least part of its action to union on amino or amide groups. Theis and Lams (1944) have studied the groups on proteins which are involved by formaldehyde and find that pH has an important influence upon the extent and nature of the reaction. Ethylene oxide and other epoxides react with amino groups as well as with sulfhydryl groups and phenolic hydroxyls. The enzyme chemists use ketene and carbon suboxide as reagents to detect the essential nature of amino groups in enzymes. Any amine reagent would presumably be effective. Carboxyl groups are also essential for activity for some enzymes and may be tied up by numerous chemicals. Epoxides such as ethylene oxide, propylene oxide, and epichlorohydrin were found by Fraenkel-Conrat (1944) to be suitable reagents for esterification of protein carboxyl groups in aqueous solutions and at room temperature. From this survey it is evident that the active groups on enzyme proteins react readily with many chemical substances and that in many cases such reactions destroy enzyme activity. In addition to the inhibitors which react with the functional group of the enzyme proteins, there is a wide variety of compounds which interfere with the prosthetic groups of the enzymes. There may be (1) competition between the protein and the inhibitor for the prosthetic group, or (2) competition between the prosthetic group and the inhibitor for the
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ORVILLE WYSS
protein. Examples of the first method of inhibition include HZS, HCN, CO, azide or hydroxylamine which react with the copper or iron containing prosthetic groups of the respiratory enzymes. The presence of such substances lowers the amount of active prosthetic groups available for union with the enzyme and consequently lowers the biological activity. Other examples are oxalate and fluoride which inhibit some Ca-requiring enzymes and citrate which inhibits some Mg-requiring enzymes. The second method for interfering with prosthetic groups is exemplified by the inhibition by Mg, of some enzymes requiring Ca as the prosthetic group, or the interference of certain flavin enzymes by acriflavin or atabrine. Competition with the substrate for place on the active enzyme surface is a type of inhibition that has received much attention in recent years. The specific inhibition of enzyme action on one compound by the presence of a structurally related compound is explained by the existence of two separate phases in the process. First the enzyme combines with the substrate and second, the enzymic change takes place within the enzymesubstrate complex. Substances,similar to the substrate in molecular configuration can combine with the enzyme but undergo no further change. Thus they inhibit the process by combining with the active groups of the enzyme. The extent of the inhibition depends on the proportion of the enzyme which thus can be rendered inert; this can be varied from no activity t o maximum activity by varying the concentration ratio of inhibitor to substrate. Competitive inhibition is best exemplified by malonic acid (COOHCHzCOOH)which interferes competitively with euccinic dehydrogenase in its oxidation of succinic acid (COOHCH&H&OOH). Another example of competitive inhibition is H2 gas competing with Ns gas for space on the surface of the nitrogen-fixing enzyme of Azotobacter (Wyss and Wilson, 1941). Better known are the competitive inhibitions caused by sulfonamides and other analogues of vitamins or amino acids. In some cases enzymes that attack a compound having an E-configuration are inhibited by the presence of the optical isomer. A complete review of competitive inhibition by metabolite antagonists is presented by Roblin (1946). Another type of enzyme inhibition is the interference with the forward progress of an enzyme-catalyzed reaction by the accumulation of t4e end products of the reaction. The effectiveness of such inhibition depends on the equilibrium conditions. It is possible in some cases to protect effectively a substrate from enzyme activity by the addition of an end product since the forward progress ceases when equilibrium is reached. Such a method is presumably of theoretical interest only, since the achievement of high concentrations of end product at the site of enzyme action would
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385
be difficult to attain with living organisms. From natural substances there has been isolated a group of enzyme inhibitors whose action is not well understood. These substances are called antienzymes. Some of them appear to be protein hydrolysis products, e.g., the anti-tryptic substance from egg white isolated by Balls and Swenson (1934). Others are proteins such as the trypsin inhibitor crystallized by Northrop (1939) and the pepsin inhibitor crystallized by Herriott (1941). These substances occur widely in nature. Mirsky (1944) found that the crystalline “trypsin inhibitor” as well as the antitrypsin of soybean origin (Ham and Sandstedt, 1944) inhibited the fibrinolytic activity of streptococci. Intestinal parasites contain substances which inhibit the action of proteolytic enzymes. A naturally occurring inhibitor of amylase action has been found in a number of cereals by Kneen and Sandstedt (1943). A fat soluble factor found in navy beans and believed to be present in many other foods strongly inhibits the activity of pancreatic amylase (Bowman, 1943).
V. APPLICATIONS The naturally occurring inhibitors are of especial interest to the food technologist as they explain the natural resistance of certain materials to autolytic enzymic destruction and undoubtedly offer some protection against the enzyme action of microorganisms. It appears likely that as more of these substances are studied and their action is better understood some may be developed that will prove useful in food preservation. Since such substances are already constituents of some foods, the problem of toxicity to the consumer may not be as formidable as in the case of other chemical agents. The many antibiotic agents which are being isolated and studied must also be considered for their possible usefulness in this field. The specificity of many of these substances does not necessarily exclude them since many food spoilage problems can be solved by restraining a single species. In this connection the synergistic action of combinations of the agents has not been studied. Of the myriad of synthetic chemical agents known to have antibacterial action only a comparatively small number are of concern to the food technologist. In the interest of sanitation and during cleaning procedures he encounters the soaps and synthetic detergents. As pointed out these compounds destroy microorganisms by virtue of their action on the cell membranes and in high concentrations they also disturb the colloidal nature of the proteins. In this class we must also place the phenols and cresols, although killing curves with these substances offer strong evidence that in the limiting concentrations microbial death may be brought about by interference with the genetic mechanism involving cell division. Unpublished experiments from the author’s laboratory indicate that the
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ORVILLE WYSB
chlorphenols also cause membrane destruction. The high activity of these compounds is due in part to their low water solubility and hence their tendency to concentrate in the lipoidal cell membrane. Strong oxidizing agents may also destroy the cell membrane, but it appears likely that TABLII I Mechanism of Action of Food Preservatives and Related chemicals Compound Cationic and anionic surface active agents Phenols, chlorphenols, naphthol sulfonates, cinnamic acid Fatty acids, alcohols, and long chain aldehydes Chloracetic acids Benzoates, chlorbenzoate, hydroxybenzoate and its esters Salicylate Borates Sulfur dioxide, sodium sulfite or persulfite Chlorine, chloramines, nitrogen trichloride, peroxides, nitrates, and other oxidizing agents Ethylene oxide and other epoxides Fluorides, fluosilicates, and fluoborates Formaldehyde Salt
Probable Action Destruction of cell membrane Denaturation of enzyme proteins(?) Destruction of cell membrane Reaction with protein in genetic mechanism(?). Destruction of cell membrane Competitive inhibition of enzymes by short chain acids Membrane action. Competitive inhibition(?) Membrane action Competition with coenzyme Membrane action. Competition with cozymase. Competitive interference with enzymic utilization of amino acids Reaction with enzyme involved in phosphate metabolism Reaction with aldehyde formed in carbohydrate dissimilation. Reduction of 5-S links in enzyme protein Destruction of sulfhydryl groups in enzyme protein (or gene protein(?)) Reaction with carboxyl and other active groups on enzyme protein Oxidizing action Interference with prosthetic groups Reaction with active groups on enzyme protein Reaction with active groups on protein of enzyme (or gene?) Precipitation of enzyme protein
inhibition of organisms by oxidizing agents, excepting such powerful agents as free chlorine, ozone and peroxide, is actually due to the oxidation of -SH enzymes to the inactive S-S form. Patents have been issued involving iodates and bromates €or the suppression of the action of proteolytic enzymes. Of the inhibiting agents used for food preservatives the fatty acids, alcohols, and aldehydes have received considerable attention. All these series are surface active and therefore are presumed to act by virtue of their
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effect on the membrane. When tested in nutrient broth the activity of the succeeding members of the fatty acid series increases with chain length until their insolubility in water becomes the limiting factor. When tested in a minimal medium, however, without amino acids or vitamins, it is observed that acetic, propionic, and butyric acids are much more active than would be expected (Wyss, 1946;Wright and Skeggs, 1946). This enhanced activity is due to the competition with certain amino acids for space on the active enzymic groups. One of the enzymes competitively inhibited appears to function in the synthesis of pantothenic acid. The competitive substrates may be 0-alanine, or in certain cases aspartic acid or glutamic acid or their breakdown products. From this one would infer that propionic acid, e.g., would have a much greater inhibiting effect in bread than would be anticipated from laboratory tests using a nutrient medium and that this enhanced action would be less pronounced in a food material containing free amino acids or pantothenic acid. Wyss et al. (1945) observed that with the short-chain fatty acids a decrease in pH enhances activity to a much greater degree than is the case with the acids with longer chain lengths. This fits in with the hypothesis of interference with intracellular enzymes by short chain members of the fatty acid series. As pointed out above, the molecular form of acids passes through the cell membrane more readily than the ionic form. Since the dissociation constant for the 3-carbon propionic acid does not differ greatly from that of the 8-carbon caprylic acid, the differential pH effect indicates that the latter acid apparently does little damage on entering the cell beyond its effect on the cell membrane, while the former exhibits an additional intracellular inhibition if the antagonistic metabolites are not present in high concentration. A similar situation exists when salicylic acid is employed as an inhibitor. Iv&novics (1942a,b) reports that in the presence of protein breakdown products about 0.1 M salicylate is required for the inhibition of the growth of an organism which is inhibited by 0.00002 M salicylate in the absence of complex nitrogenous food. Under the latter conditions the salicylate, like the short-chain fatty acids, interferes with the use of certain amino acids for the synthesis of pantothenic acid. Stanley and Schlosser (1945) present data which indicate that it is the synthesis of the pantoate portion of the pantothenate molecule which is inhibited. Salicylic acid also inhibits cozymase-conditioned reactions (v. Euler and Ahlstrom, 1943). The action is not a specific removal of the coenzyme by salicylate but more likely a competition for the protein by cozymase and the inhibitor, or at least an apparent weakening of the affinity of the apozymase for cozymase. The studies with cozymase were done with isolated enzyme systems and it is likely that in the intact cell these enzymes yill be pro-
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ORVILLE WYSS
tected except at pH values where the salicylic acid exists largely in its undissociated form (Rahn and Conn, 1944). Benzoic acid also exhibits several types of action, one of which appears to be a competition with the coenzyme for the enzyme protein. I n the case of glucose or lactic dehydrogenase the inhibition by benzoate is alleviated by the addition of coenzyme (v. Euler, 1942). Unpublished experiments from the author’s laboratory show that a much greater activity can be demonstrated in a medium low in amino acids than is observed in nutrient broth, and that under certain conditions nicotinic acid interferes with inhibition by bensoate. In a rich medium the high concentrations required for inhibition appear to interfere with the functioning of the membrane, an interference which can be enhanced by certain substitutions in the molecule to increase its heteropolar nature. Some compounds which have been employed as preservatives and function in this manner are &naphthol, its sulfonate, and various esters of benzoic acid. Boric acid and borates are known to interfere with phosphate metabolism (Pfeif€er et al., 1945). Borate assimilated in the animal body is tied up with glycerol or other polyhydroxy alcohols. Zittle (1947) demonstrated that a t very low concentrations it inhibits the enzyme, phosphomonoesterase, obtained froin intestinal mucosa. Whether this is due to the borate ion competing with the phosphate or is due to its union with the polysaccharide portion of a polysaccharide-protein complex is not evident. Fluorides also inhibit enzymes catalyzing the splitting of phosphate esters. Fluoride has been a useful tool in the elucidation of the mechanism of glycolysis as, in limiting concentrations, it is quite specific in action. At higher concentrations it inhibits a wide variety of enzymes probably by forming a reversible, inactive combination with the enzyme protein. Belfanti et al. (1935) report that calcium-requiring enzymes are inhibited by fluorides and oxalates which compete with the enzyme protein for the prosthetic group (calcium). The action of sulfur dioxide in reducing S-S linkages which are essential for enzymatic activity has been mentioned. Under some circumstances the acidity of the sulfurous acid resulting from the solution of the gas is an important factor contributing to the inhibition. The compound is a powerful inhibitor of yeasts since a t the low pH it can penetrate the cell and disrupt the normal progress of the alcohol fermentation. There the sulfite reacts with acetaldehyde making a combination which is not attacked by the enzyme and thus exhibiting another type of enzyme inhibition, viz., a competition between the enzyme and the inhibitor for the substrate. The highly reactive formaldehyde inactivates enzymes by attacking a number of the groups on the protein which may be essential for activity.
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At pH values of 5 or below, it is fixed mainly in the acid amide and imino groups. Above pH 6 it attacks the nitrogen in the imidazole ring of histidine and above pH 9.5 it blocks the free amino groups of the lysine residues. At high pH values it also reacts with sulfhydryl groups. The concentrations of formaldehyde that are required for inactivation of organisms and enzyme systems vary widely. It is possible to obtain active invertase from yeast cells treated with 0.5% formaldehyde, a concentration which destroys the fermenting enzymes. In some cases the action appears to be partially reversed by the addition of oxidizing agents. The chloracetic acids will effectively inhibit microbial growth if they are used at a low pH where they can pass through the cell membrane. There they react with sulfhydryl groups and thus inactivate enzymes. It is likely that they may, also, in a minimal medium, interfere competitively with enzymatic syntheses. Many of the compounds discussed in this section are not at present acceptable as food preservatives in this country, but they have been used recently in other countries and some are being used illegally in the United States. The reaction of ethylene oxide with carboxyl and other active groups on proteins has been mentioned. Since this is not an acid, pH has little effect upon its penetration into the cell. In water solutions, ethylene oxide hydrolyzes to ethylene glycol which is relatively nontoxic for microbial cells. The longer chain epoxides, such as propylene oxide, hydrolyze even more rapidly. A thorough study of the action of epoxides against yeasts, molds, and bacteria, and their use in food preservation is now in progress (Whelton et d.,1946). The completion of this work, which involves the effect of the agents on metabolic processes as well as its over-all effect on growth, should facilitate the interpretation of the mechanism of action of the epoxides.
VI. STJMMARY AND CONCLUSIONS
It is evident that the chemicals which have been employed as food preservatives fall into several general classes. ’ The reactive compounds which destroy functioning groups on enzyme proteins must be relatively nonstable so that they do not persist in the food and poison the consumer. Compounds of this class include formaldehyde, sulfur dioxide, ethylene oxide and peroxides, and other oxidizing agents. As preservatives they give a good initial effect but their action soon disappears; they are useful to lower the initial microbial count and thus extend the storage life of food or assist the action of other preservative measures. It is essential that the decomposition products of such compounds be carefully defined under all possible conditions so that the possibility of toxic residues is eliminated. There is a distinct need for new effective agents of this type
390
ORVILLE WYSS
to meet special requirements in the fresh food industries. Further research is needed on the epoxides and other types of oxidizing agents, especially gaseous types such as nitrogen trichloride and organic peroxides. A thorough study of reducing agents which decompose to nontoxic products would probably yield new additions to our short list of useful preservatives. Persistent enzyme poisons such a3 borates and fluorides find little favor in the food industries since the enzymes which are inactivated are essential to the consumer as well as to the microorganism. The high concentrations required for microbial inhibition indicate that they can be successfully employed only in foods which are consumed as a very small part of the diet. Consideration must be given to the use of such substances under conditions where only small amounts are actually consumed. For example, they might be effectively employed in antiseptic ice. Under some conditions they may find use for impregnating the inedible portion of a food product such as fruit rinds or the superficial layer and even the bone in meat products, Substances which have a cumulative toxicity might be used during emergencies when the time of consumption is limited to a few days or weeks. An example of such a situation might be a military campaign or a camping expedition. The requirement of new agents of this type as well as of those presently available is a thorough understanding of all phases of their toxic action so that they can be employed with the same confidence that many toxic pharmaceuticals are now being used. The chemicals which interfere with the membrane should prove useful in some instances, although some of the best agents of this type are barred because of obnoxious tastes and odors, their effect on texture, and because of additional physiological action. Certainly, of the hundreds of compounds of this type that have been prepared, some can be selected which are useful even though only under restricted conditions. With the exception of penicillin and related agents most of the chemicals which are said to destroy or inhibit microorganisms by affecting the genetic mechanism, are highly toxic to higher forms. The current emphasis on fundamental research on antibiotics should reveal information required for determining the usefulness of these products for food preservation. The competitive enzyme inhibitors such as propionic and benzoic acids appear to offer the greatest possibilities (salicylic acid is less suitable because of its additional physiological effects such as that on blood clotting). For successful food preservation these competitive inhibitors must be used for preserving foods whose content of the competing metabolite is low. Naturally they must have, at the concentrations consumed, no other marked physiological action, and other foods in the diet must contain sufficient amounts of the competing metabolite to prevent manifestations of the competitive inhibition in the consumer. Regardless of how thoroughly the theoretical as-
MICROBIAL INHIBITION BY FOOD PRESERVATIVES
391
pects of the inhibition are understood their actual employment in food can be sanctioned only after extensive pharmacologic tests. From the tremendous number of metabolite antagonists which are being synthesized or studied it may eventually be possible to select other successful preservatives for many kinds of foods. It is possible that the best food preservative will involve the use of combinations of such agents to attack a complex anabolic enzyme system at several steps. REFERENCES Anderson, E. H. 1945. Studies on the metabolism of the colorless alga, Prototheca zopfi. J. Gen.Physiol. 28, 287-327. Anson, M. L. 1941. The sulfhydryl groups of egg albumin. J. Gen. Physiol. 24, 399421. Balls, A. K., and Swenson, T. L. 1934. The antitrypsin of egg-white. J. Biol. Chem. 106, 409-419. Barron, E. S. G., and Singer, T. P. 1945. Studies on biological oxidation. XIX. Sulfhydryl enzymes in carbohydrate metabolism. J. Biol. Chem. 167, 221-240. Beadle, G. W. 1945. Biochemical genetics. Chem. Revs. 37, 15-46. Belfanti, S., Contardi, A., and Ercoli, A. 1935. Studies on the phosphataaes. Biochem. J . 29, 517-527. Bowman, D. E. 1943. Ether soluble fraction of navy beans and the digestion of starch. Science 98,308-309. 36, Chang, S. L. 1944. Destruction of microorganisms. J. Am. Wafer Works ASSOC. 1192-1207. Delbriick, M. 1941. A theory of autocatalytic synthesis of polypeptides. Cold Spring Harbm Symposia Quant. BioE. 9, 122-126. v. Euler, H. 1942. Coenzyme and inhibitors; vitamins and antivitamins. Ber. 76, 1876-1885. v. Euler, H., and Ahlstrom, L. 1943. The influence of Na salicylate on enzyme systems. 2. physiol. Chem. 279, 175-186. Fraenkel-Conrat, H. 1944, The action of 1,kpoxides on proteins. J. Biol. C h . 164, 227-238. Gale, E. F., and Taylor, E. S. 1947. The assimilation of amino acids by bacteria. 2. The action of tyrocidin and some detergent substances in releaaing amino acids from the internal environment of S t r e p t o e o m faemlis. J. Gen.Microbial. 1,77-84. Gilman, A., and Philips, F. S. 1946. The biological actions and therapeutic applicatiom of 8-chloroethyl amines and sulfides. Science 103, 409-415. Ham, W. E., and Sandstedt, R. M. 1944. A proteolytic inhibiting substance in the extract of unheated soybean meal. J. Biol. Chem. 164, 505-506. Hellerman, L. 1937. Reversible inactivations of certain hydrolytic enzymes. PhvsiO2. Revs. 17,454-484. Henry, H., and Stacey, M. 1946. Histochemistry of the gram staining reaction for microorganisms. Proc. Roy. Soc. London BUS, 391-406. Herriott, R. M. 1936. Inactivation of pepsin by iodine and the isolation of diiodotyrosine from iodinetad pepsin. J. Cen. Phys-iol. 20, 335-352. Herriott, R. M. 1941. Isolation, crystallization and properties of pepsin inhibitor. J. Gen. Physiol. 24, 325-338. Herriott, R. M., and Northrop, J. H. 1934. Crystalline acetyl derivatives of pepsin. J. Gen. Physiol. 18, 36-67.
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ORVILLE WYSS
Hotchkiss, R. D. 1946. The nature of the bactericidal action of surface active agents. Ann. N . Y . A d . Sci. 46, 479-493. Ingram, M. 1947. A theory relating the action of salts on bacterial respiration to their influence on the solubility of proteins. Proc. Roy. SOC.London 134, 181-201. IvBnovics, G. 1942a. Mechanism of the antiseptic action of salicylic acid. Naturwissmchaften 80, 104-105. Idnovics, G. 1942b. The salicylate ion aa a specific inhibitor of the biosynthesis of pantothenic acid. 2. physiol. Chem. 276, 33-55. Jacobs, M. H. 1940. Some aspecta of cell permeability to weak electrolytes. Cold SprinB Harbor Symposia Quant. Biol. 8, 30-39. Kneen, E., and Sandstedt, R. M. 1943. An amylase inhibitor from certain cereals. J. Am. Chem. Soc. 66, 1247. Krampitz, L. O., and Werkman, C. H. 1947. On the mode of action of penicillin. Arch. Bwchem. 12,57-07. Michaelis, L., and Schubert, M. P. 1938. The tl~eoryof two step oxidations involving free radicals. Chem. Revs. 22, 437-470. Mhky, I. A. 1944. Inhibition of &hemolytic streptococci fibrinolysin by trypsin inhibitor (antiprotease). Science 100, 198-200. Mitchell, P. D., and Crowe, G. R. 1947. A note on electron micrographs of normal and tyrocidine-lysed streptococci. J. Gen. Microbiol. 1, 85. Northrop, J. H. 1939. Crystalline enzymes. Columbia Univ. Press, New York. Pfeiffer, C. C., Hallman, L. F., and Gersh, I. 1945. Boric acid ointment. J. Am. Med. Assoc. 128, 266-274. Philpot, J. S. L., and Small, P. A. 1938. The action of nitrous acid on pepsin. Biochem.
J . 32,642-548.
Rahn, 0. 1945. Injury and death of bacteria by chemical agents. Biodynamics Monographs, No. 3. Normandy, Mo. Rahn, O., and Conn, J. E. 1944. Effect of increase in acidity on antiseptic efficiency.
Id.Eng. Chem. 36, 185-187.
Roblin, R. 0. 1946. Metabolite antagonists. Chem. Revs. 38, 255-375. Ryan, E. J., Schneider, L. K., and Ballentine, R. 1946. Mutations involving the requirement of uracil in Clostridium. Proc. Natl. Acad. Sci. U . S . 32, 261-271. Schnitzer, R. J., Camagni, L. J., and Buck, M. 1943. Resistance of small colony variants (Gforms) of a staphylococcus towards the bacteriostatic action of penicillin. Proc. SOC.Exptl. Bwl. Med. I S , 75-78. Singer, T. P. 1946. Enzyme inhibitors and the active groups of proteins. Brewms Digest 20, 86-8, 104-106. Stanley, P. G., and Schloseer, M. E. 1945. Biological activity of pantolactone and pantaic acid. J. Biol. C h . 161, 613-516. Steinberg, R. A., and Thorn, C. 1940. Mutations and reversions in reproductivity of aapergilli with nitrite, colchicine, and d-lysine. Proc. Natl. A d . Sci. U. S . 26, 363-366.
Stone, W.S., Wyss, O., and Haas, F. 1947. The production of mutation in Staph&cocoa aurm by irradiation of the substrate. Proc. Natl. A&. Sci. U. S . 33, 69-67.
Theis, E. R., and Lama, M. M. 1944. The protein-formaldehyde reaction. J . B i d . C h . 164, W103. duVigneaud, V., Etch, A., Pekarek, E., and Lockwood, W.W. 1931. The inactivation of crystalline insulin by cysteine and glutathione. J. Bhl. Chem. 94, 233-242. Whelton, R., Pfaff, H. J., Mrak, E. M., and Fisher, C. D. 1946. Control of microbio-
MICROBIAL INHIBITION BY FOOD PRESERVATIVES
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logical food spoilage by fumigation with epoxides. Food Industries 18, 23-25, 174-176, 318, 320. Wright, L. D.,and Skeggs, H. R. 1948. Reversal of sodium propionate inhibition of Escherichia coli with p-alanine. Arch. Biochem. 10, 383388. Wyss, 0.1946. The bacteriostatic action of short chain fat acids. J . Bact. 61, 801. Wyss, O.,Ludwig, B. J., and Joiner, R. M. 1945. The fungistatic and fungicidal action of fatty acids and related compounds. Arch. Biochem. 7, 416-426. Wyss, O., and Wilson, P. W. 1941. Mechanism of biological nitrogen fixation. PTOC. Natl. A d . Sci. U.S . 27, 162-168. Zittle, C. A. 1947. Effect of borate on a protein-polysaccharide complex, the phoe phoesterase from calf intestinal mucosa. J . Biol. Chem. 167, 287-2923.
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High-Polymer Pectins and Their Deesteriiicationl BY GEORGE L. BAKER Univereity of Deloware Agrhltural Experiment Station, Newark, Delaware CONTENTS
I. Introduction . . . . 11. Highly Polymerized Pectin
. . . 1. Composition . . . . 2. Source Material . . . 3. Extraction Methods . . 4. Quality of Products . . 111. Deesterification of Pectins . . 1. Methods of Deesterification
. . . . . , . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
a. Acid Demethylation . . b. Alkali Demethylation . . c. Enzyme Demethylation 2. Properties of Low-Ester Pectins . a. Viscosity . . . . . . b. High-SolidsGels . . , c. Low-Solids Gels . . . . 3. Uses of Low-Ester Pectins , . 4. Pectic Acid and Pectates IV. Future Considerations . . . . . References . . . . . . . .
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. 396 . 396 . 396 .399 . 401 . 405 . 406 . 408 .a9 . 410 . 411 . 413 . .
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I. INTRODUCTION The most important gelling agent for fruit products at present is pectin, a by-product itself of the fruit industry. Pectin is the viscous, colloidal substance extracted from plant tissues, usually through heating and pressing. It belongs to the group of substances known as hemicelluloses found throughout the cell walls of plant life, the larger concentrations occurring in rapidly growing surface tissues (parenchymatous cells). Botanically, the pectic substances are identified largely with the middle lamellae of the cell walls. Pectins occur in vacuoles, lamellae, and cell walls. While Vauquelin must be credited as the first to indicate some of the properties of the gelling principle in juices (Vauquelin, 1790, 1791, and 1829),Braconnot (1824)proved that the principle has acidic properties and that it exists universally in all fruits and vegetables. It was he who suggested the name pectic acid, from T V X T L S , coagulum. Names applied to 1 Published as Miscellaneous Paper No. 23 with the approval of the Director of the Delaware Agricultural Experiment Station. Contribution of the Department of Chemistry, December 28, 1946. 395
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QEORQE L. BAKER
pectin and pectic compounds have varied considerably throughout the past 120 years, but the following is an abstract of the present nomenclature adopted by the American Chemical Society in 1944 after a revision of that of 1926 (A. C. S., 1944): Pectic substances - The group designation. Protopectin - Water-insoluble parent pectic substance. Pectinic acids - Colloidal polygalacturonic acids with more than a negligible proportion of methylester groups. - Normal or acid salts of pectinic acids. Pectinates - General term for pectinic acids or pectinates caPectin pable of forming gels with sugar and acid. Pectic a'cids - Colloidal polygalacturonic acids mostly free of methyl ester groups. Pectates - Normal or acid salts of pectic acids. Full definitions and statements concerningthem will be found in the Report of the Nomenclature Committee. It is believed the chart developed by Joseph (1945) will give an understanding of pectin nomenclature better than formal definitions (Fig. 1). It will be an easy reference in case of confusion during the reading of the discussions in this paper. During the past decade considerable impetus has been given to research on pectic substances in the United States through the establishment of the various regional research laboratories of the United States Dept. of Agriculture. The results of their studies plus those of industry and established research units throughout the world have been many, so many in fact that the present discussion must be limited. Since it must be limited, it was thought best to consider those phases of research which most directly affect the food industry. There are 2 outstanding phases of study: (1)the extraction of high-grade pectin (highly polymerized pectin); and (2) the production of partially deesterified pectins or pectinic acids. A description of these 2 phases and their practical application to the food industry will be given here. 11. HIQHLY POLYMERIZED PECTIN 1.
COMPOSITION
The composition of pectin has been studied extensively by numerous investigators since Braconnot first described its acid properties (Braconnot, 1824,1825,1831,1832). Those investigators whom pectin chemists choose as giving the most valuable contributions leading to a disclosure of what is considered today as the true composition of pectin may be listed as
HIQH-POLYMER PECTINS AND THEIR DDESTERIFICATION
397
follows: Fremy (1840,1847a,1847b, 1848, 1859) who studied soluble pectins and various hydrolytic products; Wohl and von Niessen (1889)who established the presence of galactans; von Fellenberg (1914, 1918) who found that methyl ester groups were present amounting to from 9-11.5% ESTERIFIED POL f ALACTURONIDE (16.; To) - - - - - COMPLETELY \
! 16
M boxyl of this region thepretically possible but n
yet attained.
14
E
Q
1
'0
z
13 12
PECTIN
11
HIGH METHOXYL PECTINATES A id or Neutral
lo 9
PECTINIC ACID
8
d8 7
N.1
V I I LOWER LIMIT (Pectinurn)
OR PECTIN
When highly puriied and acid washed. 4ll unesterified car)oxyl groups are free.
G I 3
E
$
6
H
B 4
#
3
z
2
LOW-METEOXYL or LOW-ESTER PECTINS
LOW METHOXYL PECTINATES Acid or Neutrul
1
0
P E C T I C ACID-(or
PECTATES)
Fig. 1. Nomenclature and methoxyl content of pectic substances (Joseph, 1946).
as methyl alcohol; Ehrlich and co-workers (Ehrlich, 1917, 1930, 1932; Ehrlich and Sommerfeld, 1926;Ehrlich and Kosmahly, 1929;Ehrlich and Schubert, 1929) who through extensive studies proved the presence of galacturonic acid, methyl and acetyl groups, galactose and arabinose; and Nanji et al. (1925)who isolated what they considered as the basic unit of
398
GEORGE L. BAKER
pectin, believing it to be a Bmembered ring consisting of 4 molecules of galacturonic acid and 1 molecule each of arabinose and galactose. These last workers believed 2 or 3 of the carboxyl groups of the galacturonic acid molecules might be methylated. Smolenski (1923) and his co-workers were the first to suggest that pectins are highly polymeriaed. Their proposed chain structure of galacturonic acid units in glycosidic linkage approached the present, generally accepted chain formula suggested by Meyer and Mark (1930) after a study of X-ray analyses. Following the important work by Schneider and his associates (Henglein and Schneider, 1936; Schneider et al., 1936, 1937, 1938) upon nitrated and acetylated pectins it has been considered that the chain compound proposed by Meyer and Mark (1930) may be written as follows:
This formula shows about 75% of the carboxyl groups methylated (11.92% CHaO); however, the positions of the free carboxyl are arbitrary. In the case of the nitrated pectins with which Schneider et al. worked, acetyl groups were absent and it was possible to almost completely remove arabinose and galactose by reprecipitation with dilute alcohol. Schneider and co-workers (1936, 1937, 1938) estimated from their determinations that apple pectin has a molecular weight of 280,000; citrus pectin, 220,000; orange pectin, 150,000; and beet pectin 20,000 to 35,000. The pectins in juices are not of the best. They have been found nonhomogeneous in degree of polymerization and not as highly polymerized as pectins from pulpy tissues (Schneider et al., 1936, 1937, 1938). Juices of apples, pears, and plums contain pectins of about the same molecular weight, 25,000-35,000, and the pectins in juice from oranges have a molecular weight of about 40,000-50,000 (Svedberg and GralBn, 1938). From the last paragraph of the above review, it might be concluded that pectin, while a large, chain-like molecule, is a relatively simple substance. Anyone who has worked with it cannot agree. In nature, pectin remains a complex substance. In practical usage pectin also remains a complexity. Pectin compounds extracted from a relatively pure lemon albedo by Myers and Baker (1931,1934) were found to vary in composition with the variation of the time, temperature, and pH of extraction. Their products which were similar to commercially extracted pectins contained
HIOH-POLYMER PECTIN’S AND THEIR DEESTERIFICATION
399
galactose, arabinose, and acetyl groups in addition to the polygalacturonic acids which were found to be from about 8-14% methylated. The high degree of polymerization of their preparations was indicated by the high viscosities and the jelly grades. These are similar to the types of pectins with which the food industry must deal. Myers and Baker (1934) point out that early researchers, who considered the constitution of pectins, did their work upon products with no consideration of their jellying power. Ehrlich’s pectins, for instance, were subjected to hydrolysis and were considerably depolymerized before he analyzed them. If pectin products are carefully prepared one obtains results similar to those described in the 56th Annual Report of the New York Agricultural Experiment Station. Here it is indicated that pectin contains methoxyl groups in glucosidic linkages which are not severed by treatment with alkali and that these groups are attached to the only free aldehyde groups in the polymer, i.e., to the end of the polyuronic acid chain. This assumption allowed the calculation of around 50 uronic acid groups provided a straight-chain structure was present. Morel1 et al. (1934) also showed a methyl group in glycosidic linkage and an x-ray analysis of a sample of their polyester glycoside indicated the existence of a long-chain molecule. Analytical data (Myers and Baker, 1934) show a high degree of variability in both the arabinose and galactose content. Arabinose decreased with a lengthening of the extraction period due to hydrolysis to alcoholsoluble substances, but galactose increased with the temperature of extraction. The latter substance might have been occluded as “ballast” material upon precipitation when the larger pectin yields were obtained at higher temperatures. It has been shown (Schneider and Bock, 1937) that both of these substances are largely removed by precipitation of pectins with dilute alcohol (53%). The contention has been made (Bonner, 1936; Olsen et aE., 1939) that these 2 substances are scattered at intervals along the pectin chain molecule. Essentially, however, it has been proved that pectinic acids from the several fruit sources are colloidal polygalacturonic acids with more than a negligible proportion of methyl ester groups and that the chain of d-galacturonic acid residues of pyranose structure are linked together through positions 1 and 4 as suggested for pectic acid (Meyer and Mark, 1930; Hirst and Jones, 1938, 1939; Beaven and Jones, 1939; Smith, 1939; Luckett and Smith, 1940). 2. SOURCE MATERIAL
The production of highly polymeri~edpectin is largely dependent upon the quality of the pectic substances in the source material. The albedo or white portion of the rind of the citrus fruits constitutes a huge reservoir
400
GEORGE L. BAKER
of pectin-rich material which, in view of increased citrus production, dwarfs apple pomace by comparison. The most commonly reported data (Wilson, 1925) show moist apple pomace to contain up to 2.5% pectin, lemon pulp up to 4.0% pectin, and orange pulp up to 5.5% pectin, all on the 100-grade (jelly strength, a quality criterion) basis. However, using polyphosphates (Baker and Woodmansee, 1944; Maclay and Nielsen, 1945), larger quantities of pectin have been extracted. Maximum yields, on undried basis, vary with stage of ripeness as follows: apple pomace, 3.5%; grapefruit peels, 5%; orange peels, 7.5%; and lemon peels, 10%. Briefly, apple residues are generally prepared by grinding the whole fruit, expressing the juice under hydraulic pressures sufficient to leave only 55 to 60% moisture, and then drying the pressed pomace. Sometimes in the past, when the residues were pressed to only 75% moisture content, the pomace was run through a pomace picker and then repressed to aid in removing juice, but with modern presses this is unnecessary. The pressed, moist residues are best dried to less than 10% moisture in a current of heated air in kilns or by passing through a rotary drier. Citrus residues are generally used in the fresh, moist state, because they are available over long-seasonal periods, making drying unnecessary. Several procedures have been proposed, however, for treating fresh peels in order to remove bitter substances and prevent the loss of pectin before drying the citrus-pulp for storage. If salts of aluminum (Bosurgi and Fiedler, 1932) are present in a concentration of 0.01% while heating pecticcontaining materials to 80°C.(176°F.) with water for 1 hour, it is claimed that the pectin is retained while the fibrous, coloring, and other soluble materials are removed upon subsequent pressing. Another process (Myers, 1939b) employs a 0.1% copper salt solution in order to remove soluble impurities. These procedures may be expected to effect normal extraction methods. Since the pectinates in the treated peels are more insoluble, slightly more acid than normally required must be present for proper extraction. Loss in jelly units from pectic raw material is also claimed to be prevented by soaking in 3 4 % solution of glycerol and then drying the material (Myers and Cowgill, 1940). There is a great variation in the pectic value of source materials, much of this variation is dependent upon the quality of the fruit from which the pomace or peel is obtained and the methods of handling and storage of these by-products previous to use. Seasonal and climatic conditions have some effect. The user shouId evaluate source material by measuring viscosities of a series of extractions covering a range of conditions of time, temperature, and pH (Baker and Kneeland, 1935) or of an extract obtained by heating under standard conditions (Mehlitz, 1939). Solution viscosity depends upon the quality (grade) and the quantity (%) of pectin
HIGH-POLYMER PECTINS AND THEIR DEESTERIFICATION
401
removed through extraction. If one is working constantly with the same type of source material, formulas for an approximate evaluation of the jelly units
[(quantity of source material)1 (grade X percent)
can be developed which will prove helpful (Baker and Kneeland, 1935). The method of evaluation given by Mottern and Karr (1946) may be used, but it is subject to the criticism that extraction may not have been adjusted to optimum pH. A viscosity-pH extraction curve is preferred. Studies of changes in the pectins of moist fruit pulps during storage with HzSOs (Morris, 1934) showed increase in jellying value if the fruit is heated to destroy enzymes and then cooled before addition of the acid. Jellying power of pulps treated in the above manner increased during storage at ordinary temperatures, but not during cold storage. It is now known that the suggestion of Morris (1935) that the increase may represent a stage in the slow conversion of pectinic acid to pectic acid is correct (see under “Gels”). In storage of apple pomace with HiSOs, more than 0.2% HaSOa caused a perceptible increase of soluble pectin (Saburov and Kalebin, 1935). Recent work (Charley et al., 1942; Burroughs et al., 1944) shows an effort should be made to dry the wet pomace immediately after it is pressed or very considerable losses in pectic value will take place during even a day’s storage. Concentrations of from 250-1500 p.p.m. of SOn have a preservative effect for about a week. Probably much of the decomposition is due to naturally occurring pectic enzymes (Joslyn and Sedky, 1940). However, chemicals such as ethylene (Heid, 1941),when used to ripen citrus fruits, have been found to lower jelly unit yields of pectins. Since it is well known that laboratory-prepared samples of dried apple pomace quite generally have 2 or even 3 times the jelly unit value of commercially prepared products, it would appear that further studies toward improving the grade of the commercial products are in order. The quantity of pectins present in the various commercial products does not vary appreciably, it is the degree of polymerization which shows great variation. 3. EXTRACTION METHODS Pectic substances exist in fruits and vegetables as protopectin, pectinates, and pectates. The proportion of any one of these substances existing at any one time cannot be predicted. The highest concentration of protopectin is found in growing tissue. This is changed to soluble pectinic acids during growth or ripening of plant tissues due to the slow action of plant acids or the more rapid action of the pectic en5ymes; the latter,
402
GEORGE L. BAKER
if free to act, cause a rapid conversion to pectic acids. Reaction with any available cation takes place readily as deesterification proceeds and pectinates are formed. The pectinates consist of a heterogeneous mixture of pectin units of varying degrees of polymerization and methylation, with random cross-linkage through available polyvalent cations. Solubility varies with the pectinate formed; the sodium and potassium salts are easily soluble while salts such as those produced by calcium, iron, and aluminum are insoluble under conditions of acidity existing in most plant tissue. As the methoxyl contents of the pectic substances are decreased, their reactivity increases until finally the difficultly soluble pectates are formed. It will be understood from this that many variations can be expected from pectins extracted from vegetative tissues. The above background may be obtained by reading the published works of many investigators from Fremy (1840) down to the present time. Fremy has frequently been misquoted as considering protopectin (pectose as he called it) an insoluble calcium compound, but from his experiments it is clear he considered protopectin to be merely an insoluble neutral substance (Fremy, 1847a, 1847b), the precursor of the insoluble pectinates. Many views, pro and con, have been presented by as many investigators. These variable views are due, in large degree, to the differences in age or degree of ripeness of the parent substance with which they worked. Unpublished data obtained at the Delaware Agricultural Experiment Station indicate that 70% more jelly units can be obtained from half-grown Stayman apples than from the full-ripe apples (same weight basis). This roughly indicates the progress of pectic changes in the growing fruit. From the commercial viewpoint, the value of a pectic source material is dependent upon the number of jelly units which it will yield upon extraction. The efficiencyof extraction methods is related to the 3 factorstime, temperature, and pH, at time of extraction. Concentration of the source material in the extraction mixture is of little importance other than that it should allow the free transference of heat, the higher the temperature of extraction the greater the necessary dilution. Contrary to this, in the extraction of fruit juices from fruit pulps the ratio of fruit to water is important (Baker, 1937) in obtaining optimum color, flavor, and gelling capacity. The relative effects of the variation of time, temperature, and pH in the extraction of pectin have been extensively investigated (Myers and Baker, 1931). Jelly units, the product of the yield of pectin times its grade, extracted by heating 30 minutes a t various pH values are shown in Fig. 2. The effect of longer periods of heating a t the optimum pH given in Fig. 2 is shown by the curves in Fig. 3. These figures are representative only. In the presence of an excess of polyvalent ions, a lower pH than indicated would be found optimum. The acids used far
HIOH-POLYMER PECTINS AND THEIR DEESTERIFICATION
403
extraction may be any of those capable of producing the desired pH. Some slight variation in their action may be expected. Modifications in extraction procedure have been suggested, such as the use of alkyl or aryl esters of aliphatic or aromatic acids, xylene, chloroform, or inorganic sulfocyanides (Rosenfield, 1938) for the softening and swelling of the crude pectic-containing material and extraction a t pH 2.
1.0
2.0
0.5
Pfl Fig. 2. Curves. showing the variation in the jelly units, at various temperatures, with variation of the pH of the extracting medium; extraction period maintained constant at 30 minutes (Myers and Baker, 1931).
Since the main idea, which may be gained from the preceding paragraphs, is to arrange for the separation of the polyvalent cations from the pectinates formed by nature, several other modifications of the usual acid extraction methods may be used to accomplish this purpose. Acid-alcohol treatment may be used to convert the insoluble pectinates into water-soluble products (Hirsch, 1942) insoluble in the alcohol, but which may be solubilized later by water extraction. Protopectin may be hydrolysed in a warm solution of HaSOa catalyzed by 2% &POl on the basis of air-dry,
404
QEORQE L. BAKER
crude pectic pulp according to a German patent (Otto and W i d e r , 1942). Combinations of acids such as this have been used many times and it would be difficult to state the originator; pH is the important factor in acid extraction, heat being required for erne of solution and hydrolysis of protopectin. Probably the most important modifications in the extraction of pectin are those effected by ion-exchange procedures. Hydrogen zeolite (Myers
Fig. 3. Curves showing the variation in the jelly units, at variow temperatures, with variation of the extraction period; pH of the extracting medium maintained constant (Myers and Baker, 1931).
and Rouse, 1943) has been found one of several cationexchange materials valuable in the solubilization of pectins. Upon removal of the cation, the pectinic acid is easily solubilized in accord with tenets described in previous paragraphs. The ion-exchange material functions in 2 ways; its hydrogen replaces the polyvalent cations with hydrogen-forming pectinic acids and, since these acids are soluble, they ionize to produce pH values of 2.5-2.8 which aid, upon the application of heat, in the solubilization of other pectinates. Polyphosphates (Baker and Woodmansee, 1944; Maclay and
HIQH-POLYMER PECTINS AND THEIR DEESTERIFICATION
405
Nielsen, 1946) inactivate (deionize) certain polyvalent ions and thus aid in the extraction of pectins at pH values above 3, therefore, high temperatures and short extraction periods can be used if desired. Pectins present in apple pomace can be solubilized with as little as 2% polyphosphate (based on dry weight) while as much as 20% polyphosphate must be used with some of the citrus albedos which are high in aluminum and calcium content. In contrast t o the ion-exchange adsorbents the polyphosphates can be u d only once, therefore, a high jelly unit yield is required for their economical use. Yields from 20-100% higher than normal are customary. The polyphosphat,es should not be used when the aluminum precipitation procedures are employed with citrus pectins due to precipitation difficulties and the impossibility of satisfactory removal of the aluminum ion in the presence of the phosphates. Addition of excess calcium to solutions of pectin extracted as above will cause increased viscosity and sometimes precipitation, depending on the degree of deesterification of constituent pectin molecules. Following extraction, pectin extracts are drained and pressed free from the crude pulp. Then the pectins are recovered from the filtered extracts by clarification and precipitation with alcohol or salts (von Fellenberg, 1918; Jameson et aZ., 1924; Olsen and Stuewer, 1938; Myers, 1939a) or concentrated by vacuum evaporation. The concentrates may be used as such or precipitated with alcohol, filtered, pressed, and dried. Actually, very little has been accomplished in the way of new methods of recovery in the past 20 years. Drum- and spray-dried pectins have been produced in small volume. The drum-dried products have the disadvantage of being fluffy and voluminous, but are easily dispersed. The spray-dried pectins are of very small particle size, are generally quite hygroscopic, and are difficultto disperse unless the particle size is increased. There appears to be a need of much further research on spraydrying methods. 4. QUALITY OF PRODUCTS The quality of pectins can be approximated by viscosity measurements. The viscosity of water solutions is dependent upon several factors, mainly, concentration, pH, degree of polymerization (grade), degree of methylation, and the amount and kind of cations capable of forming salts with the pectinic acids. The effects of the concentration and the grade upon viscosities are well known (Myers and Baker, 1927, 1929; Owens, Lotzkar et al., 1944). The hydrogen-ion concentration has greater influence upon viscosity as the methoxyl content is lowered and as the concentration of metal ion is increased (Baker and Goodwin, 1939a, 1941a; Gaponenkov, 1937; Kortschak, 1939). High-methoxyl pectins in the absence of polyvalent cation show maxi-
406
GEORGE L. BAKER
mum viscosities in the pH range 5-7.
While these pectins are slightly affected by the presence of increased acid for relatively long periods of time, inweased alkali is attended with gradual demethylation and depolymerization and, consequently, a considerable change in pectic properties. In the acid range, metallic ions do not affect solution viscosity below pH 2.75 as much as above this pH. Abnormally high viscosities result, probably as a result in part by the formation of complex hydroxides, upon addition of such salts as those of copper, lead, iron, and aluminum. Excess of these heavy metal ions will cause coagulation and precipitation in certain specific pH ranges. Actually, the only true determination of grade or quality can be obtained by making up a series of 65% soluble solids sample gels under conditions which will reflect those of actual usage. Set conditions of acidity cannot be specified because the buffer capacity may vary considerably, also the optimum pH of gelation may vary. Normal preparation and usage of pectin for gel manufacture, however, may be generalized to quite an extent. For instance, the “excess-acid” method (Stuewer et al., 1934) is good for grading if one does not wish to carry out a procedure reflecting conditions of actual usage. The optimum pH of gelation for 65% soluble solids gels varies considerably with the type and age of the fruit from which the pectin is obtained. An optimum pH as high as 3.8 has been obtained with pectins extracted by heating with polyphosphates, but the general run of pectins shows an optimum pH of gelation of 3.0-3.4 when made by the usual boiling procedure with all ingredients in the mixture. A more complete discussion of gelation will be presented under the section dealing with deesterification.
111. DEESTERIFICATION OF PECTINS After von Fellenberg (1914; 1918) established the presence of methyl ester groups in pectin (9-11.5% as methyl alcohol) intensive study of the effect was initiated. A fully methylated pectin has never been obtained nor has protopectin, the water-insoluble parent pectic substance, been isolated as a fully methylated pectic substance. Von Fellenberg considered that pectin should be partially demethylated in order to form a jelly. The gradual chemical transformation from pectin through “pectosic acid” to pectic acid had been described much earlier (Fremy, 1848). Fremy found this action took place either during ripening of fruits, through the direct action of pectase, or during chemical treatment through the use of caustic soda, potash, sodium or potassium carbonate, or the use of lime, baryta, or strontia water. Von Fellenberg, however, recognized this transformation as the gradual removal of methyl ester and that as these groups
HIQH-POLYMER PECTINS AND THEIR DEESTERIFICATION
407
were split off the easier was the reaction with metallic salts. He was wrong, however, in concluding that viscosity always declines as methoxyl groups are split from the pectin molecule. Researchers in the 1920's (Sucharipa, 1923, 1925; Luers and Lochmiiller, 1927; Branfoot, 1929; and also Gaddum, 1934) quite generally interpreted their findings as indicating that the jellying pawer of pectin decreased with decrease in methoxyl content. Luers and Lochmiiller thought that pectins (pectinic acids) with less than 7.3% methoxyl do not form jellies. Myers and Baker (1927, 1929), on the other hand, concluded that the
Fig. 4. Relationship of methoxyl content and equivalent weight of pectins. (Data compiled by the author from the literature.)
methoxyl content of pectinic acids is no criterion of their jellying power. They (1931, 1934) found definite proof of this when they examined several series of pectins extracted in temperature ranges between 22" (71.6"F.) and 100°C. (212°F.) and a t pH values in the range 0.2-3.0. They concluded that jellying power dspended upon the degree of polymerization of the galacturonic acid. Their studies showed that the methyl ester groups could be removed at temperatures below 60°C. (140°F.) and at low pH values without excessive depolymerization. Bock (1943)) in an unpublished manuscript, has supported the American findings (apparently unknown to him) and also attached great importance to the degree of methylation. According to his findings, methods of extraction should be designed to dissolve out from the protopectin pectins with as high a degree
408
GEORGE L. BAKER
of polymerization as possible, preferably splitting off some of t.he met,hoxyl groups. The combining weight of pectinic acid decreases, of course, during demethylation. The use of the terms “combining weight” or “equivalent weight” is unfortunate as far as colloidal properties are concerned in that it injects confusion into terminology. The relationship of combining
Fig. 6. Demethylation during extraction of pectin at 50°C. at various pH values (Baker and Goodwin, 1941b).
weight and methoxyl content of pectinic acids is given in several articles (Olsen et al., 1939;Kaufman et al., 1942;Schultz et al., 1945). The average relationship compiled from data appearing in these articles may be shown by a curve as in Fig. 4. The combining weight of a pectin is established by the degree of demethylation (von Fellenberg, 1914, 1918;Olsen et al., 1939);a statement of methoxyl content should be sufficient to indicate the physical properties of the compound. It is the physical properties in which the food processor is interested.
1. METHODS OF DEESTERIFICATION Low-ester pectinic acids may be prepared by 3 methods: acid demethylation, alkali demethylation, and enzyme demethylation.
HIOH-POLYMER PECTIN8 AND THEIR DEEGTERIFICATION
409
Acid Demethylation Acid demethylation of pectinic acids may be accomplished during extraction, or following extraction, either with a liquid concentrate or a powdered pectinate. Removal of the methyl ester groups requires a low temperature (below 60°C.), a high hydrogen-ion concentration, and a relatively long period of time (Myers and Baker, 1931, 1934; Olsen et al., 1939; Baker and Goodwin, 1941b). a.
Fig. 6. The effect of extended acid hydrolysis upon the demethylation of high- and
low-ash prepared pectins. Curve for pectin extracted at pH 1.68 is included for purp o w of comparison (Baker and Goodwin, 1941b).
An example of deesterification during extraction at 50°C. (122°F.) is illustrated in Fig. 5 (Baker and Goodwin, 1941b). This figure shows the effect of extended periods of extraction of apple pomace at 50°C. (122°F.) at various pH values upon the methoxyl content of the dry pectin product. It will be noted from Fig. 5 that practical methods would require a relatively low pH in the case of pectic-containing source material similar to that examined. Resistance to further demethylation is found as the reaction progresses. Demethylation of a pectinate concentrate or powder will proceed at a rate dependent upon the type of pectinate. A low-ash type of pectinate
410
QEORQE L. BAKER
offers less resistance to the removal of methyl ester groups than a high-ash type. An example of the differences in the rate of removal is given in Fig. 6 (Baker and Goodwin, 1941b). The low-ash pectinate contained 0.76% ash, while the high-ash product had 10.69% ash. The pectinate extracted a t pH 1.68 is the same as shown in Fig. 5. All reactions were carried out a t pH 1.6 f 0.1. In the case of the prepared pectinates demethylated as in Fig. 6, each gram of original pectin was first dispersed with 1 mI. of 95% ethyl alcohol to aid wetting and then in 80 ml. of acid. The pectinates were thus redissolved and the reaction was practically the same as though carried out on a liquid pectinate concentrate. If more alcohol is used (Baker and Goodwin, 1941b) the pectinates are less soluble and the rate of demethylation is retarded, i.e., the higher the concentration of alcohol, the lower the rate of demethylation. Table I indicates the effect of the concentration TABLE I Demethylation of Pectin in Alcohols
I
I
Mixture
'
~~
GH~OHCC. Ha0 CC. CHIO % No. Control 1 2
3 4
..
10 30 45 60
..
80 60 45 30
9.64 3.19 4.82 5.44 5.83
~
Relative viscosity (Ostwald) 0.5% solution at 26"C.,pH 2.5
Grade
12.94 6.00
304 45
3.70
70
2.58 2.20
100 120
of alcohol on the rate of demethylation at 50°C. (122°F.) in the presence of 3.5% HC1. A reduction in the temperature of the reaction aids in the preservation of grade. Grade as used here m a y be defined as the number of pounds of sugar which one pound of pectin will support as a 66% soluble solids jelly of standard strength at 26°C. (78.8"F.)under optimum conditions of gelation. The above method of demethylation when carried out at low temperature with sufficient alcohol to prevent the actual solution of the pectin is very economical because small amounts of alcohol are required to recover the pectinate, particles of which are only swollen, never fully dispersed. b. Alkali Demethylation This method of deesterification is very old. Complete deesterification was practiced by the very early investigators (Braconnot, 1824; Fremy,
HIGH-POLYMER PECTINS AN'D THEIR DEESTERIFICATION
411
1840) by a simple addition of an alkali, but controlled, partial deesterification with alkali is a fairly recent development. McDoweIl (1941a, 1941b, 1942, 1943) WM able to control the rate of the effect of alkali by low temperatures, using 0°C. (32°F.) to +lO"C. (50°F.) temperatures for the controlled deesterification of apple pectin and from freesing to +20"C. (68°F.) upon beet pectins. Alcohol, similarly to its action with acid, retards the action of alkali in deesterifying the pectinic acids (Baker and Goodwin, 1943). Electrolytes accelerate deesterification (Lineweaver, 1945). A t the time methods of treating pectin with basic reagents for the altering of jelly setting characteristics were first developed, the alteration of these physical characteristics was not correlated with methoxyl content (Cole and Cox, 1938; Cox, 1938; Joseph, 1936; Leo el al., 1939). It was recognized at that time that care must be taken to avoid depolymerization. Other methods of alkali demethylation have been proposed such as the treatment of flaked pectin with ammonia vapors (Evans and Huber, 1945). McDowell (1942) considered alkali-deesterified pectinic acids to be intermediate in character between acid- and enzyme-treated products. Contrary t o the belief induced by previous findings (Baker and Goodwin, 1944), it is now believed (Baker and Woodmansee, 1946) that McDowell's surmise is correct. e. Enzyme Demethykztion
The action of pectase (pectin esterase) in deesterifying pectinic acida has been known for a century (Fremy, 1840, 1847). An excellent review and a careful study of the action of pectase upon pectinic acids was made by Mehlitz (1930). He used coagulation or gelling in the presence of calcium salts as an index of reaction. Today, however, one is interested in obtaining pectinic acids of definite methyl ester content without depolymerization and careful control of the reaction is necessary. The Eastern Regional Research Laboratory (Hills el al., 1942; Willaman et al., 1944) has developed very accurate control measures for the enzymic deesterification of pectinic acids using pectase isolated from ripe tomatoes (Kertesz, 1938; Willaman and Hills, 1944; Hills and Mottern, 1945; Mottern and Hills, 1946). Pectase activity is favorably influenced by cations (Lmeweaver and Ballou, 1945; MacDonell et al., 1945). Enzymic demethylation is carried out (Willaman et al., 1944) by treating a pectin solution at pH 6 and 4045°C. (104"-113°F.) with pectase which is essentially free of pectinase. Alkali is added at a s&cient rate to maintain this pH as deesterification proceeds. The amount of deesterification is calculated in terms of alkali required for neutralization after a certain number of methyl groups has been removed. When the required amount of alkali has been used up, strong acid is added until the pH is
412
QEORQE L. BAKER
adjusted to about 4. The solution is then heated to 80°C. (176°F.) to inactivate the enzyme, cooled, and precipitated with alcohol. Workers (Owens et al.,1944) at the Western Regional Research Laboratory believe that the production of viscous pectate pulp (Wilson, 1938), by treating citrus pulp a t not much above 35°C. (95°F.) with alkali a t pH 8.5, depends upon the action of the pectase naturally occurring in the raw citrus materials. This is probably the case because unpublished data (Baker and Goodwin, 1939b) show that demethylation of apple pectin, extracted under conditions which would destroy pectase, with NHlOH is very slow at an initial pH 9. Demethylation at 22°C. (71.6"F.) proceeded very slowly as follows: Start - 10.34% CHsO 30min. - 9.79% CHsO 60 min. - 9.65% CH& 9.49% CHaO 90 min. .However, 30 minutes at increasing initial pH values showed increasingly higher rates of deesterification, thus: pH 9 pH10 pH11
-
9.79% CHsO 6.60% CHaO 2.49% CHaO
Enzyme demethylation is rapid and easy to control, but the pectic product cannot yet be considered the best. The average degree of polymeriration is such that fairly high viscosities are obtained, but the proportion of low-polymer material is probably high. Likewise, the average degree of demethylation can be carefully controlled, but the proportion of low-ester material is high. Molecular-weight distribution studies of pectin nitrate &B a function of the method of deesterification have been made (Speiser and Eddy, 1946), but much more should be done on this subject. The character of low-solids gels made with pectinates prepared by various methods indicates that a much higher percentage of high molecular weight pectic units is present in normal non-deesterified pectinate or partially deesterified pectinic acid prepared by aciddeesterification than is indicated by the analysis of the nitrate pectins by Speiser and Eddy illustrated in Fig. 7. Also, an unpublished anaIysis of deesterification of pectinic acid molecules by fractional ionic precipitation (Baker and Goodwin, 1939a) and a comparison of the action of calcium on low-solids gels (Hills et al., 1942) suggest that a high percentage of low-ester pectinic acids are present in the enzymedemethylated products. The hypothesis has been proposed (Schultr et al., 1945) that esterase deeshrifies portions of the galacturonide chain while chemical action (acid or alkali) acts in a randomized manner.
HIGH-POLYMER PECTINS AND THEIR DEESTERIFICATION
2.
413
PROPERTIES OF LOW-ESTER PECTINS
The partial removal of the methyl ester influences the physical properties of pectins enormously. The method of deesterification has a considerable influence on the physical characteristics at any particular methoxyl content (Baker and Goodwin, 1944; Schults et al., 1945). Solution viscosity, the grades, the optimum pH of gelation, the pH range of geletion, the speed of gelation and the temperature at which gels will remelt are all affected by the methyl ester content of the pectins. Perhaps the most important characteristic of partially deesterified pectins is their ability to form gels with little or no sugar in the presence of metallic ions. The effect of demethylation upon pectin properties will be summarimd.
Fig. 7. Molecular-weight distribution of pectin nitrate aa a function of method of deestedcation; non-deeateded , enayme deesterified - - - - - -, acid deesterified - - - (Speiser end Eddy, 1946).
-
-
a. Viscosity
Solution viscosities of normal, high-ester pectins generally show a slight decrease on adjusting the pH to 3 after which a distinct levelling off in the effect of acid is noted. Intermediate and low-ester pectins are affected by acid in different ways depending to some extent upon the method of deesterification and t o some extent upon the source material. In general, changes in acidity affect the viscosities of solutions of these latter pectins to the greatest extent at pH 3 or somewhere between pH 1.5 and 3 (Olsen et al., 1939; Baker and Goodwin, 1941b). Fig. 8 indicates the effects of the variation of acidity upon the viscosity of 0.25% solutions of low-ester pectinates of various methoxyl content, Pectinic acids containing less than about 3.6% CHaO precipitate &B a coagulum below pH 2.
414
GEORGE L. BAKER
Heavy metal ions increase solution viscosity; the pH range of their effectiveness in producing the higher viscosities is increased with decreased methoxyl content of the pectins. For instance, the effect of me-
Fig. 8. The relative viscosity of 0.26% solutions of low-ash pectins of various methoxyl content aa dected by pH. Broken lines show the effect of 24 hours of aging at 28°C. (Baker and Goodwin, 1941b).
tallic ions upon viscosity may be traced in an inverse fashion by a consideration of dispersion in the presence of their salts at pH 3.5. At this pH copper salts will prevent dispersion of pectins of any methyl ester content, as will also aluminum and nickel. Pectin above 8% methoxyl
HIBH-POLYMER PECTINS AND THEIR DEESTERIFICATION
415
content will disperse in the presence of calcium, magnesium, iron, and manganese. As the methoxyl content is lowered the more difficult becomes the dispersion in the presence of these cations and the more viscous become any resulting solutions.
b. High-Solids Gels Pectin Grades. Grades of pectins tend to increase with deesterification to about 8% methoxyl content and then decrease (Baker and Goodwin, 1941b). Improvement in grade upon acid treatment of pectin was noted (Cole and Cox, 1938) before the effect of the acid upon deesterification was recognized (Olsen et al., 1939). The decrease in grade as the pectin is further deesterified by acid hydrolysis below about 8% methoxyl content is partially due to depolymerization, but it is also associated with precipitation of pectin during the production of 65% soluble solids gels. The decrease in grade is dependent upon how drastic the demethylation conditions are, i.e., temperature and pH in chemical methods or pectinase action in enzymic methods. Methoxyl content, contrary to the belief of earlier workers (Luers and Lochmiiller, 1927), is no criterion of jellying capacity or grade. This has been proved by several groups of workers (Myers and Baker, 1929; Bennison and Norris, 1939; Baker and Goodwin, 1944). Gels with 650/, soluble solids content can be made from pectins which have been almost completely demethylated (pectic acid) if means are provided for retardation of the coagulating effect of polyvalent cations. The most practical method of preparing such jellies thus far found is through the use of the polyphosphates. “High solids” jelly, however, made from the low-ester pectins has a tendency to have a sticky surface, an undesirable characteristic. Optimum p H of Jellying. The optimum pH of gelation at 65% soluble solids content is lower with decrease in methoxyl content. This decrease has been noted to be greater in the presence of calcium ions (Baker and Goodwin, 1941b), but may be countered through the increased use of the glassy phosphates. Buffer salts are also being studied as a means of broadening the pH range of optimum gelation. Results thus far indicate that the optimum pH may be raised to as high as 4.5 by this method. The possible pH range over which gelation will occur will be variable and will depend upon the relationship of buffer salt, metallic ion, and deionizing substances such as sodium hexametaphosphate. This is also true of the initial or maximum pH of gelation. Speed of Gelation. Setting times for 65% soluble solids pectinate gels will be influenced by methoxyl content. An analysis of earliest patents (Joseph, 1936; Cole and Cox, 1938; Cox, 1938) for the altering of the time of set of gels indicates the main factor is partial demethylation of the
416
GEORGE L. BAKER
pectins. Generally as the methyl ester is removed from high-ester pectins, the setting time of their 65% solids gels first increases and then decreases (Olsen et ul., 1939; Baker and Goodwin, 1941b). Exceptions to the rule will be found. Slow-setting pectins of high-ester content have been known. The historical background and the presence of cationic influences are important in determining setting times. Melting Tmperutures. Low-melting temperatures are characteristic of 65% solids gels made from low-ester pectinates (Baker and Goodwin, 1941b). Normally, high-methoxyl pectinate gels (65% soluble solids) are not considered as reversible gels. Reversibility (solution upon heating and regelation upon cooling) may attend high-solids gels at higher pH values, but this is more the exception than the rule. The property of reversibility has value in the bakery and confectionery industries. Melting temperatures at the optimum pH of gelation may decrease as much as 10’F. (5.6”C.) or more with a decrease of 1%in methoxyl content. At pH values above the optimum, melting temperatures tend to be very much lower. Excessive amounts of metallic ions will alter these generalizations. Lour-Solids Gels Introductw/t. Partial deesterification of pectins has opened up a broad new field for investigations of gelation. Gelation was early considered as due to the action of sugar 8 s a dehydrating agent (Tam and Baker, 1924). Now gelation of 65% soluble solids pectinate gels is explained as due to the lowering of the possible solvent action of the water in the preaence of the high concentration of sugar or a “hydrogen bonding” (Peters, 1942; Speiser, 1943), the sugar promoting an extensive network of hydrogen bonds throughout the gel. When precipitation of pectins occurred upon making jelly from low-ester pectinates the natural corrective step was to lower the sugar or soluble solids content in order to allow the full gelling action. While it was found (Baker and Goodwin, 1941b) that small amounts of calcium aided the gelling of pectin in the high-solids gels in the absence of precipitation, larger amounts of calcium tended to increase precipitation tendencies, As sugar solids were decreased in order to prevent coagulation in gels (directly due to sugar) it was found that the addition of calcium salts could be increased over the amount added to the 65% soluble solids gels. It waa apparent that calcium tended to react with the pectinate and aid gelation of low-solids gels (Baker and Goodwin, 1941b; Olsen and Fehlberg, 1943). I n later studies (Hills et aE., 1942; Baker and Goodwin, 1044) it was confirmed that increased amounts of calcium were required to produce the optimum gel as soluble solids were reduced. The increase in the calcium requirement, however, was not the c.
HIQH-POLYMER PECTINS AND TaEIR DEESTERIFICATION
417
same for pectins of various methoxyl content. -4 maximum requirement is found with pectins containing from 5.5-6.5% CHaO (see Fig. 9). The role of calcium, or other metallic ions, in gelation has been termed “ionic bonding” in contrast t o that due to sugar or similar dehydrating agent which has been considered as due to “hydrogen bonding.” Reaction of metallic ions with pectinates is not new. Precipitation which is dependent upon combining power of the free carboxyl groups of
Fig. 9. The effect of added sugar on the optimum calcium requirement of gels from pectinate8 with various methoxyl content (Baker and Goodwin, 1944).
pectins was discussed years ago (Fremy, 1848; von Fellenberg, 1918; Olsen and Stuewer, 1938; Myers, 1939a);and precipitation is in some ways similar to gelation. Gelling may be considered as a controlled step toward precipitation. The lower the methoxyl content the greater the number of free carboxyl groups and the greater the number of these latter units, the greater the possibility for “ionic bonding” to function and the less the dependency upon “hydrogen bonding.” Cross linkage “ionic-bonding,” of pectinic acid molecules has been pictured as follows (Baker and Goodwin, 1941b):
GEORGE L. BAKER
418
-00
Go H
-
0-
on
-0
coocrr,
w
00-
The above can be visualized as a basis for a 3-dimensional structure which allows the formation of low-solids gels. This structure has also been recently proposed by other workers (Henglein, 1943; Bock, 1943). It is the structure, interspersed with protopectin, that can also be visualized as present in the growing plant tissues. With these considerations in mind, it is found that precipitation of pectins in 65% soluble-solids gels occurs in the presence of a slight excess of calcium at methoxyl contents below about 7.5%. Reduction of sugar solids or the calcium ion, and increased acidity tend to offsetprecipitation tendencies. Monovalent ions and the polyphosphates aid in reducing this tendency. The reduction of sugar content, or even its absence entirely from the gel, offered the possibility of opening the most interesting of new fields-substitutions of fruit pectins for animal gelatins or for agars in the gelling field. Pectinate Requirement for Low-Solids Gels. Upon examination of several miscellaneous samples of unstandardized, acid-deesterified pectinates, it is indicated that more or less similar quantitative pectinate requirements for similar soluble-solids levels will be found at corresponding methyl ester values. The amount of pectinate has been found dependent on pectic grade upon the 05% solids basis or, in other words, it is directly influenced by molecular sim. Fig. 10 shows that pectins with above about 4.5% CHaO are required in increasing amounts as the sugar solids are lowered. As a ruIe the amount of pectina.te required is greater the higher the methoxyl content is above 4.5%. ,Exceptions to this general rule are found in gels of 50% sugar content with methoxyl contents above 7.1% CH30. GeIs containing pectinates with less than 4.5% CH30tend to lose certain desirable gel characteristics such as elasticity and to acquire a degree of stickiness. Pectinates deesterified by alkali or enzymic methods tend to behave like acid-deesterified products of a somewhat higher methoxyl content.
HIGH-POLYMER PECTINS AND THEIR DEESTERIFICATION
419
In determining the amount of pectinate required for gelation at any soluble solids level the grade and methoxyl content should be known. The methoxyl content may be determined by the saponification method of von Fellenberg, or mod&cations of same (Hinton, 1939), or by the Zeisel method upon a dried pectinate precipitated from dilute solution, or it may be determined directly on the pectinate preparation if the actual pectinate content is known. If the Zeisel method is used, no residual
Fig. 10. The d e c t of methoxyl content upon the average pectin requirement at various sugar concentrations (Baker and Goodwin, 1944).
alcohol should be present (Jansen et ab., 1944). The grade of the pectinate may be approximated by measuring the solution viscosity at pH 4.5 (Baker and Goodwin, 1944), but it should also be determined by actually making up gels of 65% soluble solids content. When the grade and methoxyl content are known, a close approximation of the amount of aciddemethylated pectinate to be used can be found from reference to Fig. 10. Calcium Requirement for Low-Solids Gels. The amount of calcium required for gelation of low-solids gels must be determined for each pectinate. In practical usage it must also be determined for each different type of fruit or fruit juice because of the influence of the fruit salts naturally present. The method of determining the calcium requirement is simple, but rather time-consuming. A brief description follows: The pectinate is dispersed into about one-half the juice to be used for the test gel. It is then heated to boiling and the other half of the juice, containing various amounts of
420
GEORGE L. BAKER
calcium salt and the necessary amount of sugar for the desired soluble solids content, also at about boiling temperature, is added to it. The solution $,adjusted with hot water to the same weight for each gel of varying calcium content in the series, then the test gels are allowed to set for a uniform length of time before determining their strength, melting temperatures, etc. If desired, gel-setting temperatures may be determined 88 the gels are cooling. These vary with the amount of added calcium, becoming higher as the calcium is increased. In practical factory usage it is best not to employ more than one-half to two-thirds of the amount of calcium required for optimum gel strength in the test gels, otherwise pregelation or curdling d the gel occurs during manufacture. Changes in Low-Solids Gels with Aging. Two major changes have occurred in 30oJ, soluble solids grape gels during aging under conditions approximating those of the store-shelf. These were increased gel strength and an increased melting temperature. The changes occurred sooner in the gels made from an apple pectinate than in those made from a lemon pectinate. The increase in st,rength seemed t o parallel a loss of elasticity and an increased brittleness in the gels (Baker et al., 1947). Since the gels appeared more like pectate gels than pectinate gels after aging, it was assumed that much of the change was due to a loss of the ester content of the pectins. The pectins, therefore, were isolated and the methoxyl values determined. About one-third of the ester content of the pectinates was found to have been lost during the aging period of approximately 18 months. The amount of change in gel characteristics during aging will undoubtedly vary with the soluble solids content of a gel. Low temperature storage aids in methyl ester retention. It appears that further research is necessary for the development of more stable pectinates, more stable low-solids gels, and upon conditions of storage and handling of such gels. 3. UBES OF LOW-EBTER PECTINS Many articles have appeared in trade journals on the uses to which low-ester pectins may be put (Baier and Wilson, 1941; Kaufman et al., 1942, 1943; McCready et al., 1944; Baker, 1944). Low-ester pectins have been associated largely with the idea of the saving of sugar in gel manufacture for spreads. Actually, their use for this purpose may be of less value than when they are used in salads and desserts of low-solids content. The fact that they can be used quite freely with fruit juices at relatively high hydrogen ion concentrations gives them a distinct advantage over other gelling agents in this particular field. If fruit and vegetable juices are gelled without dilution it is obvious the products will possess high dietetic values. It is expected that the pectinates will allow the adapta-
HIQH-POLYMER PECTINS AND THEIR DEESTERIFICATION
421
tion of continuous jelly manufacturing procedure to low-solids gels similar to that which has been proposed for high-solids gels (Reich, 1939). While there is sdicient calcium in milk to gel many of the pectinate preparations without added calcium, powdered mixtures of pectinate, calcium salt, acid, b d e r salt, and sugar have been developed (Baker and Goodwin, 1941a;Olsen and Fehlberg, 1943) which will gel milk as well as many juices. These may be used as thickening agents for sirups and sauces. While heat is required for complete solution of the ingredients of the pectinate mixtures and clarity of gels, it is not absolutely essential. The pectinates have been used with moderate success in experimental studies upon the freezing of fruits (Baker, 1941;Buck et al., 1944). The pectinates reduced “run-off” and imparted gelling value to the product so that it could be heated and would gel upon cooling. Further studies upon this particular application are under way.
4. PECTIC ACID AND PECTATES The proper use of polyphosphates, with deionizing properties, or cation exchange materials may allow much wider application of pectic acids and pectates in gelling reactions. For this reason these latter substances should be referred to again. Viscous pectates have been prepared by moderate alkaline hydrolysis of pectins or pulps and peels in the cold. After filtration and acidification, pectic acid can be produced. Its salts have remarkably high viscosities (Ehrlich, 1936). Alcoholic precipitation of sodium pectate (Wilson, 1938; Baier and Wilson, 1941) produces a stringy-fibrous pectate, many uses of which have been suggested. The deesterification of flaked pectin through use of ammonia vapors has been noted in an earlier section (Evans and Huber, 1945). As noted previously, the pectinates, having less than 4y0 CHSO, and pectates will form gels of 65% soluble solids content if the effect of cations is depressed by the use of polyphosphates (Baker and Woodmansee, 1946). The polyphosphates have previously been used in the gelation of solutions from powdered pectate mixtures (Mnookin, 1940). IV. FUTURE CONSIDERATIONS As a result of the research of the past 10 years more attention should be paid by pectin manufacturers to the quality of the pectic source materials and to retention of high quality in the event of dried storage. Further study on this phase isaeeded. Extraction is found to be largely dependent upon the ease with which the ionic bonds established by nature are broken. This breakage can be accomplished by ion-exchange materials or the glassy phosphates at rela-
422
GEORGE L. BAKER
tively high pH values without the depolymeriBing action which generally accompanies the use of acids and heat. Partial demethylation of the pectin molecule has been found valuable in altering the gel characteristics of the pectins. A randomized deesteriflcation as accomplished through the action of acid or alkali appears to produce more valuable pectinic acids for gel purposes than can be produced by enqymic action. The production of low-solids gels from partially deesterified pectinates appears at this time to be their most interesting application. The extraction of high-grade pectins and the possible preparations of many new compounds as a result of partial deesterification of pectins are two developments which should play an increasingly important role in fruit by-product utilization in the future.
REFERENCES American Chemical Society, Pectin Nomenclature Committee Report. 1944. Chem. Eng. News 22, 105-106. Baier, W. E., and Wilson, C. W. 1941. Citrus pectates-properties, manufacture, and I d . Ew. C h m . 33, 287-291. WS. Baker, G. L. 1937. Extraction and standardization in fruit jelly manufacture. Food Manuf. 12, 147-150. Baker, G. L. 1941. Pectin aa aid in freezing fruits. Food I d . 13, No. 1, 65-57; No. 2, 56, 97. Baker, G. L. 1944. Jellied fruit and vegetable sauces, postwar possibilities of lowmethoxyl pectins. Food Packer 26, No. 8, 31-32. Baker, G. L.,and Goodwin, M. W. 1939a. Viscosity of dilute pectin solutions as affected by metallic salts and pH. Delaware Agr. Expt. Stu. Bull. 216. Baker, G. L., and Goodwin, M. W. 1939b. Unpublished data. Baker, G. L., and Goodwin, M. W. 1941a. Pectin jellying composition. U. S. Patent 2,233,574. Baker, G. L., and Goodwin, M. W. 1941b. Demethylation of pectin and its effect upon jellying properties. Delaware Agr. Expt. Sta. Bull. 234. Baker, G. L., and Goodwin, M. W. 1943. Unpublished data. Baker, G. L., and Goodwin, M. W. 1944. Effect of methyl ester content of pectinates upon gel characteristics at different concentrations of sugar. Delaware Agr. Expt. Sta. Bull. 246. Baker, G. L., and Kneeland, R. F. 1935. Pectin content of raw material, optimum conditions of extraction determined by viscosity. Fruit Products J. 14, 204, 205, 210, 220. Baker, G. L., and Woodmansee, C. W. 1944. Polyphosphates in the extraction of pectin. Fruit Products J. 23, 164, 165, 185. Baker, G. L., and Woodmansee, C. W. 1946. Unpublished data. Baker, G. L., Woodmansee, C. W.,and Meschter, E. E. 1947. “Shelf-life” of lowsolids grape gels. Food Technol. 1, No. 1, 11-16. Beaven, G. H.,and Jones, J. K. N. 1939. Molecular structure of pectic acid. Chemistry & Industry 68, 363. Bennison, E. W., and Norris, F. W. 1939. The pectic substances of plants. VI. The
HIGH-POLYMER PECTINS AND THEIR DEESTERIFICATION
423
relation between jelly strength, viscosity and composition of various pectins. Bimhm. J . 33, 1443-1451. Bock, H. 1943. Theory and practice of pectin production. Translation of unpublished book obtained through courtesy of C. L. Hinton. (The German text is in the possession of the Board of Trade, Documents Unit, Lansdowne House, Berkeley Square, W. 1, London). Bonner, J. 1936. Chemistry and physiology of the pectins. Botan. Rev. 2, 475-497. Bosurgi, G., and Fiedler, K. 1932. A method for the elimination of bitter substances from the fibre of pectin-containing plant materials in general and particularly from the peel of acid fruit. Brit. Patent No. 388, 284. Braconnot, M. H. 1824. Research on an acid universally spread throughout all vegetable plants. Ann. chim. 27, 173-178. Braconnot, M. H. 1826. New observations on pectic acid. Ann. chim. 29, 96-102. Braconnot, M. H. 1831. The principles of fruit jellies in current juice. Ann. chim. 47,2~e280.
Braconnot, M. H. 1832. The existence of pectin in the barks of trees. Ann. chim. 49, 376-385.
Branfoot (Carre), M. H. 1929. A critical and historical study of the pectic substances of plants. Dept. Sci. Znd. Research (Brit.), Special Rept. NO.33. Buck, R. E., Baker, G. L., and Mottern, H. H. 1944. Pectinates improve frozen fruit. Food I d s . 16, 113-115, 147-148. Burroughs, L. F., Kieser, M. E., Pollard, A., and Steedman, J. 1944. The treatment of apple pomace prior to drying for subsequent pectin extraction. Fruit Products J . 24,4-6.
Charley, V. L. S., Burroughs, L. F., Kieser, M. E., and Steedman, J. 1942. The treatment of apple pomace prior to drying for subsequent pectin extraction. Ann. Rept. AQT.Hort. Research Sta., Long Ashton, Bristol, 89-100. Cole, G. M., and Cox, R. E. 1938. Method of treating pectin. U. S. Patent 2,109,792. Cox, R. E. 1938. Method of altering the setting time of pectin. U. S. Patent 2,133,273. Ehrlich, F. 1917. Pectins, their composition and importance. Chem. Ztg. 41,197-208. Ehrlich, F. 1930. Chemistry of pectin and its relation to the formation of incrustations of cellulose. Celltdosechaie 11, 140-151. Ehrlich, F. 1932. Pectin chemistry. 11. A typical reaction of d-galacturonic acid and of pectin. Ber. 66B, 352-358. Ehrlich, F. 1936. The pectin problem. Chem. C&T. I., 788-789. Ehrlich, F., and Kosmahly, A. 1929. The chemistry of pectins from fruit. Biochem. 2. 212, 162-239.
Ehrlich, F., and Schubert, F. 1929. Chemistry of the pectin substances: tetragalacturonic acids and d-galacturonic acid from the pectin of sugar beets. Ber. 62B, No. 8, 1974-2027. Ehrlich, F., and Sommerfeld, R. V. 1926. The composition of pectin of the sugar beet. Biochem. 2. 168, 263-323. Evans, L. H., and Huber, L. J. 1945. Process of producing pectinous gels and composition. U. S. Patent 2,380,739. Fellenberg, Th. von, 1914. On the knowledge of pectins. Mitt. Lebensm. Hyg. 6, 224-256.
Fellenberg, Th. von, 1918. Constitution of the pectin substances. Biochem. Z . 86, 118-161.
Fremy, E. 1840. The first examination of the ripening of fruits. Ann. Chem. Pharm. (Liebig) 26, 368-393.
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Fremy, E. 18478. Experiments with the gelatinous matters of vegetable plants. J . Pluarm. Cham. 12, 13-24. Fremy, E. 1847b. About gelatinous plant principles. Ann. 68, 383-387. Fremy, E. 1848. Report on the ripening of fruits. Ann. chim. phys. 24, 5-58. Fremy, E. 1859. T h e effect of lime on the utriculas tieeue of plants. Cmnpt. rend. 49, 561-564. Gaddum, L. W. 1934. T h e pectic constituents of citrus fruits. F b i d u Agr. Ezpt. Sla. BuU. 388. Gaponenkov, T. K. 1937. The effect of mineral and organic substancea on the viscosity of solutions of pectic substances. J. Gen. Chem. U.S. S . R. I , 2900-2908. Heid, J. L. 1941. Effect of ethylene treatment upon the recovery of citrus pectin. Fruit Products J . 21, 100-103, 125. Henglein, F. A. 1943. Protopectin and protocellulose. J. Mukromot. Chem. 1, 121130,from C. A. 88, 1267. Henglein, F. A., and Schneider, G. 1936. Esterhation of pectin substances. Bet. 89B, 309-324. Hills, C. H.,and Mottern, H. H. 1945. The properties of tomato pectase. Eastern Regional Research Laboratory, Philadelphia, Pa., unpublished data. Hills, C. H., White, J. W.,and Baker, G. L. 1942. Low-sugar jellying pectinates. Proc. Zmt. Food Techml. 47-68. Hinton, C. L. 1939. Fruit pectins and their chemical behavior and jellying properties. Dept. Sn'. I d . Rea. (Gt. Britain), Special Rept. No. 48. Hirsch, P. 1942. Treatment of pectinous materials. U. S. Patent 2,273,521. Hirst, E. L., and Jones, J. K. N. 1838. Pectic substances. I. The araban and pectic acid of the peanut. J. Chem. SOC.1988, 496-505. Hirst, E. L., and Jones, J. K. N. 1939. Pectic substances. 11. Isolation of an araban from the carbohydrate constituents of the peanut. J. C h . SOC.1989, 452-454. Jameson, E.,Taylor, F. N., and Wilson, C.P. 1924. Pectin product and procees of producing same. U. 9.Patent 1,497,884. Jansen, E. F., Waiabrot, S. W., and Rietz, E. 1944. Errors in the Zeisel methoxyl values for pectin due to retained alcohol. I d . Eng. C h . 16, 523-524. Joseph, G. H.1936. Method of treating pectin. U. 5. Patent 2,061,158. Joseph, G. H.1945. California Fruit Growers Exchange, private communication. Joslyn, M. A., and Sedky, A. 1940. The relative rates of destruction of pectin in macerates of various citrus fruits. Phnt Physiol. 16, 675-087. Kaufman, C. W., Fehlberg, E. R., and Olsen, A. G. 1942. Chemists adapt pectins to new industrial uses. Food I d . 14, No. 12,57;1943. Food I d . 16,No. 1,58. Kertesz, Z. I. 1938. Pectic ewymes. 11. Pectic enzymes of tomatoes. Food Research 3, 481-487. Kortschak, H.P. 1939. Electrolytes and the viscosity of pectin solutions. J. Am. Chem. SOC.81, 2313-2317. Leo, H.T.,Taylor, C. C., and Lindsey, J. W. 1939. Method of controlling certain jeIIing properties of pectin. U. S. Patent 2,173,260. Lineweaver, H. 1945. Acceleration by electrolytes of alkaline deesterificstion of pectin. J. Am. C h m . Roc. 61, 1292-1293. Lineweaver, H.,and Ballou, G. A. 1945. Effect of cations on the activity of alfalfa pectinesterme (pectaae). Arch. Bwchem. 6, 373-387. Luckett, S., and Smith, F. 1940. Constitution of pectic acid. I. Methylation of pectic acid and the isolation of the methyl ester of 2,3dimethylmethylgalacturonoside. J . Chem. SOC.1940, 1106-1114.
HIGH-POLYMER PECTINB AND THEIR DEEBTERIFICATION
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Lilers, H., and Lochmilller, K. 1927. The measurement of gelling power of fruit pectins. Kolloid-Z. 42, 164. McCready, R. M., Owens, H. S,, and Maclay, W. D. 1944. Alkali-hydrolyzed pectins are potential industrial producta. Food I d . 16, 69-71, 139-140. MacDonell, L. R., Jansen, E. F., and Lineweaver, H. 1946. Properties of orange pectiiesteraae. Arch. Biochem. 6 , 389-391. McDowell, R. H. 1941a. Influence of temperature on gel formation. Nature No. 3765, 780-781. McDowell, R. H. 1941b. Improvement in or relating to the hydrolysis of pectin. Brit. Patent 541,628. McDowell, R. H.1942. Private communication. McDowell, R. H. 1943. Pectinic acids from sugar-beet pulp. Brit. Patent 566,842. Maclay, W. D., and Nielsen, J. P. 1046. Method of extracting pectinous materials. U.8.Patent 2,378,376. Mehlitz, A. 1930. Studies on pectaee. I. Enzymic studies on favorable conditions for pectase coagulation. Bwchem. 2.221, 217-231. Mehlitz, I. A, 1939. Results of the investigation of apple residues. Vmatspjlege u. Lebemmittelforach. 2,641-690. Meyer, K. H., and Mark, H,1930. Der Aufbau der hochmolekularen Naturstoffe. Akedemische Verlagsgesebchaft, Leipzig, 219. Mnookin, N. M. 1940. Jelly and jelly manufacture. U. S. Patent 2,207,299. Morell, S., Bauer, L., and Link, K. P. 1934. The methyl glucosidea of the naturally occurring hexuronic acids. 111. Polygalacturonic acid methyl glucoaides derived from pectin. J. Biol. Chcm. 106, 1-13. Morris, T. N. 1934. Changes in the pectic substances of fruits during storage. Report Food Invest. Board Dept. Sci. Ind. Res. (at. Britain) 1988,1SS-l6l. Morris, T.N.1936. Changes in the pectin of fruits during storage. Report Food Invest. Board Dept. Sci. Id.Res. (Gt. Britain) 1984, 220-223. Mottern, H.H.,and Hills, C. H. 1946. Low ester pectin from apple pomace. Znd. Eng. Chem. 88, 1163-1166. Mottem, H.H., and Karr, E. E. 1948. Determination of pectin grade of apple pomace. Fruit Prodwta J . 26, 292-296, 313; 316. Myers, P. B. 18391%. Method of preparing pectin. U. S. Patenta 2,163,620 and 2,163,621. Myers, P. B. 1939b. Method of treating pectin containing raw materiala. u. 8. Patent 2,166,902. Myers, P. B., and Baker, G. L. 1927. The viscosity and jellying properties of pectin solutions. Dehware Agr. Ezpt. Sta. Bull. 14s. Myers, P. B., and Baker, G. L. 1929. The extraotion of pectin from pectic materifsls. Delaware Agr. Expt. Sta. Bull. 160. Myers, P. B., and Baker, G. L. 1931. The effect of temperature on the extraction of pectin. Debware Agr. Expt. Sta. Bull. 168. Myers, P. B., and Baker, G. L. 1934. The physic-chemical properties of pectin. Delaware Agr. Expt. Sta. Bull. 107. Myers, P. B., and Cowgill, W. W. 1940. Pectin and its manufacture. U. 9. Patent 2,186,472. Myers, P. B., and Rouse, A. € 1943. I. Extraction and recovery of pectin. u. s. Patent 2,323,483. Nanji, D. R., Paton, F. J., and Ling, A. R. 1926. Application of de-carboxylation to the establishment of the conetitution of pectine and t o their determination. J. SOC. them. I d . 44,253-288.
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QEORQE L. BAKER
Olsen, A. G., and Fehlberg, E. R., 1943. Pectin jelly composition. U. S. Patent 2,334,281.
Olsen, A. G., and Stuewer, R. 1938. Method of preparing pectin. U. S. Patent 2,132,577.
Olsen, A. G., Stuewer, R., Fehlberg, E., and Beach, N. M. 1939. Pectin studies, relation of combining weight to other properties of commercial pectins. Ind. Eng. Chem. 81, 1015-1020. Otto, R., and Winkler, G. 1942. Pectin recovery from plants. Ger. Patent 730,898. Owens, H. S., Lotzkar, H., Merrill, R. C., and Peterson, M. 1944. Viscosities of pectin solutions. J. Am. Chem. SOC.68, 1178-1182. Owens, H. S., McCready, R. M., and Maclay, W. D. 1944. Enzymic preparation and extraction of pectinic acids. Ind. Eng. Chem. 36, 936-938. Peters, C. A. 1942. Private communication. Reich, G. T. 1939. Manufacture of jellies and the like. U. S. Patent 2,185,064. Rosenfield, B. 1938. Extracting pectin. Brit. Patent 480,096. Saburov, N. V.,and Kalebin, M. I. 1935. Effect of sulfurous acid on apple-pulp pectin. Fruit Producle J . 14, 275-277, 280-283. Schneider, G., and Bock, H. 1937. Constitution of pectin substances. Ber. IOB, 1617-1630.
Schneider, G., and Bock, H. 1938. The determination of pectic substances. Angm. Chem. 61, 94-97. Schneider, G., and Fritschi, U. 1936. Esterification of pectin substances. 111. The molecular magnitude of pectin substances. Ber. 69B,2537-2543. Schneider, G., and Fritschi, U. 1937. Esterification of pectin substances. IV. Determination of constitution on pectin ester. Ber. IOB, 1611-1617. Schneider, G., and Ziervogel, M. 1936. Esterification of pectin substances. 11. Acetyland formyl-pectin. Ber. 69B,2530-2536. Schultz, T. H., Lotzkar, H., Owens, H. S., and Maclay, W. D. 1945. Influence of method of deesterification on the viscosity and acid behavior of pectinic acid solutions. J. Phys. Chem. 49, 554-563. Smith, F. 1939. Molecular structure of pectic acid. Chemistry & Industry 68,363-364. Smolenski, K. 1923. Pectins. Roczniki Chem. S, 86-152, from C. A . 19, 41-42. Speiser, R. 1943. Gelation. Eastern Regional Research Laboratory, Philadelphia, Pa., unpublished data. Speiser, R., and Eddy, C. R. 1946. Effect of molecular weight and method of deesterification on the gelling behavior of pectin. J. Am. Chem. SOC.68, 287-293 (1946).
Stuewer, R., Beach, N. M., and Olsen, A. G. 1934. Pectin studies. 11. Sugar-acidpectin relationships and their bearings upon routine evaluation of apple pectin. Znd. Eng. Chem.,A d . Ed. 6,143-146. Sucharipa, R. 1923. Experimental data on pectin-sugar-acid gels. J , Assoc. O f i a l Agr. Chem. 7 , 5 7 4 . Sucharipa, R. 1925. Die Pektinstoffe. Serger & Hempel, Braunschweig. Svedberg, The, and Graln, Nils. 1938. Carbohydrates of well-defined molecular weight in plant juices. Nature la,261-262. Tarr, L. W., and Baker, G. L. 1924. Fruit jellies. 11. The role of sugar. Delaware Agr. Expt. Sta. Bull. lS6. Vauquelin, M. 1790. Analyse du tamarin. Ann. chim. 6, 92-106. Vauquelin, M. 1791. Analyae de la cwse. Ann. chim. 6, 275-293. Vauquelin, M. 1829. Report on the presence of pectin in carrot roots. Ann. chim. 41, 46-61.
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Willaman, J. J., and Hills, C. H. 1944. Pectase. U. S. Patent 2,358,429. Willaman, J. J., Mottern, H. H., Hills, C. H., and Baker, 0.L. 1944. Method of preparing pectinate. U. S. Patent 2,358,430. Wilson, C. P. 1926. The manufacture of pectin, Id. Eng. C h . 17, 1065-1067. Wilson, C. W. 1938. Pectate and method of making same. U. S. Patent 2,132,065. Wohl, A., and Niessen, K. von. 1889. Ueber die durch Erhitzen mit Wasser losbaren Bestandtheile des Riibenmarks. 2. deut. Zuckerind. 59, 924.
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Author Index :no4 in parentheses indicate senior author8 of the refersncss and are included to assiat in iocating refeizncea where a particular name is not on a given page. Exam le: Akeley R. V. 294 (nee Murphy) meana that Murphy el al. w i l l be mentioned on page 294 the et af: acoountin; for dkeley. This art& can be located, under Murphy in the lint of referend. Numbera in italicr refer to the _ DBIW- on which referenoes are hated in bibliograpbies at the end of each article.
A
Atkinson, F.E.,57,58,61,104 Austin, J. J., 223 (see Stewart), 229 (see Stewart), 235 (see Stewart), 966
Aberg, B., 299, 302,303,319 Adam, W. B., 65,68,100 Adolph, W. H., 107,141 B Ahlatram, L., 387,391 hiyar, S. P., 313,319 Akeley, R. V., 294 (see Murphy), 297 (see Babkin, B. P., 134,141 Bder, W.E.,420,421,&?9 Murphy), 391 .Ibrecht, W. A., 112, 141, 312, 313, 380 Bailey, K.,6, 8,34, 207,%54 Albright, W. B., 156 (see Thompson), 101 Baird, F. D.,119,141 Baker, G.L., 398,399,400,401,402,403, Alderton, G., 169,194 404,405, 407,408,409,410,411,412, Alexander, L. M., 213 (see Harshaw), 224 413,414,415,416,417,419,420,421, (see Hamhaw), 966 Aline, E., 153 (see Snell), 900 @9, 483,494,496,4961 497 Allen, R. J. L., 259 (see Tomkins), 263 Baker, G. O.,312,313,393 (see Tomkins), 266 (see Barker; Tom- Baker, L. C.,293 (see Lampitt), 297 (see Lampitt), 391 kina), 271 (see Tomkins), 272 (see Tomkins), 275 (see Tomkins), 276 Baldwin, F. M.,132,141 (see Tomkins), 280 (eee Wager), 986, Ball, C.D.,172,173 (see Hardt), 194,197 Ballentine, R., 375 (see Ryan), 59a: 889 Allison, W. W., 276 (see Pyke), 277 (see Ballou, G. A., 411,494 Aykroyd, W. R.,109,141 Pyke), 989 AlmqUiat, H. J., 119, 141, 152, 153 (see Apes, G.B., 122,14.4 Balls, A. K., 127,141, 170, 185, 194,196, Lepkovsky), 155, 194, 198 210,964,386,3991 Ahberg, C.L.,126,146 Balog, E. G.,258, 260 (see Cruess), 271, Ames, S. R.,42 (see Ivee), 108 886, 287 Ammon, R., 183,186,194 Bandemer, 5. L., 152 (see Schaible), 154, Anderson, E. H.,382,391 900 Anderson, W. S., 310 (see Sheets), 311 (see Sheets), 312 (see Sheets), 313 Banfield, F. H., 18,414 (see Sheets), 314 (see Spire), 399,393 Barbour, A. D.,167,197 Barelare, B.,Jr., 112 (see Richter), 113 Anderesen, F. G., 380,369 (see Richter), 114 (see Richter), 118 Anson, M. L., 209,263,379,391 (see Richter), 9146 Appert, N.,66 Barger, W. R., 369 Appleman, C. O., 262, 268,282,B86 Barker, H. A., 329, 330, 331, 332 (see Amon, D.I., 313,319 Stadtman), 334, 335, 336, 338, 343, Ascham, L., 298,303(see Reder), 304,308, 345,347,348,356,380,361,362,363, 310 (see Sheets), 311 (see Sheets), 312 364,370,379 (see Sheets), 313 (see Sheeta), 314 (see Barker, J., 259 (see Tomkins), 263 (see Speirs), 399,385 Tomkins), 266, 268, 271 (see TomAamundson, V. S., 152,194,220,863 kina), 272 (see Tomkins), 275 (see Aetbury, W. T., 9, 34,208,964 Tomkins), 276 (see Tomkina), 282, Atkina, C.D.,55 (see Moore E.L.; Wied886,289 erhold), 90 (see Moore, E. L.),98 (see Moore, E.L.), 99 (me Moore, E.L.), Barnes, R. H., 130,141,141 103, 104,328 (see Moore, E.L.), 332 Barnes, W. C., 306,318 Barron, E.S. G.,381,3991 (see Moore, E.L.), 571 429
430
AUTHOR INDEX
Bartlett, E. M., 257, 268 (see Rose), 282 (see Rose), 283 (see Rows), 289 Bastron, H.,174 (see Denton), 196 Bate-Smith, E. C., 6,7, 9,10,11, 14, 15, (see Bennion), 18,30,32,34,159,167 171,172,173,174,181 (see Bennion), 196,208,218,232,234,264 Bauer, L., 399 (see Morell), 436 Baumann, C. A., 153, 196 Beach, N. M., 399 (see Olsen), 406 (see Stuewer), 408 (see Olsen), 409 (see Olsen), 413 (see Olsen), 415 (see Olsen), 416 (see Olsen), 426 Beadle, B. W., 84,100, 101, 214,264 Beadle, G.W., 375,$91 Beadles, J. R.,116,142 Bean, R., 183 (see Lineweaver), 184 (see Lineweaver), 186 (see Lineweaver), 198 Bearse, G.E.,153, 196,198 Beattie, H. G., 328,333,341,369,371, 372 Beaven, G.H., 399,@2 Beavens, E. A., 280,286, 328 (see Pederson), 333 (see Pederson), 341 (see Pederson), 371 Beckley, V. A., 258,986 Bedford, C. L., 281, 283, 286, 342, 353, 354,365,368,369 Beeson, K. C., 292, 299, 303, 305, 307, 310, 312,313, 314,319,321 Belfanti, S., 388,391 Bendall, J. R.,7,9,10,34 Benedict, F. G.,13, 34 Bennion, E.B., 167,181,196,197 Bennison, E.W., 415,4.92 Bergdoll, M. S., 263 (see Doty), 271 (see Doty), 276 (see Doty), 277 (see Doty), 288 Berger, J., 125 (see Woolley), 127 (see Woolley), 147 Bergmann, M., 208,264 Bernard, C.,7, 34 Bernfeld, P.,10,36 Bernhard, K.,152,196 Bernstein, L., 64 (see Hamner), lU2, 296 (see Hamner), 297 (see Hamner), 298, 300,303 (see Hamner), 305,308,309, 319,320 Berry, J. A., 182,196 Bessey, 0.A., 293,319 Best, C. H.,7, 11, 12,14,34,37 Best, L. R., 160 (see Stewart), 171 (see Stewart), 188 (see Stewart), 192 (see Stewart). 900 Bethel, R., .328 (see Nichols), 330, 342 (see Nichols), 344 (see Nichols), 360
(see Nichols), 362 (see Nichols), 371 Bethke, R. M., 122 (see Kick), 124, 142, 144, 152 (see Wilder), 153, 154, 196, 198,208 Beuk, J. J., 84,103 Birdseye, C., 25,38 Birmingham, J. R., 119,146 Black, A.,117 (see Voris), 118 (see Voris), 120 (see Kahlenberg), 14,147 Black, H. G.,261,$86 Black, W.H., 25,34 Block, R.J., 133, 1 4 Bloom, W.,209,210,266 Boaz, T.G., 36 Bock, H., 398 (see Schneider), 399, 407, 418,423, 426 Boelte , M.D., 120,1&,1& Boggs, M. M.,159,160 (see Fevold), 161, 164, 179 (see Fevold), 180 (see Fevold), 182, 188, 189, 190, 191, 192, 193,196,196 Bohren, B. B., 174,196,197 Bohstedt, G., 124 (see Bethke), 1.42 Bolton, W., 154, 196 Bondi, A.,306,307,319 Bonner, J., 399,423 Bornstein, B., 360 (see Mrak), 365 (see Mrak), 371 Borsook, H., 359,369 Bosshardt, D. K.,130,1-41 Bosurgi, G.,400,423 Boucher, R. V., 219 (see Murphy), 220 (see Murphy), 266 Boudreau, F. G., I@, 148 Bourne, J. A,, 280, 286 Bowman, D. E., 126,142,385,391 Boyd, J. M.,99,100,100 Braconnot, M. H., 395,396,410,423 Bradley, H.C., 31,34 Braman, W. W.,116 (see Beadles), 14.9 Branfoot (Card), M. H., 407,@3 Branion, H.D., 163,196 Brightwell, S. T.,280 (see Wager), 289 Brink, R. A.,306 (see Porter), 329 Brobeck, J. R., 106, 129, 130, 131, 136, 137,l.be, Brody, S., 123,14%' Brooks, J., 22, 36, 159 (see Bate-Smith), 163,171, 175, 182,183,184,188,196, 197 Brown, G. B., 116 (see du Vigneaud), 143 Brown, H. D., 307 (see Gum), 319 Brown, W. D., 310,319 Brunstetter. B. C.. 280 (see Caldwell). 265 (see Chdweli), 266 (see Caldwell), 269 (see Caldwell), 277 (aee Caldwell),
AUTHOR INDEX
278 (see Caldwell), 283, 986, 989 Brush, M. K., 45, 49 (see Hinman), 51 (see Hinman), 52 (see Hinman), 100, 109 Bryan, L. A., 265 (see Legault), 267 (see Legault), 271 (see Legault), 272 (see Legault), 988 Buck, M., 376 (see Schnitzer), 399 Buck, R. E., 92, 101,421, 4.93 Bull, S., 12, 36 Bullet, F., 152 (see Bernhard), 196 Bunkfeldt, R., 119 (see Krieger), 144 Burkhardt, G. J., 333 (see Schrader), 372 Burlinson, L. O., 302 (see Smith, M. C.),
323
Burmester, B. R., 152 (see Almquist), 194 Burr, G. O., 119, l& Burr, M. M., 119, 149 Burrell, R. C., 307 (see Gum), 319 Burrounhs. L. F.. 401.423 Burton, W. G., 259, '264, 268, 269, 271,
272, 273, 274, 275, 276, 278, 279, 986
Butler. 0..282. 986 Byerly, Ti C., 153 (see Titus), 901 C
Cabell, C. A., 174 (see Denton), 196 Caldwell, E., 41 (see Pressley), 45 (see Pressley), 47 (see Pressley), 55 (see Smith, M. C.), 103,104 Caldwell, J. S., 260,261,262 (see Wright), 265, 206, 268 (see Green), 269, 277, 278,279 (see Green), 280 (see Green), 282, 283, 986,987,988,990,335, 870 Callow, E. H., 11, 12, 13, 14, 15, 17, 18, 19, 36, 218, 253,964
Calvery, H. O., 163, 196 Camagni, L. J., 376 (see Schnitzer), 399 Cameron, 9.S., 360, 369 Camp, S. C., 70 (see Fenton), 101 Campbell, H., 258,262,203,272,282, 987 Cannan, R. K., 109 (see Longsworth), 198 Cannon, P. R., 115 (see Frazier), 143 Carey, E. J., 240, 243, 244, 246, 247, 251, 964 Carbon, A. J., 133, 134, 142 Carleaon, V., 82 (see Kohman), 109 Carolus, R. L.,294 (see Reder), 290 (see Reder), 298 (see Reder), 303 (see Reder), 304 (see Rgder), 308 (see Reder), 317 (see Reder), 399 Carrick, C. W., 21Q, 964 Carruth, F. E.,127, 147 Cartwright, G. E.,126, 1 4
43 1
Gary, C. A., 115, 142 Caster, A. P., 170, 200 Cederquist, D. C., 108 (see Everson), 127 (see Everson), 143 Chace, E. M., 258, 987 Chaikoff, I. L., 128 (see Chernik), 142 Chandler, J. P., 110 (see du Vigneaud),
143
Chang, S. L., 377, 391 Chargaff, E., 151, 196 Charley, V. L. S., 401, 423 Chase, S. W., 122 (see Kick), 144 Chastain, S. M., 204 (see Makower), 266 (see Makower), 988 Chaves, J. M., 332, 348, 569 Chemerda, C., 188 (see Solowey), %OO Chen, S.-M., 308, 391 Chen, T. M., 303 (see Shen), 398 Chernik, S., 128, 14.9 Child, A. M., 268 (see Rogers, M. C.), 273 (see Rogers, M. C.), 989 Christensen, P. B., 20, 36 Clark,A. J., 135, 1& Clarke, F. L., 173 (see Van der Scheer), 901
Clay, J., 110 (see Harris), 136 (see Harris),
143
Clifcorn, L. E., 41, 57, 58, 59, 60, 62, 60, 79, 84, 85, 93, 100, 101,330, 370 Cochran, H. L., 294 (see Reder), 290 (see Reder), 298 (see Fteder), 303 (see Reder), 304 (see Reder), 308 (see Reder), 310 (see Sheets), 311 (see Sheets), 312 (see Sheets), 313 (see Sheets), 314 (see Speirs), 317 (see Reder), 392, 323 Cole, G. M., 411, 415, 4.93 Collins, D. A., 10, 36 Common, R. H., 154,196 Conn, J. E., 388, 399 Conquest, V., 162, 196 Conrad, R. M., 194, 196,214 (see Schrieber), 216 (see Wagoner), 217, 218 (see Schrieber; Wagoner), 222 (see Wagoner), 253 (see Wagoner), 966 Contardi, A., 388 (see Belfanti), 391 Cook, F. C.,229 (see Wiley), 966 Cook, W. H., 32,37,192 (see Pearce), 193 (see Pearce), 199,213, 215, 216, 217, 222, 964,966 Coonen, N. H., 67 (see Guerrant), 71 (see Guerrant), 72 (see Guerrant), 73 (see Guerrant), 77 (see Guerrant), 78 (see Guerrant). 81 (see Guerrant). 101 Cori, C. F., 131, 149 Couch, J. R., 153 (see Snell), 900
432
AUTHOR INDEX
Coulter, M. D., 26 Cowgill, C.R., 117, 131, 142 Cowgill, G. R., 118 (see Street), l4Y Cowgill, W. W., 400,426 Cox, R. E., 411,415,423 Crocker, E. C., 219,964 Crone, E. B., 276 (see Pyke), 277 (see Pyke), 2889 Crowe, G. R., 379,392 Crowley, L. V., 308 (see Holmes), 310 (see Holmes), 313 (see Holmes), 314 (see Holmes), 320 Cruess, W. V., 258,260,266,271, 286, MY, 330,334, 341, 342,364,365,369, 3YO Cruickshank, E. M., 150, 152, 174, 196, 212, 213, 219, 264 Cruickshank, E. W. H., 127,f4.2 Culpepper, C.W., 66,103, 260 (see Caldwell), 261 (see Caldwell; Green), 262 (see Wright), 265 (see Celdwell), 266 (see Caldwell; Green), 268 (see Green), 269 (see Celdwell), 277 (see Caldwell), 278 (see Caldwell), 279 (see Green), 280 (see Green), 282 (see Caldwell), 283 (see Galdwell; Wright), 886, 887,988, 890, 335,370 Cunningham, E., 41 (see Thompson), 104 Curl, L. C., 328, 332, 346, 348, 349, 350, 354, 355, 356, 370 Cuthbertson, E.M., 121, 148, I43
D Dam, H., 125, 14.8 Daniel, E.P., 40, 101 Davenport, H.W., 359 (see Borsook), 369 Davidson, J. A., 152 (see Schaible), 156 (see Mallmann), 199, 200 Davies, A. F., 259,289 Davis, C. M., 109,1.48 Davis, H. P., 306 (see Hathaway), 320 Davis, M. B., 258, 260, 261, 266, 987 Davis, R.,174 (see Denton), 196 Davis, R. J., 67 (see Richardson), 104 Dawson, C. R., 276, 2887 Dawson, E. H., 168, 191, 196 Dawson, V. T., 156 (see Stuart), 165 (see Stuart), 801 Day, H. G., 119 (see McCollum), 121 (see McCollum), 125 (see McCollum), 146 De, 8. S., 126, 142 Deasy, C,L., 135 (see Haagen Smit), 143 De Felice, D., 67,101 Delbriick, M., 376, 391 Delorme, G.,314,388 Demerec, M.,375
Denny, F. E., 268,273,277,282,283,284, 287,288 Denton, C. A., 174,196 Desikachar, H. S . R., 126 (see De), 142 Deuticke, H. J., 9, 3,5 DeVancy, C. M., 153,196 Dichek, M.,117, 147 Dickinson, S.,208,254 Dicks, E. E., 156 (see St,uart), 163, 164, 165, 166, 186, 188,201 Dimick, A. L., 160 (see Fevold), 178 (see Edwards), 179 (see Fe,vold), 180 (see Fevold), 182 (see Fevold), 196 Dimiek, M. K., 117, 142 Dingemans, J. J. J., 159, 196 Doty, D. M . , 263, 271, 276,277, 288 Dove, W. F., 112, 124, 142, 294 (see Murphy), 297 (see Murphy), 321 Dowell, C. T., 358,370 Drews, H.E., 221, 266 Drill, V. A., 130, 149 Drummond, J. C., 107 (see Roberts, W.). 111, 14.8, 146 Dubois, C. W., 217,218, 264 Dubois, K.P., 128,14.2 Duckworth, J.,127 (see Cruickshank), 142 Duddles, W. J., 172 (see Ball), 194 Dudgeon, L. T., 294 (see Karikka), 296 (see Karikka), 297 (see Karikka), 308 (see Karikka), 321 Dunlop, A. P., 358,3YO Durham, H. A., 276 (see Pyke), 277 (see Pyke), 289 Dutcher, R. A., 41 (see Guerrantf, 56 (see Guerrant), 68 (see Guerrant), 82 (see Guerrant), 88 (see Guerrant), 89 (see Guerrant), 93 (see Guerrant), 94 (see Guerrant), 101,219(see Marble), 220 (see Marble). 255 Dutton, H. J., 161, 173, 174, 176, 177, 196, 196, 199
Dyche, J. K., 156 (see Shaw), 200
E Eakin, R. E., 127,143 Eckert, J. F., 123, 130, 137,146 Eddy, C.R., 412, 413,426 Eddy, C.W., 339,340, 347,350 (see Nelson), 353 (see Nelson), 370,371 Eddy, W. H., 82 (see Kohman), 108 Edgington, B. H., 122 (see Kick), 124 (see Bethke), 1.48, I & Edmond, J. B., 310 (see Sheets), 311 (see Sheets), 312 (see Sheets), 313 (see Sheets), 314 (see Speirs), 329, 323
AUTHOR INDEX
433
Edwards, B. G., 160 (see Fevold), 161, Moore. E. L.1.358 (see ., . Moore. E.L.)... 370, 371 174, 176, 177, 178, 179 (see Fevold), 180 (see Fevold), 182 (see Fevold), v. Euler. H.. 387. 388. 391 196, 196 Evans, C . d , 125,12?, 128, 143 Eheart, J. F., 294 (see Reder), 296 (see Evans, H.M., 119,143 Reder), 298 (see Reder), 303 (see Evans, L. H., 411,421,423 Reder), 304 (see Reder), 308 (see Everson, G. J., 108,127,143 Reder), 317 (see Reder), 399 Ewell, A. W., 22,23,26,36 Eheart, M. S., 298, 303 (see Reder), 304, Esell, B. D., 260 (see Caldwell), 265 (see 308,322 Caldwell), 266 (see Celdwell), 269 (see Ehrlich, F.,397,421,423 Caldwell), 277 (see Caldwell), 278 Eidt, C. C., 258 (see Davis), 260 (see (see Caldwell),283 (see Caldwell), 286 Davis), 261 (see Davis), 266 (see F Davis), 287 Elion, E., 365,370 Ellenberger, H.A., 67 (see Guerrant), 71 Fairchild, T. E., 112,146 (see Guerrant), 72 (see Guerrant), Fankuchen, I., 208, $64 73 (see Guerrant), 77 (see Guerrant), Fardig, 0.B., 41 (see Guerrant), 56 (see Guerrant), 67 (see Guerrant), 68 78 (see Guerrant), 81 (see Guerrant), (see Guerrant), 71 (see Guerrant), 101 Ellet, W. B., 310 (see Sheets), 311 (see 72 (see Guerrant), 73 (see Guerrant), 77 (see Guerrant), 78 (see Guerrant), Sheets), 312 (see Sheets), 313 (see 81 (see Guerrant), 82 (see GuerSheets), 314 (see Speirs), 322, 323 Ellis, G. H., 305, 319 rant), 86 (see Guerrant), 89 (see Guerrant), 93 (see Guerrant), 101 Ellis, N.,154,196 Ellis, N. K.,263 (see Doty), 271 (see Farish, M., 294 (see Reder), 296 (see Reder), 298 (see Reder), 303 (see Doty), 276 (see Doty), 277 (see Doty), 888 Reder), 304 (see Reder), 308 (see Ellis, N. R., 151 (see Riemenschneider), Reder), 317 (aee Reder), 3.% 153 (see Titus), 184 (see Riemen- Feaster, J. F., 84 (see Greenwood), 86 (see Greenwood), 87,88 (see Greenwood), schneider), 199, 901 Elvehjem, C. A., 41 (see Ives), 42 (see 97, 101, 102 Ives), 44 (see Ives), 45 (see Ives), 47 Fehlberg, E. R., 399 (see Olsen), 408 (see Kaufmm; Olsen), 409 (see Olsen), (see Ives), 55 (see Wagner), 64, 70 413 (see Olsen), 415 (see Olsen),416, (see Wagner), 71 (see Wagner), 72 (see Wagner), 73 (see Wagner), 77 420 (see Kaufman), 421, @4, 426 (see Wagner), 78 (see Wagner), 80 Fellenberg, Th. v., 397,405, 406,408,417, (see Wagner), 81 (see Wagner), 85 4 3 (see Wagner), 86 (see Ives), 89 (see Fellers, C. R., 67, 92, 98, 101, 103, 328 (see Esselen), 329 (see Moore, E. LJ, Wagner), 101, 102, 104, 118 (see 337 (see Moore, E. L.), 338 (see Krehl; Waisman), 121 (see Stirn), Moore, E. L.),348 (see Moore, E. L.), 1-14, 147 349 (see Esselen; Moore, E. L.), 350 Embden, G., 11, 36 (see Moore, E. L.), 356 (see Moore, Empey, W. A., 22, 32, 36 E. L.), 358 (see Moore, E. L.),370, Engel’hardt, V. A., 6, 8, 9,36 371 Englander, D. A. (Lieut.), 108,143 Fenton, F., 52, 64 (see Gleirn), 70, 101, Ercoli, A., 388 (see Belfenti), 391 217 (see Dubois), 218 (see Dubois), Erdiis, T., 7, 9,36 g6.1 Eriokson, J. O., 209 (see Neurath), 266 Ferres, H. M., 310,319 Ershoff, B., 130, 143 Ferry, R. M., Jr., 118 (see MoKibbin), 146 Esaelen, W. B., Jr., 100 (see McConnell), Fevold, H. L., 150, 158, 160, 161 (see 103,328,329 (see Moore, E. L.), 337 Boggs), 164, 169, 170, 174, 178 (see (see Moore, E. L.), 338 (see Moore, Edwards), 179, 180, 181, 182, 188, E. L.),348 (see Moore, E. L.), 349, 189,190, 191,192,193,194,196,196, 350 (see Moore, E. L.), 356 (see 198
434
AUTHOR INDEX
Fiedler, K., 400,423 Fieger, E.A., 154 (see Swenson), 201 Field, A., 362 (see Morgan), 371 Finch, A. H., 302,308 (see Jones, W. W.), 309, 312, 313, 319, 380 Finch, N., 284,288 Fincke, M. L., 79,101 Fisher, C. D., 360,365,369, 370,S Y l , 389 (see Whelton), 392 Fitch, A., 383 (see du Vigneaud), 398 Fitch, R. H., 6 (see Voegtlin), 58 Fitzgerald, G. A., 67,68 (see Jenkins), 69 (see Jenkins), 101, 109, 213,214,215, 222,232,264,266 Fletcher, D. A., 155 (see Thistle), 201 Floyd, W. W., 54, 101 Flynn, L. M., 64,102 Folger, B. B., 156, 196 Forbes, E.B., 120, 14.8, 14.4 Foster, R. L., 64 (see Heinse), 102, 295 (see Heinse), 320 Fox, F. W., 129 (see Walker, A. R. P.), 147 Fox, S. W., 172, 198 Fraenkel-Conrat, H.,383, 391 Frmke, K.W., 121, 122, 129, 143 Frankel, M., 173,196 Fraps, G. S., 54, 101, 153, 2U0, 310 (see Sheets), 311 (see Sheets), 312 (see Sheets), 313 (see Sheets), 314 (see Speirs), 322, 323 Frasier, L. E., 115, 14.9 Fremy, E., 397, 402, 406, 410, 411, 417, 423, @4
French, C. E.,117 (see Voris), 118 (see voris), 14r Friar, H.F., 260, 261, 287, 288, 364 (see Crueas), 369 Fricke, H., 375 Fried, M., 312, 319 Fritschi, U.,398 (see Schneider), 486 Fryd, C. F. M., 161, 175,196, 197 v. Fiirth, O., 7, 8, 36 Fulmer, H. L., 153 (see McFarlane, W. D.), 198 Fulton. C. O., 188, 192,197 Funk, E.M.,197 Fyler, H. M., 220 (see Asmundson), 263 G
Gaddum, L. W., 407,424 Gale, E.F., 379,5991 Gane, R., 157, 186, 188,197 Gaponenkov, T.K., 405, 424 Garrick, C. W.,174 (see Hauge), 197
Gaasner, F. X., 126 (see Wilgus), 14Y Geiger, E., 116,1-48 Gersh, I., 388 (see Pfeiffer), 392 Gibbons, N. E.,180 (see Thistle), 162 (see Thistle), 165 (see Thistle), 106 (see Thistle), 188, 192,197, 801 Gieger, M., 294 (see Reder), 296 (see Reder), 298 (see Reder), 303 (see Reder), 304 (see Reder), 308 (see Reder), 310 (see Sheets), 311 (see Sheets), 312 (see Sheets), 313 (see Sheets), 314 (see Speirs), 317 (see Reder), 322, 323 Gilks, J. L., 110,111, 1 4 Gillam, A. E., 154, 197 Gilman, A., 376,391 Giral, F., 293,319 Giroud, A,, 292, 303,319 Gleim, E.G., 64,101 Glenn, D. S., 334,341,ST0 Goettsch, M., 119 (see Pappenheimer). 1 4 Goff, H. R.. 311, 313,383 Golumbic, C., 125, 1.46 Goodwin. M. W.. 405. 408. 409. 410, 411. 412,'413,414, 415, 416, 417, 419;421; 428 Gore, H. C.,259, 263, 276, 288,336, 3Y0 Goresline, H. E., 156 (see McFarlane, V. H.), 156 (see Stuart), 165 (see Stuart), 188, 19Y, 199, 8U0, 801 Gortner, R. A., Jr., 128, 1.43 Go=, H.,132 (see Kleiber), 14.4 G r a b , N., 398, @6 Grant, G. A., 166, 192,201 Greaves, J. E., 313, 319 Greco, P. A., 188 (see Winter), 808 Green, E. L., 261, 266, 268,279,280, 288 Green, H. H., 112, 119,14.9 Green, R. G., 125, 127, 128, 1-48 Greenberg, D. M., 120,121,1@,1@, 144, 1.47
Greene, D. J., 119,141 Greene, L., 263 (see Doty), 271 (see Doty), 276 (see Doty), 277 (see Doty), 888 Greenstein, J. P., 209 (see Neuroth), 266 Greenwood, D. A., 84,86, 87, 88, 97 (see Feaster), 100, 101 Greer, L. P., 346, 362,870 Grewe, E.,156 (see Stuart), 164, 166,801 Griffith, W.H., 116, 14-3 Griffiths, A. E., 302 (see Smith, M. C.), 323 Grimball, P. C., 64 (see Heinse), 108, 295 (see Heinze), 296 (see Poole), 880,388 Griswold, R. M., 25, 26, 36
AUTHOR INDEX
435
Gross, C. R., 259 (see Nichols), 280, 889 Hamilton, T. S., 12,28 (see Mitchell), 29 (see Mitchell), 36 Groesfeld, J., 150, 197 Hamner, K. C., 64,109,292,296,297,298, Grover, D.W., 167, 197 299,300,301,302,303,305,306,307, h e r r a n t , N.B., 41,56,07,68,71,72,73, 308,309(see Bernstein), 519,390,393 77, 78, 81, 82, 86, 89, 93, 94, 101, 293 (see Vavich), 294 (see Vavich), Hampton, H. E., 312, 313, 380 Hamy, A., 313,390 383 Guggenberg, N., 100 (see McConnell), 103 Hankins, 0.G., 25, 31,36 Guilbert, H. R., 119, 132 (see Kleiber), Hansen, E.,64, 109, 295, 301, 306, 310, 390 I&, 144 Hanson, H. L., 222, 223, 229, 230, 233, Gum, 0. B., 307,319 235,251, 964,266 Gustavson, R. G., 120 (see Wilgus), 147 Hanson, 8. W. F., 161, 175, 196,197 Guthrie, J. D., 284,987,988 Harden, A., 332,334,3YO Guthrie, K. C., 3 Hardt, C.R., 172 (see Ball), 173,194,197 Gyargy, P., 128 (see du Vigneaud), 1-43 Hardy, W.B., 99 Hargreaves, F. J., 116 (see Harris), 130 H (see Harris), 1-43 Harmer, P. M., 308,320 Harris. L. J.. 116. 136.. 143. . . 293.. 320 Haag, J. R., 116,1-43 Harris; R. S.; 50,i09 HaagenSmit, A. J., 82, 101,135, 1-43 Harrison. D. L.. 222 (see Stewart). 225 Ham, F.,375,399 (see’Lowe;Stewart), 226 (see Lbwe), Haw, V. A., 330,331,332(see Stadtman), 227 (see Lowe), 228 (see Lowe), 230 334,335,336,338,343,345,347,348, (see Lowe; Stewart), 231 (see Lowe), 351,356,357(see Stadtxxhn), 358 (see 232 (see Lowe; Stewart), 234 (see Stadtman), 360 (weStadtrncLn), 362, Lowe; Stewart), 236 (see Lowe; Stew363,364(see Stadtman), 360,367(see art), 237 (see Stewart), 238 (see Stadtman), 368,370,3Y9 Stewart), 239 (see Lowe), 241 (see Habs, H., 11, 36 Stewart), 243 (see Lowe; Stewart), Hrtenni, K.D.,166 244 (see Lowe), 245 (see Lowe), 246 Hafner, F.H., 128,1& (see Lowe), 247 (see Lowe), 248 (see Haines, R. B., 21, 22, 23,24, 31,36 Stewart), 249 (see Lowe), 250 (see Hale, W.S., 213 (see Harshaw), 224 (see Lowe), 252 (see Lowe), 966,866 Hamhaw), 866 Harshaw, H. M., 206, 207, 213, 224, 966 Hall, C. E., 9,36 Hall, H.H., 156 (see Stuart), 163, 105, Hart, E. B., 121 (see Stirn), 124 (see Halpin), 1-43, 147 186, 188,901 Hall, J. A., 332, 333, 334, 342, 346, 351, Hart, G. H., 119 (see Guilbert), 1-43 Hartman, A. M., 115, 14.9 352,354,364,366, 307, 370 Hartsell, A., 284,888 Hall, J. L., 17, 18,36 Hall, L., 119 (see Zucker), 132 (see Hartsler, E. R., 293,380 Hatch, M. B., 271 (see Wiegand), 282 (see Zucker), 148 Wiegand), 283 (see Wiegand), $90 Halliday, E. G., 42 (see Hinma;n), 45 (see Brush), 49 (see Hinman), 51 (see Hin- Hathaway, I. L., 300,390 man), 62 (see Hinman), 90 (see Hauck, H. M., 98, 99, 108, 294 (see Karikka), 296 (see Karikka), 297 (see Moschette), 91 (see Moschette), 92 Kwikka), 308 (see Karikka), 321 (see Moschette), 93 (see Moschette), 94 (see Moachette), 95 (see Mo- Hauge, M. S., 219, 96.4 schette), 90 (see Moschette), lOa,108, Hauge, S. M., 174,196,197 Hawkes, C. D.,113 (see Richter), 114 (see 103 Richter), 118 (see Richter), 146 Hallman, L. F., 388 (see Pfeiffer), 399 Halpin, J. G., 124, 143, 153 (see Bau- Hawthorne, J. R., 159 (see Bate-Smith), 163, 105, 166, 107, 171, 172, 173, 181 I 1 1 8 M ) , 196 (see Bennion), 182,188,189,196,187 Ham,W. E., 385, 391 Hamburger, J. J., 55, 109, 328, 348, 349, Hay, R. L., 192,197 Hayward, J. W., 128,14.4 358, 360, 362,303,870
436
AUTHOR INDEX
Heberlein, D. G., 66, 79, 84, 85, 97, 100. 100, 102, 330, 3YO Heid, J. L., 401, 484 Heilbron, M., 154, 19Y Heiman, V.,154, 19Y,109 Heinze, P. H., 64, 102, 295, 390 Heiss, R., 22, 36 Heller, V. G., 156 (see Thompson), 801. 303, 380 Hellerman, L., 381, 391 Hemingway, A., 10,36 Henderson, J. L., 119, 144 Henglein, F. A.,398, 418, 494 Henriques, V., 36 Henry, H., 379, 391 Henry, R. E., 100 Henry, W. C., 167, 19Y Herriott, R. M., 383, 385, 391 Hibbard, A. D.,64, 108 Higgins, M. M., 42 (see Hinman), 102 Hilborn, M. T., 257, 262 (see Ross), 268 (see Ross), 282 (see Ross), 283 (see Ross), 889 Hilditch, T. P., 211, 212, 866 Hill, T. J., 122 (see Kick), 144 Hills, C. H., 411, 412, 416, 424, .bas, 42Y Hiner, R. L., 25, 31, 36 Hinman, W. F., 42, 45 (see Brush), 49, 51, 62, 90 (see Moschette), 91 (see Moschette), 92 (see Moschette), 93 (see Moschette), 94 (see Moschette), 95 (see Moschette), 96 (see Moschette), 100, 102, 103 Hinton, C. L., 407 (see Bock), 419, 4.83,
494
Hirsch, P., 403, 484 Hirschmann, D. J., 186, 19Y Hint, E. L., 399, 494 Hoagland, D.R., 313, 319 Hoagland, R., 29, 30, 31, 36 Hochberg, M., 73 (see Melnick), 109,293, 920
Hoelzel, F., 130, 14.4 Hoet, J. P., 7 (see Best), 14 (see Best), 34 Hoffert, E., 228, 266 Hoffman, 232 Holcomb, R., 206, 266 Holinger, P. H., 134, 136, 166 Hollinger, M. E., 294 (see Reder), 296 (see Reder), 298 (see Reder), 303 (see Reder), 304 (see Reder), 308 (see Reder), 317 (see Reder), 39.8 Holmes, A. D., 64 (see Schroeder), 104, 308, 310, 313, 314, 320
Holmes, C. E., 124 (see Halpk), 143, 1 3 (see Baumann), 196 Holmes, H. L., 356 Holst, W. F., 155, 198 Holt, L. E., Jr., 112 (see Richter), 113 (see Richter), 114 (see Richter), 118 (see Richter), 146 Hood, 5. L., 314 (see Parks), 321 Hoover, S. R., 170, 185, 194 hop kin^, E. W., 162 (see Steffen), 800 Hoppert, C. A., 135, 1.64 Horne, G. A., 23, 36 Horner, G., 68 (see Adam), 100 Hoskisson, W. A., 168 (see Tracy), 801 Hotchkiss, R. D., 378, 379, 398 Houghton, H. W., 213,214, 256 Howard, B. J., 229 (see Wiley), 266 Howard, L. B., 264, 265, 266, 267, 278, 280, 288
Howell, C. E., 119 (see Guilbert), i@ Hsieh, K. M., 303 (see Shen), 388 Huber, L. J., 411, 421, @3 Huddleson, J. F., 173 (see Hardt), 19Y de la Huerga, J., 118 (see Krehl), 144 Hughes, J. S., 154, 198 Hunt, C. H., 153,198 Hunter, A. S., 304, 306 Hunter, J. E., 153 (see Murphy), 199,219 (see Marble), 220 (see Marble), 266 Hutchins, M. C., 261 (see Green), 266 (see Green), 268 (see Green), 279 (see Green), 280 (see Green), 288
I Ingram, M., 19, 20, 36,380, 392 Insko, W. M., Jr., 152, 198 Irving, J. T.,129 (see Walker, A. R. P.), 147 Isbell, H. S., 358, 370 Iv&novics, G., 387, 992 Ives, M., 41, 42, 44, 45, 47, 55 (see Wagner), 86, 87 (see Feaster), 1O f , 102, 104
Ivy, A. C., 114 (see Warkentin, J.), 124 (see Warkentin, J.), 134 (see Holinger), 136 (see Holinger), 144,147
J Jack, E. L., 119 (see Henderson), 144 Jackson, C. M., 121, 128, 130, l& Jackson, J. M., 84 (see Greenwood), 86 (see Greenwood), 87 (see Greenwood), 88 (see Greenwood), 97 (see Feaster), 101
Jackson, R. W.,133,166
AUTHOR INDEX
Jackson, S. H., 306,393 Jacobs, M. B., 371 Jacobs, M. H , 382,399 Jakus, M.A., 9 (see Hall, C. EJ, 36 Jameson, E.,405,424 Janes, B. E.,295, 320 Jansen, E.F., 411 (see MacDonelI), 419, 424,496 Jefferys, E. P., 359 (see Borsook), 369 Jeffreys, C. E.P., 82 (see Haagen Smit), 101 Jenkins, R. R., 68, 69,109 Jenness, L. C., 257, 262 (see Row), 268 (see Ross), 282 (we Ross), 283 (see Ross), 289 Jensen, L. B., 23,36 Jewell, W.R.,335,380, 364,369,370 Johnson, C. M., 175, 176,198 Johnson, D. W.,114, 1 4 Johnson, H. V., 229,230,266 Johnson, J. M., 6 (see Voegtlin), 38 Johnson, L. P. V., 303, 390 Johnson, V., 123 (see Quigley), 146 Joiner, R. M., 387 (see Wyss). - .. 399s Jokl, E., 12, 14,36. Jones, E.C., 211 (see Hilditch), 212 (see Hilditch), 966 Jones, F. T.,.166, 198 Jones, G. I., 153 (see Klose), 174 (see KIose), 108 Jones, J. B., 65, 103 Jones, J. K. N., 399,499,494 Jones, W.W., 302 (see Finch), 308, 309 (see Finch), 319,390 Joseph, G.H., 396,411,415,494 Joslyn, M. A.,56,102,268,987,327,328, 337,339, 348, 349, 350,351, 352, 364, 356,357,368,359,360,362,363,367, 370, 371,401,494 Jukes, T. H., 153 (see McFarlane, W.D.), 153 (see Lepkovsky), 198, 220 (see Asmundson), 963 Jungherr, E., 119 (see Pappenheimer), 146
437
Katz, J., 352,371 Kaufman, C.W.,408, 420, 424 Keim, F. D.,306 (see Hathaway), 390 Kelly, E. H,, 134 (see Holinger), 136 (see Holinger), 144 Kelly, W. C.,301,302,323 Kennard, D.C.,124 (see Bethke), 149, 153 (see Bethke), 154 (see Bethke), 196 Kennedy, E.K., 156,900 Kertesz, Z. I., 57 (see Robinson, W.B.), 80 (see Robinson, W. B.), 104, 411,
494
Kester, E.B., 177, 178, 198 Kick, C. H., 122,1.6.6 Kies, M. W.,210,964 Kieser, M. E., 401 (see Burroughs; Charley), 493 Kilpatrick, P. W., 262, 263,272,282,287 King, C. G.,27, 30,31, 36, 70 (see Fenton), 101,293,3.91 King, F. B., 150,199 Kirch, E. Z., 300 (see Kaaki), 391 Kirchner, J. G.,82 (see Hagen Smit), 101, 135 (see Haagen Smit), 143 Kirk, M. M.,63,109 Kleiber, M., 120,132, 1.6.6 Kleinschmidt, R.V., 156,196 Kline, L., 151, 176, 176, 183 (see Lineweaver), 184 (see Lineweaver), 186 (see Lineweaver), 198 Kline, R. W.,162 (see Steffen), 164, 171, 172,198,900 Klose, A. A., 152 (see Asmundson), 153, 158, 174,194,198 Knandel, H. C.,153 (see Murphy), 199, 219 (see Marble; Murphy), 220 (see Marble; Murphy), 966 Kneeland, R. F., 400,401,499 Kneen, E.,385,399 Kodicek, E.,174 (see Cruickshank), 196 Koenig, M. C., 164,198 Koga, T., 183, 184, 185,186, 198 Kohman, E.F., 40,82, 109 K Koiaumi, T.,299, 391 v. Kolnitz, H., 121 (see Levine), 14.4 Kahlenberg, 0. J., 120,144 Kon, S. K., 112, 114, 1.6.6 Kahler, H., 6 (see Voegtlin), 38 Koonz, C. H., 25 (see Ramsbottom), 32, Kakukawa, T.,299,391 36,37, 236,966 Kalebin, M. I., 401, 426 Koppanyi, T.,361,371 Kanapaux, M. S., 64 (see Heinze), 109, Kortschak, H. P., 405, 49.4 295,298 (see Pooh), 390,322,323 Kosmahly, A., 397, 493 Karikka, K. J., 294,296, 297,308,391 Kostenko, V. D., 299, 391 Karr, E.E.,401,426 Kosterlitz, H. W.,127 (see Cruickshank), Kaski, I. J., 300,321 142 Katchalsky, A., 173,196 Kramer, A., 44, 46, 47, 48, 109, 329, 371
438
AUTHOR INDEX
Kramer, M. M., 164 (see Koenig), 198 Krampitz, L. O., 376, 399 Kratzer, F. H., 127, 14.6 Kraybill, H. R., 84 (see Beadle; Greenwood), 86 (see Greenwood), 87 (see Greenwood), 88 (see Greenwood), 97 (see Feaster), 100, 101 Krehl, W. A., 118,1 4 Kremmers, R.E., 182 (see Sell), 900 Krieger, C. H., 119,14.6 Krishnan, B. G., 109 (see Aykroyd), 141 Krumbholz, G., 26,36 Kuzmeski, J. W., 308 (see Holmes), 310 (see Holmea), 313 (see Holmea), 314 (see Holmea), 390
L Lamb, F. C.,64,66, 66, 68, 60, 61, 62, 66, 66, 67, 70, 72, 73,74, 77, 80,81, 82, 83, 86, 89, 91, 103 Lampitt, L. H., 293,297,381 Lams, M.M., 383,399 Lantz, E. M., 299,391 Larson, L., 100 Lathrop, R. E.,368 Latschar, E. E., 17 (see Hall, J. L.), 18 (see Hall, J. L.), 36 Lausten, A., 150, 169, 170, 182,196 Lawrence, A. 8. C., 4 (see Needham, J.), 9 (see Needham, J.), 37 Lawrenz, M.,128,144 Lea, C. H., 214,966 Lease, E. J., 310 (see Sheets), 311 (see Sheets), 312 (see Sheets), 313 (see Sheets), 314 (see Speirs),399,393 Lee, F. A., 64, 103 Lee, M. O., 122, 14.6 Legault, R. R., 265,267,271,272,988 Lahmann, K. B., 26,36 Leo, H. T.,411,.494 Lepkovsky, S., 119 (see Henderson), 128 (see Chernik), 14.3, 14.4, 163,198 Levine, H.,121, 14.6 Lew, M.,260 (see Cruess), 987 Lewis, H. B., 116 (see GriEth), 14.3 Lewis, J. C., 163 (see Klose), 198 Lewis, J. H., 126,144 Lewis, W. R., 263 (see Doty), 271 (see Doty), 276 (see Doty), 277 (see Doty), 988 Lightbody, H. D., 186, 197 Linderstrgjm-Lang, K., 376 Lindsey, J. W., 411 (see Leo), 494 Lineweaver, H., 161, 169, 183, 184, 186, 186, 198, 268 (see Campbell), 887, 411,494,4.36
Ling, A. R., 397 (see Nanji), 4.36 Link, K. P., 126, 144, 399 (see Morell), &6
Litwiller, E. M., 271 (see Wiegand), 282 (see Wiegand), 283 (see Wiegand), 990 Ljubimova, M. N., 8, 8, 9 (see Engel’hsrdt), 36 LO, T.-Y., 308,391 Lochmllller, K., 407, 416,496 Lockwood, W. W.,383 (see du Vigneaud), 399
Lo COCO,G., 298,391 h f f l e r , H. J., 293,391,329,331,340,347, 348, 349,360, 362,571 v. Loesecke, H.W.,78, 105, 340,3Yi Lohmann, K.,3,4,36 Lombard, P. M., 261 (see Caldwell), 282 (see Caldwell), 283 (see Caldwell),987 Long, C. N. H., 136 (see Brobeck), 1.49 Long, J. D., 360 (see Fisher), 370 Longsworth, L. G., 169, 198 Lorenz, P. W., 162 (see Almquist), 194 Lotekar, H., 406, 408 (see Schultz), 412 (see Owens; Schultz), 413 (see Schulte), 4.36 Lovern, J. A., 156,198, 268,988 Lowe, B,, 160 (see Stewart), 171 (see Stewart), 188 (see Stewart), 190 (see Payawal), 182 (see Stewart), 199,900, 222,223 (see Stewart), 225,226,227, 228,229(see Stewart), 230,231,232, 233, 234, 236 (see Stewart), 236,237 (see Stewart), 238, 239, 241 (see Stewart), 242,243,244,246,247,248, 248,250,251 (see Hanson),262,964, 966,966 Luokett, S., 399, 484 Ludwig, B. J., 387 (see Wyss), 393 Lueck, R. H., 98, 103 Lilers, H., 407, 416,496 Ludgren, H. P., 161, 169,198 Lundsgaard, E.,3, 4,36 Luak, J. L., 281,283,986 Lutikova, P. O., 188,908 Lutwak-Mann, C., 4,36 Lynn, J. M., 168 (see Dawson, E. H.), 191 (see Dawson, E. H.), 196 Lyon, A. V., 360 (see Cameron), 369 Lyon, C. B., 314 (see Parks), 391 Lyons, M., 152, 198
M MacArthur, M., 268 (see Davis), 260 (see Davis), 261 (see Davie), 266 (see Davis), 987
AUTHOR INDEX
McBryde, C. N., 29, 30, 31, 36 McCall, K.B., 118 (see Waisman), 147 McCalla, A. G.,312,313,322 McCance, R. A., 121, 124, 127, 128, 146 McCarrison, R., 109,146 McCarthy, J. F., 27, 30,31,36 McCay, C. M., 125 (see Madsen), 146 McClary, C. F., 153, 198 McClurg, B. R., 252 (see Paul), 266 McCollum, E. V., 119, 121, 125,1/16 McCollum, J. P., 300, 301,321 McConnell, J. E. W., 100, 103 McCready, R. M., 420,4.96,426 McDermott, F. A., 336,3Yl McDonald, C. H., 132 (see Baldwin), 141 MacDonell, L. R., 411,46'6 MacDowell, L. G.,55 (see Moore), 103 McDowell, R. H., 411, 426 McFarlane, V. H., 156, 188,197,198,200,
439
McMillan, T. J., 293, 321 McNally, E.H., 156,201 McWhirter, L., 294 (see Reder), 296 (see Reder), 298 (see Reder), 303 (see Reder), 304 (see Reder), 308 (see Fkder), 310 (see Sheets), 311 (see Sheets), 312 (see Sheets), 313 (see Sheets), 314 (see Speirs), 317 (see Reder), 322, 323 Madsen, H. S., 335 (see Wiegand), 372 Madsen, J., 14,36, 218,266 Madsen, L. L., 25 (see Hiner), 31,36,125,
146
Maerz, A., 328,37'1 Magoon, C. A., 66, 103,281, 288 Maillard, L. C., 170,199 Makower, B., 157,174,186,199,264,266, 288 Mallmann, W. L., 156, 199 Malmsten, H. E., 124,146 2Ood Malsch, L.,353 McFarlane, W. D., 153, 198 Mangelrr, C. E.,259,288 MeGeorge, W. T., 312,313, 319 MacInnes, D. A., 169 (see Longsworth), Mann, F. C., 131, 146 Mann, T., 4, 36 198 Mapson, L. W., 259 (see Tomkins), 263 Mack, G. L., 293,391 (see Tomkins), 266 (see Barker; TomMcKeegan, M., 222 (see Stewart), 225 (see kins), 271 (see Tomkins), 272 (see Lowe; Stewart), 226 (see Lowe), 227 Tomkins), 275 (see Tomkins), 276 (see Lowe), 228 (see Lowe), 230 (see (see Tomkins), 280 (see Wager), 286, Lowe; Stewart), 231 (see Lowe), 232 289 (see Lowe; Stewart), 234 (see Lowe; Stewart), 236 (see Lowe; Stewart), Marble, D. R., 219,220, 266 237 (see Stewart), 238 (see Stewart), Mark, H.,398,496 239 (see Lowe), 241 (see Stewart), Marks, H.P., 7 (see Best), 14 (see Best), 243 (see Lowe; Stewart), 244 (see 34 Lowe), 245 (see Lowe), 246 (see Marsh, G. L., 328,337,339, 348,349,350 (see Joslyn), 351, 352, 354, 356, 358, Lowe), 247 (see Lowe), 248 (see 359,367, 370,3Yl Stewart), 249 (see Lowe), 250 (see Marshall, J. B., 303 (see Johnson, L. P. Lowe), 252 (see Lowe), 266, 966 V.), 320 McKibbin, J. M., 118,146 Mackinney, G., 126, 147, 266, 267, 279, Matlack, M. B., 357,371 280, 287, 988, 329, 335, 336 (see Matthew, A.,336, 347, 354,362, 365, 3Yl Stadtman, E. R.), 338 (see Stadtman, Mattill, H.A., 125, 1.46 E.R.), 343 (see Stadtman, E.R.), 345 Maw, W. A., 206,220, 225, 253, 266 (see Stadtman, E. R.), 347 (see Stadt- Maximow, A. A., 209,210,866 man, E. R.), 348 (see Stadtman, E. Maxwell, M. L., 220 (see Asmundson), 263 R.), 352, 356, 357 (see Stadtman, Mayer-Groeq W., 138,146 E. RJ, 358 (see Stadtman, E. R.), Mayfield, H.L., 67 (see Richardson), 104 359, 360, 361, 362,363, 364, 367 (see Maynard, L. A., 64 (see Hamner), 67 (see Stadtman, E. R.), SYf, 378 Stimson; Zimmerman), 102,104,125 McKinnon, M. I., 281,283, 989 (see Madsen), 146, 292, 296 (see Hamner), 297 (see Hemner), 299, Mackintosh, D. L., 17 (see IJall, J. L.), 300,303,305, 307, 320, 321 18 (see Hall, J. L.), 36 McKittrick, D. S., 133, 146 Mecham, D. K., 151, 199 Maclay, W.D., 400,404,408(see Schultz), Mehlitz, A., 411, 426 412 (see Schultz), 413 (see Schultz), Mehlitz, I. A., 400,496 Meitina, R. A., 9 (see Engel'hardt), 36 420 (see McCready), 496,496
440
AUTHOR INDEX
Mellanby, E., 124, 146 Melnick, D., 73, 103, 293 (see Hochberg), 320 Melville, D. B., 128 (see du Vigneaud), 143 Menaue, P., 358, 370 Mendel, B., 186,199 Mendel, L. B., 108,112,114,117,127,1.46 Merrill, R. C., 405 (see Owens), 412 (see Owens), 426 Meschter, E. E., 420 (see Baker), 422 Metcalfe, B., 154 (see Pearce), 199 Meyer, H., 306,307,319 Meyer, K.,7,8, 38 Meyer, K. H., 10,36, 398, 426 Meyerhof, O.,4, 36 Michmlis, L., 376, 392 Miller, C. D., 108, 146 Miller, E. V., 262, 286 Miller, M. W., 153,196 Miller, V. L., 1.53 (see McClary), 198 Mirsky, I. A,, 385,392 Mitchell, H.H., 12, 28j 29, 36, 116 (see Beadles), 118.128 (see Lawrenz). .. 148.
i.d-6,14i’
’
Mitchell. H. S.. 117. lL5 Mitchell; J. H.; 310’(seeSheets), 311 (see Sheets), 312 (see Sheeta), 313 (see Sheets), 314 (see Speirs), 322,323 Mitchell, L. C., 155, 199 Mitchell, P. D., 379,392 Mnookin, N. M., 421,426 Moldtmann, H.G., 303,321 Moore, E. L., 55, 90, 98, 99, 103, 104, 328, 329, 332,337,338, 346, 348,349, 350, 364,355, 356,358,370,371 Moore, R. C., 294 (see Reder), 296 (see Reder), 298 (see Reder), 303 (see Reder), 304 (see Reder), 308 (see Reder), 310 (see Sheets), 311 (see Sheets), 312 (see Sheets), 313 (see Sheets), 314 (see Speirs), 317 (see Reder), 322, 323 Moore, R. L., 188, 197 Moran, T., 1, 3, 22, 23, 27, 31,37 Morell, S.,399, 426 Morgan, A. F., 261,288,328 (see Joslyn), 337, 348 (see Joslyn), 349 (see Joslyn), 350 (see Joslyn), 362, 371 Morgan, C.T., 123, 1.46 Morgan, J. D., 123, 1.46 Morgulis, S., 141 Morris, H.J., 183 (see Lineweaver), 184 (see Lineweaver), 186 (see Lineweaver), 198,258 (see Campbell), 287 Morris, H. P., 150, 199
Morris, T. N., 401,426 Moschette, D. S., 90, 91, 92, 93, 94, 95, 96, 103 Mottern, H.H.,350 (see Nelson), 353 (see Nelson), 371, 401, 411, 421 (see Buck), 4831 424, 425, 487 Moxon, A. L., 128 (see Dubois), 142 Moyer, J. C., 81, 103 Mrak, E.M., 258,287, 328 (see Nichols), 329, 330, 331, 332 (see Stadtman, E. RJ, 334, 335, 336 (see Stadtman, E. R.), 338 (see Stadtman, E. R.), 342 (see Nichols), 343 (see Stadtman, E. R.), 344 (see Nichols), 345 (see Stadtman, E. R.), 347 (see Stadb man, E. R.), 348 (see Stadtman, E. R.), 351, 356 (see Stadtman, E. R.), 360, 361, 362, 363, 364, 365, 370, 371, 372, 389 (see Whelton), 392 Mtiller-Thurgau, H., 268, 282, 5’88 Munsell, H.E., 41, 101 Murlin, J. R., 111, 147 Murphy, E.F., 294,297, 321 Murphy, R. R., 153, 199, 219, 220, 266 Murray, C. W., 169,198 Murray, W.T., 25, 31, 38 Mursell, J. L., 107, 113,1.46 Muth, F., 353 Myers, P. B., 398,399,400,402,403,404, 405,407, 409,415,417, 425 Mylne, A. M., 265 (see Legault), 267 (see Legault), 271 (see Legault), 272 (see Legmlt), 288
N Nanji, D. R., 397,4.96 Neal, N. P., 306 (see Porter), 392 Needham, D. M., 4, 5, 9 (see Needham, J.), 37 Needham, J., 4,9,37, 183, 185, 199 Nelson, E.K., 350,353,371 Nelson, V. E., 132 (see Baldwin), 141 Nelson, W. L., 293, 294,301, 321, 323 Nestler, R. B., 150, 153 (see DeVaney), 196, 199 Neurath, H., 208,209,266 Newman, K.R., 98, 103 Nickerson, J. T. R., 213, 214, 215, 222, 232, 264, 266 Nichols, P. F., 259, 260, 289, 328, 329, 330,335,342,344,345,347,360,362, 365, 371, 372 Nielsen, E., 264 (see Makower), 266 (me Makower), 288 Nielsen, J. P., 400, 405, 426
AUTHOR INDEX
441
Peacock, W. M., 268 (see Wright), 273 (see Wright), 282 (see Wrigh ), 283, 289, 290 Pearce, J. A., 154, 155 (see Thistle), 159, 180 (see Thistle), 161, 162 (see Thistle), 165 (see Thistle), 166 (see Thistle), 168, 192, 193, 197, 199, 201 Pearl, R., 112,146 Pearson, P. B., 153 (see Snell), 200 Pease, V. A., 258 (see Chace), 287 0 Pederson, C. S., 328, 333, 341, 369, 371, 372 Olcott, H. S., 151, 173, 181,199 Peech, M., 312,319 Olliver, M., 70,103, 293,320 Pekarek, E.,383 (see du Vigneaud), 392 Olmstead, W.H., 135, 147 Pennington, M. E., 184, 199, 207, 213, Olsen, A. G,, 182 (see Sell), 200,399,405, 222, 229 (see Wiley), 234,235, 966 406 (see Stuewer), 408,409,413,415, Pepkowitz, L. P., 300, 321 416, 417. 420 (see Kaufman). .. 421.. Peters, C. A., 416,426 4.94; 426. Peters, R. A., 113, 131,146 Olson. 0. E.. 128 (see Dubois). lL3 . Peterson, G. T., 58, 59, 60, 62, 99, 100, Oomsj A., 15,37 ' too, 101 Orent, E. R., 121, 146 Peterson, M., 405 (see Owens), 412 (see OrenbKeiles, E., 119 (see McCollum), Owens), 426 121, 125 (see McCollum), 145 Peterson, W. H., 125 (see Woolley), 127 Organ, J. G., 58, 104, 300,323 (see Woolley), 147 Orr, J. B.,110, 111, 146 Petrosini, G., 308, 322 Osborne, T. B., 108,112, 114, 127,146 Pfaff, H. J . , 389 (see Whelton), 392 Oser, B. L., 73 (see Melnick), 103, 293 Pfeiffer, C . C., 388,392 (see Hochberg), 320 Philips, F. S., 376,391 Ostern, P., 10,37 Philpot, J. S. L., 383,392 Otto, R.,404,426 Pierce, H.C., 222 (see Pennington), 266 Owens, H.S., 405,408 (see Schultz), 412, Pilcher, R. W., 98,103 420 (see McCready), 426, 426 Pittman, D.W., 313,819 Platenius, H.,65,103, 299, 322 P Pohle, K.,10, 37 Palmer, L. S., 114, 144 Pollard, A., 309,322,401 (see Burroughs), P'an, M.T., 121,144 423 Pollard, A. E., 44 (see Ives), 45 (see Ives), Pancoast, H.M., 330,342,370 Pappenheimer, A. M., 119,146 47 (see Ives), 102 Parkinson, T. L., 293 (see Lampitt), 297 Ponting, J. D., 293,321 Poole, C.F., 296,322 (see Lampitt), 321 Parks, R. Q., 298,302,305(see Bernstein), Porter, J. W., 306,322 308, 309 (see Bernstein), 314, 319, Postma, C., 15, 37 Potter, V. R., 122,129,143 320,321 Powers, J. J., 328 (see Esselen), 349 (see Passmore, R., 109 (see Aykroyd), 141 Paton, F. J., 397 (see Nanji), 426 Esselen), 370 Patterson, E. G., 118 (see McKibben), 146 Powers, R., 259 (see Nichols), 289 Patton, A. R., 126 (see Wilgus), 147, 276, Powick, W. C., 29,30,31, 36 Prater, A. N., 135 (see Haagen Smit), 1.43 277,289 Pressley, A., 41,45,47, 103 Paul, M.R.,328,371 Paul, P., 252, 266 Price, F. E., 335 (see Wiegand), 572 Procter, H. A., 11, 12,37 Pavlow, I. P., 134,145 Payawal, S. R., 189, 199 Proctor, B.E... 56,102,281, 289,341,351, . . Payne, L. F., 154,198,214(see Schrieber), 358,371 217 (see Schrieber), 218 (see Schrie- Pullv. G. N.. 340. 371 ber), 266 Put&m, F. W., 209 (see Neurath), 266 v. Nieasen, K., 397,4.97 Nightingale, G. T., 303,390 Nikolaeva, N. V., 31,37, 38,210,266 Noel, W. A,, 258 (see Chace), 259 (see Nichols), 287, 289 Noggle, G . R., 308,309,323, 324 Norris, F. W.,415,@9 Northrop, J. H., 383,385, 391, 392 Notley, V. E., 258,286
I.
442
AUTHOR INDEX
Roberts, W. (Sir),107, 146 Robertson, H. G., 185,189 Robinson, H. E., 97,98,105 Q Robinson, W. B., 67, 60,104, 293, 326 Robison, R., 332, 334, 370 Quackenbush, F. W., 153, 201 Roblin, R. O., 384, 392 Quigley, J. P., 123, 146 Roe, J. H., 358, 359, 376 Quinn, G., 360, 369, 372 Rogers, C. F., 268 (see Rogers, M. CJ, Quint, E., 118, 136, 146 273 (see Rogers, M. C.), 289 R Rogers, H. B., Jr., 259, 272, 289 Rogers, M. C., 268, 273, 289 Roleson, E. P., 360, 372 Rahn,O., 374, 388, 399 Ronzoni, E., 3,37 Ramsbottom, J. H.. 235. 266 Rose, C. S., 128 (see du Vigneaud), 14.9 Ramsbottom; J. M:, 25,'32, 36, 37 Rangnekar, Y. B., 309,322 Rose, D., 312, 313, 322 Record, P. R., 122 (see Kick), 1.44, 162 Rose, W. C., 113, 115, 146 (see Wilder), 153 (see Bethke), 196, Rosenfield, B., 403,4 6 Ross, A. F., 262, 267, 268, 270, 281, 282, 202 Reder, R., 294, 296, 288, 303, 304, 308, 283, ,889 310 (see Sheets), 311 (see Sheets), 312 Ross, E., 91, 104 (see Sheets), 313 (see Sheets), 314 (see Ross, W., 55 (see Smith, M. C.), 104 Rostovskaya, Yu. V., 70, 104 Speirs), 317,326,323 Reed, H. M., 328, 329, 335, 342, 344,345, Rouse, A. H., 404,486 347,360,362,365,571 Rowan, A. N., 10, 37 Reeve, R. M., 156,199 Rudney, H., 186,199 Reich, G. T.,421, 426 Ruschmann, G., 272, 289 Reid, D. F., 119 (see Henderson), 14 Rushton, E., 281, 289 Reid, M., 154 (see Pearce), 159 (see Rusk, H. P., 12,36 Pearce), 168, 188 (see Gibbons; This- Russell, W. C., 162,154,200 tle), 192 (see White), 193 (see Pearce), Ruth, W. A., 128 (see Lawrenz), 144 197, 199, 201, 202 Rutledge, L. F., 263, 276, 288 Reid, M. E., 303, 322 Ryan, E. J., 375, 396 Remington, R. E., 121 (see Levine), 14.4 Rendle, T., 259, 289 S Reyniers, J. A., 135, 146 Rhead, A. J., 211 (see Hilditch), 212 (see Hilditch), 266 Saburov, N. V., 401, @6 Rhodes, W. E., 259, 289 Sacks, J., 4, 57 Rice, E. E., 84, 97, 98, 103 St. John, J. L., 170, 200 Rice, K. K., 123, 146 Sair, L., 32, 37, 222, 266 Richardson, J. E., 67, lo4 Sakov, N. E., 6, 7, 37 Richardson, W. D., 235, 266 Sampson, A. W., 124, 146 Richert, P. H., 341, 364, 362, 972 Samuels, L. T., 124, 134, 146 Richter, C. P., 112, 113, 114, 118, 119, Sanborn, N. H., 56, 57, 62, lo4 122, 123, 130, 133, 137, 146 Sando, C. E., 357,871 Ridder, C., 41 (see Pressley), 45 (see Sandstedt, R. M., 385,391, 392 Pressley), 47 (see Pressley), 105 Sapun, M. P., 309, 322 Riemenschneider, R. W., 151, 184, 199 Sassaman, H. L., 124 (see Bethke), 1@, Rietr, E., 419 (see Jansen), 424 154 (see Bethke), 196 Rimington, C., 185,201 Satterfield, G. H., 64 (see Schroeder), 104 Riou, P., 314, 322 Savage, C. G., 360 (see Cameron), 369 Ritchie, A. D., 15, 37 Sayers, M. A., 114, 14'7 Rittenberg, D., 151 (see Chargaff), 196 Schaffer, N. K., 122, 144 Ritzman, E. G., 13, 34 Schaible, P. J., 152, 154, 200 Robbins, R. C., 64, lo4 Schloaser, M. E., 387,396 Roberts,J. A,, 91, lo4 Schmidt, E. C. H., Jr., 123, 146 Pyke, W. E., 276, 277, 289
AUTHORL INDEX
443
Schmitt, F. O., 9 (see Hall, C. E.), 36, Smart, H. F., 166 (see Stuart), 166 (see 207,966 Stuart), 901 Schneider, G., 398, 399, 494,496 Smith, A. M., 309, 383 Schneider, H., 119, 132, 146 Smith, E. C., 1, 22, 27, 30, 31, 36, 37 Schneider, L. K., 376 (see Ryan), 398 Smith, F., 399, 494,496 Schnetzler, E. E., 166 (see Thompson), Smith, F. G., 296, 308, 383 901 Smith, H. G., 116, 146 Smith, H. R., 329, 371 Schnitzer, R. J., 376, ,999 Schoon, J. G.,15, 37 Smith,M., 183, 185, 900,258, 987 Schrader, A., 333,378 Smith, M. C., 41 (see Pressley), 46 (see Schrieber, M. L., 214, 217, 218, 866 Pressley), 47 (see Preseley), 66, 103, Schroeder, G. M., 64, 104 104,302,323 Schubert, F., 397, 493 Smith, O., 306,s.M Schubert, M. P., 377, 399 Smith, R. M., 152, 164, 900 Schtitte, E., 183, 186,194 Smith, V. D. E., 128, 130, 144 Schultz, T. H., 408, 412, 413, 4M Smolenski, K., 398, 496 Schwartze, E. W., 126, 146 Smorodintsev, I. A,, 31, 37, 38, 210, 9.56 Scott, A. W., 281 (see Ruahton), $89 Snell, E. E., 41 (see Thompson), 10.4,127 Scott, E. M., 112, 114, 118, 136, 146 (see Eakin), 143,163, 900 Scott, W. J., 22 (see Empey), 23, 24, 36 Sollman, T., 124, 147 Scoular, F. I., 55, 104 Solomon, E. J., 123 (see Quigley), 146 Sebrell, W. H., 118, 147 Solowey, M., 188, 900,908 Somers, G. F., 293,294,301,302,381,393 Sedky, A., 401, 494 Seegers, W. H., 116,146 Sommerfeld, R. V., 397, 493 Sell, H. M., 182, 900 Sorber, D. G.,360, 379 Semb, J., 163 (see Baumann), 196 Spaulding, E. H., 188 (see Solowey), 900 Shank, D. E., 168 (see Dawson, E. H.), Spector, H., 118, 147 191 (see Dawson, E. H.), 196 Speirs, M.,294,296, 298, 303 (see Reder), Shapiro, E., 174, 181, 196 304, 308, 310 (see Sheeta), 311 (see Sharp, P. F., 170, 800 Sheeta), 312 (see Sheets), 313 (see Shaw, T. M., 156, 174, 199,900 Sheets), 314, 317, 398, 39.3 S h y , H.,134, 136, 146 Speiser, R., 412, 413, 416, 496' Sheets, 0.A., 294 (see Reder), 296 (see Sprince, H., 136, 148 Reder), 298 (see Reder), 303 (see Stacey, M.,379, $91 Reder), 304 (see Reder), 308 (see Stadtman, E., 126 Reder), 310, 311, 312, 313, 314 (see Stadtman, E. R., 329, 330, 331,332,334, 336, 336, 338, 343, 346, 347, 348, 366, Speirs), 317 (see Reder), 389, 383 367,368,369,360,361, 362, 363,364, Shelling, D. H., 123, 130, 137, 147 Shen, S. C., 4 (see Needham, J.), 9 (see 366,367, 368,3ro, 379 Needham, J.), 37 Stadtman, F. H., 369, 366, 368, 37.9 Shen, T.,303, 399 Stamberg, 0.E., 281, 283, 989 Sherman, G. D., 308, 320 Stanley, E. C.,281 (see Rushton), 989 Sherman, V. W., 281, 889 Stanley, P. G.,387, 399 Sherwood, R. M., 162, 163, BOO Stanworth, J., 68 (see Adam), 100 Shewing, J., 168 (see Trrrcy), 901 Stare, F. J., 118 (see McKibbin), 1.46 Shipston, G. T., 332 (see Stephens), 348 Stateler, E. S., 189, 900 (see Stephens), 360 (see Stephens), Steedman, J., 401 (see Burroughs; Char366 (see Stephens), 378 ley), 493 Sideris, C. P., 308, 311, 312, 313, 314, Steenbock, H., 108 (see Everson), 119,126 399, 393 (see Woolley), 127 (see Everson; Silber, R. H., 118, 147 Woolley), 132, 143, 144, 146,147 Singer, T. P., 381, 391,399 Steffee, C. H., 115 (see Frazier), 143 Sipe, G.R., 152, 900 Steffen, A. H., 162,900 Skeggs, H. R., 387,393 Steinberg, R. A., 376,392 Sluder, J. C., 281, 989 Steiner, G., 27, 28, 29, 38 Small, P. A., 383, 398 Steinhauser, H., 162 (see Bernhard), 196
444
AUTHOR INDEX
Stephens, J. W., 332, 348, 350, 366, 378 Stern, R. M., 66 (see Guerrant), 67 (see Guerrant), 68 (see Guerrant), 71 (see Guersant), 72 (see Guerrant), 73 (see Guerrant), 77 (see Guerrant), 78 (see Guerrant), 81 (see Guerrant), 82 (see Guerrant), 86 (see Guerrant), 89 (see Guerrant), 101, 293 (see Vavich), 294 (see Vavich), 5$?5 Stewart, G. F., 158, 160, 164, 171, 188, 18B,190 (see Payawal), 192,198,199, 800,808,221, 222, 223, 225, 226 (see Lowe), 227 (see Lowe), 228 (see Lowe), 229,230,231(see Lowe), 232, 233,234, 235, 236,237, 238, 239 (see Lowe), 241,243,244 (see Lowe), 245 (see Lowe), 246 (see Lowe), 247 (see Lowe), 248, 249 (see Lowe), 250 (see Lowe), 261 (see Hanson), 252 (see Lowe), 864,866,866 Stiles, G. W., Jr., 229 (see Wiley), 866 Stimson, C. R., 67,104 Stirn, F. E.,121, 147 Stitt, F., 156,800 Stokstad, E.L. R., 119,141 Stone, W. S.,375,398 Stotr, E., 57 (see Robinson, W. B.), 60 (see Robinson, W. B.), 81, 103, 104, 293,388 Stout, P. R., 358 (see Dunlop), 370 Strachan, C. C., 57, 58,61,104,258 (see Davie), 260 (see Davis), 261 (see Davis), 266 (see Davis), 887 Strandine, E. J., 25 (see Ramsbottom), 37 Street, H. R., 118,147 Strickland, A. G. R., 82 (see Haagen Smit), 101 Strong, F. M., 41 (see Ives), 42 (see Ives), 44 (see Ives), 45 (see Ives), 47 (see Ives), 55 (see Wagner), 70 (see Wagner), 71 (see Wagner), 72 (see Wagner), 73 (see Wagner), 77 (see Wagner), 78 (see Wagner), 80 (see Wagner, 81 (see Wagner), 85 (see Wagner), 86 (see Ives), 89 (see Wagner), 108, 104, 306 (see Porter), 388 Stuart, L. S., 156,163,164,165,166,186, 188,801 Stuewer, R., 399 (see Olsen), 405, 406, 408 (see Olsen), 409 (see Olsen), 413 (see Olsen), 415 (see Olsen), 416 (see Olsen), 417, 4B6 Subrahmanyam, V., 126 (see De), 1 4 Sucharipa, R., 407,426 Sugawara, T., 302,303,383 Sumner, E.E., 111, 147
Sundararajan, A. R., 109 (see Aykroyd), 141 Sure, B., 117,147 Svedberg, T., 398,&6 Swandish, S.,358 (see Dunlop), 570 Sweetman, M. D., 268, 273, 889 Swenson, A. D.,185,801 Swenson, T. L., 127, 141, 154, 170, 196, 213 (see Harshaw), 224 (see Harshaw), 866,385,391 Swift, R. W., 117 (see Voris), 118 (see Voris), 147 SzentcGyorgyi, A., 8, 9, 10,38, 208, 209, 240,866
T Trtlburt, W. F., 265 (see Legault), 267 (see Legault), 271 (see Legault), 272 (see Legault), 888 Tanner, F. W., 23, 38 Tarr, L. W., 416,486 Taylor, C.C., 411 (see Leo), 424 Taylor, E.S.,379,391 Taylor, F. H. L., 126, 1.64 Taylor, F. N.,406 (see Jameson), 484 Taylor, L. W., 153 (see Lepkovsky), 198 Taylor, M. W.,152 (see Russell), 154,800 Temmer, O., 356 (see Stadtman, E. R.), 357 (see Stadtman, E. R.), 358 (see Stadtman, E.R.), 364 (see Stadtman, E.R.), 367 (see Stadtman, E.R.), 379 Temperton, H., 154,801 Tepperman, J., 136 (see Brobeck), 148 Tessier, H., 154 (see Pearce), 199 Thayer, S., 118 (see McKibbin), 1.46 Theis, E. R.,383,392 Thiessen, E.J., 304,323 Thistle, M. W., 155, 159, 160, 161, 162, 165, 166, 188, 192, 199,8U1,808 Thom, C.,376,898 Thomas, B. H.,153,801 Thompson, A. H., 333 (see Schrader), 37'8 Thompson, M. L., 41,104 Thompson, R. B., 152, 156,801 Thompson, W.L.,184, 185, 801 Thornton, N. C.,268, 273, 277, 282, 283, 284 (see Denny), 887,288 Titus, H. W., 151 (see Riemenschneider), 153, 184 (see Riemenschneider), 196, 196, 199, 801 Todhunter, E. N., 64,104,293,381 Tomkins, R. G., 22 (see Moran), 37, 269, 263, 266, 271, 272, 275, 280 (see Wager), 886,889 Tompkins, M. D.. 87 (see Feaster), 101
AUTHOR INDEX
445
W Torre, L. M., 293, 319 Tracy, P. H., 168, 101 Wade, B. L., 64 (see Heime), 109, 295, Tremblay, F. T., 310, 313, 393 390,393 Treader, C. J., Jr., 280, 261, 268, 989 Treealer, D. K., 25, 31, 38, 63, 64 (see Wager, H. G., 259 (see Tomkins), 263 (see Tomkins), 266 (see Barker; Tomkins), Gleim), 67 (see Stimson; Zimmer271 (see Tomkine), 272 ( b e Tomman), 68 (see Jenkins), 69 (see kine), 275 (see Tomkine), 276 (see Jenkins), 70 (see Fenton), 101, 101, Tomkine), 280,986, ,989 104, 217 (see Dubois), 218 (see Dubois), 964,293, 3911328,333, 341, Wagner, J. R., 41 (see Ivea), 55, 70, 71, 72, 73, 77, 78, 80, 81, 85, 86 (see 379 Ives), 89, 101, 104 Tufts,E. V., 120, 143, 147 Wagoner, C. E., 216, 217, 218, 222, 253, Turner, A. W., 162, 196 966 Wahab, A., 357, 359, 37d U Waisbrot, 8.W., 419 (see Jansen), 494 Waisman, H. A., 118,147 Upp, C. W., 152, 154 (see Sweneon), 901 Waldo, G. F., 64, 109, 301, 380 Walker, A. R. P., 129, 147 V Walker, H. A., 152 (see Russell),100 Walker, J. ,C., 296, 308, 383 Vail, G. E., 214 (see Schrieber), 210 (see Walker, J. W., 138, 146 Wagoner), 217, 218 (see Schrieber; Wang, T., 309, 393 Wagoner), 222 (see Wagoner), 253 Wang, Y. L., 174 (see Cruickshank), 196 Ward, A., 116 (see Harris), 136 (see (see wagoner), 966 Harris), 143 Vandecaveye, S. C., 310, 312, 313, 383 Ward, W. H., 151, 169, 194, 198 Van der Scheer, J., 173, 901 Van Holten, P., 261,888,364 (see Crueea), Warkentin, J., 114, 124, 147 Warkentin, L., 114 (see Warkentin, J.), 369 124 (see Warkentin, J.), 147 Van Horn, C. W., 302 (see Finch), 308 (see Jones, W. W.), 309 (see Finch), Warner, K. F., 25, 26, 34, 38 Warnock, G. M., 127 (see Cruiclrshank), 319,390 van Manen, E., 185,801 Warren, R. C., 369 (see Boreook), 369 Vauquelin, M., 395,496' Vavich, M. G., 41 (see Guerrant), 56 (see Watanabe, A. J., 84, 104 Guerrant), 67 (see Guerrant), 68 (see Watson, A. J., 156 (see McFarlane, V. H.), 188 (see McFarlane, V. H.), 198 Guerrant), 71 (see Guerrant), 72 (see Guerrant), 73 (see Guerrant), 77 Weaet, C. A., 120, 147, 352, 356, 369, 371 (see Guerrant), 78 (see Guerrant), Weber, H. H., 7, 8, 38 81 (see Guerrant), 82 (see Guerrant), Webster, G. L., 300 (see Kaaki), 391 86 (see Guerrant), 89 (see Guerrant), Weiesenbock, K., 303,383 93 (see Guerrant), 94 (see Guerrant), Weissenbock, M., 303, 383 Werkman, C. H., 375,398 101,293, 294, 393 Veitch, F. P., Jr., 351 (see Koppanyi), 371 Wetzel, G. H., 162 (see Steffen), 100 Veldhuis, M. K., 328 (see Curl), 332 (see Wharton, M. A., 25, 26, 36 Curl), 346 (see Curl), 348 (see Curl), Wheeler, K. A., 328 (see Beattie), 333 ( ~ e e Beattie), 341 (see Beattie), 369 349 (see Curl), 350 (see Curl), 354 (see Curl), 355 (see Curl), 356 (see Whelton, R., 389, 999 Whitacre, J., 310 (see Sheeta), 311 (See Curl), 370 Sheets), 312 (see Sheeta), 318 (see Vickery, J. R., 22 (see Empey), 36 Sheeta), 314 (see Speirs), 399, 393 du Vigneaud, V., 110, 128, 143, 383, 399 Whitcombe. J.. 64.103 Vivino, A. E., 351 (see Koppanyi), 371 White, A., ii4; 147 Voegtlin, C., 6, 38 White, J. W., 411 (see Hills), 412 (860 Volodkewich, N. N., 25, 27, 36,38 Hills),416 (see Hills), 424 Voris, L., 117, 118, 147 White, W. H., 165 (see Thistle), 166, 188, Vorkoeper, A. R., 156 (see Shaw), do0
446
AUTEIOF4 INDEX
192,,901, M9,213, 215,216,217,$64 Whiteman, E. F., 268 (see Wright), 273 (see Wright), 282 (see Wright), 283 (seeoWright), 990 Whiteman, T. M., 262 (see Wright), 268 (see Wright), 273 (see Wright), 282 (see Wright), 283 (see Wright), 890 Whiteside, A. G. O., 306,393 Widdowson, E.M., l21,124,127,128,Zlj6 Wiederhold, E., 55, 90 (see Moore), 98 (see Moore), 99 (see Moore), 103,104, 328 (see Curl; Moore, E. L.), 332 (see Curl; Moore, E. L.), 346 (see Curl), 348 (see Curl), 349 (see Curl), 350 (see Curl), 354 (see Curl), 355 (see Curl), 356 (see Curl), 370, 371 Wiegand, E. H., 271, 282, 283, 990, 335, 378 Wilder, 0.H. M., 122 (see Kick), 144, 152, 153 (see Bethke), lQ6,909 Wilder, W., 122 (see Kick), 1 4 Wiley, W. H., 229,966 Wilgus, H.S.,Jr., 121,126,147 Wilhelm, L. A., 154,197,909 Willaman, J. J., 411,497 Willard, H.,55, fO4 William, R. D.,135, f47 Williams, R. J., 127 (see Eakin), 143 Wills, R. F., 209,222,224,229,230,232, 248,249,251,966 Wilson, C. P., 332 (see Stephens), 348 (see Stephens), 350 (see Stephens), 351, 364,385(see Stephens), 378,400,405 (see Jarneeon), 494,497 Wilson, C. V., 25 (see Black), 34 Wilson, C. W.,412,420,421,499,497 Wilson, P. W., 384,393 Winkler, C. A., 17,25,38 Winkler, G.,404,486 Winter, A. R.,153 (see Hunt), 188, 198, 909
Wintrobe, M. M., 126, l.@ Wissler, R. W., 115 (see Frazier), f& Withers, W. A., 127, 147 Witmer, E.,222 (see Pennington), 966 Wittwer, S. H., 311,313,393 Wodicka, V. O., 284,N O Wohl, A., 397,487
Wohlgemuth, J., 151, 183, 185,$03 Wokee, F., 300,393 Wokes, G.,58, fO4 Wood, E. A., 168 (see Dawson, E. HJ, 191 (see Dawson, E.H.), 196 Woodcock, A. R., 188 (see Thistle), 192 (see Thistle), 901 Woodmansee, C. W., 400, 404, 411, 420 (see Baker), 421, &9 Wooley, J. G.,118,147 Woolley, D. W., 125,127,135,147,f@ Woolridge, R. L., 115 (see Frazier), 143 Wright, L. D., 387,393 Wright, R. C., 262,268,273,282,283, 890 Wyckoff, R. W. G., 173 (see Van der Scheer), 901 Wynd, F. L.,308,309,393, 394 Wyss, O.,375, 384, 387,399,393
Y Yarnell, S. H., 310 (see Sheets), 311 (see Sheets), 312 (see Sheets), 313 (see Sheets), 314 (see Speirs), 399,393 Young, G. A., 303 (see Johnson, L. P. V.), 390
Yo&g,- H. Y., 308, 311, 312, 313, 314, 398, 888
Young,.M., 119 (see Zucker), 132 (see Zucker), 148 Young, P. T., 113,130,148
Z Zagaevsky, J. S., 188, m8 Zeit. W.. 247. 264 Zepplin,'M., '42 (see Ives), 108 Ziervogel, M., 398 (see Schneider), 426 Ziff, N., 151 (see Chargaff), 196 Zimmerley, H. H., 310 {see Sheets), 311 (see Sheets), 314 (see Speirs),398, 393 Z i e r m a n , H. M., 118 (see Street), 1.4'7 Zimmerman, R. L.,28 (see Mitchell), 29 (see Mitchell), 36 Zimmerman, W. I., 67,fO4 Zittle, C.A,, 388,393 Zscheile, F. P.,174 (see Hauge), 197 Zucker, T.F., 119. 132,1.6s
Subject Index A Acetic acid, microbial inhibition by, 387 Acatyhminopolysacchde, action of lysozyme on, 377-378 h t y l groups in pectin, 397-399 Acriflavin, inhibition of enzyme activity by, 384 Actin, extraction from muscle, 8 relation to meat structure, 208 Actomyosin, formation during rigor mortis, 9 relation to meat structure, 208 Adenoaine diphosphate, formation during rigor mortis, 6 Adenoeine triphosphate, breakdown in muscle, 4-10 in phosphorylation of glucose, 131 relation to meat structure, 208 Adenylic acid, action in muscle, 4 Adrenalectomy, effect on rats, 123 Aerosol 0. T., action as surface-active agent, 379 &nine, effect on dried egg, 172 palanine in relation to microbial inhibition, 387 Alcohols aa food preservatives, 386, 388 Aldehydes as food preservatives, 386,388389
Alfalfa, dehydrated, Palatability of, 124 phosphorus content of, effect of fertilizers on, 313 Aluminum, effect on low-ester pectins, 414 salts, effect on pectin viscosity, 406 use in pectin extraction, 400,405 Amide nitrogen in fruit products, relation to browning, 352 Amino acids, effect on browning in dehydrated potatoes, 262, 273-275,
Antitrypsin in egg albumen, 127, 169 Appetite, aa indication of nutritional need in humans, 107-111 decrease, caused by thiamine deficiency, 117
depressants, adaptation to, 128-129 counteraction by nutrients, 127-128 destruction of physical and chemical means, 127 effect of amino acids on, 115-116 effect of deleterious compounds on, 124 endocrines on, 122-124 fat-soluble vitamins on, 119 fats on, 118-119 minerals on, 119-122 proteins on, 113-115 water-soluble vitamins on, 116-1 18 influence of brain centers on, 136 caloric output on, 129 enzymes on, 131 flavor on, 135 gastro-intestinal tract on, 134 growth on, 130 internal environment on, 130 taste mechanism on, 137 tissue irritability on, 133 physiological basis for, 107-141 psychological us. physiological factors affecting, 141 Apples, dried, chemical changes during browning in, 347-351 color changes during browning in, 328 effect of temperature on browning in, 330, 333-334
influence of oxygen on deterioration of, 342, 344, 345 minimizing browning in, 366 rate of browning in, 335 immature, jelly units of pectin from, 402 juice, ascorbic acid loss during bottling,
277-278, 285
paminobenzoic acid content of canned pineapple, 82 Amino nitrogen in fruit products, changes during browning, 351-353 Ammonia, use in demethylation of pectin, 411
Amylaee in eggs, 183 Aniline, effect on browning in orange juice, 354
Antibiotics, possible use as food preserve tives, 385 Antiemymes, inhibition by, 385
63-64
chemical changes during browning in, 349-350
color changes during browning in, 328 pectin in, 398 pectin, deesterification of, 411 demethylation of, 409-412 pectinate, changes in gels made from, 420 pulp, pectin in, 400-401 Apricots, canned, nutrients in, 43, 48-49 vitamin retention in, 82
447
448
SUBJECT INDEX
concentrate, minimizing browning in, 365 dried, chemical changes during browning in, 347-348,351-362 effect of temperature on browning of, 329-330,334 influence of oxygen on deterioration of, 342-345 minimizing browning in, 360-365 rate of browning in, 335 sirup, chemical changes during browning in, 357-358 minimizing browning in, 366 Arabinose, effect on dried egg, 172 in pectin, 397-399 Arginine in fruit products, relationship to browning, 353 Arsenic, counteraction of selenium poisoning by, 128 Arsenic compounds, inhibition of enzymes by, 382 Artichokes, effectof blanching on, 66 Ascorbic acid content. of canned foods. 41,43, 50 effect of pH on stability of, 301 foods. effect of heat sterilization on. 89 fruit,’effect of raw product handling, 82 fruits and vegetables; effect of storage, 65,90-97 in fruit products, effect of oxygen on loss of, 337,340-341,345 temperature on loss of, 330-334 on browning reactions, 327,337,348351,356, 358 loss of in relation to browning, 330332,348-351,362 . juices, effect of canning, 62-63 tin us. glass, 98-100 plants, effect of fertilizers on, 307-310 rainfall on, 306305 temperature on, 303-304 influence of climate on, 295-299 light on, 299-303,318 vegetables, effect of blanching on, 67-79 loss in potatoes during drying, 261,266 methods of aetermining, 292-294 oxidaae in apple juice, 82 sampling methods for, 294-295 Asparagine in fruit products, relation to browning, 353-354 Asparagus, B vitamins in, 42,44,45 canned nutrients in, 43,46-50 thiamine content of, 66 effect of storage on vitamin content of, 65
Aspartic acid in fruit products, relation to browning, 352-353 relation to microbial inhibition, 387 AspergiUua sydowi, mycelium of, effect of ’ in diet of rats, 125-127 Atabrine, inhibition of enzymes by, 384 Avidin in egg albumen, destruction by heat, 127 Azide, inhibition of enzymes by, 384 Aziotobacter, competitive inhibition of enzyme of, 384
B BaciUua brevk?, extraction of tyrocidine from, 378 Bacteria. See under proper name. intestinal, effect on appetite, 134-135 Bacteriostasis, effect of pH,on, 380-381 Barium chloride, microbial mutations caused by, 376 Barley, phosphorus content of, effect of fertilizers on, 313 Beans, baked, canned, nutrients in, 43, 4649 green, ascorbic acid content of, effect of 8eaaon on, 295 B vitamins in, 42,44,45 canned, nutrients in, 43,46-50 effect of blanching on vitamins in, 68, 72-73, 79, 81 storage on vitamin content of, 65 navy, antiamylase in, 126 plants, ascorbic acid content of, effect of light on, 299 Beef, acid formation in, post mortem, 3-8 aging and storing, 21-33 control of mold and bacteria on, 18-21 “dark-cutting,” 17 effecta of freezing on, 31,33 enzymes in relation to rigor mortis, 21 factors influencing color of, 16-18 tenderness of, 24-33 pH of, 15-16 rigor mortis in, 3-21 Beets, canned, nutrients in, 43,46-50 in children’s diet, 109 pectin, deesterification of, 411 Benzoic acid, microbial inhibition by, 388, 390 rendered nontoxic in body, 127 Beriberi, caused by eating white bread, 110-111 prevention of with toddy yeaat, 107 Biotin deficiency, 118 in canned foods, 42, 44-45,47
SUBJECT INDEX
449
Bismuth compounds, inhibition of eneffect on low-ester p e c t h , 416 zymes by, 382 intake by parathyroidectomized rats, Biaulfite, inhibition of browning in pota123 toes by, 261 lactate in diet of rats, 112 Blackberries, canned, nutrients in, 43,46pigs fed diet deficient in, 111 49 Caloric output, relatiomhip to appetite, Blanching, effect on vitamins in vege129-130 tables, 66-82 Canned foods, mineral composition of, 4.4, electronic, effect on vitamin losses, 81 48 steam us. water, 67-82 Canning, effecton vitamin content, 63-89 Blueberries, canned, nutrients in, 43, 46- Cantaloupes, ascorbic acid content of, ef49 fect of nitrogen on, 309 Borate, interference with phosphate mesunlight on, a02 tabolism by, 386,388 Caprylic acid, effecton cell membrane, 387 Boric acid, interference with phosphate Carbohydrate metabolism, effect on appemetabolism by, 388 tite, 131, 132 Boron, effect of on vitamins in plants, 308 Carbon dioxide, formation during brownin plants, effect on absorption of other ing ip fruit producta, 344-345, elements, 314 346-348, 360-362,366, 368, 360 Bread, microbial inhibition in by propionic packaging dehydrated potatoes in, 271-272 acid, 387 Broccoli, calcium content of, effect of limeproduction in dehydrated potatoes, stone on, 310 276-276 Bromoacetate, inhibition of enzymes by, monoxide, inhibition of enzyme activity 382 by, 384 Browning in fruit products, 325-369 suboxide, w e in detecting amino groups, chemical changes during, 346-360 383 color measurement of, 328-329 Carboxyl groups, esterification of by epoxides, 383 factors influencing color development during, 329-346 in pectin, 398 inhibition of, 380-369 Carotenese in egm, 183 reaction, active aldehyde theory of, 327 Carotene content, of canned foods, 41-44, 47,60 ascorbic acid theory of, 327 Maillard theory of, 327,351,364 effect of storage on, 92-96, 97 Brussels sprouts, effect of blanching on,68 fresh vegetables, effect of storage on,66 Buckwheat, fluorescent dye found in, 126 peaches, effect of heat sterilization on, Butter in diets, 109 89 Butyhmine, effect on browning in orange plants, effect of climate on, 305-306 juice, 364 fertilizers on, 308-309 Butyric acid, microbial inhibition by, 387 retention during blanching of vegetables, 67-68 C Carrots, B vitrrmine in, 42,44,46 canned, nutrients content of, 43,46-60 Cabbage, aacorbic acid content of, effect of carotene oontent of, effect of nitrogen on, 309 fertilizers on, 309 sebBon and location on, 295-297 season on, 306 temperature on, 306 effectof blanching on, 77 Cdcium, amount required for gelation of in children’s diet, 109 low-aolids gels, 416-420 nutrient content of, effect of blanching chloride, effect of treating potatoes with, on, 67,77 272 Caaein in diet of rats, 112, 114 content of canned foods, 44,48 Catalaae content of peas, effect of blanching on, 69 plants, effect of fertilizers on, 310314,318-319 in eggs, 183 decreased absorption of in ricketa, 132 Cathepein in relation to aging of poultry meat, 210 deficiency, 120
450
SUBJECT INDEX
Cauliflower, effect of blanching on, 66
Cellulose,action of intestinal bacteria on, 136
Cephalin content of dried eggs, 175-178 of egg yolk, 161 Cereals, a m y b inhibitor in, 386 in diets, 110 Cetyltrimethylammoniumbromide, action as surface-active agent, 379 Cherries, canned, nutrients in, 43, 46-49 vitamin retention in, 82 Chicory, effect of blanching on, 66 Chloracetic acid, microbial inhibition by, 386, 389
Chlorbenzoatq microbial inhibition by, 386
Chlorine deficiency, 121 interference with cell membrane by, 377 Chlorphenola, destruction of bacterial membrane by, 386 Choline deficiency, 118 in fruit products, relationship to browning, 363 Cholinesterase in eggs, 183 Cinnamic acid, microbial inhibition by, 386
Citrate, inhibition of enzymes by, 384 Citric acid, effect on browning in fruit products, 358, 367 Citrus fruits. See grapefruit, lemon, lime, orange. Cobalt content of planta, effect of fertilizer~on, 314 deficiency, 121 Cod liver oil, effect of feeding on palatability of poultry meat, 219-220 in diet of rats, 112 Collagen in poultry meat, 211 Collards, calcium content of, effect of lime stone on, 310 Conalbumin, 150 Copper content of plants, effect of fertilizem on, 314 effect on ascorbic acid in canned grapefruit juice, 66 tomato juice, 61, 62 ion, inhibition of enzymes by, 382 salta, effect on pectins, 400,406,414 Coprophagy caused by phosphonts deficiency, 119 Corn, carotene content of, 306 “pellagragenic” factor in, 126 sweet, B vitamin in, 42,44,46 canned, nutrients in, 43, 46-60 Cottonseed meal, effect on chicks, 124 Pi@, 124
Cranberries, dried, chemical changes during browning in, 347-348 influence of oxygen on deterioration of, 344-345 merusurement of color changes during browning in, 328 Cranberry juice, chemical changes during browning in, 349-350 measurement of color changes during browning in, 328 Creatine, formation during rigor mortis, 3 phosphate, breakdown during rigor mortis, 3, 10 Cucumber, ascorbic acid content of, effect of fertilizers on, 309 Currant juice, effect of temperature on changea in, 333 influence of oxygen on deterioration of, 341
measurement of color changes during browning in, 328 Currants, influence of oxygen on deterioration of, 341 Cyanide, effect on dehydrated potatoes, 272
Cysteine, effect in dried egg, 172 Cystine, effect of reducing agents on, 382 on appetite, 116 D Depmcreatization, effecton rats, 123 Desoxycorticoeterone acetate, effect on appetite, 123 Dextrin in diet of rats, 114 Dichlorophenolindophenol, titration of ascorbic acid with, 293 Dithionate, inactivation of &sulfide enzymes by, 382 “Dopa” oxidase in eggs, 183
E
Egg albumen, 160-151,
163-166, 167, 164, 169-172, 176, 178182, 185-186 antitrypsin in, 385 Eggs, 149-202 chemical composition of, 160-162 influence of hen’a diet on, 162-154 dried, chemical changes during storage of, 168-187 cooking propertiea of, 158, 167-168, 191-194
criteria of quality and deterioration in, 167-168 ewyme wtivity in, 182-187
461
BUBJECT INDEX
factors affecting storage life of, 164 166
fatty acids in, 174-180, 187 mineral content of, 161 palatability of, 150, 168-169,192-194 physical properties of, 166-167 retentioh of quality in, 187-194 solubility of, 159-161, 164-187 vitamin A loss during storage of, 1 7 4 181
vitamin content of, 153 Egg white, see egg albumen Em yolk. 1W-151,163-166,167,164,168-
Formaldehyde, inactivation of disulfide enzymes by, 382 emymes by, 386, 388-389 reaction with amino and amide groups, 383, 386
Fructose, effect in dried egg, 172 potato powder during storage, 269270
loiq in dehydrated potatoea, 274 Fruit, see under individual name. Furfuraldehyde, formation during browning, 367-369
169, 176, 179-188, 193
Encephalmalacia in chicks, cause of, 126 EntcMnebo huatol!ttictr, action of chlorine on cysts of, 377 Enzyme inhibitors, naturally occurring, in food preservation, 386 Enrymea, disulfide, inactivation of by reducing agents, 382-383 inhibition of, 380-386 Epichlorohydrin, esterification of carboxyl groups by, 383 Esterme, action on galacturonide chain of pectin, 412 Ether, destruction of yeaat cells by, 377 Ethylene, effect on pectins, 401 Ethylene oxide, reaction with amino groups, 383, 386 with carboxyl groups, 386, 389 with phenolic hydroxyls, 383 with sulfhydryl groups, 383
F Fata, rancid, injurious compounda in, 125 Fatty acids aa food preservativea, 386-388 in eggs, 161 Fhh, raw,thiamine-destroying enzyme in, 126
Fishmeal, effect of feeding on palatability of poultry meat, 219-220 Flour, bleached, toxic compound in, 124 Fluoborates, microbial inhibition by, 386 Fluorescence of dried egg extracts, 169162, 173
Fluoride, inhibition of enaymes by, 884 microbial inhibition by, 388,388, 390 Fluorine, effect in diet, 122 in diet, adaptation of rats to, 128 Fluoailicatee, microbd inhibition by, 386 “Folio acid” content of canned fooda, 42, 44, 46, 47
Food preservatives, cleesee of, 389-391 toxioity of, 389-391
0
Galactose in pectin, 397-399 Galacturonic acid, effect on browning in fruit products, 367 presence in pectin proved, 307 Gene interference, microbial inhibition by, 376-376
Glucose, effectin dried egg, 172-173 metabolism of, 131 need by central nervous system, 131 relation to browning in dehydrated pohtOes, 269-270, 273-274
Glutamic acid in fruit products, leaking from cella treated with detargents, 379 relation to browning, 362 relation to microbial inhibition, 387 Glycine, effect on browning in potato chips, 277 in dried egg, 172173 Glycogen, breakdown in muscle, 3 content of muscle, 11 poultry meat, effect of feeding on,218 effect of exercise on content in muscle, 11
fatigue on content in muscle, 14 feeding on content in muscle, 13 Goat meat in diet, 109 Gossypol in diet, 124-126 Grape concentrate, browning in, 341, 364, 362
Grapefruit, canned, nutrients in, 43,4849 juice, eacorbic acid content of, effect of nitrogen on, 308 B v i t a m h in, 42,44,46 Grapefruit juice, canned, ascorbic acid content of, 6 4 4 6 nutrients in, 43,4843 peels, pectin in, 400 phoephom oontent of, effect of fertih r s on, 313 Grape gels, changea in during aging, 42Q
452
SUBJECT INDEX
Grape juice, browning in, 328,333-334 chemical changes during browning in, 349-360 Gum guaiac, as antioxidant in dried egg, 181
H Halogens, oxidation of sulfhydryl enzymes by, 381 Heat sterilization, effect on ascorbic acid, 89 carotene, 89 niacin, 87-89 ribofiavin, 87-89 thiamine, 84,89 Hexadecenoic acid in poultry fat, 212 Hexokinaae, relationship to carbohydrate metabolism, 131 Hexose diphosphate, formation during rigor mortis, 5 monophosphate, formation during breakdown of glycogen, 4-6 sugars, effect on browning in dehydrated potatoes, 273-274 Hippuricase in eggs, 183 Histidine, action of formaldehyde on, 389 Histozyme in eggs, 183 Humin nitrogen in fruit products, relation to browning, 352 Hydrocyasic acid, inactivation of disul6de enzymes by, 382-383 interference with prosthetic groups of the ensymee by, 384 Hydrogen, inhibition of enzymes by, 384 peroxide, oxidation of sulfhydryl enzymes by, 381 sulfide, inactivation of disulfide enzymes by, 382 interference with prosthetic groups of the enzymes by, 384 Hydroxybemoate, microbial inhibition by, 386 Hydroxylamine, effect on browning in dehydrated potatoes, 272 inhibition of enzymes by, 384 Hypoglycemia, relation to appetite, 131 Hypophosphites, effect on appetite, 133 Hypophysectomy, effect on rata, 122 Hypothalamus, relation to appetite, 137 Hypoxanthine, formation from adenosine triphosphate, 10
I Insulin, biological activity of, 383 effect on awetite. 123 Inveztase of :&tit cells, 389
Iodine content of plants, effect of fertilizers on, 314 deficiency, 121 inhibition of enzymes by, 383 Iodoacettate, inhibition of enzymes by, 382 Iron content, of canned foods, 44,49 of plants, effect of fertilizers on, 313314 effect on low-ester pectins, 416 salta, effect on pectin Viscosity, 406 Isoaacorbyl palmitate, as antioxidant in dried egg, 181
J Jelly units of pectin solutions, 401-404
K Ksle, ascorbic acid content of, effect of sunlight on, 300 calcium content of, effect of limeatone on, 310 effect of blanching on, 77 iron content of, effect of fertilizers on, 314 Ketene, acetylation of amino and phenolic groups by, 383
L Lactic acid, dehydrogenase, inhibition of, 388 effect on pH of beef muscle, 6 post-mortem formation in beef, 3,6-8 Lactose, effect in dried egg, 173 Lard in diet of rats, 114 Lead ealts, effect on pectin viscosity, 406 Lecithinsee in eggs, 183 Lecithin content of egg yolk, 151 Lemon albedo, pectin from, 398-399 concentrate, minimizing browning in, 364,366 juice, measurement of color changes during browning in, 328 pectinate, changes in gels made from, 420 peels, pectin in, 400 pulp, pectin in, 400 “Lethal mutations,” microbial inhibition by, 376-376 Lettuce, ascorbic acid content of, effect of fertilizers on, 310 in children’s diet, 109 Lime beana, w e d , nutrients in, 43, 4649
453
SUBJECT INDEX
thiamine content of, 66 Linoleic acid in poultry fat, 212 Linseed meal, effect on chicks, 124 pigs, 124 Lip- in eggs, 183 Lipoprotein in egg yolk, 160-161 Lipovitellenin in egg yolk, 160-161, 182 Liver, unknown nutrient in, 114, 130 Liver powder intake by depancreatinized rats, 123 Livetin in egg yolk, 150-169 Lysine, action of formaldehyde on, 389 losa from detergent-treated cells, 379 Lysosyme, destruction of cell membrane by, 377-378 in egg albumen, 150, 183
M Magnesium deficiency, 120 effect on low-eater pectins, 416 inhibition of enzymes by, 384 Maillard reaction, browning in dehydrated potatoes due to, 278 Malic acid, effect on browning in fruit products, 358, 367 Malonic acid, inhibition of enzymes by, 384 Marlganese content of plants, deficiency, 121 effect of fertilizers on, 314 effect on ascorbic acid content of plants, 307-310 calcium absorption by plants, 312 Mannose, effect on dried egg, 172 Marine products, nutrients in, 41, 4549 Meat. See beef, pork, lamb, poultry. in diet, 110 Mereaptide-forming agents, inhibition of enzymes by, 382 Mercury ions, inhibition of enzymes by, 382 Metabolite antagonists, competitive inhibition of enzymes by, 384 Metaphosphates, effect on appetite, 133 Metaphosphoric acid, use to inhibit oxidation of ascorbic acid, 293-294 Methionine, effect on appetite, 133, 135136 chicks, 128 in diet of rats, 116 Methoxyl groups in pectin, 397, 399,405, 407410,413-418 pectinic acids, relation of combining weight to, 408 Methylene dihydroxycoumarin, interfer-
ence with prothrombin formation, 126 Methyl esters in pectin, 397,399,406-107, 409-411, 413-414, 416,420 Microbial inhibition by ultraviolet snd X-ray radiations, 376 through interference with cell membrane, 377-380 enzymes, 380-386 genetic mechanism, 374-377 Milk, dried, in diets, 108 gelation of by pectinate mixtures, 421 in diets, 109-110 Mineral content of plants, effect of fertilizers on, 310-314 Mineral content of vegetables, effect of blanching on, 68 Molds, action of epoxides on, 389 Mushrooms, canned, nutrients in, 43, 4649 Mustard gas, chromosomal breaks produced by, 376 Myosin, action on adenosine triphosphate, 6 in relation to meat structure, 207-208 Myristic acid in poultry fat, 212
N Naphthaleneacetic acid, methyl ester of, use to inhibit sprouting in potatoes, 284 &naphthol, microbial inhibition by, 388 Naphthol sulfonates, microbial inhibition by, 386 Niacin deficiency, 118 content of canned foods, effect of heat sterilization on, 87-89 storage on, 92,94-95,97-98 vegetables, effect of blanching on, 8081 counteraction of “pellagragenic” factor by, 125 Nickel,’ effect on low-ester pectins, 414 Nicotinic acid. See niacin. Nitrite, inactivation of enzymes by, 383 microbial mutations caused by, 376 Nitrogen, effect on ascorbic acid content of plants, 308-309 calcium absorption by plants, 311312 gas, packaging dehydrated potatoes in, 271-272 trichloride, microbml inhibition by, 386 Nutrienta, variability in reaction to, 140
454
SUBJECT INDEX
0
Oat plants, mcorbic acid content of, effect bf 'soil propertiee on, 308 carotene content of, effect of soil properties on, 309 OIeic acid in poultry fat, 212, 210, 217 Olive oil in diet of rats, 112 Onions, ascorbic acid content of, effect of fertilizers on, 309 Orange, concentrate, minimiaingbrowning in, 362, 304, 366 juice, browning in, 328-329, 332-334, 336-341, 362, 365
canned, ascorbic acid content of, 56 nutrients in, 43, 46-50 tin us. glass containers for, 98-100 chemical changes in during browning, 331, 346. 349-350, 352-359 pectin in, 398
peel, pectin in, 400 pulp, pectin in, 400 Orthophosphates, effect on appetite, 133 Osteophagia caused by phosphorus deficiency, 119 Ovalbumin, 150 Ovoglobulins, 150 Ovomucoid, 150 Ovomucoidase in egg albumen, 183 Oxalate, inhibition of enzymes by, 384,388 Oxalic acid, effect on browning in fruit products, 368 Oxidizing agents., inhibition of enzymes by, 381-382 Oxygen absorption by dehydrated potatoes, 275-276 Oxyhemoglobin in poultry meat, 209 Oaone, oxidation of sulfhydryl ewymes by, 381
P Palmitic acid in poultry fat, 212 Pancreatic amylase, inhibition by antienzymes, 385 Pantothenic acid content, of canned foods, 4147
effect of storage on, 93-95, 97-98 of foods, effect of heat sterilization on, 87-88
inability of rats to recognize, 118 in eggs, 163 inhibition of enzyme functioning in synthesis of, 387 Papain in poultry meat, 210
Parathyroidectomy, effeat on rats, 123 Parsnips, effect of blanching on, 68 Peaohea, B vitamins in, 42,44,45 aanned, nutrients in, 43, 46-50 Vitamin retention in, 83, 92 dried, chemical changes during browning in, 361 effect of temperature. on browning in, 329, 334
steam peeling on ascorbia acid content of, 83 Peach juice, ascorbic acid loss in bottling, lye
u8.
63
Pear juice, pectin in, 398 Pears, canned, nutrients in, 43,4649 dried, browning in, 335, 305 effect of temperature on browning in, 329
infiuence of oxygen on deterioration of, 342 Peas, rrscorbic acid content of, effect of fertilizers on, 310 sunlight on, 310 €3 vitamins in, 42, 44,46 canned, nutrients in, 43, 46-60 thiamine content of, 60 effect of blanching on vitamin content, 68-72
Pectaee, deeeterification of pectinic acids by, 411412 Pectates, use in gelling reactions, 421 Pectin, aoetylated, 398 apple, molecular weight of, 398 beet, molecular weight of, 398 changes in during storage of fruit pulps, 401
growing fruit, 402 citrus, moleculw weight of, 398 deesterification of, 406412 fruit, substitution for animal gelatins, 418
gelation, high-solids, preparation of, 416-416
by low-ester pectins, 416420 low-solids. calcium requirement for, 416-418, 419-420
low-solids, changes in, with aging, 4ao low-solids, pectinate requirement for, 418-419
optimum pH of, 413, 415, 416 speed of, 413, 415416 grades of, in relation to deesterification, 413, 415
highly polymeriied, 394405 composition of, 396-398 jellying power of, 407
SUBJECT INDEX
low-ester, effect of acidity changes on viscosity of, 413-414 of heavy metal ions on viscosity of, 414-415 properties of, 413-420 uses of, 420-421 methods of drying, 405 nitrated, 398 nomenclature for, 395-396, 397 orange, molecular weight of, 398 polymerization of in commercial products, 401 precipitation of, 399,416-418 transformation to pectic acid, 406 viscosity of, 405406 Pectinates, composition of, 402 mixtures, use of to gel milk, 421 rate of demethylation of, 409-410 use in freezing fruits, 421 Pectinic acids, composition of, 397, 399 Penicillin, inhibition of cell division by, 375 ribonucleic acid dissimilation by, 375 Peppers, ascorbic acid content of, effect of light on, 299 canned, nutrients in, 43,46-49 Pepsin, inactivation of, 383 inhibitor, effect on enzyme, 385 in poultry meat, 210-211,251 Permanganate, oxidation of sulfhydryl enzymes by, 381 Peroxidase in eggs, 183 microbial inhibition by, 386 pH, effect on browning in potato powder, 272 inhibition of enzymes by, 380-381 of canned foods, relation to vitamin loases, 92 of dehydrated potatoes, relation to thorescence, 276 relation to pectin extraction, 402-406 Phenols, activity as surface-active agents, 378, 385-386 Phosphatase in egg yolk, 184 Phosphate esters, appearance in destruction of cell membrane, 378 role in carbohydrate metabolism, 131133 Phospholipids in egg yolk, 150-151 Phosphomonoesterase, inhibition of, 388 Phosphorus content, of canned foods, 44, 48,49 deficiency in rats, 119-120 of plants, effect of fertilizers on, 312-313 Phytic acid, interference with calcium absorption, 124
455
Phytin in bread, adaptation of man to, 129 Pimentos, canned, nutrients in, 43, 46-49 Pineapple, ascorbic acid content of, effect of iron on, 308 calcium contcnt of, effect of fertilizers on, 311-312 canned, nutrients in, 46-49 vitamins in winter vs. summer packs, 82 retention during storage, 92 flavor components of, 135-136 iron content of, effect of fertilizers on, 313-314 juice, ascorbic acid content of, correlation of total acidity with, 303304 effectof light on, 303, 305 effectof temperature on, 303 canned, nutrients in, 43,46-50 vitamin retention during storage, 92 phosphorus content of, effect of fertilizers on, 313 Pituitary hormone, effect on appetite, 122 Plum juice, pectin in, 398 Polygalacturonic acids in pectin, 399 in pectinic acid, 399 Polyphosphates, use in gelling reactions with pectic acid and pectates, 421 making gels from pectic acid, 415 pectin extraction, 400,404-405 Pork, canned, effect of storage on B vitamin content, 97-98 luncheon meat, retention of B-vitamins in. 86-88 Potassium chloride in diet of rats, 112 deficiency, 120-121 effect on ascorbic acid content of plants, 308,310 efYect on calcium absorption by plants, 311-312 d t s , use of to form pectic acid, 406 Potato chips, deterioration in, 259, ?73, 286 flour, rate of darkening of, 264 Potatoes, ascorbic acid content of, effect of location and season on, 296297 effect of nitrogen on, 309 effectof rainfall on, 304 effect of sunlight on, 302 browning in during drying, 259-263 canned, deterioration in, 259,286 cooking quality in relation to reducing sugars, 283 dehydrated, browning during storage, 263-272, 273-279, 285
456
BWBJECT INDEX
browning in, effect of sulsting on, 260-281
of raw material factors on, 281-283 chemical changes during storage, 273279 compression of, 271-272, 281 defects in, 257-288 development of defects in during storage, 263-272 effect of moisture content on, 263288, 278, 280-281 sugars on, 280-284 packaging on storage life of, 271272 raw material factors on storage life Of, 288-271 aulfiting on, 266-288, 278-280 temperature on, 283-266 ractors influencing deterioration of, 259-273 “off-flavom” developed in during storage, 278 vitamin lossea in, 281 effect of blanching on, 68, 77 temperature and moisture content on browning in, 259-260 French fried, deterioradon in, 259, 273, 288 in diet, 109-110 inhibition of sprouting in, 284 phosphorus content of, effect of fertilizers on, 313 reducing sugar content of, 281-284 in relation to cooking quality, 283 silage made from, 272 solamine in, 124 sweet, canned, nutrients in, 43, 48-50 Poultry, chemical composition of edible portion of, 206-207 disintegration of-fibersduring aging of, 247-251 factors Muencing flavor of, 219-224 juicinese of, 225-230 tenderneas of, 230-232 fat changes in during storage, 213 composition of, 211-212 effect of diet on composition of, 213 iodine value of, 212-213 rancidity in, 214-216 stability of, in-Silu, 214-218 frozen, factors influencingfirrvor of, 222224 histological changes in during aging of, 238-251 meat, post-mortem changes in, 232-234 muscle, glycogen content Of, 233, 263
histology of, 209-211, 234-253 proteins of, 207-211 tenderness and histological changes during aging, 251-253 rigor mortis in, 232234 nodea in, 245-247 turbulence in fibers of, 244 Procainesteraae in eggs, 183 Propionic acid, microbial inhibition by, 387,390
Propyleoe oxide, esterification of carboxyl groups by, 383 hydrolysis in foods, 389 Proteaee in eggs, 183 Protein content of vegetables, effect of bhnching, 88 Protein denaturants, microbial inhibition by, 380 Protein utilization, relationship of growth to, 130 Proteins, affinity of surfaceactive ions for, 379 injurious compounds in, 127 Protopectin in growing tissue, 401 Protozoa, hunger in, 133 Prunes, Italian, canned, nutrients in, 43, 4649
Paeudomonas fluoreseas in eggs, 188 Pyridoxine content of canned foods, 42, 46, 47
deficiency, 117-118 Pyrophosphates, effect on appetite, 133 Pyruvic acid, abnormal metabolization of, 113, 131
R Raspberries, aacorbic acid content of, effect of sunlight on, 301 influence of oxygen on deterioration of, 341
Raspberry juice, ascorbic acid loss in bottling, 83 deterioration of, 328, 333, 341 effect of temperature on changes in, 333 Reducing agents, inhibition of enzyme activity by, 382-383 Reindeer meat, life sustained by, 109 Reaazurin reduction test for egg pulp, 186187
Ribo&vin content, of canned foods, 4147 effect of heat sterilization on, 87-89 effect of storage on, 93-95, 97-98 deficiency, 117-1 18 e5ciency of food utibation increased by, 117
SUBJECT INDEX
of eggs, 153 of plants, effect of climate on, 307 retention in canned tomato products, 93-95 of vegetables, effect of blanching on, 8081 effect of storage on, 64-65 Ribonucleic acid, inhibiting dissimilation by penicillin, 376 Rice, phosphorus content of, effect of fertilizers on, 313 polished, deficiencies caused by, 109 Rigor mortis, theory of, 8-10 Rutabagas. See Swedes. Rye, effect on chicks, 124 plants, carotene content of, effect of fertilizers on, 309
457
effect of blanching on vitamin content of, 67-68,73, 77, 81 effect of storage on vitamin content of, 65 phosphorus content of, effect of fertilizers on, 313 Stachydrine in fruit products, relationship to browning, 353 Stearic acid in poultry fat, 212 Storage of canned foods, effect on vitamin content, 89-98 Strawberries, aacorbic acid content of, effect of sunlight on, 301 iniluence of oxygen on deterioration of, 341 Strawberry juice, ascorbic acid loes in bottling, 63 deterioration of, 328,333, 341 S Streptococci, inhibition by antitrypain, 385 Saccharine, preference for in relation to Strontium hydroxide, we of to form pectic acid, 406 blood glucose, 138 Succinic acid, competitive interference in Salicylaae in eggs, 183 oxidation of, 384 Salicylic acid, microbial inhibition by, dehydrogenase, competitive interference 387-388,390 with by malonic acid, 384 Salmon, B vitamins in, 42,44,45 Sucrose, effect on browning in potatoes Salmonella ih eggs, 188 during storage, 269-270,273-274 Sauerkraut, canned, nutrienta in, 43, 46effect in dried egg, 173 50 in diet of rats, 112 Selenium, counteraction of toxicity of, 128 Sudangrass, ascorbic acid content of, effect effect in diets, 121-122 of manganese on, 308 Silver ions, inhibition of enzymes by, 382 Sodium carbonate, use of to form pectic Sugar, preference for in relation to blood glucose, 138 acid, 406 reducing, effect on browning in potatoes, chloride, effect of treating potatoes with 262-263, 268-271, 273-274, 277before blanching, 272 278,285 in diet of rats, 112 in fruit. changes inhibition of proteinases by, 380 -. - during- browning, 3541-357 deficiency, 120 hexametaphosphate, relation to gelation Sugars in vegetables, effect of blanching on, 68 pH of pectin, 415 hydroxide, use of to form pectic acid, Sulfhydryl enzymes, poisons for, 381-382 S a t i n g potatoes, effect on browning after 406 dehydration, 266-268, 278-279, phosphate in diet of rats, 112 suhite, microbial inhibition by, 386 285 Solamine in potatoes, 124 Sulfocyanides, use in pectin extraction, 403 Soybean, antitrypsin in, 385 injurious substances in, 108-109, 125- Sulfonamides, competitive inhibition of enzymes by, 384 126 Spinach, ascorbic acid content of, 52 Sulfur dioxide, effect of on browning of dehydrated potatoes, 260-261, effect of fertilizers on, 308 266-268,279 effect of light on, 299 on ascorbic acid loes in orange juice, B vitamins in, 42,44, 45 348-349, 362 calcium content of, effect of location on, carbon dioxide production in dried 31Ck311 fruit, 347, 360 canned, nutrients in, 43,46-50
458
SUBJECT INDEX
canned, effect of storage on vitamin measurement of browning in dried apricots, 329 content of, 94-96 nutrients in, 43, 46-50 on pectin in apple pulp, 401 carotene content of, effect of sunlight inactivation of disulfide enzymes by, on, 305 382-383, 386 juice, ascorbic acid of, 56 influence on browning in dried fruit, canned, ascorbic acid content of, 56360-364 inhibition of enzymes by, 386, 388 63 carotene content of, 57 loss in dried apricots, 330,342-345,380Tomato juice, canned, effectof oxygen on, 362 5840 Sulfuric acid, use to inhibit oxidation of effect of storage on, 93-97 ascorbic acid, 293 vitamin content of, 93-95,97 Sulfurous acid, storage of fruit pulps with, niacin content of, 56, 57 401 nutrients in, 43,46-49 Supersonicwaves, destruction of microbial riboflavin content of, 56,57 agents by, 373 thiamine content of, 56,57 Surface-activeagents, action on cell memtin us. glaas containers for, 98-99 brane by, 378-379 leaves, ascorbic acid content of, effect of Surfaceactive ions, affinity for proteins, sunlight on, 302 379 pectase from, 411 Swedes, effect of blanching on, 68 Sweet clover, toxic compound in, 125 Tributyrinw in eggs, 183 Triosephosphate, formation during rigor mortis, 5 T Trypsin inhibitor. effects on microbiological agents, 385 Tartaric acid, effect on browning in fruit in relation to aging of poultry meat, 210 Tryptophane, effectin diet of rat, 115 products, 358 Taste perception for sweetness, depression effect on browning in orange juice, 354 Turnips, carotene content of, effect of ferof. 138 Thiamine content, of canned citrus juices, tilizers on, 309 greens, ascorbic acid content of, effect effect of storage on, 92 of fO&, 41-46, 60 of light on, 302 location and season on, 296, 298effect of can size on, 86,88 299 of heat sterilization on, 84-89 of storage on. 92-98 rainfall on, 304 deficiency, 113, 117, 131 temperature on, 303 destruction in potatoes by sulfiting, 261 calcium content of, effectof fertilizers of eggs, 153 on, 310-311 canned, nutrients in, 43, 46-49 of plants, effect of climate on, 306-307 of vegetables, effect of blanching on, carotene content of, effect of location 79-81 on, 306 requirements of rat, 116-117 of moisture on, 306 Thyroidectomy, effect on rats, 124 of season on, 305-306 Thyroxine, effect of feeding to animals, iron content of, effect of fertilizers on, 314 130 phosphorus content of, effect of fertia-tocopherol, as antioxidant in dried egg, 181 lizers on, 313 Tyrocidine, damage to cell membrane by, deficiency, 119 destruction by rancid fats, 125 378 379 Tomatoes, ascorbic acid content of, effect of fertilizers on, 307-309 v of location and season on, 296-298 of sunlight on, 300-301 Vegetables, canned, factors controlling of temperature on, 303 mineral and vitamin content of, 291-292 B vitsmins in, 42, 44,45
469
SUBJECT INDEX
canning, effect of storage on, 64-65 time of harvest on, 64-65 Vitamin A, content of dried eggs, loss during storage, 174, 181 of eggs, effect of hen’s diet on, 163 deficiency, 111, 119 Vitamin B1. See thiamine. Vitamin B,. See riboflavin. Vitamin C. See also ascorbic acid. content of plants, influence of climte on, 295-307 of fertilizers on, 307-310 Vitamin D deficiency, 119 in eggs aa affected by hen’s diet, 153 utilization of phosphorus induced by, 132 Vitamin E. See a-tocopherol. Vitamin K, counteraction of hemorrhagic tendencies by, 125 deficiency, 119 effect on appetite, 116-119 in canned foods, 39-100 distribution between solid and liquid portions, 44-49 effect of blanching on, 68-82 of canning on, 52-89 of grading and cutting on, 66 of preparation on, 49-62 of storage on, 89-98 of type of container on, 98-100
factors influencing stability of, 53 Vitellin in egg yolk, 150, 169
W Wheat bran, action of intestinal bacteria on, 135 calcium content of, effect of fertilizers on, 312 germ oil in diet of rats, 112 phosphorus content of, effect of fertilizers on, 313 thiamine content of, effect of location on, 306-307
Y Yeeat, action of epoxides on, 389 of formaldehyde on. 389 of sulfur dioxide on, 388 destruction of cells by ether, 377 in diet of rats, 112, 114 intake by depancreatinized rats, 123 reduction of growth rate by mustard gm, 376
z Zinc content, deficiency, 121 effect on sscorbic acid content of t o m toes, 308 of plants, effect of fertilizers on, 314
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