Advances in Food Research, Volume 24

ADVANCES IN FOOD RESEARCH VOLUME 24

Contributors to This Volume

Sally Hudson Arnold W . Duane Brown Reiner Hamm Klaus Hofmann Walter M. Urbain Jonathan W. White, Jr. Robert L. Wickremasinghe

ADVANCES IN FOOD RESEARCH VOLUME 24

Edited by C. 0. CHICHESTER The Nutrition Foundation, Inc. New York. New York and University of Rhode Island Kingston. Rho& Island

E. M. MRAK

G . F. STEWART

University of California Davis, California

University of Calif(omia Davis, California

Editorial Board S. LEPKOVSKY EDWARD SELTZER W . M. URBAIN J . R. VICKERY

E. C. BATE-SMITH J . HAWTHORN M. A. JOSLYN J . F. KEFFORD

1978

ACADEMIC PRESS

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CONTENTS Contributors to Volume 24 . . . . . . . . . . . . . . . . . . . . . . . . .

vii

Sulfhydryl and Disulfide Groups in Meats Klaus Hofmann and Reiner H a m

I. 11 .

111. I \. . V. VI . VIl . VrII . IX .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods for the Determination of SH and SS Groups . . ................... SH Groups in Muscle Proteins and Their Role in the Fu n of Muscle . . . . . . . . SH and SS Content of Meats and Meat Fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intluence of Processing on the SH and SS Groups of Meat . . . . . . . . . . . . . . . . . . . . Influence of the SH Groups on the Shelf Life of Meat and Meat Products . . . . . . . . Toxicological Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . ........................................

2 3 30 43 58 84 84 86 88 88

Histamine (7) Toxicity from Fish Products Sally Hudson Arnold and W . Duane Brown

I. 11. I11. IV . V.

Nature of the Problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of Formation of Histamine in Fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detection and Determination of Levels of Histamine in Fish . . . . . . . . . . . . . . . . . . . Relationship o f Spoilage to Histarnine Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . Unresolved Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . ................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

114 121 130 135 139 147

Food Irradiation Walter M . Urbain

I. I1 I11 IV . V. VI . VII

Introduction-Historical Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... ....... Radiation and Radiation Sources . . . . . . . . . . . . . . . . General Effects of Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications of Food Irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Economics of Food Irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wholesomeness of Irradiated Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ The Future of Food Irradiation . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

155

163 168 174 205 209 213 216

V

vi

CONTENTS

lea Robert L. Wickremasinghe I. U. UI . IV . V. V. VI. VI.

vu .

VIII . IX . X.

......_......... Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Composition of Tea .. . . . . . . . . . .... Changes during the Processing of Tea . . . . .. .. .......... . . Organoleptic Properties .. . ....... . .. .. . .. ....... . .. .. ........ . .. . Storage of Tea . . . . . . . . .. ..... . . . . . . . . . . . . ................. Potential By-Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical Effects .. .. .. .. .. .. .. . . . . . . . Host Plant-Pest Relationships .................... Instant Tea . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . Additional Research Needs . . . . . . . . . . . . . . ........................ ........................ References . . . . . . . ............................. .. ... .... .. ... ......... References.

229 232 25 I 263 266 268 269 27 1 272 213 273

Honey Jonathan W. W. White, Jr. Introduction ........................... Production and Processing ....................... Market Forms of Honey. . . . . Analysis and Composition Compositio . . . . . . . . . . . . . . . Analysis Physical Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . Storage of Honey . . . . .. .. . . . . . . . vn. Nutritive Value . ....................... ......................... VUI . Uses . . . . . . . . . . . . VUI IX , Standards, Specifications, an IX X. Research Needs . .................... X References.. . . . . . . . . . . . . .. . . . . . . .................... References

288 289 295 291 333 344 352 354 358 363 364

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

375

I. U. U. Ill. IV. V. V VI. VI.

I

CONTRIBUTORS TO VOLUME 24 Numbers in parentheses indicate the pages on which the authors' contributions begin.

SALLY HUDSON ARNOLD, Institute of Marine Resources, Department of Food Science and Technology, University of California, Davis, California 95616 (113) W. DUANE BROWN, Institute of Marine Resources, Department of Food Science and Technology, University of California, Davis, California 95616 (113) REINER HAMM, Bundesanstalt fur Fleischforschung, Kulmbach, Germany ( I ) KLAUS HOFMA", Bundesanstalt fur Fleischforschung, Kulmbach, Germany (1)

WALTER M. URBAIN ,* Michigan State University, East Lansing, Michigan 48824 (155) JONATHAN W. WHITE, JR., Eastern Regional Research Center, Philadelphia, Pennsylvania I91 18 (287) ROBERT L. WICKREMASINGHE, Tea Research Institute of Sri Lanka, Coombs, Talawakelle, Sri Lunka (229)

St.

*Present address: 10645 Welk Drive, Sun City, Arizona 85351.

vii

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ADVANCFS IN FOOD RL-%ARCH

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VOI .

24

SULFHYDRYL AND DlSULFlDE GROUPS IN MEATS* KLAUS HOFMANN AND REINER HAMM

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Methods for the Determination of SH and SS Group:, . . . . . . . . . . . . . . . . . . A . General Problems in the Determination of SH Groups in Soluble and Insoluble Proteins . . . . . . . . . . . . . . . . . . . . . .................... B . Methods for the Determination of SH Group Meats . . . . . . . . . . . . . . C . Determination of SS Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. SH Groups in Muscle Proteins and Their Role in the Function of A . Myofibrillar Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Proteins of the Sarcoplasniic Reticulum (SR) . . . . . . . . . . . . . . . . . . . . . . C . Proteins of the Sarcolemma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . Proteins of Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Proteins of the Sarcoplasmic Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . SH and SS Content of Meats and Meat Fractions . . . . . . . . . . . . . . . . . . . . . . A . SH and SS Content of Muscles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Cysteine plus Cystine Content of Muscles .............. C . SH Content of Organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . Fdctim Influencing the SH Content of Raw Meat . . . . . . . . . . . . . . . . . . . V . Influence of Processing on the SH and SS Groups of Meat . . . A . Influence of Heating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Freezing and Frozen Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Freeze-Drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . Curing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Ripening of Dry Sausages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F . Smoking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . Irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V1 . lnfluence of the SH Groups on the Shelf Life of Meat and Meat Products . . VII . Toxicological Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX . Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 3 3 6 28 30 31 41 41 42 42 43 4.5 50 52 5.5 58 58 13 77 77 80 80 81 84 84 86 88 88

.

*Dedicated to Professor Dr . Alfons Schiiberl Hannover (Germany). a pioneer in the chemistry of organic sulfur compounds .

Copyright 0 1978 by Academic Press Inc . All righn of reproduction in any form rebewed. ISBN 0-12-016424-8

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2

KLAUS H O F M A N N A N D REINER H A M M

I. INTRODUCTION Reviews on the occurrence, properties, and functional importance of SH groups in biological systems were recently presented in three comprehensive monographs (Jocelyn, 1972; Friedman, 1973; J. M. Tortschinski, 1974). However, the research on SH and SS groups in muscle tissue used for food has not yet been reviewed, although a considerable amount of work has been done in this field. SH groups are usually considered to be the most reactive functional groups in proteins (Wallenfels and Streffer, 1964); but under certain conditions the reactivity of these groups can be more or less inhibited. It is understandable, therefore, that SH groups in proteins have attracted the attention of many research workers and that the role of SH groups in proteins has been the subject of a large number of investigations. SH groups can easily be oxidized to SS groups, the SH/SS redox equilibrium 2 R-SH

+t

0 2

R-SS-R

+ H20

or 2 R-S- - 2e

* R-S-S-R

being of great biological importance. Consequently, in any discussion of the role of SH groups, a consideration of SS groups must also be included. It is the purpose of this review to discuss the methods for the determination of SH and SS groups in proteins and to assess the importance of these groups in meat quality and meat processing. Most of the work taken into consideration here is related to red meats (muscles from cattle, pigs, and sheep) and poultry; fish is only occasionally mentioned. Amino acids, cysteine and cystine, are the carriers of the SH and SS groups in proteins. Knowledge on the reactions of these amino acids is therefore indispensable for an understanding of the reactivity of SH and SS groups in biological systems. Most of the SH (and SS) groups in meat are located in the muscle proteins (see Table V). Because only a small proportion of these groups exists as low molecular SHES compounds, mainly glutathion, the research discussed in this review is primarily related to protein SH. Cysteine and cystine content is of great importance for the nutritive value of meat, as it is for most foods. Although cysteine and cystine do not belong to the essential amino acids, a deficiency of “total cystine” (sum of cysteine and cystine) in nutrition increases the requirement of one of these essential amino acids, methionine, which can be metabolized to cysteine. The methionine content of food proteins also limits their nutritive value. For this reason, a sufficient

SULFHYDRYL AND DISULFIDE GROUPS IN MEATS

3

supply of “total cysteine” helps to spare methionine in the intermediate metabolism. Any destruction of cysteine or cystine during the treatment of foods as indicated by the disappearance of SH plus SS groups represents a detrimental effect on nutritive value. It is evident, therefore, that studies of SH (and SS) changes during the storage and processing of meat are of particular interest for the nutritionist. It should be mentioned that reliable information on changes in the SH and SS content of proteins can be obtained by direct determination of these groups in the intact system. This approach is preferable to amino acid determination after hydrolysis because the problem of an exact determination of these amino acids after hydrolysis by acids or enzymes has not yet been satisfactorily solved. The importance of SH and SS groups in sensoric quality as well as for the processing of foods has been investigated for many years. This research has related mainly to milk and other dairy products (particularly cheese), cereals, doughs, and beer (Hofmann, 197 la). Corresponding work on meats started about 15 years ago. The assay of SH and SS groups is extremely difficult for a number of reasons. We will, therefore, discuss the various methods of SH and SS analysis available and examine the interpretation of the results of each in detail. Our lack of knowledge on the role of SH and SS groups in meat is mainly due to the difficulties in the determination of protein SH. It is our hope that the gaps and contradictions in our understanding of SH and SS groups which are presented in this review will initiate further research in this field.

II.

METHODS FOR THE DETERMINATION OF SH AND SS GROUPS

This chapter will present a review of methods which are used or which may be useful for the quantitative assay of SH and SS groups in animal tissues and muscle proteins. Histometrical methods for the demonstration of the location of SH groups using dyes are briefly discussed. A. GENERAL PROBLEMS IN THE DETERMINATION OF SH GROUPS IN SOLUBLE AND INSOLUBLE PROTEINS The complicated problems of SH assay in proteins have been thoroughly presented and critically discussed in several review articles (e.g., Cecil and McPhee, 1959; Benesch and Benesch, 1962; Leach, 1966; Hofmann and Hamm, 1974a; Ashworth, 1976). With nondissolved proteins, e.g., muscle homogenates, additional difficulties arise which should be examined in detail (Hamm and Hofmann, 1966a). It has been observed that, in certain proteins in the native state, the total

4

KLAUS HOFMANN AND REINER HAMM

number of sulfhydryl groups is not available to chemical reagents. Not all proteins, however, demonstrate this behavior. Moreover, the number of SH groups which react depends on several internal and external factors: the nature of the protein, the presence or absence of denaturing agents, temperature, pH value, the kind and concentration of SH reagent used, and the time of reaction. There are, therefore, several degrees of availability of protein SH groups. The difficulties in SH determination in proteins are due partly to the methods themselves and partly to differences in the reactivity of those groups. Many times the SH values determined are lower than the actual SH content; in some cases they are higher. For the confirmation of a certain result, several reagents should be applied for SH determination as has been recommended by Benesch and Benesch (1962). If the results of these various analyses differ from each other, additional investigation is necessary. In Table I the different factors leading to incorrect results are summarized. An incomplete reaction of protein SH groups can be due to three different reasons: (a) steric hindrance of SH groups by the specific structure of the protein, (b) interaction of SH with other functional groups (combined function), and (c) repulsion between hydrophilic reagents and hydrophobic groups (particularly TABLE I FACTORS LEADING TO WRONG RESULTS IN THE DETERMINATION OF SH GROUPS IN PROTEINS"

SH value too low Nonreactive (inaccessible, inavaiI able, masked, hidden) SH groupsb Incomplete reaction (slowly reacting groups; reaction time is too short. e.g., at direct titration)

SH groups are partially oxidized by oxygen (air) before or during the determination (SH -+ SS)

SH value too high Unspecific SH reagent (reaction or complex formation with other functional groups of protein) Interference by substances which consume SH reagent (e.g., ascorbic acid if an oxidizing reagent is used; or J-, Br- and S2- at titration with AgNO,) Cleavage of SS to SH groups by the SH reagent or by hydrolysis

After Hofmann and Hamm (1974a). The reasons for an unsufficient reactivity of SH groups need only a brief summary in the text because they were discussed in detail by Hofmann and Hamm (1974a). a

SULFHYDRYL AND DISULFIDE GROUPS IN MbATS

5

alkyl residues) located in the vicinity of SH groups (hydrophobic environment). Denaturing agents (urea, guanidine, dodecyl sulfate) or heating usually cause an elimination of these inhibiting influences. This might be due to an unfolding of the peptide chains of protein which makes the SH groups accessible to SH reagents. Because of the powerful reducing field required for such a reaction (Chibnall, 1943), the old supposition that SH groups are produced by the actual cleavage of the S-S linkage is no longer satisfactory. Chibnall(l943) suggested a hydrolytic cleavage of thiol ester linkages (R-CO-S-R’) during denaturation, but this type of linkage has not yet been demonstrated in proteins. In addition, the assumption that the formation of free SH groups during denaturation is due to an opening of thiazoline ring systems present in proteins (Linderstr@m-Langand Jacobsen, 1941) has not been confirmed by studies with model systems (Martin el al., 1959; Kolthoff and Shore 1964; Hofmann, I966a). According to another hypothesis, proteins contain isothiuronium residues which can react with amino groups to cause SH formation (Brush ef al., 1963). It is certain that the binding of some SH reagents (such as Ag+ ions or PCMB) to easily available SH groups can result in a denaturation of the protein which makes other hidden SH groups easily available (Bocchini et al., 1967; Jeckel and Pfleiderer, 1969). The denaturation of a protein usually makes the SH groups more easily oxidizable by oxygen. This can be prevented by the addition of EDTA (Sakai and Dan, 1959; Calcutt and Doxey, 1962). which sequesters catalyzing traces of heavy metals; 0.02 M EDTA is sufficient for the protection of SH groups (Sedlak and Lindsay, 1968) although 0.2 M EDTA was normally used in the preparation of homogenates (Tarnowski et al., 1965). According to their reactivity, protein SH groups are usually divided into three categories: fast reacting, slowly reacting, and nonreacting. However, this schematic classification is quite arbitrary. It does not take into consideration that the reactivity of an SH group is not an absolute property of this group. This reactivity depends essentially on the type of reagent used as well as other factors, e.g., pH or buffer systems. For this reason, very different SH values are often obtained after the reaction of the native protein with different reagents (Cecil and McPhee, 1959). The same is true for SH determination in meat (see Section IV). A great number of SH reagents are available (more than one hundred), which indicates the lack of a universally applicable method which is satisfying in every respect. The choice of method depends on the type of investigation in question because the determination of SH groups can have several different purposes: (1) Determination of total cystine content (cysteine plus half cystine) after complete reduction of SS groups in the course of the analysis of amino acids. Such an SH assay with the nonhydrolyzed protein prevents losses of cysteine and cystine from occurring during hydrolysis (Friedman, 1974) as already mentioned. (2) Investigation of the role of SH groups in the biological function of proteins.

6

KLAUS HOFMANN A N D REINER HAMM

(3) Study of the process of protein denaturation connected with a change (usually an increase) in the reactivity of SH groups. (4) Study of the reaction of protein SH groups with lead, mercury, other heavy metals, toxins, carcinogenic compounds, and other substances which are of relevance for environmental pollution, health hazards, and residue analysis. In this respect the protective effects of and the detoxification by SH groups in foods are of particular interest. Considering these different purposes of SH research, it is not always necessary to determine the total amount of SH groups in proteins; in some cases the determination of only the easily available SH groups of the native protein by a convenient reagent is intended (e.g., in cases 2 and 3 above). But other factors are also important for the correct selection of an SH assay method. Not all SH reagents are suitable for the SH determination in proteins, and only a small part of the reagents suggested for the SH determination in proteins can be used for the unsoluble proteins of muscle tissue (Hamm and Hofmann, 1966a). Reagents which can be used for studies on meat must fulfill the following requirements: (1) Because organic solvents cause the denaturation of muscle proteins, the SH reagent must be soluble in water. (2) The pH of the reagent solution should be approximately 7 in order to prevent denaturation by an acid or a base. (3) It must be possible to measure the excess of a reagent or a soluble reaction product after the reaction. (4) The SH reagent or the reaction product has to be stable because the reaction with undissolved proteins requires a relatively long amount of time. (5) The method has to be sensitive because of the low SH content of animal tissues. The determination of SS groups is usually based on the determination of SH groups before and after reduction of SS to SH. Here, both the complete reduction of all disulfide groups as well as the prevention of an oxidation of SH groups formed are necessary. Generally, a complete unfolding of the proteins by denaturing agents (e.g., urea, guanidinium hydrochloride) is important in order to provide a complete reduction of the S S groups.

B. METHODS FOR THE DETERMINATION OF SH GROUPS IN MEATS The number of reagents that are used or are suitable for the determination of SH groups in proteins, peptides, and amino acids is so great that it is not possible to discuss all of the possibilities in this review. Fortunately, this is also not necessary because of several excellent publications. A review on the function and analysis of SH groups in proteins was given by Heide (1955), who described in detail a modem assay based on amperometric titration with silver nitrate. Vol-

SULFHYDRYL AND DlSULFlDE GROUPS IN MEATS

7

uminous reports were presented by Cecil and McPhee (1959), Cecil (1963), and Leach (1966). Benesch and Benesch (1962) discussed the problems in the determination of SH groups, particularly in regard to the mercaptide formation with AgNO, and mercury compounds. The comprehensive publication of Lumper and Zahn ( I 965) includes important analytical aspects of the biochemistry of the disulfide exchange. Kakai: and Vejddek (1 974) summarized methods for the photometric determination of SH and SS groups, including a discussion of interfering factors. The application of organic mercury compounds for the chemical analysis of sulfur compounds (including SH and SS groups in proteins) was described by Wronski (1965). A further review article on the determination of SH and SS groups in proteins was published by Mesrob and Holesovsky (1967). The most recent information can be found in the monographs of Jocelyn (1972), Friedman (1973), and Ashworth (1976). This review first discusses the methods for the determination of SH and S S groups in meats and muscle proteins. Furthermore, those methods are considered which seem to be generally applicable for the determination of SH groups in insoluble material but which have been used thus far only for other biological systems such as flour or keratine. The determination of SH groups after complete reduction of SS groups allows the determination of the content of “total cystine” (cysteine plus cystine) without hydrolysis of the protein. The problems in cystine determination associated with the hydrolysis of proteins are described by Friedman (1974): “Direct assay of cystine by ion-exchange chromatography usually gives low values.. . . Consequently, many attempts have been made to change cystine residues to acid stable derivatives. . . . However, most of these attempts do not prevent complete destruction of the modified residues during acid hydrolysis, and the new derivatives are sometimes incompletely resolved on the chromatogram of an amino acid analyzer.” A further disadvantage in the application of protein hydrolysis is the fact that in most cases it is impossible to differentiate between SH groups and SS groups originally present. In this review the discussion of SH assay is therefore focused on methods applicable to the intact, nonhydrolized proteins. Such procedures present the additional advantage of allowing the study of the process of denaturation by following the changes in the reactivity of SH groups. The availability of the SH groups of a protein depends on its state in the protein structure and on the type of reagent used. Usually a denaturing agent [concentrated solutions of urea, guanidinium hydrochloride or, in our opinion the most suitable, diluted sodium dodecyl sulfate (also named lauryl sulfate or Duponol) solutions] has to be used in order to determine the total quantity of protein SH groups. With Ag+ ions, however, all SH groups can also be determined in the native meat protein ( H a m and Hofmann, 1965).

8

KLAUS H O F M A N N A N D REINER H A M M

The SH reagents can be divided into (1) oxidizing agents (2 RSH + RSSR), (2) mercaptide-forming agents (RSH .+ RSMe’, (RS)2Me”), (3) alkylating agents (RSH + RSR), and (4)other reagents. The reactions are usually measured either by spectrophotometry (including fluorometry) or by titration. The end-point of titration is mostly determined by means of electrometric methods (potentiometry, polarography , or amperometry). 1. Oxidizing Agents, Including Disiilfides

In most earlier research oxidizing agents known from oxidimetric methods were most often used, including iodine, ferricyanide, o-iodosobenzoate, porphyridin, 2,6-dichlorophenol-indophenol(Barron, 1951) and phosphoric tungstic acid (Folin and Morenzi, 1929).The desired reaction 2 RSH

- 2e + R-SS-R + 2H+

does not always occur in a stoichometric way because the sulfur of the SH group can be oxidized to a valence higher than that of disulfide. Furthermore, the influence of other reducing substances present in biological material (e.g., ascorbic acid or thiamine) can interfere in the reaction. More recently, the use of N-bromosuccinimide for the determination of cysteine and cystine has been described (Thibert et al., 1969;Bachhawat el af., 1973).However, this reagent is not highly specific. A different type of oxidation is the reaction of SH groups with disulfides encompassing an SH-SS exchange: R’SH

+ R-SS-R + R‘SS-R + R-SH

Numerous methods for the determination of SH groups in proteins are based on this type of reaction. Mirsky and Anson (1935)have treated protein SH groups with cystine and determined the obtained cysteine by reaction with phosphoric tungstic acid. Protein-bound SH groups in tissue slices can be visualized with 2,2’-dihydroxy-6,6’-dinaphtyl disulfide; a procedure introduced by Barnett and Seligman (1952). After the SH-SS exchange, the naphtol residue bound to the protein is coupled with tetrazotized diorthoanisidine to a red dye. It is also possible to extract the excess reagent from the tissue and to determine it after coupling to the red dye (Flesch et al., 1954). Ellman (1959) introduced the disulfide 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB, Ellman’s reagent) which is now widely used for the determination of SH and SS groups in tissues (Ellman. 1959;Gabay et al., 1968;Khan el al., 1968;Sedlak and Lindsay, 1968;Dube, 1969; Caldwell and Lineweaver, 1969; Dzinleski et al., 1969; Yuan, 1970; Randall and Bratzler, 1970;Buttkus, 1971;Bowers, 1972;Boyne and Ellman, 1972;Dub6 et al., 1972;Habeeb, 1972;Hay et al., 1973;Miller and Spencer,

9

SULFHYDRYL AND DISULFIDE GROUPS I N MEATS

197.5; Usunov and Zolova, 1976). In basic solution, the disulfide DTNB (I) reacts quickly with dissociated SH groups in proteins as follows:

COO-

COO-

+

protein-SS &NO2-

[-S&NOz -

-

S

COO-

e p Q

-

(11)

H s -&Noz (111)

The resulting anion of the mesomeric 3-carboxy-4-nitro-thio-phenolate(II)* imparts the solution a lemon-yellow color which can be photometrically measured at 412 nm. The advantages of this reagent are its high specificity for SH groups and the high sensitivity of the resulting color reaction (eM = 13,600). As Klotz and Carver (1961) have pointed out, the stoichemistry of this method is not clear because Ellman's reagent (E-SS-E) can react also in another way: 2 Protein-SH

+ E-SS-E

---f

Protein-SS-Protein

+ 2E-SH

But this possibility involves no disadvantage for SH determination, because, in this case, 1 mole of the yellow thiophenolate is also formed from 1 mole proteinSH. The same is true if the mixed disulfide, primarily formed, reacts with a further protein SH group (Habeeb, 1972): Protein-SS-E

+ Protein-SH

-

Protein-SS-Protein

+ E-SH

The reaction of DTNB with protein SH groups is faster and more complete when the protein is allowed to be denatured by 8 M urea solution (Srere, 196.5). *Only by utilizing this nomenclature is it clear that the SH group is present in the ionized state (as thiophenolate). This is important because the nondissociated reaction product (111) is colorless (it fades by acidification). Consequently the Ellman's reagent should be termed bis(3-carboxy-4nitrophenyl) disulfide.

10

KLAUS HOFMANN AND REINER HAMM

Therefore, the determination of total protein sulfhydryl requires that the protein be denatured, preferably with sodium dodecylsulfate (Diez et al., 1964). The pH of the reaction mixture has a marked effect on the rate of color development. At pH 8, color development is complete in 5 minutes for all proteins tested (Beveridge ef d., 1974). Although the color reaction also occurs in solutions of weak acidity (above 4.7, Sedlak and Lindsay, 1968), pH values not lower than 8 are required in order to obtain complete reaction; Ellman (1959) suggested pH 8.2. Higher pH values accelerate the SH disulfide exchange (Lumper and Zahn, 1965), but they also induce a hydrolytic cleavage of the reagent causing a strong increase of the blank. According to our own experience, the original light-yellow color continuously deepens even in solutions of DTNB in phosphate or tris buffer of pH 7.5. We therefore recommend solutions of DTNB in ethanol, without addition of buffer, which are stable for a longer time (Hofmann and Bliichel, unpublished observations). Sedlak and Lindsay (1968) used methanol as a solvent for DTNB probably for the same reason. Calvin (1954) has shown that in basic solution two symmetrical disulfides can react with each other and form a mixed disulfide. In the case of the protein disulfide (PSSP) and Ellman’s reagent (ESSE), the reaction would be as follows: PSSP

+ ESSE + 2 PSSE

This reaction would not influence the results of the estimation of SH groups with ESSE because no thiolate anion (ES-) is formed. Furthermore, after Robyt ef al. (1971), mixed disulfides may also be formed by a series of reactions between ESSE and proteins which contain SH and SS groups. After the initial reaction PS-

+ ESSE + PSSE + ES

the released thiol ES- reacts with protein SS*, forming a second molecule PSSE and PS- which reacts again with ESSE. Altogether 3 moles PSSE and 1 mole ES- result. The derivatized protein PSSE can be separated, and the addition of a thiol (dithiothreitol) or adjustment of the pH to 10.5 releases a corresponding amount of ES- which can be measured at 412 nm. With this method Robyt et al. (1971) estimated the number of SH and SS groups in several proteins. Although ES- reacts immediately with protein SS groups, the number of ES- ions released is equal to the number of protein SH groups. Thus SS groups do not limit the specificity of the estimation of SH groups by Ellman’s reagent as it was postulated by Jocelyn (1972). Diez et al. (1964) concluded from their experiments that Ellman’s reagent in comparison with SH reagents such as N-ethylmaleimide, p-chloromercuri*In contrast, Weitzman (1975) reported, that thionitrobenzoate (ES-) would not react with disulfide groups in proteins. Therefore, the postulated mechanism of reaction seems to be in question.

SULFHYDRYL AND DISULFIDE GROUPS IN MEATS

11

benzoate, and iodine is the most useful reagent for routine determination of the total content of protein SH groups. A turbidity in the reaction solution can be eliminated either by filtration (Sedlak and Lindsay, 1968; Boyne and Ellman, 1972) or by centrifugation in the ultracentrifuge (Hofmann and Bliichel, unpublished observations). The precipitatin of proteins by TCA is not recommended (Dube, 1969). Dube found that the yellow color of the solution, obtained after reaction of GSH with DTNB, disappears after the addition of TCA, but comes back completely after adjusting the pH to 8; however, this is not the case in reaction mixtures of meat proteins and Ellman’s reagent because the formed carboxy-nitro-thiophenolateis partly adsorbed by the precipitate. This can be recognized by the strong yellow color of the washed precipitate after adjusting to pH 8 (Hofmann and Bluchel, unpublished observations). Since the reagent itself, as well as the tissue suspension after separation of the insoluble constituents show some absorbance, both blanks have to be measured separately and to be subtracted from the absorbance of the sample solution. Contrary to Dube (1969), we found that, in the determination of SH groups in meat with DTNB, myoglobin does not seriously disturb the measurement because the absorbance caused by myoglobin is extremely small, particularly with pork. Even with beef, the blank absorbance amounts to not more than 10% of the total absorbance (Hofmann and Bliichel, unpublished observations). It is often necessary to differentiate between protein-bound and nonbound SH groups in tissues. The latter groups are soluble in TCA and can be determined after the precipitation of proteins; the former can be calculated as the difference between the total SH content, measured after denaturation of the proteins by SDS, and the nonprotein SH content. Furthermore, the content of “available protein SH” can be determined by DTNB using the nondenatured sample (Habeeb, 1972). Under special conditions, DTNB reacts quickly with nonprotein SH and slowly with protein SH so that both types of SH groups can be determined in the same mixture. After Gabay et al. (1968), tissue homogenates are allowed to react with DTNB for 2 minutes at pH 6.8 giving nonprotein SH, and for 20 minutes at pH 7.6 giving total SH. Protein SH is then estimated by subtracting nonprotein SH from total SH. Boyne and Ellman (1972) described a kinetic analysis which allows the differentiation between (a) soluble, rapidly reactive SH (GSH-like), (b) soluble, slowly reactive SH (BSA-like*), (c) soluble, unreactive SH, (d) insoluble, reactive SH, and (e) insoluble, unreactive SH. Butterworth et al. (1967) suggest another method for the determination of protein SH in the presence of nonprotein SH: from the mixed protein disulfide, which is separated from the system after reaction with DTNB, the thiophenolate anion is released by the addition of dithiothreitol and then measured. *BSA

=

bovine serum albumin.

12

KLAUS HOFMANN AND REINER HAMM

Under the conditions used in the SH determination, DTNB also reacts with sulfite, thiosulfate (Man and Bryant, 1974), hydrogen sulfite, cyanide, and sulfide (Benedict and Stedman, 1970); therefore, such compounds interfere in the SH determination. Principally, all substances carrying a sulfur containing anion at pH 8 react with DTNB. Thiamine is another interfering factor which has not been taken into consideration up to now. In basic solution a cleavage of the thiamine ring occurs resulting in the formation of a S - group (Zima and Williams, 1940; Vogel and Knobloch, 1953):

By the reaction of thiamine with DTNB at pH values as low as 8.2 (i.e,, under the conditions of the SH determination), a yellow color is obtained. Actually, this reaction can be used for the determination of thiamine (Hofmann, 1974~). However, the thiamine content of meat is not sufficient to cause a noticeable error in the SH determination by DTNB. The maximum thiamine content of meat is about 0.9 mg per 100 gm of meat (Schweigert and Payne, 1956), whereas the total SH content varies between 60 and 80 mg SH per 100 gm of meat (see Section IV, A). For certain investigations, DTNB cannot be used. An example is the study of the reaction of patulin with the SH groups of meat (Hofmann et al., 1971): Patulin

+ RSH

-+

Patulin-SR

Following an addition of DTNB to measure the SH concentration after a certain time of reaction, first an equivalent amount of E-SH is obtained (see above). The E-SH, however, can react with patulin, and, consequently, the SH content measured in this manner will be too low. The determination of SH groups with other disulfides was suggested by Bitny-Szlachto et al. (1963), Drabikowski and Bitny-Szlachto (1963, I964), Kakol et al. (1964), Drabikowski and Nowak (1965), Grassetti and Murray (1969), Kakol (197I), and Swatditat and Tsen (1972). The use of thiamine disulfide (Kiermeier and Hamed, 1962; Kohno, 1965, 1966) allows a particularly sensitive SH assay: The thiamine formed by the SH-SS exchange is measured with the fluorometric thiochrome method. Here the relatively great error (+ 10%) of the thiochrorne method is a disadvantage. Bis(p-nitropheny1)-disulfide(Maier, 1969) is suitable for the determination of the sum of volatile mercaptans and hydrogen sulfide which arise, e.g., during heating of meat (see Section V, A, 4).

SULFHYDRYL AND DISULFlDE GROUPS I N MEATS

13

2 . Mercaptide-Forming Reagents and Amperometric Titration Unsatisfactory results with oxidation methods lead to the study of reactions with heavy metals, which react with SH compounds forming undissociated mercaptides in which the H of the SH group is replaced by a heavy metal. Reagents frequently used for the determination of SH groups in tissues are silver salts, mercury salts, and organic mercury compounds of the composition R-HgX(R = alkyl or aryl residue; X = halogen or OH). The procedures preferred are titrations with amperometric or potentiometric end-point determination using platinum or dropping mercury electrodes. This type of indication is based on the reducibility of heavy metal ions at the surface of the electrode. AS to the theory and application of the use of electrometric methods in biochemistry, we refer to the special literature (Kolthoff and Lingane, 1952; Konopik, 1953; Ewing, 1960; Brezina and Zuman, 1956; Purdy, 1965). a. The Amperometric Titration with Silver Nitrate. The amperometric titration of SH compounds with AgNO, was introduced by Kolthoff and Harris (1946) and first applied to the investigation of proteins by Benesch and Benesch (1948). Benesch et al. (1955) improved this assay essentially by using tris(hydroxymethy1)aminomethane as a buffering agent instead of ammonia and by the use of Hg/HgO/Ba(OH), reference electrode. Due to the potential of this electrode (-0.1 V against saturated calomel electrode), air oxygen cannot be reduced at the indicator electrode and, therefore, a complete elimination of oxygen (which could not be realized) is unnecessary (Kolthoff et al., 1965b). (Another reason for eliminating oxygen is the prevention of SH oxidation; such an oxidation. however, can be extensively prevented by addition of EDTA.) From that time, the amperometric titration with AgNO, was widely used for the determination of SH-(and SS-) groups in proteins and tissues. Important work on the methodology-other than animal tissues-was carried out by Rosenberg el a f . (1950), Heide (1953, Kolthoff et al. ( I 957, 1965b), Stauff and Duden (1958), Carter (1959), Staib andTurba (1956), Gruen and Harrap (1971), Harrap and Gruen (1971) (Ag/S specific ion electrode), and Mildner et ul. (1972). The great stability of the titration agent AgNO,, the simplicity of the apparatus (which can be easily built in each laboratory), the exact end-point determination (see Fig. I ) , and the small number of interfering factors involved are the reasons that this method was applied to practically all SH-containing materials including tissues. Animal tissues which have been investigated include liver, kidney, heart (Bhattacharya, 1958, 1959; Lastovskaya, 1969; Pavlyuk and Genyk, 1970), tumor tissues (Neogy et al.. 1961a,b), nerves (Krasnov, 1962), myofibrils from skeletal muscle (Hofmann, 1964, 1971a; Hamm and Hofmann, 1965, 1 9 6 6 ~Tinbergen, ; 1970), whole muscle tissue (Krylova and Kusnezowa, 1964; Hamm and Hofmann, 1965; Lastovskaya, 1969; Hofmann et al., 1969, 1974; Bolshakov and Mitrofanov, 1970; Bognar, 1971a; Hofmann, 1971b; Bow-

14

KLAUS HOFMANN AND REINER HAMM

FIG. 1. Amperometric titration curves: (A) Direct titration of SH groups with silver nitrate. Values of current (marked by circles) recorded 30 seconds after each addition of the reagent (0.1 ml AgNO,). (B) Titration of SH protein as in A, but with a continuous registration of the current (notice the decrease in current after the early additions of reagent which indicates the retarded reaction of the SH groups of protein). (C) AgNO, is titrated with an SH compound or KJ (reverse SH titration).

ers, 1972; Kortz, 1973; RaheliC et al., 1974), canned meat products (Bem et al., 1970; SusiC et al., 1974), and freeze dried meat (Potthast, 1972). Furthermore, the SH and S S groups in wheat flour were determined by amperometric (Rohrlich and Essner, 1966) and potentiometric titration (Kiihbauch and Wunsch, 1971). When compared with the amperometric titration, the potentiometric titration has two notable disadvantages: (a) the end-point determination is less exact because the point of inflection of the S-shaped titration curve is not very sharp, and (b) the air oxygen has to be removed as completely as possible because the use of a stronger negative electrode (calomel electrode) is necessary, the potential of which allows the reduction of oxygen. Several authors used the amperometric titration with AgN03 for the determination of SH groups in the isolated muscle proteins such as actin, myosin, tropomyosin, etc. (Azzoneef al., 1956; Staib and Turba, 1956; Tortschinski, 1959; Poglasov and Baev, 1960; Kofman, 1963; Berg et al., 1965; Hamm and Hofmann, 1965; Lytvynenko er al., 1963; Lusty and Fasold, 1969; Yuan, 1970; Brennock and Read, 1972; Hofmann, 1972a). The principle of the amperometric titration is as follows: a platinum wire indicator electrode is placed in the solution to be titrated; an electric connection is made with the reference electrode. The diffusion current flowing through the cell (caused by free Ag+ ions) is read on a microamperometer. During the titration of SH groups with silver nitrate, the current is near zero until the end-point because the Ag+ ions added are still being consumed by the formation of silver mercaptide: R-SH

+ Ag+ * R-SAg + Ht

After the end-point is reached, the diffusion current of silver rises because of an excess of silver ions in the solution. This diffusion current is proportional to the concentration of silver ions. By plotting the current reading during the titration

SULFHYDRYL AND DISULFIDE GROUPS IN MEATS

15

against the volume of silver nitrate solution, two straight lines are obtained which intersect at the end-point (Fig. 1A). By replacing the microamperometer with the recorder of a polarograph, a stepwise course of the current is obtained corresponding to the added portions of silver nitrate solution (Fig. 1B) (Beneschet af., 1955; Hofmann, 1972b). Instead of the rotating platinum electrodes which were originally preferred (Kolthoff and Harris, 1946), the use of a more simple stationary platinum electrode is recommended (Staib and Turba, 1956; Carter, 1959; Hofmann , 1964; Mallik, 1965; Hamm and Hofmann, 1966c) whereby the medium is agitated by means of a magnetic stirrer or by the use of a rotating beaker (King and Morris, 1967). The stationary electrode is even more sensitive than the rotating platinum electrode (Mallik, 1965; Richmond and Somers, 1966). If only small volumes of fluid are available for the demonstration, the titration can be carried out using a vibrating platinum electrode (Rosenberg et af., 1950). In addition to the platinum electrode, an AgS specific electrode was also suggested (Gruen and Harrap, 1971; Harrap and Gruen, 1971). Figure 2 shows the schema of a titrations apparatus with a stationary indicator electrode (after Staib and Turba, 1956; modified by Hamm and Hofmann, 1966~).The apparatus is only slightly susceptible to interference; the platinum electrode has to be cleaned only after a great number of titrations by dipping it into warm 20% nitric acid (contrary to information in the literature, e.g., Leach, 1966, p. 19). A direct titration of tissue homogenates is difficult because tissue particles adhere to the indicator electrode and cause an irregular influence on the diffusion current. Therefore, no regular titration curves are obtained (Hamm and Hofmann, 1966c; Bolshakov and Mitrofanov, 1970; Pavlyuk and Genyk, 1970). The direct titration of dissolved proteins with slowly reacting SH groups also implies problems. These difficulties can be overcome by the “indirect titration”: In this procedure an excess of AgNO, is first added to the sample; after the time required for a complete reaction, the excess of AgNO, is titrated with a SH compound (Neogy et al., 1961 a; Lusty and Fasoid, 1969; Bolshakov and Mitrofanov, 1970) or with KJ (Hofmann, 1969, 1971b; Bem et af., 1970; SusiC et af., 1974) (see titration curve Fig. 1C). The “double-indirect titration” procedure (Hofmann, 1964; Hamm and Hofmann, 1966c), which also allows the use of SH reagents such as NEM or PCMB (Hofmann and Hamm, 1967b; Hofmann, 197 Ic), is even more variable. The applicability of phenylmercuric acetate for this method was also examined (Mildner et al., 1972). This method of “double-indirect titration” should be briefly described because of its general applicability. The meat or protein sample, which should contain between 0.2 and 0.8 pmole SH (e.g., 25 mg tissue) is first incubated with I .O pmole of the SH reagent (AgNO,, PCMB, NEM, etc.). After the reaction, the mixture is filtered and 1.0 pmole of SHglutathion is added to the filtrate. The glutathion remaining after reaction with the excessive SH reagent is finally titrated amperometrically with IOp3M AgNO,

16

KLAUS HOFMANN AND REINER HAMM

iE 'I

17-

-4 IndKO tor

Reference electrode

2 3

FIG. 2 . Schema of the apparatus for the amperometric titration of SH groups (Hamm and Hofmann. 1966~).( I ) Indicator electrode (glass tube with platinum wire. 0.5 mm in diameter and I cm in free length). (2) Reference electrode (platinum wire, 0.5 mm in diameter and 2 cm in free length. dipped in mercury which is covered by a thin layer of HgO and Ba(OH), and a solution saturated by both substances. (3) Conductive connection (saturated KCI solution). (4) Porous diaphragm (clay). (5) Measuring instrument. (6) Magnetic stirrer with a constant speed of rotation. (7) Coated magnetic rod. The platinum wires of the two electrodes can be connected with the measuring instrument by either a drop of mercury or by welding them together directly. The content of the electrode vessels is 100 ml each; the volume of the sample solution 36 ml. Through the open tube in the top of the titration vessel, an inert gas can be led into the sample solution by means of a small rubber tube.

solution in tris buffer pH 7.4 (Fig. 3). As Fig. 3 shows, the consumption of AgNO, in the double-indirect titration procedure corresponds exactly to the amount of SH present. Therefore, the result is the same as would be obtained with a direct titration, if that method could be applied at all. Another essential advantage of this procedure is the fact that the titration of GSH with Ag+ ions results in an ideal titration curve, as shown in Fig. IA and B. With the direct titration of proteins, however, curves are sometimes obtained, the first part of which is not horizontal but at a slight incline. The problem of the evaluation of such titration curves was recently discussed by Hofmann and Hamm (1974b). We have applied this method to investigations about the role of SH groups in meat during heating, curing, freezing, storage, etc. (Hofmann, 1971d). The method has also been successfully used by other research workers for the deter-

17

SULFNYDRYL A N D DISULFIDE GROUPS IN MEATS

I

I

1 - 1

SH reagent ( 1 ml lO-3M

21

prnole)

protein SH (eg. 0 6 p r n ) remaining

m -

SH reagent (04p m f

SH glutathion (1 mllO-3 M

91 pm)

remaining SH glutathion (0.6 +m)

AgN03 consumed during titration (0.6+m)

L ’

I

I

1 pMd

1

*

FIG. 3 . Principle of the determination of SH groups by the double-indirect titration method (Hofmann. 1964; Hamm and Hofmann, 1 9 6 6 ~ )At . top of illustration, SH reagent. is valid for SH reagents which bind I SH group per molecule. c . g . . AgNO,. NEM. o r PCMB.

mination of SH groups in meat and myofibrils (Tinbergen, 1970; Bognar, I97 1 a; Potthast. 1972; Bolshakov and Mitrofanov, 1970; RaheliC et al., 1974). Doubleindirect titration permits a great variety in both reaction conditions and in the types of the SH reagent primarily used. The application of different reagents using the same technique is recommended for checking results obtained with one special reagent, for example AgNO,. This possibility is of particular interest because the specificity of SH reagents is not always certain. As a result, the specificity of Ag+ ions for SH groups in proteins has often been questioned (cf. Kolthoff and Stricks. 1950; Cecil and McPhee, 1959; Benesch and Benesch, 1962; Leach, 1966; Burton 1958), although this criticism was founded on investigations with low molecular compounds (mainly with cysteine). This problem was discussed in detail by Hofmann and Hamm (1974a). They concluded that results obtained with low molecular SH compounds cannot simply be transferred to proteins. On the contrary, studies using proteins with defined SH and SS content showed that under the conditions of the amperometric titration (tris buffer pH 7.4), Ag+ ions can be considered as being specific for protein SH groups (Hofmann and Hamm, 1975). It should be mentioned, however. that the direct titration of SH proteins with AgNO, in the presence of 8 M urea may lead to overly high SH values. This occurs because of a cleavage of SS groups with the excess of silver ions as Kolthoff et al. (1965b) found with bovine serum albumine. In absence of urea, however, they obtained correct SH values. In order to increase the specificity of the titration, a “blank” titration has been carried o u t using an excess of PCMB to block the protein SH groups (Bhattacharya, 1958, 1959). But the “blank” obtained in this way seems to be questionable because PCMB reacts more slowly and less completely with the SH groups of tissue than

18

KLAUS HOFMANN AND REINER HAMM

AgNO, (Hamm and Hofmann, 1967). Therefore, instead of a real blank, it is the SH groups unavailable for PCMB which are determined. From results obtained with several cysteine derivatives it can be concluded that Ag+ ions and mercaptide form a complex: R-SAg = x Ag+ -+ (R-SAg)Ag$

Therefore, the possibility of an excess of Ag+ consumption during the titration depends on the conformation of the SH compound. Complex formation apparently occurs if a cysteine derivative of Type I or I1 is present, while Type 111 does not form complexes (after Hofmann and Hamm, 1974a). +

HaN-CH-CGR

I

r* SH

Type 1

R-NH-CH-COO-

I

TH2

R-HN-CH-C&R

I

CHp

I

SH

SH

Type I1

Type 111

The use of AgNO, for the SH determination in muscle tissue does not result in inflated SH values as has been shown by comparison with other SH reagents (Tinbergen, 1970; Hofmann, 1 9 7 1 ~ cf. ; Section IV, A). During elaboration of the procedure for the SH determination in tissues by amperometric titration with AgN03 (described previously), Hamm and Hofmann (1 966c) investigated several possible influences. The results are as follows: (1) Between 10" and 40" C, the temperature of the GSH solution during titration has no influence on the titration end-point.* At 5"C, however, an elevated end-point was observed. (2) The titration of GSH in 8 M urea leads to the same result as the titration in the absence of urea. (3) An increase in the excess of AgN0, (over SH) from 100 to 200% does not influence the result of the indirect titration of myofibrils. (4) After 1 hour's reaction of AgNO, with myofibrils and subsequent titration, the same results are obtained whether these procedures are carried out in the presence or in the absence of air (under nitrogen). (5) Denaturation of the myofibrils with 8 M urea does not result in a significant increase of SH groups; therefore, all SH groups present in the native muscle proteins seem to react with AgNO,. (6) The recovery of GSH added to myofibrils and to the total tissue was 97% and 100% respectively. The accuracy of this amperometric titration is 97-98% (Krasnov, 1962). The error in the estimation of SH in animal tissues with this method has been deter*This was also found by Hoch and Vallee (1960).

SULFHYDRYL AND DISULFIDE GROUPS IN MEATS

19

mined to be from I .5% (Pavlyuk and Genyk, 1970) to 3% (Hamm and Hofmann, 1966~). The presence of Ca2+,Mg’+, Zn”, Fe3+, NO,-, Pod3-, lactate, and ATP at levels found in meat and meat products does not influence the amperometric titration of GSH with AgNO,. However, the presence of Cu2+and Mn2+ ions at higher levels results in low SH values, probably by a catalytic oxidation of the SH groups which cannot be prevented by the addition of 0.5% EDTA (Hofmann, 1970). Concentrations of sodium chloride higher than 0.1 M cause a change in the normal titration curve and, therefore, make the determination of the end-point less exact. Titrations in a 0.6 M KCI solution also result in abnormal curves; thus the amperometric titration of actomyosin in this solution is not useful (Hofmann, 1970). Finally it should be mentioned that free amino acids other than cysteine, which are always present in tissues, do not interfere in the amperometric titration (Benesch er al., 1955; Hofmann and Hamm, 1975). The same is valid for NAD, hemin chloride, ascorbic acid, and oxidized glutathion (Benesch et al., 1955). b. Mercury Compounds as SH Reagents. Numerous Hg compounds have been suggested for the determination of SH groups in proteins; we will discuss only the most important of these compounds. In addition to Hg2+ salts, organic mercury compounds have been used. Here the organic residue R‘ in reaction ( I ) contains hydrophilic groups for increasing the solubility in water and chromophoric groups for producing a measurable color. The reactions of protein SH (I) with an excess of the mercurial reagent (11) are as follows:

+

(I)

x R-SH

(2)

2~ R-SH

(1)

(X

+ Y)

R’-Hg+

+ (X + y) Hg2+ (W

-H+ + - H+ 4

x R-S-Hg-R‘ x (R-S)2Hg

(111)

+ yR’-Hg+

+ y Hg2+ (IV)

Three different principles are used for measuring these reactions: (a) Determination of the reaction product (111) in reaction (1) and (2): (i) Measurement of the increase in optical density at 250-255 nm which occurs if p-chloromercuribenzoate (PCMB) reacts with SH groups (Boyer, 1954). This method is only possible if the SH protein is dissolved and if the solution remains clear during reaction with PCMB. In order to correct for slight changes in opacity, the difference in readings at 255 and 320 nm was used (Yasui et al., 1968). By stepwise addition of the mercurial, a spectrophotometric titration can also be carried out (Katz and Mommaerts, 1962; Tonomura and Yoshimura, 1962; Arai and Watanabe, 1968). (ii) By coloring the tissue or insoluble proteins with 1-(4-chloromercuriphenylazo)-naphtol-2 (Flesch and Kun, 1950; Burley, 1954; Szydlowska et al., 1967), a subsequent, semiquantitative evaluation is possible. Further reagents suggested

20

KLAUS HOFMANN AND REINER HAMM

for labeling SH groups are 2-chloro-mercuri-4-phenylazophenol and 2-chloromercuri-4-(p-nitrophenylazo)-phenol(Chang and Liener, 1964), and fluorescein1,3,6,8-tetramercuric acetate as a sensitive spot reagent (Havir et al., 1966). (iii) Reaction of the SH proteins with I4C-PCMB and measurement of the radioactivity of the product (Erwin and Pedersen, 1968; Krabow and Golosby, 1971). (b) Determination of the reagent in excess (IV): ( i ) Measurement of the absorption of the reagent used, e.g., of PCMB at 232 nm after separation from the insoluble proteins (Hamm and Hofmann, 1967). (ii) Transformation of the reagent in excess into a colored complex, e.g., by the reaction of PCMB with dithizon (Fridovich and Handler, 1957; Sasago et al., 1963), or the reaction of o-hydroxymercuribenzoic acid with thiofluorescein (absorption maximum of 588 nm) (Wronski, 1967). ( i i i ) Titration of the reagent in excess with cysteine after reaction with the tissue using sodium nitroprusside as an indicator. The end-point of the titration is the appearance of a red color (MacDonnell et al., 1951; Zahn et al., 1962). Another possibility is the potentiometric titration of the excess of PCMB with cysteine (Calcutt and Doxey, 1959, 1961; Calcutt, 1961; Calcutt et al., 1961; Doxey, 1961) and the indirect back-titration of excessive SH reagents such as phenyl mercuriacetate (Mildner et al., 1972) and other suitable Hg reagents using the technique described by Hamm and Hofmann (1966~). (iv) Polarographic determination of the reagent in excess such as CH,HgJ or CH,HgCl (Maclaren et al., 1960; Leach, 1960a,b; Hird and Yates, 1961; Jamieson et al., 1963; Forbes and Hamlin, 1968; Mrowetz and Klostermeyer, 1972; Mrowetz et al., 1972; Marsalova and Roozen, 1973). (c) Estimation of reagent consumed (11) minus (IV): Direct titration of the protein SH groups in the presence of sodium nitroprusside until the purple color disappears. Reagents suggested for this titration are CH,HgNO, (Katchalski et al., 1957; Barany et al., 1964; Dworschak, 1970), NEM (Tsao and Bailey, 1953; Connell, 1957), PCMB (Connell, 1960a,b) and phenyl mercurihydroxyde (Meichelbeck, 1963). The titrations must be carried out at a temperature near 0°C because of the instability of the color. Furthermore, titrations with o-hydroxy mercuribenzoic acid and thiofluorescein (with which the color changes from blue to clear) (Wronski, 1963) or with salyrganic acid (mersalyl) and azopyridine (pyridine-2-azo-p-dimethylaniline)(Klotz and Carver, 1961; Ehrlich, 1967; Parker and Kilbert, 1970) as indicators are possible. Amperometric titration with HgClz is often used because of its very exact end-point determination (Kolthoff et al., 1957, I965b; Oganessjan and Dschanibekova, 1958; Matsumoto et al., 1960; Sullivan et al., 1963). Amperometric titrations can also be carried out with methylmercuric and ethylmercuric chloride (Kolthoff and Tan, 1965; Kolthoff et al., 1965b). Two advantages

SULFHYDRYL AND DISULFIDE GROUPS IN MEATS

21

of these two mercurials are the ease of preparation of extremely pure ethyl mercuric salts and the high level of water solubility of methylmercurinitrate. A disadvantage of the use of Hg2+ salts for SH determination is the fact that it is not certain whether the bivalent Hg cation reacts with one SH group only, forming RSHgX(X = halogen). or with two SH groups, forming (RS),Hg (Benesch and Benesch, 1962). Therefore, the application of organic mercurials, which can react with one SH group only, is generally preferred. PCMB is the most frequently employed compound of this type. It is extremely stable and, contrary to most of the other mercurials, has a low level of toxicity. Its low solubility in water and the difficulty of preparation of PCMB of high purity are disadvantages of this reagent (Benesch and Benesch, 1962). Impurities in commercial preparations can cause a catalytic oxidation of SH groups as has been demonstrated in the following example. When Bendall (personal communication) tried to block a part of the SH groups of the isolated myosin with PCMB, he found that the amount of SH groups decreased more strongly than had been expected from the amount of PCMB added. Hofmann and Bendall (unpublished observations) used the following experiment in order to find the reason for this phenomenon: They added to GSH solution in tris buffer pH 7.4 (a) unpurified PCMB, (b) purified PCMB (twice recrystallized in NaOH), and (c) unpurified PCMB and EDTA (final concentration 0.01 M ) . The molar proportion SH: PCMB was 4: 1 in all cases. A stream of air was passed through each solution for 30 minutes. Before and after aeration the SH content in an aliquot of the solutions was determined by Ellman’s reagent. The solution of the unpurified PCMB (without EDTA) showed a decrease in the SH content of 21%, the other two solutions a decrease of 2 and I % , respectively. This result indicates that the unpurified preparation contained traces of heavy metals which are known to catalyze the oxidation of SH groups. The spectrophotometric assay of SH groups in myofibrils by PCMB (estimation of the excess of the reagent by measuring the optical density at 232 nm) involves a relatively high error factor (12.6%) (Hamm and Hofmann, 1967); therefore, this method does not allow an exact SH determination in tissue or in myofibrils. Finally some methods should be mentioned which may be of interest for analytical as well as for preparative purposes. An insoluble reagent formed by the binding of PCMB to Dowex-2 resin has been used for the selective removal of thiols from solutions or from tissue homogenates (McCormack er al., 1960). It was possible to remove the bound thiol from the reagent by exchange with other SH compounds. This reagent might be valuable for concentrating or isolating these thiols. An organomercurial-polysaccharide has been synthesized and successfully applied to the separation of protein mixtures into an SH-fraction and a fraction containing no SH groups (Eldjam and Jellum, 1963). This material has been applied as a purification step in the isolation of SH enzymes as well as for concentrating dilute solutions of these enzymes. It is particularly well suited for

22

KLAUS HOFMANN AND REINER HAMM

the chromatographic fractionation of individual SH proteins. Furthermore, an organomercurial resin has been prepared which is capable of binding low molecular SH compounds and SH proteins, which can then be recovered almost quantitatively by elution with cysteine (Liener, 1967).

3.

N-Ethylmaleimideand Its Derivatives

N-ethylmaleimide (NEM) contains a reactive double bond causing an absorption maximum in the UV range of 300-302 nm. At pH values around 7, an addition of SH groups to the double bond occurs.

R-SH

+

R-S,H

HC=CH O(L,,,CO I

I -

C-CH,

I

1

,co

oc, I?

During this reaction the absorption maximum in the UV disappears because of the transition of the double bond into a single bond. The measurement of this decrease in absorbence at 300 nm permits a quantitative determination of SH groups. Principal studies on this method were camed out by Friedmann (1952), Gregory (1955), Alexander (1958), Roberts and Rouser (1958), and Leslie (1965). The formation of additive compounds by the reaction of thiols with NEM was demonstrated by Smyth et al. (1960) and Lee and Samuels (1961). The extreme stability of the C-S bond in the reaction product is an important advantage of this method. But NEM also shows some disadvantages which limit its application: (a) the sensitivity of the measurement is relatively small (eM = 620 at 300 mm); (b) in an aqueous solution, NEM is gradually disintegrated because of a hydrolytic cleavage of the CO-NH bond and the product of this hydrolysis reacts very slowly with SH groups (Gregory, 1955); (c) under certain conditions, several non-SH containing amino acids may react with NEM (Riggs, 1961), therefore, NEM is not always specific; (d) if proteins are not removed from the reaction mixture before SH determination, high blanks may appear because of the high absorbance of proteins around 300 nm. Nevertheless, NEM is widely used for blocking or determining protein SH. According to our experience, NEM is a suitable SH reagent provided that there is not too great an excess of the reagent and that the reaction time is not too long. Leslie et al., (1962) found NEM suitable for the determination of mercapto groups in proteins when they were denatured. Using NEM for the determination of SH groups in tissue and muscle proteins, Hamm and Hofmann (1966b) came to the following conclusion. The titration after Tsao and Bailey (1953) with sodium nitroprusside as an indicator is not appropriate because the change in color is indistinct due to the slow

SULFHYDRYL AND DISULFIDE GROUPS IN MEATS

23

reaction of the SH groups. But the following spectrophotometric procedure is quite suitable: 0.5 gm myofibrils of finely minced tissue (containing about 0.1 gm protein) is weighed into a 12-ml test tube and mixed with 5 ml 0.1 M phosphate buffer pH 6.0 and 5 m12 10-3M aqueous NEM solution. After shaking for 2 hours at 2 5 T , the main part of the proteins is removed by centrifuging at 15,000 X g for 5 minutes. The remaining proteins are precipitated in 5 ml of the clear supernatant by the addition of 1 ml 20% TCA. After centrifugation (15,000 X g for 5 minutes), the optical density of the supernatant is measured against a blank at 300 nm. In the calculation of the SH content, the partial hydrolysis of NEM (decrease in absorbence 0.003) and the moisture content of the sample (0.4. lop3 1) have to be taken into consideration. The SH content is calculated from the extinction E measured in a 1-cm cuvette by the following equation: pM01 SH = 10.00 - 20.12 x (E + 0.003). The presence of TCA does not interfere in the measurement of optical density. The protein precipitate does not include measurable amounts of NEM, provided that the water insoluble proteins are removed before as described above. With this method 3.5 moles SH/105 gm protein were found in myofibrils, whereas with AgN03 9.2 moles SH/l05 gm protein were determined in the same preparation. Therefore, NEM reacts only with a part of the SH groups which are defined as “easily available SH groups” of muscle proteins. A preceding denaturation of proteins, either by 8 M urea or by heating (cf. Section V,A, l), results in an increase in SH groups reacting with NEM; but the total number of SH groups in myofibrils which react with AgNO, cannot be achieved (Hamm and Hofmann , 1966b). An almost equal number of SH groups detectable with AgNO, can be determined by reaction with NEM if the NEM reacts at pH 7.4instead of pH 6.0 and if the subsequent SH determination is carried out by indirect amperometric titration (Hofmann, 1 9 7 1 ~ )It. follows from this result that at a higher pH the SH groups are more easily available for NEM. But the determination of the partially available SH groups with NEM at pH 6.0 is still of value because it allows one to follow the process of protein denaturation (unfolding) (see Section V,A, 1). Weitzman and Tyler (1971) found that NEM gives a well-defined polarographic reduction wave and that measurement of this can form the basis of a more sensitive procedure for estimating SH groups. Numerous N-substitution products of maleimide were synthetized in order to obtain derivatives which are more sensitive for optical measurement than NEM. These derivatives cannot be discussed in detail. Some of them were used for studying protein conformation because the reaction products of SH groups with NEM derivatives are resistant against protein hydrolysis by acids. Holbrook et af. (1966) studied the influence of the size of the substituents in N-substituted maleimides on their reactivity against SH groups in proteins. They found that the larger the substituent the slower the reaction. Colored or fluorescent compounds were obtained by the

24

KLAUS HOFMANN A N D REINER HAMM

introduction of chromophoric groups into the NEM molecule (Witter and Tuppy, 1960; Riordan and Vallee, 1972; Kanaoka et a / . , 1967, 1968, 1973; Sekine and Ando, 1972; Nara and Tuzimura, 1973). Such derivatives can be used for the histochemical demonstration of SH groups (Tsou et al., 1955). Furthermore, NEM and its derivatives were labeled by substituents containing I4C (Kielley and Barnett, 1961; Tkachuk and Hlynka, 1963; Lee and Samuels, 1964; Lee and Lai, 1967), 3?S and 35S(Merz et a l . , 1965). Maleimides were used for the determination of SH groups in myofibrils (Hamni and Hofmann, 1965; Hofmann, 1971 c), poultry muscles (Gawronski et a l . , 1967), fish muscle (Connell, 1957), and in muscle proteins, particularly in myosin and actin (Tsao and Bailey, 1953; Kielley and Barnett, 1961; Katz and Mommaerts, 1962; Groschel-Stewart and Turba, 1963; Martonosi, 1968; Seidel, 1969). According to Schoberl (1958), in addition to maleimides, vinylsulfones, which also bear reactive double bonds, are very suitable for the blocking and determination of protein SH groups, e.g., in keratins. 4 . Other SH Reagents Of the reaction types not described in the preceding sections, the nitroprusside reaction has been known for the longest time. The sensitivity of its reaction with SH groups in alkaline solution forming a red color is very great: (Fe(CN),NO]'-

+ R-S- + [Fe(CN),NO.S-R]'

In a strong alkaline solution, the color fades very quickly, while in an ammoniacal solution, it is quite stable. However, the color stability is insufficient for an exact quantitative determination of SH groups. Nevertheless, sodium nitroprusside has often been used for the assay of SH groups in meat (Chajuss and Spencer, 1962b; Khan et a l . , 1963; Khan and van den Berg, 1964, 1965; Motoc and Banu, 1968; Davidkovli and Davidek, 1971; Khan and Nakamura, 1971). Numerous efforts for increasing the color stability with additives or variation of reaction conditions have had only limited success. An addition of cyanide improves the color stability (Schoberl and H a m , 1948); but in this case a cleavage of SS groups occurs which might result in overly high SH values. Sodium nitroprusside has also been used as an indicator for the titration of SH groups of proteins with other SH reagents (Connell, 1960a,b); but such procedures imply problems which must not be overlooked. The discoloration of the protein-SH-nitroprusside complex in such titration methods is due to a replacement of the nitroprusside anion by the added SH reagent: [Fe(CN),NO. S-Protein]

+ reagent

-

[Fe(CN),NO]'-

+ reagent-S-Protein

SULFHYDRYL A N D DISULFIDE GROUPS IN MEATS

25

The sodium nitroprusside often reacts with only a limited number of the protein SH groups. Consequently, the change in color from red to almost clear will have already occurred if the titration reagent has reacted with the SH groups that participate in the nitroprusside color complex of the protein. So, rather than all the SH groups reacting with the titration reagent, only the groups which react with sodium nitroprusside are determined. Sodium nitroprusside is specific for SH groups in proteins as well as generally in animal tissues. The sodium nitroprusside reaction provides an excellent and very sensitive spot test for the qualitative indication of SH groups. With meats, the red myoglobin color disturbs the test; but with washed muscle tissue, myofibrils. or with meat which is pale by nature (e.g., poultry or fish muscle), good results are obtained. The SH test with sodium nitroprusside can be carried out also in ZnCI, solution (instead of ammonia) which causes a low acidification of the reaction mixture (Pohloudek-Fabini and Papke, 1964). Myofibrils take o n an intensively pink color, whereas, with solutions of SH compounds such as cysteine or glutathion, a pink precipitate is formed. The use of B mixture of I % sodium nitroprusside solution and a 30% ZnClz solution has been recommended (Hofmann, 1965a). An intensive color reaction is obtained with native muscle proteins. Serum albumin or egg albumin reacts only after denaturation by urea or heating (Hofmann, 1965a, 1966a). Contrary to the SH test in ammoniacal solution, creatine and acetone do not interfere. Sulfite and thioesters, however. do react. The presence of sufficient amounts of ammonium ions or EDTA prevents the color reaction of nitroprusside ZnC1, by the formation of Zn complexes (Hofmann, 1965a). This result suggests that zinc participates in the color formation. The spot test with iodine azide represents a very sensitive SH reaction (see Feigl, 1960): 2NaN,

+ JB

('")

>

3 N4

+ 2 NaJ

This reaction, which occurs in a sufficient rate only in presence of SH groups, can be recognized by the formation of small gas bubbles (nitrogen). SH reagents which have been known for a long time are iodoacetamide, iodoacetate, and iodoacetic acid. They are used for the alkylation of SH groups of the cysteine residues in proteins. After hydrolysis of the S-alkylated protein the cysteine residues are present as carboxymethyl cysteine which can be determined in the course of the amino acid analysis. Under certain conditions these reagents can react with methionine, histidine, and lysine residues of proteins (Gundlach ez ul., 1959). In many investigations, iodoacetamide, iodoacetate, and iodoacetic acid have been used for blocking and determination of SH groups in muscle proteins (Mirsky and Anson, 1935; Mirsky, 1936; Barany and Barany. 1959; Barany et al., 1964; Stracher, 1964).

26

K L A U S H O F M A N N A N D REINER H A M M

4-Iodobutanesulfonate was also recommended as an SH alkylating agent in the analysis of amino acids (Jermyn, 1966). 4,4’-Bisdimethylaminodiphenylcarbinol is a new, highly sensitive SH reagent, which shows in an acidic solution an intensive blue color (cM = 70800 at 612 nm). The product of the reaction between the dye cation and SH groups is colorless, probably because of the following reaction (Rohrbach et al., 1973): 1+

r

colorless

This decrease of optical density caused by SH groups can be used for the quantitative determination of protein SH. Urea and cyanate disturb this reaction because they also cause a discoloration; 4 M guanidine, however, does not interfere, Another sensitive test for cysteine, glutathion, and other SH compounds is possible by means of chloranil (I), bromanil (11). or 2, 3 dichloronaphtoquinone (111) (Hofmann, 1965b).

(11

(11)

(1x1)

For this test, the SH containing solution is alkalized by the addition of K,CO, and shaken with a solution of I, 11, or 111 in chloroform. After the separation of the aqueous and organic phases, the colored reaction products appear in one of these two phases (see Table 11). Disulfides and SH-free amino acids do not interfere in this reaction. The color of the solutions is very stable and is suitable for quantitative SH determination (Hofmann, 1965b).

27

SULFHYDRYL AND DISULFIDE GROUPS I N MhATS TABLE I1 COLOR REACTION OF HALOGENIZED QUINONES WITH SH COMPOUNDS I N ALKALINE (K,C03) SOLUTION“.”

SH compound

Chloranil bromanil

2.3-Dichloronaphtoquinone

Cysteine Glutathion Thioglycolic acid Eth ylmercaptane Thiamine (SH form) Thiamine pyrophosphate

Green (H,O) Reddish brown (H,O) Yellow (H,O) Red (CHCI:,) Reddish brown (CHCI:,) Yellowish brown (H,O)

Yellow (H,O) Pink (H,O) Orange (H,O) Yellow (CHCl3) Yellow (CHCI,) Pink (H,O)

(’ From Hofrnann (1965b). The color appears in the phase indicated in parentheses

Fontana et al. (1968) suggested azobenzene-2-sulfenyl bromide as an SH reagent specifically for cysteinyl residues; in this reaction asymmetric disulfids are formed:

/ S -Br

/

S-S-R

Friedman (1973, 1974) found that protein SH groups can be transferred quantitatively to acid-stable S-P-pyridyl ethyl cysteine residues by reaction with 4-vinylpyridine. The corresponding cysteine derivatives released by hydrolysis elute as discrete peaks from an amino acid analyzer. A related assay was also developed in which half-cystine residues are changed to S-P-(2-quinolylethyl)cystine residues. These side chains can be estimated by ultraviolet spectroscopy in intact or hydrolyzed proteins. Recently new fluorescent reagents, N-dansylaziridine and NBD-chloride (7-chloro-4-nitrobenzo-2-oxa1,3-diazole) have been described as selectively reacting with protein thiols (Scouten et al., 1974; Price and Cohn, 1975). These reagents may be used to label SH portions of proteins, to differentiate between buried and exposed sulfhydryls, and to determine the nature of the region (hydrophobic, hydrophilic) surrounding a given sulfhydryl group. In certain instances, the possibility of a specific determination of the content of reduced and oxydized glutathione in tissues is of interest. This can be done by means of enzymatic spectrophotometric methods (Klotsch and Bergmeyer, 1962; Lack and Smith, 1964; Srivastava and Beutler, 1968; Tietze, 1969).

28

KLAUS HOFMANN AND REINER HAMM

C.

DETERMINATION OF SS GROUPS

Usually the determination of disulfide groups in protein is carried out in two steps: (1) by reduction or cleavage of the SS groups to SH groups, and (2) determination of the SH groups formed. Since the methods for the determination of SH groups have been discussed, we will only consider the reduction of S S groups here. The reagents frequently used for this purpose are: (a) mercaptans such as P-mercaptoethanol (Anfinsen and Haber, 1961 ; Thompson and O’Donnell, 1961; Christian and Schur, 1965; Habeeb, 1972; Beveridge et al., 1974), thioglycolate (Katchalski et al., 1957; Sela et a/., 1959; White, 1960; Leach and O’Donnell, 1961), dithiothreitol (Cleland, 1964, 1968) or dithioerythritol (Habeeb, 1972); (b) sodium borhydride (Stahl and Sigga, 1957; Moore et al., 1960; Stauff and Duden, 1958; Brown, 1960; Seon et al., 1965; Cavallini ef al., 1966; Glaseretal., 1970); (c) sulfite (Kolthoff e t a l . , 1958, 1965a; Carter, 1959; Christian and Schur. 1965; Rohrlich and Essner, 1966); (d) cyanide (Grote, 1931; Wronski, 1964; Roberts and Rouser, 1958; De Marco ef al., 1966); (e) Tri-n-butylphosphine (Harrap and Gruen, 197I); and (f) electrolytical reduction (Leach ef al., 1965, see also Friedman, 1973). The corresponding reaction mechanisms are as follows: (a) 2 R-SH + Prot.-SS-Prot. + 2 Prot.-SH + R-SS-R Prot.-SS-Prot. 4 2 Prot.-S- H, or* (b) 2 H4 R-SS-R NaBH, 3 H,O + 8 R-SH + NaH,BO, (c) SOa2- Prot.-SS-Prot. 4 Prot.-SSOg Prot.-S(d) CNRot.-SS-Prot. 4 Prot.-SCN + Prot.-S(e) Bu,P Prot.-SS-Prot. H,O 4 2 Pro[.-SH + Bu,PO (9 2e Rot.-SS-Prot. -+ 2 hot.-.!-

+

+ + + + +

+

+

+

+

The reagents most frequently used are types a, b, and c. Mercaptans also are often added to proteins in order to protect SH groups. For a complete reduction of protein SS groups, the presence of a denaturing agent such as 8 M urea, 4 M quanidine, or 0.5% SDS solution and a considerable excess of the reducing agent are generally necessary. Therefore, in most cases, after reduction but before the SH determination, the excess of the reducing agent must be removed. This is an easy process in the case of NaBH,; by acidification of the alkaline reaction mixture, the reagent is completely destroyed. An excess of mercaptans has to be eliminated, e.g., by precipitation of the proteins. Sulfite has often been used for the cleavage of SS groups and subsequent determination of SH groups by amperometric titration with AgN03, which is not disturbed by an excess of sulfite. *The reaction mechanism seems to be unclear. In the first reaction, hydrogen is released; in thc second, no hydrogen appears (see Jocelyn, 1972; Friedman, 1973).

SULFHYDRYL A N D DISULFIDE GROUPS IN MEATS

29

The fact, however, that, after reaction of SS with sulfite, only one SH group is obtained is disadvantageous (see reaction c). Well-proved methods for the SS determination in proteins using NaBH,, P-mercaptoethanol, and dithioerythritol as reducing agents and subsequent SH determination with Ellman's reagent are described by Habeeb (1972). Reduction of SS by NaBH, has been shown to be convenient for the determination of SS in animal tissues (Hamm and Hofmann. 1965, 1966c; Dubi, 1969; Bognar, 1971a; Habeeb, 1972; Dubeet ul., 1972). A description of the SS determination in tissue (after Hamm and Hofmann, 1966c) follows: The sample, containing about 5 mg protein (e.g., 25 mg minced muscle tissue) is weighed into a conical centrifuge tube (10 ml), mixed with 0.5 ml reducing agent (0.12 gm NaBH, in 5 ml of 8 M urea), and kept for 1 hour at room temperature. Foaming can be prevented by the addition of a drop of octanol or by putting traces of a silicon defoamer on the glass wall of the tube. Then the NaBH, is destroyed by a stepwise addition of 0.15 M HNO, until a pH of 6 . 7 4 . 4 is obtained. After keeping the mixture under nitrogen for 30 minutes, 28 ml of water and 5 ml of a tris buffer pH 7.4 are added. In this mixture the SH groups are determined by amperometric titration. The use of hydrogen selenide allows a direct histochemical demonstration of SS groups (Olszewska et al., 1967); the sections on the slides are saturated with water vapor and treated with gaseous H,Se for 2 hours. The areas containing SS groups stain yellow-brown. The test is based on the reaction: R-SS-R

+ H,Se -+

2 R-SH

+ Se

The presence of thiol groups does not influence the results of this reaction. Another method which is appropriate for the determination of SS groups is based on the fluorescence quenching of fluorescein-mercury(I1)-acetate by SS groups (Karush et al., 1964). SH groups, which also react, are blocked beforehand with iodoacetamide. Maeda et ul. (1970) developed a method for visualization of cystinecontaining peptides in peptide maps. The chromatogram is sprayed with an NaBH, solution in ethanol and the excess of NaBH, is decomposed by dipping it in acid. The paper is dried, neutralized by exposure to ammonia vapors, and then sprayed with Ellman's reagent. Yellow spots appear immediately. These can be eluted and analyzed to establish the amino acid composition of the cystinecontaining peptides. A combination of the methods for demonstrating SH groups by means of 2,2'-dihydroxy-6,6'-dinaphthyldisulfideor Fast Blue B (Gabler and Scheuner. 1966) with Mercury Orange permits simultaneous color differentiation between SH groups (blue or purple) and cystine disulfide bonds (red-orange) (Szydlowska and Junikiewicz, 1973).

30

KLAUS HOFMANN A N D REINER HAMM

Ill. SH GROUPS IN MUSCLE PROTEINS AND THEIR ROLE IN THE FUNCTION OF MUSCLE The muscle cell consists of the myofibrils, the sarcoplasmic reticulum, and some cell organelles such as nuclei, mitochrondia, lysosomes, and ribosomes. This structural material is embedded in the fluid matrix of the sarcoplasma. The cell is surrounded by the cell wall, the sarcolemma. The structural cell elements including the membranes are to a large extent built up by proteins. A great variety of other proteins is dissolved in the sarcoplasmic matrix o r loosely attached to cell structures. The bulk of the sulfhydryl groups in muscle is bound to proteins. Nonprotein thiol is rather low and consists largely of glutathion ( I .5 pmole/gm tissue in rabbit skeletal muscle) (Jocelyn, 1972; see also Section IV,A). Most of the numerous proteins of the muscle cell contain sulfhydryl groups of physiological importance. It is not the purpose of this review to discuss in detail the role of SH groups in muscle physiology. For a better understanding of the reactions that occur in muscle as a food, however, some knowledge of the most important facts from the extensive field of muscle research might be necessary, particularly in future meat research, because thus far not much use has been made of the results from this research. With regard to the great number of different protein SH groups in the muscle, to the numerous effects of these sulfhydryls on enzyme activity, protein interactions, membrane transport reactions, etc., it might be very difficult to elucidate the importance of a particular type of sulfhydryl for meat quality or in changes in meat protein. Most of the information on the biochemical and physiological role of SH groups in muscle proteins has been obtained by studying the effect of blocking these groups with specific reagents during enzyme activity or protein interactions. Chemical modification of sulfhydryls has been extensively used for elucidating the structure of the active sites of adenosinetriphosphatase (ATPase) and other enzymes. In this type of study, the chemical modification must cause stoichiometric inactivation, and there must have been specific protection by the substrate or by competitive inhibitors against the inactivation. Even so, the possibility remains that this particular modification occurs other than at the catalytic site and that specific protecting agents induce a change in confinnation at a location other than the catalytic site (Tonomura, 1973). These points have not always been taken into account. Hence, not all the results mentioned in this chapter can be regarded as irrevocable. A.

MYOFIBRILLAR PROTEINS

The proteins which build up the myofibril are shown in Tables I11 and IV. Although all myofibrillar proteins are related to the contractile mechanism of the

SULFHYDRYL A N D DlSULFlDE GROUPS IN MEATS

31

muscle fiber, their functions are distinctly specialized (Table 111). The fundamental process of contraction is known to be carried out by myosin and actin, but these two proteins alone cannot bring about the contraction process of living muscle. The recognition of the physiological role of tropomyosin and troponin has further stimulated the discovery of new myofibrillar proteins. On the whole, the idea of regulatory proteins (proteins that enable myosin and actin to perform the contraction-relaxation cycle under physiological conditions) has been established (Ebashi and Nonomura, 1973).

TABLE 111 MYOFIBRILLAR PROTEINS OF THE SKELETAL MUSCLE"

'

/Myosin

Contractile proteins

\

Actin

I /

Troponin Functional Regulatory proteins

Structural M-Protein 'I

After Ebashi and Nonomura ( 1973)

TABLE IV CONTENTOFCONTRACTION AND REGULATORY PROTEINS IN RABBIT MYOFIBRIL"

Proteins

Percentage of protein by weight

Myosin Actin Tropomyoain Troponin &-Actinin P -Actinin M-Protein

55-60 20

'I

4.5

3 -5 1-2

-0.5 -0,s

After Ebashi and Nonornura (1973)

32

KLAUS HOFMANN A N D REINER HAMM

I , SH and SS Group Content of Myofbrillar Proteins Table V shows the data for SH and SS content of the single myofibrillar proteins. All myofibrillar proteins contain SH groups. Disulfide groups have been found only in tropomyosin and troponin. No reliable data for SH groups could be found in the literature for p-actinin. The results of the estimates of SH groups in myofibrils are listed in Table VI. Some of the values determined in native myofibrils with NEM, PCMB, and DTNB obviously do not represent the total SH content but rather the SH groups available under the conditions used in the reactions. The average SH content of the different myofibrils is 9.1 moles SH/I05gm protein (values lower than 6 were omitted). This value corresponds very well to the sum of SH groups of the single myofibrillar proteins, calculated from the values of Table VII. As the average SH content of whole muscles was 10.2 (pork) and 10.5 (beef) (see comments on Tables IX and X), it is evident that the SH groups in meat are generally about 90% bound to myofibrils. Table VII provides the figures for mg SH and SS content of the major proteins per 100 gm of the total myofibrillar protein. These TABLE V SULFHYDRYL AND DlSULFlDE GROUPS IN MYOFIBRILLAR PROTEINS. EQUIVALENTS OF SH AND SS GROUPS PER lo5 gm PROTEIN

Protein

Molecular weight

SH

SS

8-9!,.c.d.?J

0u.d.~

8.4' 7.3' 8.6' 7 - 10"

0

SH

+ SS"

~~~~~~~~~~~

Myosin

HMM

220.000

320-360,000

8.4'

7.4" 10' 6.4"

Subfragment I Subfragment 2 LMM Actom yosin

4b.P

n.n"

0 0 0'

7 .5' ) I 8.5'

Actin

Actinin

46.OO0-47.000

I2"."

-95,000

13' 30.3" 7.8' 9-1 I " 5.1'

O"."

7.W 9.3'

33

SULFHYDRYL AND DlSULFlDE GROUPS I N MEATS TABLE V

Protein Tropomyosin

Molecular weight

( L ontinued)

SH

SS

SH

+ SS"

3-5" 4.31 4.7"

70.000 ( o r 35.000)

3. I'

Troponin

8 1 .OOO

5" 5.4"'

Troponin I

24,000

13.9.' 12.0"

Troponin T

37.000

Troponin C

1 8 .OOO-20 .OW

M-Protein

165.000

I .6.r 0. I " 0.4'' 8.9' 5.0'' 3.8" 4.4""

I"

3.7' 4" 4.3" 4.6' 4"

10"

12.2'

10.2''

" Usually determined as cysteine acid by amino acid analysis. in some cases also as SH after reduction 0 1 SS. " Ebashi and Nonomura (1973). ' Woods and Hartley (1967). " Bariny P r t i / . (1964). " Tonomura (1973). Robson and Zeece (1973). "Connel (1961). * Buttkus (1971). a Brennock and Read ( 1972). Hofmann (l972a). Ehrlich (1967); Hamm and Hofmann ( 1965). Staib and Turba (1956). ''I Kofman (1963). " Drabikowski and Nowak (1970). " Carsten (1966). Martonosi (1968). Cohen et a / . (1973). Suzuki P I N / . (1973). Yasui et a / . (1968). Hodges and Smillie (1970). ' Hodges and Smillie (1972). ' Ebashi er d.(1968). Arai and Watanabe (1968). Wilkinson et cd. (1972). ' Greaser EI a / . (1973). Schaub et a / . (1972). "" Masaki and Takaiti (1974).

'

'

34

KLAUS HOFMANN AND REINBR HAMM TABLE VI ESTIMATES OF SH GROUPS IN MYOFlBRlLS

SH reagent

Moles SH (per lo5 gm protein)

Original SH data"

AgNOS NEM NEM PCMB PCMB DTNB DTNB DTNB DTNB DTNB CH3HgN03 o-iodobenzoic acid

8.5-9.0 9.5 7.8-12.9" 3.5-4.0 8.9-9.7

85-90 pmoleslgm protein (1) 3.14 mg/gm protein (2) 2.59-4.27 mg/gm protein (3) 1.16-1.31 mglgm protein" (2) 2.95-3.21 mglgm protein (3) 5.4 moles/105 gm protein (4) 3.06-3.09 mg/gm protein (3) 0.59 pmolelmg N ( 5 ) 3.0-3.9 moles/105 gm protein (6) 8.6-9.6 moles/105 gm protein" (6) 60-65 pmoledgm protein ( I ) 85-90 pmoledgrn protein' ( I ) 8.8 mole/105 gm protein (7) 2.75-4.32 mg/gm protein (3)

''

5.4

9.2-9.3 9.4 3.0-3.9 8.6-9.6 6.0-6.5 8.5-9.0 8.8 8.3-13. I

Numbers in parentheses correspond to the following investigations: ( I ) Tinbergen (1970): (2) Hofmann and Hamrn (1966); ( 3 ) Hofmann, Miiller. and Baudisch (unpublished observations); (4) Arai and Watanabe (1968); (5) Khan rt al. (1968); (6) Hay rr a / . (1973); (7) Barany rt ( I / . ( I 964). " Mean value: 10.3 t 1.26 (n = 20). ' Reaction at pH 6.0. " Reaction at pH 7.4. " Myofibrila denatured. "

TABLE VII DISTRIBUTION OF THE SULFHYDRYL AND DISULFIDE GROUPS UPON THE MAJOR MYOFIBRILLAR PROTEINS

Protein Myosin Actin Tropomyosin Troponin

mg SH/100 gm total myofibrillar protein

%

mg SS/lOO gm total myofibrillar protein

%

I56 70 3

65 29

-

-

-

1

10

4

12 11

48 52

SULFHYDRYL AND DISULFIDE GROUPS IN MEATS

35

figures were obtained by combining the mean values of Tables Wand V. As Table VII shows, more than 95% of the sulfhydryls of myofibrils are located in the actinmyosin system, whereas all disulfide bonds are built into the tropomyosintroponin system.

a . Myosin. There are about forty-two SH groups in the myosin molecule. Many authors have found that the number of SH groups in myosin which are titratable with PCMB is approximately equal to the halfcystine content found by amino acid analysis. Myosin probably, therefore, contains no SS bridge (Tonomura, 1973). In Table V,the values for the myosin subunit heavy meromyosin (HMM). including its components, subfragments 1 and 2 and the subunit light meromyosin (LMM), are also listed. The amino acid sequence around the SH residues of myosin has been extensively studied. The conclusion that myosin contains at least 16 and probably between 20 and 22 unique thiol sequences indicates that the molecule consists of two chemically equivalent components (Weeds and Hartley, 1968). There is remarkable agreement that purified myosin has three distinguishable light chains (C,, Cqr C3). SH groups seem to participate in the interaction between the chains, because the blocking of SH groups of myosin with 5,5'dithiobis(2-nitrobenzoic acid) (DTNB) tends to release light chain C,. C, and C3 appear to have similar peptide chains. each containing a single cysteine peptide; C2 contains two cysteine residues. The amino acid sequence of these peptides has been clarified (Taylor, 1972). b. Actin. The SH groups of actin, which are titratable with PCMB or 2.3dicarboxy-4-iodoacetamide azobenzene are found to be approximately equal to the number of halfcystines in the molecules as determined by amino acid analysis. Like myosin, actin contains no SS-bridge (Tonomura, 1973). Only 1 mole of SH per mole of G-actin binds rapidly with SH reagents, and subsequently there is a slower reaction. The same holds true for F-actin: Only I mole per mole G-actin unit binds with SH reagents, while the remainder reacts even more slowly than with G-actin (Tonomura, 1973). Several SH-containing peptides have been isolated from the tryptic hydrolysates of actin (Young, 1969). Now the positions of the reactive and not available cysteine SH groups in the peptide chain of the G-actin molecule have been elucidated (Elzinga and Collins, 1973).

c. Tropomyosin. The molecular weight of tropomyosin was found to be about 70,000. In the presence of reducing agents, e.g., of P-rnercaptoethanol, tropomyosin dissociates into two similar subunits, each of the molecular weight of about 34,000. It has been suggested that a disulfide bond is involved in the association of the two subunits (Tonomura, 1973). In the procedures for isolation

36

KLAUS HOFMANN AND REINER HAMM

of tropomyosin, a reducing environment is usually provided in order to prevent oxidation. Therefore, it is not yet clear whether the tropomyosin is present in the myofibril as the SS-lined dimere or as the SH monomeres. In samples of rabbit tropomyosin from different animals, Woods (1968) found, in the absence of SH reagents, molecular weights between 40,000 and 85,700. After treatment with mercaptoethanol, molecular weights around 34,000 were obtained. Therefore, in some preparations, SS bridges seem to occur, while, in other preparations, they do not. d . Troponin. From the 5 moles SH/105 gm troponin, 1.8-2.5 do not react with NEM (Ebashi et al., 1968). In Table V the halfcystine (SH SS) figures of the three troponin subunits TN-I, TN-T, and TN-C are listed. TN-I inhibits ATPase activity of myosin, TN-T binds to tropomyosin, and TN-C binds Ca2+. In the troponin, these subunits are supposed to be present in the molar proportion 1:I : I . If this proportion of the three troponins is correct, the halfcystine figures obtained by Greaser et ul. (1973) (Table V) should result in 3.9 moles SH + SSlmole troponin. Drabikowski and Nowak (1970), however, found 8.1 moles SH + SSlmole. The latter figure seems to be more probable because it corresponds to the sum of SH and SS groups obtained by others (Table V).

+

2 . Sulfhydryl Groups Involved in the Function of Myofibrillar Proteins Several hundred publications deal with the role of SH groups of myofibrillar proteins in enzymatic activities, in the interaction with ions, substrates, and other proteins, and in the process of muscular contraction and relaxation. It is impossible to quote all these papers in this brief review. But in addition to the more recent research work, research papers and review articles will be cited which mediate the access to all the other literature. a . Myosin. SH groups are important for the adenosine triphosphatase (ATPase) activity of myosin because SH reagents modify this enzyme in various ways (Bendall, 1969; Young, 1969; Ebashi and Nonomura, 1973; Seidel and Gergely, 1973; Tonomura, 1973; Taylor, 1972). Fifty percent of the ATPase activity is lost when five groups of 42 SH groups per molecule are blocked, and inactivation is complete if seven per molecule are blocked. These groups are located in the head region of the molecule, probably fairly close to the active site. It is known that there are two kinds of particularly reactive cysteine residues per subunit of myosin, generally referred to as S, and S2 (or SH1 and SH2). These groups are located at or near the active site of the HMM moiety and near the binding site for ATP. Blocking of the S, sulfhydryls results in an increase in the Ca2+- activated

SULFHYDRYL AND DISULFIDE GROUPS IN MEATS

37

ATPase activity with a simultaneous loss in the K+ (EDTA) activated ATPase. Blocking of both sulfhydryls, S, and S2. eliminates both types of ATPases. If the S2 groups alone are modified, the enzyme activity is similar to that of the &-modified myosin; namely, Ca-activated ATPase is activated and K+ (EDTA) activity is lost. The requirement of both sulfhydryls for the inhibition of myosin ATPase in the presence of MgATP has also been demonstrated (Seidel, 1972; Reisler et al., 1974a). Measurements of the binding of nucleoside di- and triphosphates by native, S, or S,-blocked myosin revealed that all three forms bind ATP equally well. Thus, although SH blocking does not alter the binding of a substrate. it does interrupt the catalytic process. In the resting muscle, myosin contains bound MgATP which decreases its reactivity with NEM, an effect which is not reproduced by adenosine diphosphate or any other nucleotide (Barany and Barany, 1973). It has been suggested that maximum inhibition and maximum activation of ATP hydrolysis in vivo occurs in the millimolar range of Mg2+ through the formation and disruption of a cyclic MgATP ternary complex with myosin, involving coordination with the S, and S2 sites. This idea could be confirmed by crosslinking the two essential SH groups with a bifunctional dimaleimide reagent (Reisler et al., 1974b). The results of Petuskova (1973) also indicate a paired arrangement of these SH groups. Petaskova suggested that the role of SH groups in myosin probably consists of maintaining the structural integrity of the molecule and not in the direct participation in the hydrolysis of ATP. The S, groups react more rapidly with NEM or PCMB than the S2 groups. There is only one S , and S, group per heavy chain of myosin. The S, and S2 groups are generally presumed to be in subfragment 1 of the HMM. Phenolic SH reagents were found to activate Ca2+-ATPase and to inhibit K+(EDTA)-ATPase of both HMM and subfragment I , which make up the head of the myosin molecule. The S,-blocking of subfragment 1 proceeds much faster than that of HMM. ATP-induced conformational changes around the active site of myosin and HMM which were caused by the modification of the S2 group were preserved in subfragment 1. Therefore, the effect of phenolic SH reagents on myosin ATPase and that of ATP on the conformation around S2 cannot be interpreted in terms of subunit-subunit interaction (Kameyama et al., 1974). The reaction of myosin SH groups with DTNB liberates a single class of light chains of 18,000 daltons from HMM without a significant loss of ATPase activity. The other light chains. however, cannot be removed without a reduction in such activity (Weeds and Frank, 1973). Kakol (1971) suggested that the SH groups of the light chains of myosin are essential in preserving ATPase activity. The sequences reported for peptides containing the S, and S2 groups, however, are different from the sequences around the SH groups of the light chains (Taylor, 1972). Myosin contains about 2 moles of “intrinsic” Ca2+which cannot be removed

38

KLAUS HOFMANN A N D REINER HAMM

by the usual purification methods but which can be irreversibly removed by treatment with PCMB followed by P-mercaptoethanol. The latter reagent completely removes the PCMB from the myosin so that ATPase activity is completely recovered. The ATPase activity of actomyosin reconstituted from this myosin and actin is very different from that containing Ca2+ (Tonomura, 1973). In addition to ATPase activity, the ability of myosin to combine with actin is essential for the process of muscular contraction. SH groups are involved in this interaction between myosin and actin (Needham, 1973; Tonomura, 1973). Since myosin masked with certain SH reagents can bind with actin, even though its ATPase activity has been completely eliminated, the active sites for two functions must be different. Other results, however, are not consistent with this idea. Out of the 15 SH groups contained in 200,000 gm myosin, two are necessary for ATPase activity, while three are essential for actin combination; one of these groups is supposed to take part in both phenomena. The effect of F-actin on S,-blocked HMM (with NEM) as shown by Kameyama and Sekine (1973) suggests that the S2 region may be involved in the successive and cyclic conformation changes in the contractile protein system thought to occur in the sliding process of filaments. Other experiments have revealed an involvement of the S, group in the actin binding of myosin, because the binding of actin to myosin causes a conformational change of myosin by influencing the S, groups (Seidel, 1973). Therefore, it is not surprising that, at physiological ionic strength, the binding of actin to myosin has the same effect as the activation of myosin ATPase by the modification of the S, group (Burke et al., 1974). Treatment of actomyosin in the absence of salt, i.e., in the gel state, revealed that actin protects the S, group from reaction with NEM. That the Mg-activated ATPase also remains unaffected implies that the same sulfhydryls are necessary for the functioning of the Mg-activated reaction when the ionic forces involved in the overall interactions are in effect. In the presence of salt and Ca2+,however, treatment with increasing NEM concentrations produces stepwise inactivation of both Ca- and Mg-activated ATPases. The protein unit of one myosin and two actins may react first with about 7-8 NEM molecules without effect (Bkiny and Merrifield, 1973; Schaub and Watterson, 1973). 6 . Acfin. Sulfhydryl groups are also involved in the functional properties of actin (Drabikowski and Bitny-Szlachto, 1964). It was shown that mercurials inhibit the polymerization of G-actin to F-actin and also cause the release of bound nucleotides. By the use of various SH reagents, it was possible to separate these processes and to show that the actin molecule contains at least three kinds of SH groups: The first, which apparently is not connected with any specific property of actin, reacts directly with NEM; the second kind is involved in the polymerization of this protein; and the third is more or less directly connected with the nucleotide binding. It was suggested that two SH groups per mole are required for binding ATP.

SULFHYDRYL AND DISULFIDE GROUPS I N MEATS

39

The different effects obtained by blocking the same SH groups of G-actin with various SH reagents on polymerization, however, lead to two questions: Can different SH groups be assigned to polymerization and nucleotide binding sites and are the SH reagents specific (Taylor, 1972). From spin labeling experiments, it has been concluded that, in addition to tyrosine, lysine, and histidine, sulfhydryls are involved in the configurational changes during polymerization of actin. Laki and Alving (1973) found that one SH group of the G-actin moiety of F-actin participates in the dephosphorylation of ATP by actin. Modification of actin by treatment with SH reagents (PCMB, iodoacetamide, NEM) did not interfere with actin-myosin combination. Modification of HMM with PCMB, however, inhibited combination. These results of Heazlitt et al. (1973) confirmed the observation of Kuschinsky and Turba (195 I ) that while SH groups from myosin are necessary for the symplex formation of actin and myosin, those from actin are not.

c. “Natural Actomyosin, Troponin, Tropomyosin. “Natural actomyosin,” extracted from muscle fibers or myofibrils, is a complex of the proteins actin, myosin, tropomyosin, troponin, and some minor protein components. This complex represents the contractile system of the myofibril. A mixture of tropomyosin and troponin (“natural tropomyosin”), when added to a purified system of actin and myosin, confers upon the ATPase activity of the latter system the extreme sensitivity against Ca2+ ions which is a marked feature of the intact fiber and fibrillar preparations. Pure tropomyosin, even with SH groups carefully protected against oxidation, fails to do so (Bendall, 1969). SH groups are involved in the Ca-sensitizing effect of the tropomyosintroponin system on myosin (Daniel and Hartshorne, 1972). SH reagents can remove the Ca-sensitive response of “natural actomyosin. This effect has been shown both by the measurement of ATPase activity and by superprecipitation. In either case the inhibitory effect of the troponin-tropomyosin complex in the absence of Ca2+ is blocked. A similar effect has also been shown with muscle fiber in that the reaction with NEM caused tension development under Ca2+-free conditions which normally favored relaxation (Kuriyama et al., I97 1). Troponin was originally considered as a likely site of the critical SH group, and experiments with PCMB suggested that this was the case. However, it has been shown that PCMB can be transferred between different muscle proteins; one therefore cannot be certain of this hypothesis. From labeling experiments with NEM it was concluded that the critical SH site might be on the myosin molecule, and PCMB or NEM treatment of troponin did not produce a marked effect on either its Ca-sensitizing activity or Ca-binding activity (Ebashi and Nonomura, 1973). Hartshorne and Daniel (1970) also came to the conclusion that SH groups of troponin are not essential for its function. So the postulation of Fuchs (1971) that Ca-sensitive SH groups exist at a site of troponin, which is essential for its regulatory function, may be incorrect. I’

”

40

KLAUS HOFMANN A N D REINER H A M M

The integrity of certain SH sites on myosin seems to be essential for the normal Ca-sensitizing effect of “natural actomyosin.” Daniel and Hartshorne (1972) demonstrated that the SH groups, which are essential for Ca sensitivity of the normal “natural actomyosin,” are located in the heavy chains of the myosin molecule and that the critical SH groups are not identical with the S, sulfhydryl groups of myosin. Seidel and Gergely ( 1 973) also concluded from spin labeling experiments that the SH groups, whose blocking has been shown to abolish Ca-sensitivity, are not the S, groups. SH groups appear to play no essential role in the attachment of troponintropomyosin to myosin. As to tropomyosin, carboxymethylation of the SH groups reduced the inhibitory effect of this protein on the Ca-stimulated ATPase of desensitized actomyosin but did not effect the Mg-stimulated ATPase (Cummins and Perry, 1973). The supposition that SH groups of tropomyosin may be involved in the polymerization of this protein could nor be confirmed by Drabikowski and Nowak (1 965). d. Actinins. The substitution of about half of the SH groups of myosin by 2-aminoethyl isothiuronium makes the resulting actomyosin unresponsive for a-actinin (no activation of ATPase activity or of the turbidity response by a-actinin); the same substitution of actin does not affect the response to a-actinin. Either substitution diminishes the binding of c-w-actinin to actomyosin; neither substitution abolishes contractility measured in terms of gel syneresis (Seraydarian et al., 1968). PCMB did not affect the shortening in length of F-actin particles by p-actinin (Maruyama, 1971).

e . Muscle Fibers. In the presence of Ca and Mg ions, ATP is split rapidly by the actomyosin filaments. The free energy from this process is used for contraction and for the development of power through the mediation of the sliding of actin and myosin filaments over one another. By modifying the enzyme sites on the actin and myosin filaments (e.g., by the addition of the SH reagent salyrgan to a fiber which develops tension in the presence of ATP, Mg’+ and Ca2+), the ATPase activity and the tension immediately drop to zero and the fiber releases. This effect of salyrgan can be reversed by the addition of cysteine, restoring the SH groups on the enzyme center once more and allowing ATP to split and tension to be redeveloped (Bendall, 1969).

B. PROTEINS OF THE SARCOPLASMIC RETICULUM (SR) Several different protein SH groups are present in the SR. Some of them are clearly involved in the ATPase activity and the Ca-accumulation function of this

SULFHYDRYL A N D DISULFIDE GROUPS IN MEATS

41

system. It is believed that seven proteins are components of the Ca-transport system of SR, such as ATPase, proteolipid, calsequestrin, 54,000 dalton protein, and "acidic proteins." The proteolipid contains 24 halfcystine equivalents per lo5 gni protein. the 54,000 dalton protein 7.4/105 gm (detected as cysteic acid) (MacLennan p t al., 1973). Hasselbach and Seraydarian (1966) demonstrated that lo5 gm of the vesicular protein contained seven equivalents. of which three reacted readily with NEM without the impairment of Ca transport or of ATPase activity. Loss of the extra ATP splitting associated with Ca uptake, as well as loss of Ca transport and storage. followed blockage of the other four SH equivalents. The SH groups are located on the outer surface of the transporting membranes. According to Panet and Selinger (1 970). ten SH equivalents per 1 O5 gm membrane protein of the SR are titratable by DTNB in the absence of sodium dodecyl sulfate (SDS); however, in the presence of SDS, 14 SH groups were found. ATP protected SH groups essential for the ATPase activity of the SR. The asymmetric distribution of proteins in the SR membranes, containing 10-12 mole SH groups per lo5 gm protein. became symmetric if more than four SH groups were blocked by 2-chloromercuri-4-dinitrophenol: At this point, the Ca-dependent ATPase of the SR was completely inhibited, but Mg-dependent ATPase was slightly activated and Ca transport was inhibited (Dupont and Hasselbach, 1973). PCMB and p-chloromercuribenzene sulfonic acid (CMBS) increased the rate of Ca efflux from the whole frog muscle. While PCMB appeared to inhibit SH groups in the terminal cysternae of SR (causing a fractionating of the muscle twitch), CMBS seemed to act primarily at the surface sites with limited access to the cysternae (Kirsten and Kuperman. 19704. The muscle showed increased rigor tension irz v i m when incubated with 1 mM NEM, and Ca efflux from the whole muscle was increased. NEM apparently produces rigor by inhibition of Ca uptake through the SR (Kirsten and Kuperman, 1970b). So the effect of NEM o n muscle physiology seems to be detemiined not only by its reaction with myosin SH groups as mentioned above but also by the reaction with the SH groups of SR. C.

PROTEINS OF THE SARCOLEMMA

Modification of the SH groups of the sarcolenima with DTNB did not affect the Ca-dependent ATPase, but it decreased the Mg-dependent ATPase of these membranes. When the reagent was added in the absence of ATP, both enzymes were inhibited whether the divalent cations were present or not. Cysteine or dithiothreitol reversed this enzyme inhibition. Modification of the sarcolemma SH groups by NEM strongly inhibited the activity of both ATPases in the presence of ATP, and fully inhibited them in the absence of ATP. This inhibition was

42

KLAUS HOFMANN A N D REINER HAMM

not reversed by cysteine or dithiothreitol (Gimmelreikh and Koval, 1973). The activity of the Na+/K+-stimulatedATPase of the sarcolemma is inhibited by such SH reagents as PCMB or NEM (Matsushima, 1974). D.

PROTEINS OF MITOCHONDRIA

A substantial number of SH groups are present in the mitochondrial membranes (Jocelyn, 1972). Protein SH groups are involved in the electron transport. The flavoprotein and cytochrome components of the electron transport chain possess SH groups. The SH groups of NADH dehydrogenase appear to be mainly structural and not to have a catalytic function in accepting electrons. The SH groups of succinic dehydrogenase may be both structural and catalytic. The transfer of electrons from NADH dehydrogenase to coenzyme Q may require SH groups; the further transfer to cytochrome c does not, although the enzyme complex concerned contains several SH groups. The cytochrome itself contains SH and SS groups. Cytochrome oxydase contains some SH groups, but it is not known whether they are required for activity. Various observations have implicated SH groups in the yet unresolved mechanism of oxidative phosphorylation. Oxidative phosphorylation is inhibited not only by NEM and DTNB but also by arsenicals, suggesting that SH groups are required. The passive membrane transport of inorganic phosphate by a special mechanism, unlike the other mitochondrial exchange systems, is inhibited by SH-combining agents such as DTNB, mercurials, or NEM. As to the active transport, Ca2+ accumulation is partly inhibited by SH reagents such as mercurials, but the Na+/K+-exchange is stimulated for unknown reasons. The effect of mercurials on the ATPase of mitochondria varies from a stimulation at a low concentration to an inhibition at a higher concentration. E.

PROTEINS OF THE SARCOPLASMIC MATRIX

The supernatant, which is obtained after centrifugation of a muscle homogenate at about 100,000 X g, contains a great number of dissolved albumins and globulins. Not all of these contain SH groups. So it is known that no SH groups are present in the myoglobin from the mammalian skeletal muscles. SH groups are, however, involved in the activity of enzymes of the glycolytic chain. Glycolysis is inhibited by the SH reagent iodoacetate at a concentration which does not affect contraction (Jocelyn, 1972). Some of the SH dependent enzymes have been isolated. They include phosphofructokinase, glyceraldehyde-3phosphate dehydrogenase, lactate dehydrogenase, and phosphorylase. In addi-

43

SULFHYDRYL A N D DISULFIDE GROUPS I N MEATS

tion to these glycolytic enzymes, creatine phosphotransferase requires SH groups for its activity.

IV. SH AND SS CONTENT OF MEATS AND MEAT FRACTIONS The SS content of tissues is generally rather low. Therefore, most investigations have dealt with the estimation of the SH content only. Much confusion exists in literature concerning the term “SH content in tissue,” because there are different kinds of SH groups which are not always clearly differentiated from each other. Ellman (1959). who introduced DTNB for the assay of SH groups in biological material, entitled his publication, “Tissue Sulfhydryl Groups.” However, only the SH content in the tissue extracts was estimated; no similar estimate was made for whole tissues. Other authors (e.g., Khan) have used the term “SH groups in meat” quite generally, although only the nonprotein SH content of extracts was estimated. Therefore, it is absolutely necessary to explain what type of SH groups were estimated in each case. The total SH content of meat is distributed on protein and nonprotein substances as well. The different possible fractions are shown in the scheme of Table VIII. Boyne and Ellman (1972) used the term “total soluble SH” to refer to the sum of the SH content in the soluble proteins and in the nonprotein fraction. In the

TABLE VIII SCHEME OF THE DISTRIBUTION OF SH GROUPS IN MUSCLE TISSUE

Water insoluble proteins (e.g.. myofibrils)

e

-reactive SH slowly reacting SH

‘masked SH

,reactive

Water soluble proteins (e.g., sarcoplasmic proteins)

k

\

.Protein SH

SH

- total slowly reacting SH

masked SH cysteine SH

Nonprotein glutathione S H

SH

44

KLAUS HOFMANN A N D REINER HAMM

following tables, the total SH content of meat is generally given. In some cases, one cannot be sure if all SH groups or only a part of them were estimated by the reagent used. Therefore, the kind of reagent-whenever indicated in the literature-has been listed as well. Very different dimensions are used in literature for expressing the SH content in meat. Therefore, the original SH data were transformed to milligrams of SH/100 gm meat and/or to moles of SH/1OS gm protein for better comparison. Values given in parentheses correspond only approximately to the original data, because in these cases the protein contents were not indicated and the mean protein content of lean meat, which is about 18%, was taken as a basis for the calculation. In the following scheme, the factors used for the transformation of the original data are given.

[

mgSH 100 grn meat

X

1'

3.024

percent protein ~

[

1-

mole~H lo5 gm protein

3.024 x

[

mgSH

1

gm protein

097

The question of how the SH content in whole meat can best be expressed has no easy answer. It is not completely correct to relate the total SH content in whole meat to the protein content because a part of the SH groups in meat is not bound to protein. On the other hand, when the whole tissue (as wet weight) is used as a reference standard, the SH values are influenced by differences in concentrations of water, fat, and connective tissues (which are virtually free of SH groups), and the variations in the SH content of different meats can be attributed to changes in these factors. The answer to the question of what reference unit is to be employed depends on the kind and the aim of the investigations involved. There are cases in which statements can be contradictory depending on whether the results refer to meat or to protein. For instance, the SH content in cow and bull muscle was found to be 87.3 and 79.3 mg SH/l00 gm tissue, respectively, or 11.8 and 12.8 moles SH/105 gm protein, respectively. In the first case, the conclusion would be that the SH content in cow muscle was higher than in bull muscle; in the second case, it would seem to be lower. The reason for this apparent contradiction is the different protein content of both meats. This exam-

45

SULFHYDRYL AND DISULFIDE GROUPS IN MEATS

ple clearly shows that conclusions and comparisons of SH results must be drawn very carefully. There are many factors that influence the SH content of raw meat. It is not surprising, therefore, to find a considerable variation in the results of different literature in this field. These influences are discussed separately in Section IV,

D. A.

SH AND SS CONTENT OF MUSCLES

Values are listed for total SH content in pork and beef (Tables 1X and X), for muscles of various meat and test animals (Table XI), for nonprotein SH contents in muscles (Table XII) and for SS groups in muscles (Table XIII). Comrnenrs on Table IX: The SH figures in porcine muscles estimated with different SH reagents vary from 8 . 3 to 12.9 moles SH/I05 gm protein (49.6 to

TABLE IX SH GROUPS 1N PORCINE MUSCLES

Muscle

long. dorsib long. dorsi long. dorsi" long. dorsi" long. dorsi long. dorsi long. dorsi long. dorsi semimembr .'' semimembr. semimembr. psoas

SH reagent

&NO,

AgNO, DTNB NEM" NEMJ DTNB NEM' PNSS.*

SH (mg) per 100 gm tissue

Original SH data" 1 .SO pmolesll00 mg meat 1 I .6 moles/105 g prot. (2) 73.6 mg/lOO gm meat (3) 74.0 mg/100 gin meat (3) 3.14 mg/gm protein (4) 8.48 moles/lP gni protein 12.89 moles/105 gni protein 2.89 mglgm protein (4) 3.44 mg/gm protein (4) 1 1 .OX moles/105 gm protein 3.05 mg/gm protein (4) 283 pg/gm tissue (5)

(I)

(2) (2)

(2)

49.6 71 .O 73.6 74.0 (56.5) 50.5 76.7 (52.0) (61.9) 64.5 (54.9) 28.3

SH (moles) per lo5 gm protein (8.3) 11.6 11.2 11.3 9.5 8.5 12.9 8.7 10.4 11.1

9.2 (4.8)

Numbers in parentheses correspond to the following investigations: ( I ) Krylova and Kusnezowa (1964); (2) Hofmann. Bliichel. Miiller. Baudisch, and Hiinim (unpublished observations): (3) Hofmann CI ul. (1974): (4) Fischer and Hamm (1975); (5) Motoc and Banu (1968). 'I

M . /ongis.simits dorsi. Vacuum packaged after slaughter. " Air packaged after slaughter. In presence of dodecylsulfate. Reaction at pH 7.4. 'I M . semimembr-uriuc.eus. 'I Sodium nitroprusside. I'

46

KLAUS HOFMANN AND REINER HAMM

76.7 mg SHll00 gm tissue). The value of 4.8 moles SH, which was estimated with sodium nitroprusside, obviously does not represent the entire SH content. The average value is 10.2 moles SH/105 gm protein (62.2 mg SH/lOO gm tissue) when the low value is excluded. Figures estimated with AgNO, vary from 8.3 to 11.6, those estimated with NEM under different conditions from 8.7 to 12.9 and those estimated with Ellman’s reagent (DTNB) from 8.5 to 11.1 moles SH/105 gm protein. This shows that the values found with AgNO, are in the same range as the values found with NEM (at pH 7.4) and DTNB. No significant differences seem to exist between the SH contents of longissimus dorsi and semimembranaceus muscles. Comments on Table X : The values lower than 8 moles SH/105 gm protein do not seem to represent the normal total SH content in bovine muscle (even in some cases when AgNO, is used as a reagent). It is well known that sodium nitroprusside and NEM at pH 6.0 react only with one part of all SH groups in proteins (see Section 11, B,3 and 4). From the other values it can be concluded that the values for beef muscles vary from 8.5 to 12.1 moles SH/105gm protein (50.5-87.3 mg SH/l00 gm tissue); the mean value is 10.5 (65.9 respectively). These figures are virtually in the same range as those for pork. Therefore, contrary to the statement of Krylova and Kusnezowa (1 964), the SH content of pork is not generally higher than the SH content of beef. In addition there are no considerable differences in SH content between cow and bull muscles, between longissirnus dorsi and supra spinam muscle, or between vacuum-packed and air-packed muscles shortly after slaughter. The number of SH groups in calf muscles varying between 8.4 and 10.4 moles SH/1O5 gm protein (52.7-75.6 mg SH/lOO grn tissues) seems to be somewhat lower than those in beef. However, Krylova and Kusnezowa (1964) found that beef from animals at age 9-10 months gave higher SH values than that from animals at age 16-18 months. The differences in the SH content of longissimus dorsi and diaphragm muscles of calves were found to be significant (P < 0.01) (Fischer, Hofmann, and Hamm, unpublished observations). Comments on Table X I : All SH values estimated with PCMB, iodoacetate, and sodium nitroprusside (3.2-6.7 moles) are lower than those found with Ellman’s reagent or AgN03 (9.0-1 1.9 moles). The latter, which probably represent the total SH content in muscles, are within the range of the values estimated for pork and beef (see Tables IX and X). Therefore, the skeletal muscles of different animals (pigs, cattle, chickens, rats) contain approximately equal amounts of SH groups. The SH values of rabbit, rat, and frog muscles estimated with iodoacetate were also found to be comparable. The figure of 21.4 moles SH found by Caldwell and Lineweaver (1969) is certainly too high and is therefore not included in this discussion. Comments on Table XII: The content of nonprotein SH [respectively of glutathione (GSH)] in muscles varies in a wide range from 1.1 to 7.6 mg SH/100 gm meat (average 3.7). This may be due to the fact that the GSH content of

SULFHYDRYL AND DISULFIDE GROUPS IN MEATS

47

TABLE X SH GROUPS IN BOVINE MUSCLES

Sample. Muscle Beef, long. dorsi" Beef, long. dorsi" Beef, long. dorsi" Beef, ('?) muscle Beef, long. dorsi" Beef, long. dorsi' Beef, long. dorsi Beef, long. dorsi Beef, long. dorsi Beef, long. dorsi Beef, long. dorsi Beef, long. dorsi Beef, supra spinam Beef, psoas Bull, long. dorsi Cow, long. dorsi Calf, long. dorsi Calf, semimembr.' Calf. diaphragma Calf, long. dorsi Calf, semimembr. Calf, diaphragrna

SH reagent

Original SH data"

SH (mg) per 100 gm tissue

SH (moles) per 105 gm protein

8.8 moles SH/105 gm protein ( I ) 1.36 pmolesl100 mg meat (2) 1.03 pmolesll00 mg meat (2) 1.1 . moleslmg meat (3) 69.0 mg/IOO gm meat (4) 68.0 mgl100 gm meat (4) 11.25 moles/l05 gm protein (5) 61 pg/IOO mg tissue (6) 3.1-3.6 moles/105 gm protein (6) 106.0-1 15.8 pmoles/gm protein (7) 84.9-94.0 pmoles/gm protein (7) 9.11 moleslgm protein (5) 79.8 mgllOO gm meat (8) 273 pglgm tissue (9) 79.3 mgllOO gm meat (5) 87.3 mgllOO gm meat ( 5 ) 75.61 mg/IOO gm meat (10) 69.55 mg/lOO gm meat (10) 56.59 mgl100 gm meat (10) 9.08 moles/105gm protein (10) 8.99 moles/l05 gm protein (10) 8.42 molesil05 gm protein (10)

(52.4) 45.0 34.1 36.7 69.0 68.0 (67.0) 61.0 17.0-19.2 (63.1 -68.9) (50.5-55.9) 54.2 79.8 27.3 79.3 87.3 75.6 69.6 56.6 66.1 67.1 52.7

8.8 (7.6) (5.7) (6.2) 11.2 10.9 11.3 10.3 3.1-3.6 10.6-1 1.6 8.5-9.4 9.11 ( I 1.20) (4.54) 12.1 11.8 10.4 9.3 9.1 9.1 9.0 8.4

Numbers in parentheses correspond to the following investigations: ( I ) Hamm and Hofmann (1965); (2) Krylova and Kusnezowa (1964); (3) Bolshakov and Mitrofanov ( I 970); (4) Hofmann e r al. (1974): ( 5 ) Hofmann. Bliichel, Muller, Baudisch, and Hamm (unpublished observations); (6) Hamm and Hofmann (1966b); (7) Dzinleski er al. (1969); (8) Bognar (1971a); (9) Motoc and Banu (1968); (10) Fischer, Hofmann, and Hamm (unpublished observations). 'I M. longissimus dorsi . From 9-10-month-old animals. ' From 16-18-month-old animals. After slaughter. vacuum packaged. After slaughter, air packaged. " Reaction at pH 6.8. Reaction at pH 6.0; the meat samples were minced in different ways. i Sodium nitroprusside. j M . semimembrunaceus. ' Reaction at pH 7.4. 'I

('

'

TABLE XI SH GROUPS IN MUSCLES OF VARIOUS ANIMALS, INCLUDING TEST ANIMALS

Sample. Muscle"

SH reagent

Chicken, bright m. Chicken, dark m. Chicken, breast Chicken, breast Chicken, breast Chicken, pect. major Turkey, breast Turkey, breast Rabbit, unknown Halibut, whole m. Rat, gastrocn . Rat, unknown Frog, unknown

AgN03 AgNO, DTNB DTNB PCMB PrUSS.C DTNB AgN4 iodoac ." iodoac . DTNB DTNB iodoac.

Original SH data"

10.1 x moledmg protein (1) 11.1 x moleslmg protein ( 1 ) 0.4144.564 pmole SH/mg N (8) 1.34pmoles SH/mg N (9) 4.45moles x 10-8/mg protein (2) moles x IO-Vgm tissue (3) 5.8d-9.9r 0.491mmole/gm muscle (4) 2.73mmoles X 10-4/gm muscle (4) 0.79% cysteine in protein (5) 0.81 B cysteine in protein (5) 21.5pmoles/gm tissue (6) 1.63 mmoles/100 gm tissue (7) 0.724% cysteine in protein (5)

SH (mg) per 100 gm tissue

(60.1) (66.1) 39.3-53.7 (26.5) 19.2-32.7 1624' 0.W (38.8) (39.8) 71.1 53.9 (35.5)

per

SH (moles) lo5gm protein

10.1 11.1

6.6-9.0 21.4 4.5 3.2-5.5 6.5 6.7 (11.9) (9.0) 6.0

~~~~~

Abbreviations: m. = muscle; pect. = M . pectoralis; gastrocn. = M. gasrrocnemius. investigations: (1) Bolshakov and Mitrofanov (1970); (2)Bolshakov er af. (1972); (3)Chajuss and Spencer (1962b); (4) Bowers (1972); (5) Mirsky (1936);(6)Boyne and Ellman (1972);(7)Sedlak and Lindsay (1968);(8) Miller and Spencer (1975);(9)Caidwell and Lineweaver ( 1969). Sodium nitroprusside. " 0 hours postmortem. " 72 hours postmortem. The original data are obviously not correct. iodoacetate.

* Numbers in parentheses correspond to the following

'

49

SULFHYDRYL AND DlSULFlDE GROUPS IN MLATS TABLE XI1 NONPROTEIN SH A N D GSH CONTENT OF SKELETAL MUSCLES 01. MEAT A N D TEST ANIMALS

Sample

SH reagent

Pork Beef Beef Lamb Chicken" Chicken" Chic ken Rat Rat

ASNOS AgNOz DTNB HgC12 Prusside' Prusside Prusside DTNB DTNB

Original data"

2.6 mg SH/100 gm meat (1) 1.9 mg SHil00 gm meat ( I ) 3.94 mg GSH/gm protein (2) 15 rng CySH/100 gm tissue (3) 238" pg GSH/gm muscle (4) 395 eg GSH/gm muscle (4) 0.33"'pmoIc SH/gin muscle (5) 0.07 mmole SH/100 gni tissue (6) I .96 pmole SH/gm tissue (7)

SH (mg) per 100 gm meat

2.6 1.9 (7.6) (4.1)

2.6 4.3 1.1

2.3 6.5

Numbers in parentheses correspond to the following investigations: ( 1 ) Hofmann ef ul. (1974); ( 2 ) Dub6 ef ul. (1972); (3) Oganessjan and Dschani-

I'

bekova (1958); (4) Khan and van den Berg (1965); (5) Khan and Nakamura (1971); (6) Sedlak and Lindsay (1968); (7) Boyne and Ellman (1972). " Breast muscle. Sodium nitroprusside. " These data obviously represent nonprotein SH, although not characterized as such. " Leg muscle. This value was taken from a graph I'

'

animal tissues is influenced by several factors (see Section IV, D). Nonprotein SH related to total SH content of muscles (about 65 mg/100 gm tissue) varies from 2 to 12 %. Oganesjan and Dschanibekova (1958) found 12-16% nonprotein SH. Using the corresponding data given in Tables IX-XI, the following values for the nonprotein SH content of skeletal muscles were calculated: pork, 3%. beef, 4% (Hofmann et al., 1974), and rat, 4% (Sedlak and Lindsay, 1968). Hornsey (1959) found in the filtrate of the heat-cleared "emulsions" of pork leg muscle in water only 0 . 6 4 . 7 mg SH/100 gm meat. These values are low in comparison to those given in Table XII. The nonprotein SH groups were probably partly oxidized to SS groups during the heat treatment. The nonprotein SS was estimated to be 0.8-1.1 mg SS/l00 gm meat, and the sum of the SH and SS values in meat ( I .6-1.7 mg/lOO gm) corresponds quite reasonably t o some of the SH values listed in Table XII. Comnients on Table X I U : The SS content in muscles found by several investigators varies from 6.1 to 22.9 mg SS/loO gm meat (0.5-2.0 moles SS/105 gm protein). This significant variation may be due to the different degrees of oxidation of the SH groups. On the other hand, the SS content in muscle tissue can

50

KLAUS HOFMANN AND REINER HAMM TABLE XI11 SS GROUPS" IN MUSCLE

Sample, Muscle

Original data"

SS (mg) per 100 gm meat

SS (moles) per lo5 gm protein

Pork, long. dorsi" Pork, semimembr." Pork, long. dorsi Pork. long. dorsi Beef, long. dorsi Beef, long. dorsi Beef, supra spinam Calf, long. dorsi Calf, semimembr. Calf, diaphragm

I .27 mg SS/gm protein (1) 0.95 mg SS/gm protein (1) 6. I mg SS/IOO gm meat (2) 0.83 mg SS/gm protein (3) 1 . 1 1 mg SS/gm protein (3) 7.4 mg SS/lOO gm meat (2) I .23 mg SS/gm protein (4) 0.78 mg SSlgm protein ( 5 ) 0.67 mg SS/gm protein ( 5 ) 1.19 mg SS/gm protein ( 5 )

(22.9) (17. I ) 6 .I 14.9 20.0 7.4 (22. I ) (14.0) (12.1) (2 1.4)

2.0 I .5 (0.5) I .3 1.7 (0.6) I .9 I .2 I .o I .9

" For the assay of the SS values listed, NaBH, was used to reduce SS to SH and AgNO, was used to titrate the SH groups formed. " Numbers in parentheses correspond to the following investigators: ( I ) Fischer and Hamm (1975); (2) Hofmann, Baudisch. and Hamm (unpublished observations); (3) Hofmann, Muller. and Hamm (unpublished observations); (4) Bognar (1971a); ( 5 ) Fischer. Hofmann, and Hamm (unpublished observations). '' M. longissirnus dursi. M. semimembruniireus.

''

depend on the age of the animals tested. For example Oeriu (1962, 1964) reported that the aging process in animals (guinea pig, dog, rabbit, rat) leads to an increase in the SS content in blood and in several tissues. However, the variation in the SS content in beef and veal is so great that a similar tendency cannot be recognized. It should be emphasized that in any case the SS content is only a small proportion of the cysteine plus cystine content in muscles (see Table XIV).

B . CYSTEINE PLUS CYSTINE CONTENT OF MUSCLES Cysteine is the only amino acid in proteins which contains the SH group. Therefore, the cystine content can be estimated by determination of the SH content. As cystine can be reduced (e.g., with sodium borhydride according to the reaction CySSCy 2 H + 2 CySH), the content of cysteine plus cystine (total cystine) can be determined after the reduction of the protein. Table XIV shows the values for the (total) cyctine content of muscles. These estimations were carried out either by means of amino acid analysis after hydrolysis or by using the assay of SH (SH + SS/2) after reducing the SS groups. The factors for the transformation of the different terms are given in the following schema:

+

51

SULFHYDRYL AND DISULFIDE CROUPS I N MEATS mg SH red. [gm protein]

x 0 363 -+

1

gm cystine 0 120x mole (SH + SSI2) gm protein] + 105 gm protein

[

TABLE XIV CYSTEINE PLUS CYSTINE CONTENT (CALCULATED AS CYSTINE) OF MUSCLES

Sample

Assay

Beef Beef Beef Beef Beef Beef" Beef" Pork Pork Pork Lamb

Amino acid Amino acid Amino acid SH + SSIZ" SH + SSl2" SH + SS/2b SH + SW2" SH + SSI2" SH + SSI2" Amino acid Amino acid

Original data'' 1.0-1.3 gm cystindl6 gm N (1) 1.2-1.5 gm cystine/l6 gm N (2) 1.35 % protein (3) 4.93 mg SHlgm protein (4) 12.1 moles SH/IOs gm protein (5) 10.9-12.1 moles SH/105 gm protein ( 5 ) 10.7-10.8 moles SH/IOs gm protein (5) 12.4 moles SH/105 gm protein ( 5 ) 4.424.45 mg SH red.'/gm protein (6) 1.31 70protein ( 3 ) I .35 9% protein (3)

Cystine (gm) per 100 gm protein

I .o-I .3 I .2-1.5 1.35 I .79 1.45 1.31 - I .46 I .29-1.30 1.49 1.60-1.62 1.31 1.35

Numbers in parentheses correspond to the following investigators: ( I ) Bigwood (1960, cited in Bigwood. 1972); (2) Greenwood et ul. (1951); (3) Schweigert and Payne (1956); (4)Bognar (1971a); (5) Hofmann, Miiller, and Hamm (unpublished observations); (6) Fischer and Hamm (1975). Using AgN03. I ' Freeze-dried meat. " Using NEM. 1.03 x SS [mg]. SH red. = SH

+

The sum of SH and reduced SS is called SH red (calculation see footnotee, Table XIV). The values of the total cystine content in muscles estimated by amino acid analysis (Table XIV) vary from 1 .O to 1.35 gm/l00 gm protein, and the values obtained by determination of the SH groups vary from 1.29 to 1.79. The higher values for SH groups in the nonhydrolyzed protein may be explained by the fact that during hydrolysis cystine is partly destroyed (see Section 11, C ) , and in this respect the results obtained with myofibrils may be interesting. After reduction of the myofibrils with NaBH4 at different temperatures (20"-70°C), 4.26-4.43 mg SH/gm protein was determined, corresponding to I .55-1.60 gm total cystine/lO gm protein (Hofmann, 1964). On the other hand, the amino acid analysis after acid hydrolysis of the same material with 6 N HCl led to a value of 1.2 gm cystine/l00 gm protein, again demonstrating the detrimental effect of protein hydrolysis. Consequently, the most probable mean value of the cysteine plus

52

KLAUS HOFMANN AND REINER HAMM

cystine content of beef, pork, and lamb muscle seems to be 1.5 gm cystinef100 gm protein. This value corresponds to 12.5 moles SH/105 gm protein or 4.1 mg SH/gm protein. C. SH CONTENT OF ORGANS There is a great confusion in the literature in presenting SH values. In the earlier papers, very often the symbols pg, 7 , and u were used which should mean gm. However, the corresponding values would be too low by a factor of 103 in comparison to the values given in the more recent literature or to all the values for muscle tissue (Table IX-XI). Therefore the mentioned symbols were interpreted as gm. The total SH groups content in liver is listed in Table XV, and in several other inner organs in Table XVI. The nonprotein SH content in these organs are listed in Table XVII. The following comments are provided for these tables. Comments on Table XV: The SH content of the liver of pig, rabbit, mouse, and rat varies from 23.4 to 98.6 mg SH/lOO gm tissue. It should be mentioned that the average SH content of liver (47.2 mg SH) is lower than that of muscles (65 mg SH). This is somewhat surprising, but liver is rich only in nonprotein SH TABLE XV ESTIMATES OF SH GROUPS IN LIVER OF MEAT A N D TEST ANIMALS

Sample

SH reagent

Original SH data","

Rabbit Rabbit Mouse Mouse Mouse Mouse Rat Rat Rat Rat Rat

Ferricyanide CMPAN' FCMB PCMB Ferricyanide CMPAN

46.5 ?/I00 mg (I) 45.2 ? / I 0 0 mg ( I ) 23.4 ull00 mg (2) 30-35 $100 mg (3) 31.3-52.9-y/lOO mg (1) 35.6-59.4y/IOO mg (1) 55.4 mg/i00 gm (4) 2.03 mmole/100 gm (5) 29.8fimoleslgm (6) 35.648.4?/I00 gm ( I ) 36.7-53.8y/IOO gm (I)

DTNB DTNB Ferricyanide CMPAN

SH (mg) per 100 gm tissue

46.5 45.2 23.4 30-35 31.3-52.9 35.6-59.4 55.4 67.0 98.6 35.6-48.4 36.7-53.8

Numbers in parentheses correspond to the following investigators: ( I ) Flesch and Kun (1950);(2)Calcutt and Doxey (1959);(3) Calcutt (1961);(4)Bhattacharya (1959);(5)Sedlak and Lindsay (1968);(6)Boyne and Ellman (1972). * Related to tissue wet weight. However, some of the given dimensions are obviously not correct (see preliminary remark to Section 1V.C). CMPAN = I -(4-chloroniercuriphenylazo)-naphthol-2. a

53

SULFHYDRYL AND DlSULFlDE GROUPS I N MEATS TABLE XVI ESTIMATES OF SH GROUPS IN INNER ORGANS" OF TEST ANIMALS ~~

Sample, Organ

SH reagent

Original SH data".'

Rat. heat Rat. heart Rat. heart Rat, h e m Mouse. heart Mouse. heart Rat. kidney Rat, kidney Rat, kidney Mouse. kidney Mouse. kidney Mouse. kidney Rat. brain Rat. brain Rat, brain Mouse. brain Mouse, brain

DTNB Ferricyan ide CMPANd Ferricyanide CMPAN &NO, Ferric yanide CMPAN Ferricyanidc CMPAN PCMB &NO, Ferricyanide CMPAN Ferricyanide CMPAN

31.2 nig/100 grn (I) 1.54 mmoles/lOo grn (2) 20.1 y/lW mg (3) 48.9y/100 rng (3) 17.2 yll00 mg (3) 41.9 y/100 rng (3) 48.3 rng/100 gm (I) 25.2 $100 mg (3) 56.4y/IOO rng (3) 20.4y/lOO mg (3) 54.3 y/l00 rng (3) 9.9 v/lOO rng (4) 4.9prnoleslgrn ( 5 ) 15.6y/lOO mg (3) 59.2y/lOO mg (3) 14.0y/100 mg (3) 52.7 y/lW rng (3)

~

SH (nig) per 100 gni tissue 31.2 50.9 20.I 48.9

17.2 41.9 48.3 25.2 56.4 20.4 54.3 9.9 16.2 15.6 59.2 14.0 52.7

For liver. see Table X V . correspond to the following investigators: ( I ) Bhattacharya (1959);(2) Sedlak and Lindsay (1968);(3) Flesch and Kun (1950);(4)Calcutt and Doxey ( 1959);(5)Gabay et ul. ( 1 968). See Note b to Table XV. CMPAN = 1 -(4-chloromercuriphenylazo)-naphthol-2.

* Numbers in parentheses

compounds (see comments on Table XVlI). The SH contents in liver of the different animals, as far as they were cstimated by the same author, do not diffcr considerably. Commmfs on Table X V I : The SH values listed in Table XVI are very different from each other due to the use of different SH reagents. The following ranges were found for rat and mouse: heart 17.2-50.9, kidney 9.9-56.4, and brain 14.0-59.2 mg SH/100 gm tissue. It is obvious that the SH content of these inner organs is generally lower than that of muscles. In fact, even the maximum value for organs is lower than the average value for muscles (65 mg SH/lOO gm tissue). Comments on Table XVII: The highest nonprotein SH contents for all organs were found in liver (average value 22.7 mg SH/lOO gm tissue) and the lowest in heart, stomach, diaphragm, and lung (4.9-8.6 mg SH/100 gm tissue). The values for kidney lie in between. It is noteworthy that the nonprotein SH content of the organs of trained animals is generally higher than that of untrained ani-

54

KLAUS HOFMANN AND REINER HAMM TABLE XVII NONPROTEIN SH AND GSH CONTENT IN ORGANS OF TEST ANIMALS

SH (mg) per ~

Animal -

Pig Rabbit Mouse Rat Rat Rat Rat Rabbit Rat Rat Rat Rat Dogd Dog' Rat Rat Dogd Dog' Dogd Dog' Dogd Dogp Dogd Dog"

Organ

SH reagent

Liver Liver Liver Liver Liver Liver Liver Liverb Kidney Kidney Spleen Brain Brain Brain Heart Heart Heart Heart Stomach Stomach Diaphragm Diaphragm Lung Lung

PCMB PCMB PCMB PCMB AgNO 3 DTNB DTNB DTNB &NO, PCMB PCMB DTNB lodine Iodine DTNB Iodine Iodine Iodine Iodine Iodine Iodine Iodine Iodine

Original data" 21.4 pg/IOO mg ( I ) 25.2 pg/lOO mg ( I ) 32.4 pg/IOO mg ( I ) 26.4 pg/lOO mg (1) 26.4 mg SH/100 gm tissue (2) 0.10 mmole SH/100 gm tissue (3) 6.00 pmoles SH/gm tissue (4) 8.1 pmolesc/gm tissue ( 5 ) 1 I .3 mg SH/100 grn tissue (2) 18.2 pgll00 mg ( I ) 9.6 pg/l00 mg (1) 2.6 pmoles SH/gm tissue (6) 45.6 mg % GSH (7) 56.6 mg % GSH (7) 0.22 mmol SH/lOO gm tissue (3) 7.2 mg SH/100 gm tissue (2) 54.0 mg % GSH (7) 69.3 mg % GSH (7) 49.5 mg % GSH (7) 53.9 mg % GSH (7) 56.8 mg % GSH (7) 65.1 mg % GSH (7) 4.57 mg % GSH (7) 55.2 mg % GSH (7)

100 gm tissue

21.4 25.2 32.4 26.4 26.4 3.3 19.8 26.8 11.3 18.2 9.6 8.6 4.9 6. I 7.3 7.2 5.8 7.5 5.3 5.8 6. I 7.0 4.9 5.9

Concerning the dimensions of the original datas see Note b to Table XV. Numbers in parentheses correspond to the following investigators: ( I ) Calcutt and Doxey (1962); (2) Bhattacharya (1959); (3) Sedlak and Lindsay (1968); (4) Boyne and Ellman (1972);( 5 ) Ellman (1959);(6) Gabay et al. (1968);(7) Wachholder and Uhlenbrook (1935). * Extracted with 5% TCA. The values for water and alcohol extracts were much lower. The original dimension (mmoles) is obviously not correct. Untrained. ' Trained.

mals. Since the nonprotein SH content in liver, kidney, and heart is markedly higher than the average nonprotein SH content in muscles, the relation of nonprotein SH to total SH is higher in these organs than in muscles. Bhattacharya (1959) showed that rat liver contains 48% and that both kidney and heart contain 23% nonprotein SH (related to the total SH content). By summarizing the SH contents given in Tables IX-XVII, we can conclude that in most cases the total SH as well as the nonprotein SH contents of the same

SULFHYDRYL AND DISULFIDE GROUPS IN MEATS

55

organs of different species vary less than the SH contents of different organs of the same species.

D. FACTORS INFLUENCING THE SH CONTENT OF RAW MEAT In this section, some natural factors that can possibly influence the SH content of meat will be discussed. The influences of processing, freezing, and other treatments are discussed separately. 1. Postmortem Aging

Chajuss and Spencer (l962b) reported a rapid decrease in the SH group content of chicken muscle during the development of rigor mortis. Gawronski et al. ( 1 967) also observed a decrease in the SH content and in the ratio of SH to SS in excised chicken breast muscle during the first hours postmortem. After Pavlovskij and Grigoreva (1966) and Golovkin and Korzhemanova (l973), the number of SH groups in muscle decreases during rigor and then increases again during relaxation. Motoc and Banu (1968) reported that the SH content of muscles would decrease due to a denaturation of the myosin. On the other hand, Kolodziejczyk (1965) found an increase of free SH groups in pork and beef during aging. These apparent contradictions may be due to differences in the availability of the SH groups based on the use of different types of reagents in the methods of measurement. (More work has to be done in order to elucidate the role of SH groups in the presence of rigor mortis and aging.) In order to find out whether the preparation of tissue was responsible for this variation in results, Miller and Spencer (1975) investigated aged samples of chicken muscle prepared in the different ways described by the other investigators. Analysis of variance showed that the SH concentration did not change significantly with aging time within any of the homogenate preparation methods. Caldwell and Lineweaver (1969) have found that no change in the SH content of chicken breast muscle occurs during postmortem aging. Furthermore, determination of the SH content with AgNO, has indicated that the postmortem storage at +2"C of beef and pork for up to 1 1 days does not change the SH content of muscle tissue (Hofmann and Schael, 1966; Hofmann et al., 1969). Further studies with different skeletal muscles of pork and beef from numerous animals have confirmed this result (Hofmann, 1971d). Finally, Hay et al. (1972, 1973) found that the aging of chicken muscle fibrils was not accompanied by a decrease in the total SH content; this indicates that there has been no oxidation of SH groups throughout the aging process. It should be mentioned that significant changes in the SH content were never found until at least 5 days after preparation in numerous cases when the SH content of myofibrils of pork and beef muscles was controlled during cold storage (Hofmann and Hamm, unpublished observations).

56

K L A U S H O F M A N N A N D REINER H A M M

2. Other Factors a. Meat Quality. Fischer and Hamm (1975) investigated the influence of the quality of pork on the number of SH groups which react with NEM (easily available SH groups) and with AgNO, (total SH groups content). The so-called pH, value, 45 minutes postmortem, and the water-holding capacity of the meat were measured as criteria of meat quality. The number of easily available SH groups decreased significantly with increasing PSE (pale, soft, exudative) conditions, i.e., with an increasing rate of glycolysis postmortem, but no correlation between total SH or SS groups content of the tissue and PSE properties was observed. In accordance with these results, Usunov and Zolova (1976) reported that rapidly reacting SH groups were drastically reduced in PSE muscle. It seems possible that the decrease in these easily available SH groups is attributable to the masking of the SH groups of myofibrillar proteins by sarcoplasmatic proteins, precipitated at low pH values on the myofibrils (Fischer and Hamm, 1975). Bendall and Wismer-Pedersen ( I 962) give a similar explanation for the finding that in PSE muscle a smaller amount of charged groups were titratable than in normal muscle.

b. Variation in rhr GSH Content. The content of SH glutathion (GSH) in muscles and organs is not constant but is, in vivo, depending on numerous metabolic factors which have been comprehensively discussed in the review of Santavy (1965). Therefore, it may be sufficient to summarize only a few of the most important influences on the GSH content and, consequently, the total SH content of tissues. GSH activates several enzyme systems which participate in the metabolism of carbohydrates. It is also important for aerobic glycolysis and is a cofactor of some dehydrogenases. The ATPase activity of myosin in the muscle is increased by GSH. Furthermore, GSH may play an important role in maintaining the activity or in the reactivation of several SH enzymes. Thus it is not surprising that the GSH level in muscles and organs is increased by training. During stress the GSH content in muscles decreases (more with untrained animals than with trained animals). The GSH level in muscle and liver is influenced by the vitamins C and BP,which are involved in the carbohydrate metabolism. c. fnfluencr ofAge. With the increasing age of animals, an accumulation of disulfide groups in tissues has been observed (Oeriu, 1962; Harisch and Schole, 1974). Lastovskaya (1969) reported that the SH content of rat liver decreased slightly during the first 12 months of age and significantly during 1-2 years of age. However, Harisch and Schole (1974) found that the GSH content of rat liver increased continuously with age, causing a rise in SH and SS groups as well.

SULFHYDRYL AND DISULFIDE GROUPS I N MEATS

57

The reactivity and the SH content of myogen A of rats were reported to increase with age, especially in young animals. This protein showed the highest enzyme activity in older rats (Goldshtein and Khilko. 1969). A significant age associated decline in the SH content of serum albumin has been demonstrated in both men and rats (Leto er a l . , 1970). I t seems that the effect of age on the SH and SS content depends on the kind of organs and proteins investigated as well. Some results concerning beef are mentioned in Section IV,A. An increased synthesis of GSH has been shown to be associated with growth hormones (Shacter and Law, 1956). SH groups are involved in cell division and play an important role in carcinogenesis (for review, see Harington, 1967). d. Deboned Meat. The production of volatilc sulfur compounds during storage in mechanically deboned poultry meat has been investigated by O’Palka (1973). In this study he found that cysteine destruction was closely correlated with methyl mercaptane production. This was an unexpected result, because Cascy et al. (1965) and Grill et u f . (1967) have shown methionine to be a precursor of methylmercaptane. The formation of volatile S-containing compounds (I6 were estimated) during storage of deboned meat is probably due to bacterial activities, because many types of microorganism are present in poultry products as a result of contamination (Kraft, 1971).

e . Unsaturated Fatty Acids. Robinson (1966) reported that SH groups are able to react with unsaturated fatty acids, the SH group probably being added to the double bond. Furthermore, SH groups can be oxidized by fatty peroxides, which may be formed during the storage of meat. f. T r u c ~ of ~ sHeavy Metals. Finally the possibility of traces of heavy metals (which are absorbed by meat animals from the environment) decreasing the SH content of meat will be examined. This question may be considered for lead, one of the widest spread trace metals, which can react with SH groups: 2 R - SH

+ Pb’+

-2H’

Pb (RS),

The maximum Pb levels in beef, veal. and pork were reported (Holm, 1976) to be 0.53 I , 0.372. and 0.158 ppm, respectively. The highest values for beef and calf liver were 0.40 and 0.26 ppm Pb, respectively. As a comparison with the SH content of meat shows, these Pb levels are very low. For instance. 0.5 ppni Pb is equal to 0.05 mg Pb in 100 gm meat, which contains about 65 mg SH. Consequently, such Pb2+ levels cannot bind more than 0.024% of the SH groups in muscle (calculated in moles). Therefore, traces of lead which can occur in meats are neglegible in respect to the determination of SH groups. The same may be valid for traces of other heavy metals. An exception is the presence of Cu2+ ions,

58

KLAUS HOFMANN A N D REINER HAMM

which are known to catalyze the autoxidation of SH groups, and which, therefore, can have a strong influence on the results of SH determination.

V. INFLUENCE OF PROCESSING ON THE SH AND SS GROUPS OF MEAT A . INFLUENCE OF HEATING Heating of meat is accompanied by changes in appearance, smell, taste, texture, and nutritive value. The cysteine and cystine moieties of proteins are particularly involved in these alterations. Therefore, the changes in and possible reactions of SH and SS groups in meat proteins during heating must be considered. The thermal formation of volatile sulfur compounds, which are important flavor components, will also be discussed.

I.

Effect of Heat Denaturation on the Availability of SH Groups

Biochemists define denaturation as a change in the specific steric conformation of a protein, i.e., a change in the secondary and tertiary structure without a chemical modification of the amino acids (Fasold and Turba, 1959). Thus, denaturation is a physical process, not a chemical one. One has to be careful in using the term “denaturation” instead of “heating,” because stronger heating results not only in denaturation but also in chemical modifications of the proteins such as reactions of the functional groups and the cleavage of covalent linkages. According to the definition of denaturation mentioned above, the oxidation of SH to SS groups and the reduction of SS to SH groups should not be called “denaturation” because these reactions are chemical modifications; this is also true for the inactivation of SH enzymes caused by auto-oxidation. Therefore, the oxidation of SH-groups by heating will be discussed in the next chapter. Dry heating of myofibrils for 30 minutes at temperatures from 30” to 70°C under both nitrogen and air resulted in an irreversible increase in the SH groups reacting with NEM at pH 6.0, as shown by Fig. 4 (Hamm and Hofmann, 1965). Using the same method for measuring the SH-groups, Schrott (1974) also observed a substantial increase in the available SH groups in actomyosin (from 1.41 to 2.41 mg SH/gm protein) when it was heated to 65°C. The SH increase was explained by the fact that NEM reacts only with a part of the SH groups in the native meat protein and that heating causes an unfolding of the protein molecule during heat denaturation (Haurowitz, 1950), making more SH groups available for NEM (Hamm and Hofmann, 1965). The release of reactive protein SH groups, which were hidden within the native folded protein

SULFHYDRYL A N D DISULFIDE GROUPS I N MEATS

59

FIG. 4. Effect of heating (30 minutes) myofibrils on the amount of SH groups reacting with NEM at pH 6.0 (Hamm and Hofmann, 1965).

structure by denaturation. was first discussed in the case of ovalbumine (Anson, 1945). An increase in the heating temperature to 120°C did not cause a further increase in the available SH groups, even though only about 70% of the SH groups are available to NEM after heating to 70°C. The total number of SH groups is obtained by reaction with AgNO, (see Fig. 4 and Table XVIII). As Fig. 4 shows, oxygen has no remarkable effect on the number of SH groups available for NEM in raw or mildly heated meat. The role of SH groups in the temperature-induced denaturation of muscle proteins was investigated by Jacobsen and Henderson ( 1 973). With actomyosin they found, after heating the protein to 60°C. a marked irreversible increase in the sulfhydryl groups which were titratable with PCMB. These authors did not give any explanation for this increase; but there is no doubt that it may also be due to an unfolding of the protein molecules induced by heating. TABLE XVIII SH LEVELS IN MYOFIBRILS AFTER 30 MINUTES HEATING"."

SH reagent

30°C

50°C

70°C

AgNOa DTNB NEM

8.5-9.0

8.4-9.0

8.5-9.0

6.04.5 3.5

6.5 5.0

8.5 7.0

"

In moles SH/105 gm protein

* After Tinbergen (1970).

60

KLAUS HOFMANN A N D REINER H A M M

Tinbergen (1970) studied the influence of heating myofibrils (from 30" to 70°C) on the number of SH groups reacting with different SH reagents. His results are in good agreement with those obtained by Hamm and Hofmann (1965); they demonstrate that the number of SH groups which react easily with NEM and DTNB increases with increasing temperature, whereas the total number of SH groups (reacting with AgNO,) remains unchanged (see Table XVIII). The heating of whole meat in a superhigh frequency electromagnetic field to an internal temperature of 65°C also led to an increase in the number of SH groups (Malyutin, 1969). Frying meat decreased the total SH content but increased the number of easily reacting SH groups titratable with CH3HgN03. using nitroprusside as an indicator (Dworschak, 1969). Increases in the available SH groups were also found after the mild heating of pork (Randall and Bratzler, 1970), turkey breast muscle (Bowers, 1972), and frog sartorius muscle (Kovaleva, 1967). Contrary to the results reported here, Dub6 (1969) found a decrease in the number of available SH groups using Ellman's reagent after cooking myofibrillar extracts of beef at temperatures between 60' and 90°C. He suggested that this disagreement might be due to the fact that in these experiments the proteins were already denatured by urea, and thus heating did not allow any further conformational change that would have made more SH groups of the molecules available to the reagent. However, not only was there no increase, but rather a decrease in the SH groups (about 6% at 60°C and 16% at 70"), which was explained by the assumption that the SH groups could be oxidized into SS groups. In the case of heating the meat to 70"C, this explanation does not agree with the results of Hofmann (1964) (see Fig. 5), Samejima et al. (1969), Tinbergen (1 970) and Bognar (1 97 I a), who showed that there is no decrease in the total SH content in meat proteins after heating to that temperature. Samejima ef at. (1969) found that neither disulfide reducing nor SH-blocking reagents prevented the heat coagulation of myosin. This supports the conclusion of Hamm and Hofmann (1965) that the heat coagulation of myofibrillar proteins is not due to an oxidation of SH to SS groups but to an intermolecular association of other sidechains of the protein molecules. However, we have to concede that the protein SH groups in different meats and myofibrils may not always react in the same way. It is well known that traces of certain heavy metals are able to catalyze the autoxidation of SH groups. Hence, differing contents of such trace elements in meat could result in an oxidation of SH groups to a different extent (for further discussion of the previous results see next section). Malyutin ( 1 969) found a decrease in the SH groups in meat after heating to 85"C, suggesting an aggregation of proteins. SH groups have indeed been shown to be involved in this heat aggregation of proteins (Connell, 1960a; Jaenicke, 1965a,b). The reaction

SULFHYDRYL AND DISULFIDE GROUPS IN MEATS

61

mechanism is suggested to be a SH-SS exchange according to the following equation (Lumper and Zahn, 1965):

HS-Prot/i

f

' S

HS-Prot

__

SH HS-Prot-SS-Prot /

However, as this reaction demonstrates, the SH-SS exchange would not lead to a change in the number of SH and SS groups. The decrease of SH groups established after heating does not necessarily indicate protein aggregation caused by SH-SS exchange. But it may be that the SH groups formed are less reactive than those originally present. No investigations have been attempted as yet to test this hypothesis. It should be pointed out that denaturation alone is not able to increase the number of S S groups. The formation of SS groups is, in any case, the result of an oxidation of SH groups. Neither can the reverse reaction, namely, the formation of SH from SS, be caused by denaturation itself alone, as has been postulated previously (Grau, 1968). It may be concluded that heat denaturation of meat proteins leads to an increase of available SH groups, depending on the temperature applied. There obviously exists a wide temperature range rather than one special temperature in which SH groups, previously masked in the native state, are exposed. This finding is in accordance with denaturation therrnoprofiles of beef muscle tissue which show that molecular changes occur between 49" and 94°C (Karmas and De Marco, 1970). The total amount of SH groups in meat with AgNO, is not effected by heating up to 70°C (Hofmann, 1964; Hamm and Hofmann, 1965; Kovaleva, 1967;

Lob 5 0 5 0 7 0 9 0

1X)OC

FIG. S. Effect of heating myofibrils (30 minutes) on the amount of SH groups reacting with AgNO, at pH 7.4 (Hofmann, 1964).

62

KLAUS HOFMANN AND REINER HAMM

Tinbergen, 1970; Bowers, 1972), whereas at higher temperatures the SH content falls (Fig. 5). The curve in Fig. 5 is quite similar to the temperature-SH curve for actomyosin presented by Hamm and Hofmann (1965; in Fig. 2 of the publication the word “myofibrils” should be corrected to read “actomyosin”). The decrease of SH at higher temperatures is due to oxidation and will, therefore, be discussed in the next section.

2 . Influence of Heating on the Total SH and SS Content Dube (1969) observed that the SH content of myofibrillar extracts of beef decreased on cooking at temperatures up to 90°C. The SH values of the heated and then reduced samples obtained after treatment with NaBH, were lower than the SH values of the raw samples. It is not clear whether the reduction of the disulfide groups was incomplete or if other oxidation products which cannot be reduced by NaBH, were formed during heating. Krylova and Kusnezowa (1 964) heated pork and beef for 30 minutes at 75” to 80°C. Using an amperometric titration with AgNO,, they found a decrease in the SH groups ranging from 27 to 29%. Figure 5 also shows that heating to temperatures higher than 70°C decreases the number of SH groups in muscle proteins. In the presence of air, the SH content dropped more than when exposed to pure nitrogen. At 120”C, the decrease under air was 40%, under nitrogen 25%;at the same time, the SS content rose by 36% and 20% respectively. These changes evidenced that the SH groups were mainly oxidized into SS groups (Hofmann, 1964; Hamm and Hofmann, 1965). Since the oxidation also occurred with the exclusion of atmospheric oxygen, it was suggested that oxidation in the presence of nitrogen is due to residual molecular oxygen included in or bound to the sample, from which it can hardly be removed. In experiments of Schweigertet al. (1949), the total cystine content of pork and lamb has proved stable during normal cooking, but not during heating at 120°C. As is evident from percentages mentioned above, there is a difference between the decrease in SH and the increase in SS (about 5%). which means a loss of the total cystine (CySH CySSCy) content. After heating the samples for 5 hours, the deficit reached 26%and 13% under air and nitrogen, respectively (Hamm and Hofmann, 1965). Bognar (1971a) found a loss of as much as 33% total cystine after the heating of beef for 1 hour at 120°C. He was able to show that this loss was due to the formation of cysteine acid. That the decrease stated by Bognar was more pronounced than the decrease in the case of myofibrils may be attributed to the different experimental conditions: The beef was heated in water in which the oxidation was probably more effective than in the dry-heated myofibrils. The formation of lanthionine, which is also discussed as a possible product of

+

SULFHYDRYL AND DlSULFlDE GROUPS IN MEATS

63

cystine destruction, seems to be unlikely under the conditions used because it occurs only in a basic medium (Hupf and Springer, 1971). Marchenko (1968) found that sterilization of beef and lamb at 120°C lowered the SH content by 62% and 50%, respectively, whereas the SS content increased for 72% and 62%, respectively. However, this hardly explains why in this case the increase in SS was higher than the decrease in SH. Bem rt al. ( I 970) and SusjC rt al. ( 1 974) extensively investigated the influence of the canning of meat which contained various amounts of nitrite, nitrate, and ascorbic acid. It was to be expected that nitrite affects the SH content because of the possible formation of nitrosothiols (Mirna and Hofmann, 1969). Canned meats with the highest addition of nitrite (0.35%) exhibited both the lowest SH content and highest SS content. Nitrate and ascorbic acid did not influence the SH content. The SH content of canned meats which were sterilized at 1 10”-115”C was much lower than that of meat pasteurized at 76°C. The total cystine content of the high-temperature canned meat products was also reduced; consequently, the nutritive value of the meat protein was lowered (see also Section V, A, 3). During the storage of both the sterilized and the pasteurized meats, the SH content decreased continuously, demonstrating that the reaction between the SH groups and nitrite advanced. Finally, a paper published by Khan and van den Berg (1965) in which the use of incorrect terms caused some misunderstanding will be briefly discussed. The authors talk about “sulfhydrylgroup content of muscle proteins,” but it is evident from the procedure described in a previous paper (Khan et a l . , 1963) that in all cases the SH group assay was carried out in an extract of muscle obtained by using 2.25% metaphosphoric acid. Such an extract. free of protein, contains only about 3% (see Section IV) of the total meat SH, mainly as GSH. Therefore, the statement of these authors that the cooking of chicken muscle decreased the SH content of “muscle protein” by about 50% is not correct. 3 . Influence clf the Thermal Destruction of Cysteine Plus Cystine (Total Cysrine) on the Nutririw Value of Meat Protein As was shown in the previous section. heating induces losses of total cystine when meat is heated to high temperatures. This has already been demonstrated in previous work by means of amino acid analysis (Beuk et ul., 1948; Donoso et ul., 1962). However. hydrolysis of the proteins causes losses of total cystine as well. It was later shown (Bognar, 1971a, b) that heating beef for I hour at 120°C caused an average loss of 28.3% methionine and 15.9% total cystine. The decrease in the other amino acids ranged from 5.0 to 8.9% (the amino acids of the meat broth were included). The relatively high losses of the sulfur amino acid content clearly demonstrates the sensitivity of these amino acids to wet heating.

64

KLAUS HOFMANN A N D REINER HAMM

Bjarnason and Carpenter (1970) studied the mechanism of heat damage in proteins using bovine plasma albumin as a model. The protein, which contained 14% moisture, lost 50% cystine when heated for 27 hours at I 15°C. Miller er al. (1965) found a similar loss (60%) for vacuum-dried cod after heating for 27 hours at 1 16°C. Methionine and cysteine plus cystine are involved in many essential functions of every living cell. This should be noted with respect to the fact that the deficiency of the sulfur-containing amino acids in human diets is a critical problem of worldwide importance (Allaway and Thompson, 1966). The nutritive value of meat protein is limited by its content of methionine and cysteine plus cystine (Donoso et a l . , 1962; Hofmann, 1966b). Therefore, any damage of total cystine diminishes irrevocably the nutritive value of meat protein (see Section I). In the heat treatment of meat during cooking and processing, the thermal sensitivity of the sulfur-containing amino acids must therefore be taken into consideration. Time and temperature of heating should not exceed certain limits (Hofmann 1966b, 1 9 7 2 ~ )There . is a formula which states that the time necessary for killing microorganisms can be shortened to a tenth when the temperature is increased by 10°C (Beuk et al., 1948). On the other hand, the rate of chemical reactions (including the damage of amino acids) is only increased about 3-fold by the same increase in temperature (van't Hoff's rule). Thus, heating the meat at a higher temperature for a shorter time (high-short heating) is preferable to heating it longer at a lower temperature. Moreover, in order to reduce the time necessary for sterilization, the cans should not be too large and should be as flat as possible. In the presence of fluid constituents (as in cans containing goulash, beef and pork in their own juices, sausages in brine, etc.), the time of heating can be shortened by rotating the cans (rotation sterilization; Rievel and Reuter, 1955; Heidtmann, 1966; Wirth, 1967; Christiansen, 1968). The quality of canned products can be improved by estimating and using the so-called F-values (Takacs ef al., 1969; Heidtmann, 1970). A loss of total cystine may be compensated by the addition of cysteine (N. N., 1970). It may also be of interest to note that it is possible to introduce SH groups into proteins and, therefore, to increase their SH content (Schoberl, 1948). Perhaps in this way the nutritive value of proteins may be improved. 4. Release of Hydrogen Sulfide During Heating

During the heating of meat numerous volatile compounds which contribute to the formation of meat flavor are split off (Hornstein et a l . , 1960; Brennan and Bernhard, 1964). Some of those volatiles are sulfur-containing compounds; the simplest and most investigated one is hydrogen sulfide. In low concentrations,

SULFHYDRYL A N D DlSULFlDE GROUPS IN MEATS

65

H,S is usually associated with high mean food acceptance scores (Olson rt d., 1959). For the determination of H,S which is released during heating, Hamm and Hofmann (1965; for details, see Hofmann, 1964, 1967) used a modified method of Marbach and Doty ( I 956). The H,S passes into a trap containing NaOH and is determined by photometric measurement (670 nm) of the blue color which develops after reaction with p-aminodimethylaniline and FeCI, in HCI. Optimum conditions for the formation of the dye and its measurement are realized by a moderate excess of FeCl, and by an HC1-concentration of I-3%. Under these conditions, the solution of the dye formed is stable (Hofmann and Hamm, 1967a). For estimation of H,S formed during the production of canned meat, test tubes, called Drager-Riihrchen, have proved useful (Bloeck Y t ul.. 1970). The odor threshold of this compound in water is as low as 10 ppb (Pippen and Mecchi. 1969). In low concentration, H,S has a favorable effect on the meat aroma and probably contributes to the flavor of all heated proteinaceous foods such as chicken, beef, fish, eggs, and milk. At higher concentrations. the objectionable odor of H,S is detrimental to the flavor (Johnson and Vickery, 1964). Fraczak and Pajdowski ( 1 955) first studied the development of H,S during the thermal processing of meat. Their results suggested that 80°C might be the temperature at which the formation of H2S in remarkable amounts begins. The aniount of H2S produced increases drastically with increasing temperature (Fraczak and Pajdowski, 1955; Parr and Levett, 1969). Hamni and Hofmann (1965) showed that the release of H,S induced by heating myofibrils increases exponentically with rising temperature (Fig. 6). The amount ofH,S formed by heating the myofibrils at 120°C for 30 minutes ranged from 16.8 to 18.7 p g H,S/gm protein. This is almost the same amount as was formed during the heating of total muscle tissue (18.4 to 19.3 pg). Therefore, at least 90% of H2S released during heating originates from myofibrillar proteins. This result agrees with the findings of Mecchi ef a / . (1964). With adipose tissue, the reverse was found; i.e., approximately 2% times as much H,S was evolved from the water soluble as from the water insoluble fractions (Pepper and Pearson, 1969). It was demonstrated that, after blocking the SH groups by Ag+ or NEM, myofibrils did not release detectable amounts of H,S during heating (Hamni and Hofmann. 1965).This evidenced that hydrogen sulfide originates from the protein SH groups rather than from disulfide groups or methionine. The same conclusion was reached by Parr and Levett (1969) in the case of chicken meat, whereas Fraczak and Pajdowski (1955) and Mecchi c’t a / . (1964) postulated that H2S might be split off from cysteine and cystine as well. An additional source of the development of H,S induced by the heating of meat is thiamin (Dwivedi and Arnold. 1971). The reported decrease of the SH content due to the formation H2S is relatively small

66

KLAUS HOFMANN AND REINER HAMM

0

70

90

110 120

O C

FIG. 6 Effect of heating (30 minutes) on the formation of H,S from inyofibrils (Hamm and Hofmann. 1965).

(2 to 3%). Therefore, it is not surprising that maximum H,S production at 120°C was reached only after more than 15 hours (Lendvai et al., 1973). The oxidation of SH groups during the heating of meat does not prevent the development of H,S, because enough residual SH groups are still present. Parr and Levett (1969) reported a disappearance of free H,S from freshly cooked meat left standing, which was mainly due to oxidation by atmospheric oxygen. After Sowa (1968), the amount of H,S does not increase significantly when the temperature is extended above 120°C. During the heating of meat with microwaves (27.4 mHz) the H,S formation was reduced (Sowa, 1968). Bloeck et al. (1970) studied the production of H2S during sterilization of fish (sardines). They found the same curve given in Fig. 6. The transformation of this curve into a half-logarithmic coordinate system resulted in a straight line, enabling the prediction of the amount of H,S formed, whereupon the calculation was brought about analogously to determine the F-value. It was found that using the “high-short heating” procedure limited the amount of H2S formed during sterilization (Bloeck er al., 1970). In general, the release of hydrogen sulfide during the sterilization of meat is a serious problem. Possible disadvantages are corrosion of the cans, discoloration of the cans (marbling) and of the content, and an unfavorable or even offensive smell when the can is opened. The marbling (see Andrae, 1969; Dahlke, 1969; Gruenwedel and Patnik, 1971) is due to the formation of iron sulfide or tin sulfide. In canned meats, which are only pasteurized, the activity of surviving microorganism may contribute to the H2S formation (Baumgartner and Baum, 1960). Cheftel (1958) established that metal ions such as Fe2+, Fe3+, A13+, Sn2+,and Sn4+,which may be exposed by corrosion effects, are able to accellerate catalytically the release of H,S. Furthermore, the pH value has a strong effect on the formation of H,S caused not only by heating but also by

SULFHYDRYL AND DISULFIDE GROUPS I N MEATS

67

bacterial action. High pH values support the release of H,S during the heating of meat (Johnson and Vickery, 1964; Krylova and Marchenko, 1969). Figure 7 shows the amount of H2S released from heated meats as influenced by the pH value. Irrespective of the type of meat used (mutton, beef, or pork), or of whether or not the pH was adjusted by artificial or natural means, all H2S values were located on the same curve, suggesting that the same mechanism was acting in each case. Muscles from animals in poor condition sometimes contain only small amounts of glycogen at the time of death; consequently, only a small amount of lactic acid arises postmortem, and, therefore, the ultimate pH of the meat is high. Such meat produces more H,S during heating than does the meat from normal animals. A decrease in carcass grade is also accompanied by an increase of both pH and H,S production (Johnson and Vickery, 1964). The addition of polyphosphate increased the concentration of H,S in canned broiler meat (Rao et al., 1975), a result which could not be attributed to the increase in pH caused by the addition of phosphate. Furthermore, it was found that the heating of meat with a high fat content produced significantly more H,S than the heating of lean meat (Kunsman and Riley, 1975).

PH

FIG. 7 . The effect of variation of pH on H,S content of volatilea produced by heating meat (Johnson and Vickery, 1964). (0. mutton; 0 , beef; W . pork.)

In the H,S assay, the hydrogen sulfide formed by heating of meat usually is transferred into the solution of the reagent. Marchenko and Kosenjasheva (1974) developed a procedure for the separate determination of the levels of hydrogen sulfide and mercaptans which are volatilized during meat cooking and those which remain in cooked meat. They found that, after heating meat to 80°C. the majority of the volatile sulfur compounds remained in the cooked meat. The hydrogen sulfide content in cooked pork was discovered to be 1.2-1.3

68

KLAUS HOFMANN AND REINER HAMM

times as much as that in cooked beef. This finding may be due to the fact that the pH value of the pork proved to be higher than that of the beef. The reaction mechanism of the H,S-formation is not yet fully understood. Schoberl (1941) attributed the development of H,S brought about by the influence of hot water on wool keratin to a hydrolytic splitting of disulfide bonds, which proceeds in two steps (Schoberl and Eck, 1936): R-CHZ-SS-CHZ-R + HZO -+ R-CHZ-SH + HOS-CHZ-R HOS-CHZ-R -,HZS + CHO-R

The same mechanism was discussed by Bjarnason and Carpenter (1970) in regard to the release of H2S during the heating of bovine plasma albumin. This reaction, however, needs an alkaline medium which is normally not present in meat. For the formation of hydrogen sulfide from SH groups, p-elimination (a) and hydrolysis (b) are possible reaction mechanisms. -NH,

-

-NH,

,,CH-CH2-SH

-co

-Cd

C=CH,

+

H,S

(a)

-NH,

,,CH-CH,-SH

-co

+HO -m, CH-W-OH 2 , -co

+

H,S

Fraczak and Pajdowski (1955) suggested a trimolecular reaction as an explanation for hydrogen sulfide formation; but trimolecular reactions are very rare; making this an unlikely possibility. In addition to hydrogen sulfide, small amounts of volatile mercaptans, thioethers, and other sulfur-containing conipounds are formed during heating and contribute to meat flavor. The best preventive way for reducing undesirable amounts of H2S and mercaptans which develop during sterilization is to select suitable meat which has been handled and stored under hygeinic conditions. Additionally thio-acceptors (e.g., zinc oxide) may be used as components of the inner lacquer of tins (Nehring, 1968; Bloeck ef al., 1970). Also the addition of weak acids such as lactic acid (Johnson and Vickery, 1964), ascorbic acid, or citric acid reduces the development of H,S (Hofmann, 1974a). The thermal formation of H,S and the oxidation of SH groups to SS groups in meat mentioned above necessarily result in a shift of the redox-potential, which is remarkably influenced by the SH/SS system (Hofmann, 1974b). The measurement of the redox-potential (Leistner and Wirth, 1965; Hofmann, 1974b) can be used to evaluate the quality of meat products. It is

SULFHYDRYL A N D DISULFIDE GROUPS IN MEATS

69

believed that each type of product has a characteristic redox potential range (Wirth and Leistner, 1970). 5 . Formation of Further S-Containing Flavor Components

Most of the typical flavor in meats is developed during heating. Raw meat. such as beef, pork, lamb or chicken, has little flavor (Crocker, 1948). The tlavnr of heated meat consists of a great number of different chemical components which are not present in raw meat but are developed from “precursors” by the influence of heat. None of the components identified in meat aroma has been described as uniquely “meaty” (Wasserman, 1972). The chemical pathways involved in the formation of meat flavor compounds during heating include Maillard browning reactions, fatty acid oxidation, and the formation of some low molecular volatile compounds, such as ammonia and hydrogen sulfide. But they also include inter- and intramolecular cyclization, as well as numerous mechanisms which are made possible by the reactivity of substances such as rnercaptans, hydrogen sulfide, ammonia. and other intermediates, especially at high temperatures (Wilson er a f . , 1973). Some of the first investigators who demonstrated the importance of several sulfur-containing volatiles for the flavor of meat were Yueh and Strong (1960) and Minor et al. (1965). The latter were able to show that the removal of the sulfur-containing components resulted in an almost total loss of meat flavor. The ability of fat to dissolve S-containing substances during cooking was demonstrated by Pippen et a / . (1969), who found that the fat of cooked poultry contains more sulfur than does the fat of raw poultry. Furthermore, it was shown that a reaction between H,S and acetaldehyde was involved, and that such reactions between H2S and carbonyls in fat could occur quite generally (Pippen and Mecchi, 1969). During the last few years, many authors have observed the formation of sulfur-containing compounds during the heating of meat which may be relevant to meat flavor (Kato et al., 1973; Mulders, 1973; Mussinan and Katz, 1973; Led1 and Severin, 1973; Scanlan et a / . , 1973; Schune, 1974; Garbusov et a / . , 1976; for reviews, see Herz and Chang, 1970; Schwimmer and Friedman, 1972; Wasserman, 1972). Boelens et a / . (1974) identified the reaction products between fatty aldehyds. H2S, thiols, and ammonia, which are normal constituents of meat, using a combination of gas chromatography and mass spectrometry. In addition. the organoleptic aspects of the reaction mixtures were discussed. Among the substances formed there were several heterocyclic S-compounds such as alkylated trithianes (I), oxadithianes (I]), dioxathianes (111), trithiolanes (IV), dithiazines (V). thiadiazines (VI), and some aliphatic thio compounds, particularly mercapto-thio-ethers (VII), dimercapto-thioethers (VIII), thiodisulfides (IX). thioaldehydes (X), and thioalkenes (XI and XII) (in the formulas the alkyl residues are omitted):

70

KLAUS HOFMANN A N D REINER HAMM

I

I

I

0

-CH-S I

-S-

S-

(IX)

-CH-CH,-CHO

I S-

4

-CH-CH=CH-SI

-CH=CH-CH

S-

(X)

(XI)

7\

S-

(XI0

Wilson et al. ( 1 973) identified forty-six sulfur-containing chemical compounds present in the volatiles of pressure-cooked beef. The main components were alkyl sulfides and alkyl disulfides, thiophenes, and sulfur-containing heterocycles (trithianes, trithiolanes, thiadiazines, and thiazoles). In addition to cysteine, cystine, and methionine, thiamine can also be a precursor of the volatile S-containing aroma components of meat: The formation of H,S (Dwivedi and Arnold, 1971), several thiazoles, and thiophenes (Arnold et al., 1969; Dwivedi and Arnold, 1972, 1973; Dwivedi et al., 1972, 1973) have been reported to be a result of the thermal degradation of thiamine. In spite of the fact that the content of carbohydrates in meat is very low, these substances may still be involved in the formation of the sulfur-containing aroma components of meat. Thus, Morton et al. (1960) found that the reactions of cysteine and other amino acids with sugar produced a flavor with a basic meat character. The reaction of cysteine with derivatives of hydroxy-dihydrofuranone (XIII) resulted in the formation of roasted meat flavor (van den Ouweland and Peer, 1975). Dihydrofuranones may be formed by the degradation of ribose-5-phosphate and have been found in a natural beef broth (Tonsbeek et al., 1968). The initial stage in the reaction of cysteine with the dihydrofuranones involves a substitution of the ring oxygen by sulfur giving derivatives of hydroxy-dihydrothiophenone (XIV). In this reaction, cysteine acts as an H,S donor. The odor components finally were identified as being derivatives of thiophene (XV), rnercapthothiophene (XVI), mercaptofurane (XVII), and mercaptodihydrofuranone (XVIII). Mulders (1973) heated mixtures of cysteine, cystine, and ribose for 24 hours

SULFHYDRYL AND DISULFIDE GROUPS IN MEATS

71

under reflux at 125°C and also identified volatile sulfur compounds such as thiophenes, thiazoles, trithione, alkylthiols, hydrogen sulfide, and carbon disulfide. Arroyo and Lillard (1970) heated mixtures of glucose and each of the sulfur-containing amino acids (methionine, cysteine, and cystine) for 2 hours at 98" C. However, none of these mixtures emitted an odor associated with meat flavor. On the other hand, a meat aroma for ready-to-eat meals could be produced by the reaction of monosaccharides with cysteine or cystine (N. N., 197 la). It may be concluded that the formation of meat flavor is not only due to Maillard-type reactions, the formation of ketones, aldehydes, amines, and heterocyclic N-compounds; it is also due to reactions in which sulfur is included in several ways. A number of patents exist for simulated meat aroma based on heating mixtures of a sulfur-containing compound, amino acids, and carbonyl compounds (Wasserman, 1972). A few patents have specified heating thiamine with various amino acids (Giacino, 1970; Yamamoto et u l . , 1970). It should be mentioned that volatile aroma components are involved not only in meat smell but also in meat taste, because the volatile compounds are, of course, soluble to some extent in meat juices and, especially, in the melted fats. This may be the reason for the fact that meat which contains a certain degree of fat gives a more aromatic taste after frying than does lean meat. It would be interesting to learn whether foreign metals (e.g., Pb, Hg, Zn, Cd) or other possible residues in meat, which are able to react with sulfur compounds, can influence the development of sulfur-containing aroma components during the heating of meat and thus influence meat flavor. As far as is known. this problem has not yet been investigated. 6 . The Texture of Meat as Influenced by Disuuide Groups Formed During Heating The ways in which meat texture is generally influenced by heating have already been discussed by Laakkonen (1973) in his review. One of the factors

72

KLAUS HOFMANN A N D REINER H A M M

that may influence the tenderness* of meat is the possible formation of disulfide bonds between protein chains induced by heating. Because this aspect is not considered in the review mentioned, it should be discussed in this section. Dub6 (1969) described the change in the texture of meat due to heat as follows: “It has been observed that upon heating meat develops a kind of rigidity that, in some ways, may compare with the resistance due to rigor. This hardening of the muscle tissue during heating is a reaction in which many substructural elements may be involved such as the proteins of the myofibrils with their chemically reactive groups. These elements may during the process contribute to the formation of different kinds of cross-bindings that might tighten the structure and increase the resistance to shear.” There is very little known about the chemical nature of the binding forces between actin and myosin during contraction. Szent-Gyorgyi (1966) did not support the assumption that the sulfhydryldisulfide bonds could be involved in the reaction between the two contractile proteins. [But SH groups of myosin are necessary for the myosin-actin interaction (see Section 111, A , 2, a).] Since the linkage can be broken by pyrophosphate and magnesium, Szent-Gyorgi suggested that there is probably an electrostatic interaction. It also seems that the analogy between muscle contraction and the hardening of muscle during heating is a superficial one. Therefore, the possibility of the tenderness of meat being influenced by the heat-induced formation of disulfide linkages should be considered independently of the process of muscular contraction. There are findings that demonstrate that the formation of disulfide groups does influence the texture of meat. Dodge and Stadelman (1959) observed that carcasses aged in air were less tender than those aged in water because of the greater opportunity for oxidation in the air. Treatment of muscle with potassium iodate, which is able to oxidize SH to SS, resulted in an increase of the shear values. The same result was found by Hird and Yates (1961) using several oxidizing agents. Chajuss and Spencer (1962a,b) exposed chicken muscles to a solution of sodium sulfite which is known to split SS bonds. After cooking these pretreated muscles, the shear values were found to be less than those of muscles stored in water only. However, treatment with hydrogen sulfite, surprisingly, did not significantly influence the shear values compared to treatment with sulfite (Chajuss and Spencer, 1962a). This finding signals caution in our interpretation of results obtained using sulfite. Because the authors did not use any buffer for preparing the solution, the solution of sodium sulfite reacted alkaline as a result of hydrolysis. Thus, this observation might be attributed to a swelling effect of the high pH of the solution. There is almost certainly a hardening effect of the oxidizing agents however. Such an effect has been suggested by Connell (1957). He emphasized the importance of cross-links between the protein peptide chains in *In this connection it may be of interest to note that the toughness of meat may be decreased by the injection of cysteine into the blood vessels of an animal 15 to 30 minutes before slaughtering. This treatment causes an activation of the proteolytic enzymes (N. N.. 1971b).

SULFHYDRYL AND DISULFIDE GROUPS I N MEATS

73

relation to texture and demonstrated that the SH content of dried fish is less than that of fresh products, assuming the fornmaticin of disulfide cross-links. In addition. Dube (1969) found that the decreasc in SH and the increase in SS in beef muscle proteins induced by heating were accompanied by an increase in the shear values of the muscle. Model experiments concerning the texture of SS-crosslinked gelatin gels were carried out by Okamoto et ctl. (1973). The relation between sensory properties, physical characteristics. and the forces maintaining gel structure were studied for gelatin thiolated t o 6.6 moles SS/I05gm protein. It was found that hardness was affected mainly by temperature, brittleness by disulfide bond content. The hardness was generally attributed t o hydrogen bonding, and the brittleness was attributed largely to disulfide bonding. Transferred to meat, this result would mean that an increase in the number of disulfide groups does not render the meat tougher but more crisp; in other words, the niasticatability of the meat would be influenced favorably rather than unfavorably by the formation of disulfide groups during heating. Indeed, it was observed that heating meat paste for a few minutes led to a product of gum-like consistency, whereas heating for a longer period of time so that the number of disulfide groups was drastically increased, resulted in the paste’s becoming increasingly crisper and more brittle (Hofmann and Hamm, unpublished observations). With whole meat, of course. the connective tissue also plays an important role for the tenderness or toughness. The significance of thiol and disulfide groups in the determination of the rheological properties of dough has been recognized by several workers (Frater et al.. 1960; Blocksma, 1972; Ewart, 1972; Jones et d.,1974). It may be that the thiol-disulfide system is also relevant to the rheological behavior of meat emulsions in the production of sausages. but no corresponding experiments have been carried out as yet.

B. FREEZING AND FROZEN STORAGE Husaini and Alm (1955) investigated the influence of the frozen storage (-4” to -20°C for 130 days) of egg white and cod fillets on the number of “masked” SH groups. Amperometric titration with AgN03 was used for the egg white and ferricyanide and o-iodosobenzoic acid for the fish muscle. The difference between the total SH content of proteins determined after the addition of a denaturing agent (dodecyl sulfate) and the amount of SH groups available in the proteins’ native state represented the number of masked SH groups. In the case of egg white, the values of masked groups showed a decrease during the first 28 days; they then began to rise and after 48 days became more or less constant. In cod muscle, the masked SH groups reached a minimum value after 50 days of frozen storage. Again, the masked SH groups then began to rise. After 90 days, the values were not constant but still varied. However. the number of masked SH

74

KLAUS H O F M A N N A N D REINER HAMM

groups finally decreased in all cases, or, in other words, the number of available SH groups increased during frozen storage. This result seems to show that the frozen storage of protein leads to protein denaturation, resulting in a release of reactable SH groups. Grau (1968) also reported that freezing meat would cause an increase in the SH groups, but no literature reference was given. According to Dzinleski et al. (1969). the amount of SH groups of proteins in both frozen beef muscle and drip increased during frozen storage (3 months at -18°C). The SH groups were determined with Ellman’s reagent. The authors postulated that frozen storage caused physical changes in the muscle tissue which resulted in a release of the reactive SH groups upon defrosting. The increases ranged from 41% to 103%. The maximum values found were 16-17 moles SH/105 gm protein. These values are certainly too high, because the total SH content of all investigated meats was found to be 10-12 moles SH/105gm protein, the maximum value being 14 moles SH (see Section IV, A). Nevertheless, it might be possible that the denaturation caused by freezing or frozen storage increases the number of SH groups which react with Ellman’s reagent, because this reagent does not seem to react with all SH groups of the native meat proteins (see Section 11, B, I ) . Connell (1960b), however, was not able to detect changes in either the easily reactable or the total SH groups in cod flesh during frozen storage for up to 3 years at - 14”, -22”, and -29°C. In an investigation by Hofmann et al. (1974) with lean beef and pork (musculus long. dorsi), one part of the samples was frozen in vacuum-sealed plastic bags, while the remainder was packaged in presence of air; both groups of samples were stored at -19°C for up to 24 months. While no significant changes in total SH content were found, the nonprotein SH content decreased (this will be discussed later in connection with results of Khan et al., 1963). Rahelid et al. (1974) also found no change in the total SH content of beef which was stored at - 18°C for I year. The SH content of pork, however, decreased considerably after 6 months. In these experiments, the stored meat samples were not protected against the influence of atmospheric oxygen by sealing them in plastic bags as was done in the experiments of Khan rt al. (1963) and Hofmann et al. (1974). Hence, the decrease in the SH content of pork might have been induced by the formation of fatty acid peroxides (less probable for beef). The oxidation of cysteine to cystine by autoxidizing lipids is well known (Wedemeyer and Dollar, 1964). A decrease of free SH groups in the protein of fish muscle (Sacramento blackfish) by freezing and to a greater extent during frozen storage was found by Mao and Sterling (1970). Khan et al. (1963) stated that a decrease in the “sulfhydryl group content of muscles” takes place during frozen storage of raw chicken muscle. This term is entirely misleading: In this investigation, “sulfhydryl group content of musc1es”refers to the SH content of a metaphosphoric acid extract of the muscle (containing only the nonprotein SH groups) rather than the total SH content of the

SULFHYDRYL A N D DISULFIDE GROUPS IN MEATS

75

muscle. Unfortunately this fact was ignored during the discussion of the results both in this and in later publications (Khan, 1965, 1966; Khan and van den Berg, 1965; Khan and Nakamura, 1971). At the end of 2 years the remaining SH content was found to be about 40% of the initial (nonprotein SH) value at - 10°C storage temperature and about 70% at - 18°C storage temperature. It was suggested “that the destruction of sulfhydryl groups can be used as an index of protein damage during frozen storage’’ (Khan e t a / . , 1963). However, in light of the fact that the SH groups of the protein were not actually estimated, this conclusion does not seem to be justified. During frozen storage, proteolytic enzymes are still active, causing an increase in the products of protein breakdown (Partmann, 1972). These products can be estimated by the Fohn-Ciocalteu reagent (sodium p-naphthoquinone 4-sulfonate) (Khan et d . , 1963; Khan and van den Berg, 1964). The ratio of SH groups and the Fohn-Ciocalteu reagent-positive nitrogen compounds of both breast and leg muscle tissue decreased progressively with time of frozen storage. Khan (1965), therefore, proposed the use of this ratio as a “quality index” for frozen stored poultry. This SH/N ratio decreased after 2 years of frozen storage at - 18”C, from about 2.8 to 1.9 for leg muscle and from about 1.8 to 0.9 for breast muscle.* The SH content was expressed as pg glutathionigm muscle, the N content as pg tyrosin-N/gm muscle. Davidkova and Davidek (1971) applied the index on pork, observing a decrease in the ratio from about 2.3 to 0.6* after 60 weeks of frozen storage at - 18°C. When the meat was stored at -S”C, the value of 0.6 was reached after only 2 months. In most cases, the meat was no more acceptable when the index fell below 0.8. Hofmann et a/. (1974) found a decrease of nonprotein SH and an increase of nonprotein N in both beef and pork after 2 years frozen storage at -19°C. The mean values ranged from 2.2 to 1.6 mg SH/lOO gm meat and from 3.4 to 5.4 gm N x 6.0/1OO gm meat. Consequently the SH/N ratio decreased by about 5070,corresponding to the values given by Khan et al. (1963). However, it seems to be questionable whether this change in the SH/N ratio is reliable for evaluating a loss in meat quality under practical conditions, because the ratio can vary in fresh meat in a wide range (1.2 to 2.2) (after Davidkova and Davidek, 1971). Furthermore, the initial SH content of frozen stored meat will normally not be known (in our opinion it should be) so that it would most often be impractical to judge meat quality by means of the SH/N ratio. Khan and van den Berg (1965) also stated that the frozen storage of cooked meat results in a gradual decrease in the SH content. Because the tenderness of the meat decreased simultaneously, it was concluded “that loss in this sulfhydryl-group content during storage may serve as an index of tenderness.” It should be pointed out that this statement was also based on the estimation of the nonprotein SH content. Since tenderness of meat is related to the properties of the *These values were taken from Fig. I in Davidkovi and Davidek (1971).

76

KLAUS H O F M A N N A N D REINER H A M M

structural proteins, this conclusion seems to be somewhat questionable. In our opinion, the results obtained with cooked meat indicate that the decrease in SH groups must be caused by an autoxidation (no participation of enzymes). However, Khan et al. (1968) attributed the decrease in the SH groups mainly to the so-called cryodenaturation, first postulated by Levitt (1962, 1966). We feel it is necessary to discuss this hypothesis in detail because it has often been adapted and discussed rather uncritically (Gaff, 1966; Khan et al., 1968; Partmann. 1968; DubC, 1969; Buttkus, 1974). Levitt (1966) points out “that SS groups are formed. and this has been explained by the SH SS hypothesis, according to which intermolecular SS bonds form between protein molecules during injurious freezing, leading to denaturation of proteins.” However, from the chemical point of view, the formation of SS from SH cannot occur without oxidation. This fact has not even been mentioned in the discussion. We read further: “That this postulated chemical change can be induced or at least accelerated by freezing has been shown in the case of the model system thiogel. When this SH-protein is frozen, intermolecular SS formation is greatly accelerated as compared with unfrozen gel. . . .” It should be pointed out that no experimental evidence was cited for an increase in SS groups induced by freezing. For that reason the following statement can be considered only as conjecture: “Thiogel . . . provides a system in which intermolecular SS bonds formation can be readily detected and measured quantitatively by the simple method of determining its melting point” (Levitt, 1965). Khan et al. (1968) also investigated the influence of frozen storage on SH groups content of chicken myofibrillar proteins using Ellman’s reagent. In fresh samples, the total SH content was found to be 0.59 pmole/mg N (equal to 9.44 moles SH/105 gm protein) and 0.15 pmole after storage at -5°C for 10 weeks. This unusually high storage temperature may have supported the autoxidation of SH groups. The decrease of protein solubility and the loss of tenderness in meat which is generally observed after freezing or frozen storage may be explained by an aggregation of protein molecules, whereby the native state may sometimes be maintained. This aggregation may be brought about by the formation of hydrophobic bonds, hydrogen bonds, ion bindings, and disulfide linkages (Jaenicke, 1964, 1965a,b). The last mentioned may be formed by a sulfhydryl-disulfide exchange reaction (Fig. 8). This principle mechanism of aggregation may be valid not only for mildly heated proteins, for which it was studied, but aIso for protein systems stored at lower temperatures. In this case, of course, the reaction velocity will be lower; however, long-time storage may cause the SH-SS-exchange reaction to some extent. The freezing of meat and other tissues, if it is done correctly, does not necessarily cause the denaturation of the proteins.

SULFHYDRYL A N D DISULFIDE GROUPS IN MEATS

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FIG. 8. Sulfhydryl-disulfide exchange as a possible mechanism for the aggregation of nativc protein molecules.

C.

FREEZE-DRYING

Connell (1957) investigated the influence of dehydration on the texture and the

SH content of fish muscle. In fresh cod, he found 0.38 gm SH/100 gm protein using o-iodoso-benzoic acid, and 0.14 gm SH/100 gm protein using N ethyl-nialeimide (NEM). The corresponding values of the freeze-dried material were 0.30 gm SH and 0.13 gni SH, respectively. Both air-drying and vacuum contact-plate drying of cod yielded nearly the same results. Connell concluded: “The results do suggest in a general way that the sulfphydryl content of the dried products is less than that of fresh fish, a finding which is substantiated by the evidence from solubility measurements that disulphide links are formed on drying.” However, the latter statement has not been proved directly because experiments to determine SS groups had not been carried out. Connell further states that “the number of experiments is not sufficient to give a conclusive answer.” Potthast ( 1 972) studied the influence of freeze drying and the effect of storage of freeze-dried pork on SH content by means of an indirect amperometric titration method using NEM and AgN03. The results showed that the easily available SH groups. which were detected with NEM. clearly decreased, whereas the total SH content did not change significantly. This finding indicates that during the freeze-drying process the protein structure becomes tighter. There seems to be none of the unfolding of protein molecules (which results in an increase of the easily detectable SH groups) that Hamm and Hofmann (1965) found in the case of the heat denaturation of meat protein. The storage of freeze-dried mcat at 4 0 4 5 % relative humidity did not result in any change of available SH groups, although a decrease in the enzyme activity (Hamm, 1964; Yasui and Hashimoto, 1966) of some muscle proteins does take place during storage. Potthast has therefore suggested that this denaturation effect is not caused by unfolding the protein chains but by the blocking or chemical changes of the active centers.

D. CURING The chemical reactions involved in the transformation of niyoglobin (Mb) to nitrosomyoglobin (NOMb) after addition of nitrate or nitrite to meat, giving the meat-stable pink color of cured meat, are extremely complicated and have not yet been elucidated in complete detail. The different steps of the formation of NOMb

78

KLAUS HOFMANN AND REINER HAMM

may be formulated in a very simplified way: reduction

reduction

(bacterial)

(chemical)

NO,-NOC-NO

t Mb

-NOMb

There is no doubt that the SH groups of meat participate in this reaction chain. Theoretically the SH groups can agitate in three ways: (a) reduction of NO,- to NO, (b) protection of Mb against oxidation to metmyoglobin (MetMb), and (c) formation of Mb by reduction of MetMb (Fe3+ +. Fez+). The role of SH groups in changes in meat color and in the curing process has been studied by numerous workers (Watts and Lehmann, 1952; Watts etal., 1955; Kelley and Watts, 1957; Hornsey, 1959; Tarladgis, 1962a,b; Stewart et al., 1965; Szakaly, 1966; Reith and Szakaly, 1967; Fox and Ackerman, 1968; Mirna and Hofmann, 1969; Olsman and Krol, 1972; Kortz, 1973: Kubberad et af.,1974). The formation of cured meat color can be accelerated by ascorbic acid (or ascorbate) which is probably due to its reducing effect and/or to the protection of the SH groups against auto-oxidation. The first investigations on the relation of free SH groups to cured meat color were carried out by Watts et a / . (1955). They heated a model mixture containing 50% egg white (delivering SH groups), 0.4% hemoglobin (so-called meat pigment), and 0.1 % sodium nitrite in phosphate buffer pH 5.8. For comparison they added ascorbic acid to a second sample of the mixture and iodoacetamide (for blocking the SH groups) to a third sample before heating. The development of the color was most intense in the mixture containing ascorbic acid and was least intense in the mixture with iodoacetamide. The authors concluded: “The reduction of metmyoglobin and nitrite necebsary for the formation of the cured meat pigment may be brought about by SH groups of muscle protein rather than by reducing enzyme systems. In the absence of ascorbic acid, formation of the pink cured meat pigment parallels the appearance of free sulfhydryl groups.” However, because of the far-reaching consequences of this statement, it must be noted that the model system used here is very different from actual conditions in meat. Rather than myoglobin, the pigment of meat color, hemoglobin, whose oxygen affinity is very different from that of myoglobin, was used. On the other hand, the reactivity of the SH groups in egg albumin is very different from the reactivity of the SH groups in meat protein: In myofibrils the SH groups are already reactive in the native state (e.g., with sodium nitroprusside), whereas there are no SH groups detectable in native egg albumin (Hofmann, 1964, 1966; Hofmann and Hamm, 1966, 1975). Watts et al. (1955) stated further, “Whereas nitrite protects the cured meat pigment from oxidation in the presence of protein SH groups it accelerates oxidation in their absence. This interesting statement does not seem to be valid for meat because meat proteins always contain free SH groups in the native as well as in the denatured state. Kelley and Watts (1957) found that in addition to ascorbic acid, cysteine and glutathione were capable of catalyzing the production of nitric oxide hemoglobin and of protecting the sur”

SULFHYDRYL AND DISULFIDE GROUPS I N MEATS

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face of cured meat from fading by light. The color of cured meat is also influencecl by SH groups which are not bound to proteins. Hornsey (1959) found a positive correlation between the color intensity of cured pork and its content of cysteine plus cystine and the ratio of cysteinelcystine as well. The following results demonstrate that SH groups can react directly with nitric acid, which may be an intermediate product during the formation of NOMb. It is well known that nitric acid can react with SH compounds of forming nitrosothiols (Beckurts and Frerichs, 1906). Ashworth and Keller (1967) established that while secondary and tertiary thiols would react in this way, thiols, such as cysteine and glutathione, would not. Nevertheless, Saville (1958) found that at pH 2- 3 a quantitative reaction between nitrite and cysteine takes place with the formation of the corresponding nitroso compound (S-nitroso-cysteine). The amount of nitrosothiol formed decreased with increasing pH value. In order to find out whether a formation of nitrosothiols is possible in meat at its natural pH (5-6). Mirna and Hofmann (1969) carried out experiments with meat and SH solutions: The addition of nitrite to minced beef and pork in amounts nearly equimolar to the SH content caused a decrease in both components of about 20-30% during storage for 1-2 weeks at +2"C and pH 5.6-5.8; in meat to which no nitrite was added the SH content proved to be stable during 12 days of storage at 2-3°C (Hofmann, 1971d). Thus, the decrease of SH in the presence of nitrite might be due to a reaction of the SH groups with nitrite or nitric acid. This explanation is supported by the observation of Olsman and Krol (I 972) that a smaller loss of nitrite occurred when the SH groups in meat had been previously b1ock:d by an SH reagent. A study of the reaction of nitrite with glutathione and with b.ysteine in watery solutions at different pH values led to the following resulis (Mirna and Hofmann, 1969): ( 1 ) At pH 7.4, no reaction takes place. At pH 2.3, the reaction is nearly complete after 15 minutes at 23°C. At pH 5.0 and IOO"C, only 16-18% of the SH groups of glutathione had reacted after 15 minutes. The pH dependence of the reaction shows that it is the free nitric acid rather than the nitric ion which reacts with the SH groups: R-SH

+ HO-NO

+

R-S-NO

+ H20

(2) The nitrosothiols are unstable. The half-life periods of S-nitroso cysteine and S-nitroso glutathione have been estimated to be 2 and 3 hours, respectively, at pH 5.5. (3) The nitrosothiol of cysteine was isolated in the form of red crystals. In water this compound is slowly decomposed under the splitting off of a gas (NO), while unsoluble cystine simultaneously precipitates. The decomposition is probably caused by the following reaction: 2 CyS-NO

+

CySSCy

+ 2 NO

80

KLAUS HOFMANN AND REINER HAMM

Because the NO group can be split off easily from the nitrosothiols formed, it was suggested that the role of the SH groups in the formation of the cured meat color may perhaps consist of a transfer of the NO group from the nitrosothiol primarily formed to the myoglobin (Mirna and Hofmann, 1969): 2 Mb

+ 2 Protein-S-NO + 2 NOMb + Protein-S-S-Protein

Kubberpd et al. (1974) studied the reaction of nitrite with the SH groups of myosin. The reaction was found to depend on the pH and the temperature much the same as the reaction with the low molecular SH compounds. The rate of the reaction was low under conditions similar to those in meat. However, curing takes days and therefore the reaction may occur to some extent. The formation of S-nitrosothiols has been also discussed by Gilbert ef ul. (I 975). Kortz (1973) presented a hypothetical mechanism for the role of the different SH fractions of meat in the development of cured meat color. According to this hypothesis, nonprotein SH compounds act as intermediates between myoglobin and the SH groups of the water-soluble protein fraction.

E.

RIPENING OF DRY SAUSAGES

Sandholm ef al. (1972) estimated the number of SH groups in dry sausages during the process of ripening using amperometric titration with AgNO,. The SH groups increased up to the twentieth day and declined by the twenty-ninth day to almost the initial value. The increase was assumed to be attributable to a bacterial reduction of the SS groups of the proteins in the sausages. However. this cannot be the case: The SS content in meat is low in relation to the SH content (see Section VI, A and comments to Table XIII) so that the SH content, after the proposed reduction of SS groups, could not rise to a value many times that of the initial SH content. The increase reported by Sandholm ef al. was higher than tenfold (for example, the values for the initial and maximum SH contents were 46 and 540 pmoles SH/gm wet weight, respectively). Furthermore, it must be noted that all these values were far too high in comparison with similar values given by other investigators (meat contains about 65 mg SH/IOO gm tissue, whereas the values reported here corresponded to 152 and 1786 mg SH/100 gm tissue respectively). Therefore, either the procedure of determination followed or the kind of calculation used may have been incorrect.

F.

SMOKING

There are a few results available concerning the question of how the constituents of smoke influence the SH groups of meat. Smoke contains about 300 different components (review: Mohler and Baumann, 1968), some of them very reactive. Among these are numerous phenols, aldehydes, and ketones (Tilgner,

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1967) which are able to react with SH groups (see Cecil and McPhee, 1959; Friedman, 1973; Stauffer, 1974). The carcinogenic. benzo[a]pyren. can also react with SH groups (Harington, 1967; Reske. 1971). Because these reactive compounds occur in smoke, it is not surprising that smoking decreases the total SH content of meat (Randall, 1969; Randall and Bratzler. 1970). The number of SH groups in heated but unsmoked pork was (converted) 12.0 moles SH/105 gm protein; in the heated-smoked samples, the value decreased to 7.0 moles SH (smokehouse conditions of 60°C. 45% R.H.. 2.25 hours). This decrease was attributed to interactions of the smoke with various reactive groups of the meat proteins (Randall and Bratzler, 1970). Krylova and Kusnezowa ( 1 964) observed a drastic decrease in the SH groups found in smoke-cured meat. After smoking, only about 40% of the initial SH groups remained; the phenolic fraction was more active and the basic fraction was less active.

G . IRRADIATION Barron (1946) postulated that ionizing radiation (X rays. alpha, beta, and gamma rays) would rapidly oxidize the thiol groups of cells. This oxidation was explained by the formation of oxidizing radicals when water is radiated in the presence of oxygen. This hypothesis has been supported by the finding that several thiol enzymes are inhibited by radiation, and that this inhibition was prevented or reversed on the addition of glutathione (Barron. 1951). Both the oxidation of thiols to disulfides and the reduction of disulfides to thiols have been observed by several authors during irradiation with ultraviolet light. Degradation of thiols and disulfides can also occur (for reviews, see Cecil and McPhee, 1959; Friedman, 1973). In aqueous solution, cysteine is oxidized to cystine under the influence of gamma radiation (Whitcher et a / . , 1953; AI-Thannon, 1968; Owen and Brown, 1969). Furthermore, hydrogen, hydrogen sulfide, hydroperoxide. and alanine was formed, depending on the pH, the concentration of oxygen, and the SH content in the cysteine solution radiated (Trumbore, 1967). Radiation of solutions containing cysteine and cystine produced also SO,. alkanes, and dimethyldisulfide (Merritt. 1966). When meat is irradiated with doses of gamma rays at the level required for the destruction of the most resistant microorganisms, one can see the development of irradiation odors and off-flavor (Wick, 1965) which may lower food acceptance and values. It is generally recognized that relatively high doses of this irradiation are accompanied by the denaturation of proteins, splitting of protein molecules, or the association of these molecular fractions (reported by Fujimaki ct al., 1961). By trapping the volatiles from irradiated meat in a solution of lead, zinc, and mercuric salts, Batzer and Doty ( I 955) obtained precipitates containing sulfur

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compounds thought to be the source of some of the undesirable odors in irradiated meat. Fractionation of some of the off-odors of beef revealed that they arise from water soluble compounds, mainly from glutathione (Batzer and Doty, 1955). Methods for estimating the amount of hydrogen sulfide and methyl mercaptane after gamma radiation of meat (based on the color reaction with N,N-dimethylp-phenylene-diamine) have been described by Marbach and Doty ( 1956), Batzer and Doty (1955), and Sliwinski and Doty (1958). Small doses below 0.8 mrad, which are used for radiopasteurization, did not influence the sensory qualities of meat, but the higher doses of I .5-5 mrad necessary for radiosterilization had a considerable detrimental effect. The specific undesirable flavor produced was due to the formation of sulfide, mercaptans, carbonyl compounds, and others (Palmin, 1970). The better method of preservation is, therefore, a combination of low dose radiation and heat processing. Several SH-containing compounds have a radioprotective effect on animal organisms, but the mechanism of this action is not yet completely understood (Modig, 1969). Graevsky er al. ( I 969) hypothesize that the radioprotective influence exerted by SH compounds is determined only by their SH groups. Experiments demonstrating distinct radiosensibilitation of mammalian cells and bacteria after blocking the SH groups by NEM or PCMB are in agreement with this conception. The treatment of meat with gamma rays (1.5-3.0 mrad) caused a moderate decrease in SH-glutathione, which was intensified drastically by atmospheric oxygen (Palmin and Breger, 1963). The freezing of minced pork and beef before irradiation with 5 mrad at various temperatures significantly protected SHglutathione, particularly if very low temperatures were used (Coleby et al., 1961). It seems that the freezing of water rather than the low temperature itself is responsible for the protection effect observed. In the investigations of Griinewald (1969), the cysteine content of freeze-dried beef and pork and of fresh beef did not decrease during radiation with less than 1 mrad; radiation with 5 mrad invariably caused a reduction in cysteine and to some extent an increase of the cystine content. On the other hand, the sum of cysteine plus cystine decreased. This shows that radiation with high doses lead to the oxidation of cysteine and to the decomposition of total cysteine plus cystine. However, in the case of fresh beef, cryogenic temperatures prevented these losses of cysteine by radiation. This finding coincides with the results of sensoric tests obtained by other investigators (Coleby et al., I96 1 ; Harland et al., 1967). In other cases the radiation of meat did not cause drastic changes in the SH content. Kardashev et al. (1970) treated fresh river sheatfish and Baltic cod with gamma rays (0.3 to 2 mrad and 0.5 to 10 mrad, respectively). This irradiation produced no change in the contents of SH groups in either the actomyosin

SULFHYDRYL AND DISULFIDE GROUPS IN MEATS

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fraction or in the watersoluble fraction of muscle proteins. Fujimaki et al. (1961) treated meat (rabbit muscle) with gamma rays (about 4 mrad) before and after rigor mortis. There was no significant difference between the content of the sulfhydryl groups of actin from both irradiated and control meat samples. According to the investigations of Hamm et al. (1975), radiation of vacuum-packed lean pork with doses of 0.2, 1 and 5 mrad at 4°C did not significantly change the total SH content of the whole tissue. In this case, the unchanged SH content may be explained by the exclusion of atmospheric oxygen so that an oxidation of SH groups induced by radiation cannot take place. Metlitskii et al. (1968) stated that the radiation of pork, beef, and turkey with 3 mrad did not cause a significant decomposition of cysteine. Hedin et al. (1961) came to the conclusion that X irradiation decreased the SH content of a gelatinelike glucoprotein fraction of beef which produced “wet dog” odor when irradiated. The SH assay was carried out with mercuric chloride and Ellman’s reagent. It was clearly shown that mercuric chloride did not react with the SH groups of the irradiated protein, whereas Ellman’s reagent did. Using the latter reagent, the SH content decreased only by about 7% (from 1.37 to I .27 pmoleslgm). Furthermore, the amino acid analyses of the protein hydrolysate gave nearly the same cysteic acid values for both the irradiated and the untreated samples. There is no doubt that, generally, irradiation of meat causes formation of volatile hydrogen sulfide and mercaptans at the expense of the SH content. However, these amounts are very low in comparison with the total SH content of meat (less than 1%). This can be explained by the finding mentioned previously that the volatile compounds are formed solely from the water soluble components of meat, which are present in minor amounts. Kraybill et al. (1960) reported that gamma irradiation (to 9.3 mrad) resulted in an increase in the SH and SS contents of raw skim milk. Ultraviolet irradiation effected a similar increase in SS content, but no changes in SS bonds. To explain these findings, several mechanisms were discussed based on a degradation of methionine. However, it seems to us much more likely that the increase of available SH groups is due to the denaturation of the proteins, known to be accompanied by an increase of the availability of masked SH groups (see Section V, A, 1). The results of McArdle and Desrosier (1953, who found a liberation of SH groups in irradiated solutions of casein and egg albumin, may be explained in the same way. Neuwirt et al. (1964) found that, after treating rat liver nuclei with a dose of 750 roentgen, there was a significant decrease in protein disulfide groups but no decrease or increase in protein SH groups. In addition, they point out that, after ray treatment, no decrease in the activity of any SH-enzyme has ever been demonstrated.

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VI.

INFLUENCE OF THE SH GROUPS ON THE SHELF LIFE OF MEAT AND MEAT PRODUCTS

In the search for new compounds for the preservation of meat and meat products, it has been observed that several SH compounds, for example, cysteine and glutathione, are able to potentiate the microbial inhibition effect of known food preservatives such as sorbic acid, benzoic acid, fatty acids, and others. Mixtures with these SH compounds have proved to be effective for the preservation of meat, fish, and other foods (Troller, 1966). It is well known that SH compounds have an antioxidative effect which retards the rancidity of fats (Maloney et al., 1966). This effect may be due to the decrease in the redox potential by SH compounds. A direct inhibition of microorganisms by SH compounds would seem to be unlikely because the growth of microorganisms is usually inhibited by SHblocking reagents (Zsolnai, 1970). Cooked meat becomes rancid more quickly than unheated meat because the SH content in cooked meat is reduced as a consequence of the heating effect (Hofmann, 1964; Bognar, 197 1a). This preservation effect of the SH groups in meat may be of limited importance; nevertheless, it seems to be worthwhile to prevent SH oxidation during meat processing as far as possible.

VII. TOXICOLOGICAL ASPECTS SH groups are able to bind toxic heavy metals [Pb2+, (see V, D,2,f), Hg2+, and others] and may therefore play an important role in detoxification reactions (Clarkson, 1971). On the other hand, experiments with rats have shown that an increase in the protein content of diets resulted in a significant increase in the retention of lead (Milev et af., 1970). In this case the lead was probably also bound to the protein SH groups of the feed. Therefore, it is difficult to predict generally whether under practical conditions the SH groups in foods have a positive or negative effect on the contamination of living organism. Furthermore, SH groups interact with numerous carcinogens such as polycyclic aromatic hydrocarbons (PAH), hormones, hepacarcinogenic substances (e.g., certain aminoazo dyes and amines, carbon tetrachloride, aflatoxins, thioacetamide, and ethionine), alkylating agents, nitroso compounds, 4-nitrochinoline N-oxide, lactones, quinones, metals, metalic derivatives, and arsenic (for review, see Harington, 1967). Therefore, SH groups in organisms and foods are important for anticarcinogenesis (for review, see Reske, 1971). Nemoto el al. (1975) reported direct evidence that conjugation with SH-glutathion is a significant

SULFHYDRYL A N D DISULFIDE GROUPS I N MEATS

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100

-

5 80

E

x 60 U,

L

ul a

C

-

w

'0 A0 C

g

20

0 0

1

2

3 4 Tirnc(hrsl

5

24

FIG. 9. Reaction of GSH with patulin in equirnolar amounts at different pH values (20°C). (A: pH 7.4; B : pH 6.0; C: pH 5.0. (After Hofmann ct d..1971 .)

mechanism for the detoxification of the epoxides of PAH, which are mutagenic and may be the carcinogenic forms of the PAH. Hofmann et al. (1 97 1 ) observed that patulin (a potent mycotoxin produced under certain conditions by molds found on meat and meat products) reacts with SH-glutathion. In chick embryo and rabbit or mice skin test. the reaction product formed was proved to be nontoxic. The kinetics of the reaction of GSH and Patulin at different pH values are shown in Fig. 9. It was concluded that, at the pH of meat and meat products, most of the patulin produced by molds might be inactivated by the SH groups occurring in meat. It is also suggested that, in bread, the mycotoxin is inactivated by SHcontaining substances after a prolonged incubation period (Reiss, 1973). Another possibility of detoxification, which has not yet been discussed, is the reaction of SH groups with nitrite (Mirna and Hofmann, 1969). This may be a competitive reaction to the formation of carcinogenic nitrosamines from nitrite and amines. Finally it should be mentioned that there exists a protective action of sulfur compounds such as cysteine and glutathione against acetaldehyde toxicity arising from heavy consumption of alcohol and heavy cigarette smoking (Sprince et d., 1975). As these examples demonstrate, SH groups may be of importance in the detoxification mechanisms of the organism. As we have seen in Section V , the amino acids, cysteine, cystine, and methionine, the exclusive sources for SH compounds in the metabolism, are not very stable under meat processing. For this reason, careful preservation may be of importance not only for the nutritive value but also for the protection of the organism against several classes of toxic substances.

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VIII.

SUMMARY

Methods for the determination of SH and SS groups in meats and their advantages and disadvantages are discussed. The choice of determination method depends on the type of investigation in question. Numerous problems which may arise in the determination of SH groups in proteins are discussed in detail. Protein SH groups may react quickly, slowly or not at all (“masked” SH groups) depending on the type of reagent, on the protein’s state, and on the conditions of reaction. Meat contains both protein SH (soluble and unsoluble) and nonprotein SH groups. Results found in the literature do not always show clearly whether it is the total SH or the SH content only in a soluble fraction that was determined. This differentiation is important because most SH groups are bound to the water insoluble myofibrillar proteins. One of the most convenient and accurate techniques for the determination of SH groups in meat is a “double-indirect’’ amperometric titration method: this enables the application of different SH reagents (AgNO,, NEM, PCMB, etc.). The determination of SS groups is usually based on the determination of SH groups before and after the reduction of SS (preferably with sodium borhydride) to SH. The procedures are briefly described. The sum of SH and SS represents the “total cystine” (cysteine plus cystine) content. Tables are presented listing the amounts of SH and SS groups in meats (muscles and inner organs), in myofibrils, and in isolated muscle proteins. Although the results given in literature vary considerably (depending on the method of assay or on the type of material investigated), the following general conclusions may be drawn: the average SH contents of pork and beef muscle are nearly equal (62 and 66 mg SH/100gm tissue, or 10.2 and 10.5 moles SH/105 gm protein, respectively); the average SH contents of skeletal muscles from different species are very similar to each other; the nonprotein SH content in muscles varies in a wide range; muscles usually contain 3 to 4% nonprotein SH as compared to the total SH content; the average SH content of myofibrils prepared from the muscles of different animals is 9.1 moles SH/105 gni protein (this value corresponding very closely to the sum of SH groups of the single myofibrillar proteins). The SS content of muscles varies from 0.5 to 2.0 moles SS/I05 gm protein. This high variation may be due to a different degree of oxidation of the SH groups. The SS content may also depend on an animal’s age. The average total SH content of liver (47 mg SH/IOO gm tissue) and of other organs (such as kidney, heart, and brain) is lower than the average SH content of muscles; however, the nonprotein SH content (mainly glutathione) is much higher in liver and in other organs than in muscles. In general, the total SH as well as the nonprotein SH contents of the same organs of different species are less variable than the SH contents of different organs of the same species. Factors which influence the available SH content of raw meat may be training

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and stress (influencing the content of glutathione), the age of animals, heavy metals, postmortem aging, and others. The role of the SH groups in muscle proteins (including the proteins of the sarcoplasmatic reticulum, sarcolemma, mitochondria, and sarcoplasmic matrix) in their physiological functions (ATPase activity, contraction, rigor mortis, and interaction of myofibrillar proteins) and in the tenderness of meat are discussed. Heating meat to 70°C increases the availability of SH groups for several SH reagents (DTNB, NEM, PCMB) as a result of denaturation; however, the total SH content does not change. At temperatures higher than 70°C the SH content decreases, chiefly because of an oxidation to SS groups. High temperatures as used for sterilization may also cause a loss of the cysteine plus cystine content, resulting in a decrease in the nutritive value of meat proteins. During heating, hydrogen sulfide and numerous other sulfur-containing volatile compounds contributing to meat flavor, are split off from the sulfur-containing amino acids of meat. The texture of meat is influenced by the formation of SS groups during heating. It seems more probable that SS groups influence brittleness rather than hardness, and that, therefore, an increase in SS groups does not render the meat tougher but crispier. Although the results to be found in literature are not uniform, freezing and frozen storage does not remarkably influence the total SH content of meat; however, there is a decrease in the nonprotein SH content during long-term frozen storage. As the nonprotein N content increases, the SH/N ratio decreases simultaneously. This ratio has been proposed as a quality index for frozen stored meat. The question of its usefulness is discussed. There is no experimental evidence for the statement that the freezing of proteins would cause an increase in SS groups. Freeze drying of meat does not decrease the SH content estimated with AgNO,, but the SH groups which are easily available for NEM decreased, demonstrating that the protein structure becomes tighter during freeze drying. It was demonstrated that SH groups are involved in the curing process. Different ways in which SH groups can react during the formation of the cured meat pigment, nitrosomyoglobin, are discussed. Smoking decreases the SH content of meat products because smoke contains many compounds (phenols, aldehydes, ketones) which are able to react with SH groups. Irradiation of meat causes the formation of hydrogen sulfide and volatile mercaptans at the expense of the SH content, but these amounts are low (< 1%) related to the total SH content. In the presence of atmospheric oxygen, higher doses of gamma radiation leads to an SH decrease. The freezing of meat before irradiation protects the SH groups. Several SH compounds are able to potentiate the microbial inhibition effect of known food preservatives. Because SH groups can bind toxic elements, toxic

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compounds, and carcinogens, SH groups in organisms and in foods are important for detoxification mechanisms. Therefore, careful preservation in order to maintain the cysteine content may be of importance not only for the nutritive value but also for the protection of the organism against several toxic substances.

IX.

RESEARCH NEEDS

( I ) Are the relation of SH to SS and the total SH plus SS content of muscle tissues of meat animals significantly influenced by the age of these animals? This question should be investigated with a number of younger and older animals sufficient for statistical evidence, or, more preferably, with the same animals at different stages of age using biopsy samples. (2) The binding forces participating in muscle contraction and rigor mortis are not yet understood. Is the SH-SS exchange reaction involved in these processes? (3) Is the SH-SS system relevant for the rheological behavior of minced meats and meat emulsions'? (4) Several hypothetical mechanisms have been discussed regarding the question of the way in which SH groups are involved in the curing process. What is the actual mechanism? (5) Do traces of metals such as Pb. Hg, Cu, Zn, Cd, and other possible residues in meat which are able to react with SH groups influence the development of sulfur containing volatiles, and, therefore, the development of meat flavor? ( 6 ) Does the introduction of SH groups into proteins (thiolation) improve the nutritive value of proteins which are poor in sulfur content?

ACKNOWLEDGMENTS We wish to express our sincere thanks to Mr. Erich Bliichcl and Mrs. E. Hofmann for their assistance during the preparation of this manuscript.

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Benesch. R., and Benesch, R. E. 1962. Determination of SH groups in proteins. Methods Biochcm. Anal. 10, 43. Benesch, R. E., Lardy, H. A.. and Benesch. R. 1955. The sulfhydryl groups of crystalline proteins. I. Some albumins, enzymes and hemoglobins. J . B i d . Chem. 216, 663. Bennett. H. S., and Watts. R. M. 1958. The cytochemical demonstration and measurement of sulfhydryl groups by azo-aryl mercaptide coupling with special reference to mercury orange. I n “General Cytochemical Methods” (J. F. Danielli. ed.). Vol. I . p. 317. Academic Press, New York. Berg. J . N . , Lebedeva, N . A , , Markina, J. A , . and Ivanov, I . I . 1965. EinfluBdes hohen Druckes auf einige Eigenschaften des Myosins. Biokhimiyu 30, 277. [Abstr.: Chem. Zentralbl. 137, No. 3 1 1428 (1966).] Beuk. J . F.. Chornock, F. W., and Rice, E. E. 1948. The effect of severe heat treatment upon the amino acids of fresh and cured pork. J . B i d . Chem. 180, 1243. Beveridge, T..Toma, S. J . . and Nakai, S. 1974. Determination of SH- and SS-groups in some food proteins using Ellman’s Reagent. J . Food Sci. 39, 49. Bhattacharya, S . K. 1958. Total sulphydryl (SH) content of blood and tissues. Biochem. J . 69, 43. Bhattacharya, S. K. 1959. Amperometric determination of sulphydryl content of blood and tissues. Nature (London) 183, 1327. Bigwood, E. J. 1972. Amino acid patterns of animal and vegetable proteins4ommon features and diversities. In “Protein and Amino Acid Function” (E. J . Bigwood, ed.), p. 238. Pergamon, Oxford. Bitny-Szlachto. S . . Kosinski, J . . and Niedzielska, M. 1963. Determination of sulfhydryl groups with 2,4-dinitrophenyl-2-hydroxyethyI disulfide. Acta Pol. Phurm. 20, 365. Bjarnason, J . . and Carpenter, K . J . 1970. Mechanism of heat damage in proteins. 11. Chemical changes in pure proteins. Br. J . Nurr. 24, 313. Blocksma. A. H. 1972. The relation between the thiol and disulfide contents of dough and its rheological properties. Cereal Chem. 49, 104. Bloeck, S . . Hofling, E., Baur, R . , and Susin, U. 1970. “Verhalten von H,S-abspaltenden Fullgutern in Steralcon.” Rep. No. 1310. AIusuisse Forschungsinst.. Neuhausen, Switzerland. Bocchini. V., Aloito, M. R., and Najjar, V. A. 1967. Sulfhydrylgruppen der Phosphoglucomutase aus Kaninchenmuskel. Biochemistry 6 , 313. [Abstr.: Chem. Zmtralbl. 139, No. 5-1235 ( I 968). ] Boelens, M . . van der Lindr. L. M.. de Valois. P. J . , van Dort, H. M . . and Takken. H. J . 1974. Organic sulfur compounds from fatty aldehyds, hydrogen sulfide. thiols and ammonia as flavor constituents. J . Agric. Food Chem. 22, 1071. Bognar, A. I97 la. Beitrag zur Ermittlung des ernahrungsphysiologischen Wertes von Fleisch in Abhsngigkeit von der thermischen Behandlung. PhD. Thesis, Univ. Hohenheim, Hohenheim, Germany. Bognar, A. 1971b. EinfluB der thermischen Behandlung auf den Gehalt an Aminosauren in Rindfleisch. Erniihr.-Umsch. 18, 200. Bolshakov, A. S . . and Mitrofanov, N . S . 1970. Evaluation of sulphydryl groups in meat by amperometric back titration (in Russ.). Prikl. Biokhim. Mikrobiol. 6, 606. Bolshakov, A . S . . Karpeev. J . J . . Mitrofanov, N. S . , and Khlebnikov, V. J. 1972. Determination of inertly reacting sulfhydryl groups in meat by their reaction with p-chloromercuribenzoate (in Russ.). Prik/. Biokhim. Mikrobiol. 8, 367. [Chem. Abstr. 77, 6 0 1 5 1 ~(1972).] Bowers, J . A. 1972. Eating quality, sulfhydryl content and TBA [2-thiobarbituric acid] values of turkey breast muscle. J . Agric. Food Chem. 20, 706. Boyer, P. D. 1954. Spectrophotometric study of the reaction of protein sulfhydryl groups with organic mercurials. J . Am. Chem. Soc. 76, 4331.

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Taylor. E. W. 1972. Chemistry of muscle contraction. Anriir. Rri,. Biochern. 41, 577. Thibert. R . J . . Sarwar. M.. and Carroll. J . E. 1969. The simultaneous determination ofcycteine and cystine using N-bromosuccinin~ide:Application in an enzymatic system+ystine reductax. Mikrm,him. Acta p. 615. Thompson. E. 0. P.. and O'Donnell. 1. J. 1961. Quantitative reduction of disulphidc bonds in proteins using high concentrations of niercaptocthanol. Bioc.him. Biophys. Actu 53, 447 Tietze. F. 1969. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Applications to mammalian blood and other lissues. A n d . Eiochem. 27, 502. Tilgner. D. J. 1967. Wirkung des Rlucherns und wirksame Suhstanzen in Riucherrauch. F/eisr.hwirtschufi 47, 373. Tinbergen. 8.J. 1970. Sulfhydryl groups in meat proteins. P r w . Eur. Meer. Meat Res. Workers, 16th. Varrra. Bulgnriu 1, 576. Tkachuk. R.. and Hlynka, 1. 1963. Reactions of flour protein sulfhydryl with N-ethylmaleimidc and iodate. Ci,rcw/ Chrnt. 40, 704. Tononiura, Y . 1973. "Muscle Proteins. Muscle Contraction and Cation Transport." Univ. Park Press, Baltimore, Maryland. Tononiura. Y . . and Yoshiniura. J . 1962. Binding of p-~.hIoromcrcuribcnzoateto actin. J. Bior,hem. (Tokyo) 51, 259. Tonsheek. C. H. T., Planckcn. A. J . . and van der Weerdhof. T. 1968. Components contributing to beet" flavour. J. Agric. Food C h o n . 16, 1016. Tonschinski. J . M. 1959. Untersuchung der SH-Gruppen des Myosins durch aniperonietrische Titration. Ukr. Biokherrr. Z h 31, 589. [Ahstr.: C h m . Zontr-dbl. 4581 (1960).I Tonschinski, J . M. 1974. "Sulthydryl and disulfide groups of proteins." Consultants Bureau, New York. Troller. J . A. 1966. Sulfhydryl-containing potentiating agents of food preservatives. U.S. Patent 3,276,881. [Chern. Ahrtr. 65, 20757h (1966).] Trumhore. C. N. 1967. "Studies in Chemical Radiation Protection Agents." Contract AT (30-I ), p. 3383. Dcleware, Univ.. Newark. Cited i n Griinewald (1969). Tsao. T. C.. and Bailey. K . 1953. Thecxtraction. purification and some chemical properties of actin. B w c h i n i . B i o p h y , Acrir 11, 102. Tsou. K.-C.. Barnett, R. J.. and Seligman. A . M . 1955. Darstellung einiger NNaphtyl-( 1 )-maleinimidc als Reagcnticn fur Sulfhydrylgruppen. J. Am. Chcnr. So<,. 77, 461 3 . [Ahstr.: Chem. Zi~ntrulhl.127, 1290 (1956).] Usunov. G . . and Zolova. L. 1976. Untersuchungen dcr SH-Gruppen und dcr Milchslurc i n der PSE-Muskulatur von Schweinen. F/risc,h~r,irtschtlfi~[,h~~ 56, 253. Van den Ouwrland. G. A . M.. and Peer, H. G . 1975. Components contributing to beef flavor. Volatile compounds by the reaction of 4-hydroxy-S-methyl-3(2H j-furanone and its thio analog with hydrogen sulfide. J. Agric. Food Cherii. 23, SO I . Vogel. H . . and Knobloch. H. 1953. Die wasserl(is1ichen Vitamine. I n "Chemie und Technik der Vitamine." Vol. 2. p. 13. Enkc. Stuttgart. Wachholder. K . . and Uhlcnbrook. K . 1935. Stcigcrung de5 Gehaltes dcr Organe an rrduziercnden Substanzen (Glutathion und Ascorhinslurc) ini Training. Pfluger.s Arch. Gearrnte Ph,wio/. Menschen Tierc 236, 20. Wallcnfeh. K . . and Streffcr. C. 1964. Chemische Rcaktivitk von Pmtcincn. Colloy. Ges. Phwiol. Chnn. 14, 6. Wasscrnian. A . E. 1972. Thcrnially produced tlavorcomponeiih in the aroma of meat and poultry. J. AgriL,. Food Chrm. 20, 737. Watts. B. M.. and Lehmann, B. T. 1952. The effect of ascorbic acid on the oxidation of hemoglobin and the formation of nitric oxide hemoglohin. Food Rcs. 11, 100.

110

KLAUS HOFMANN AND REINER HAMM

Watts, B. M., Erdman, A. M., and Wentworth, J. 1955.Relation of free sulfhydryl groups to cured meat color. J . Agric. Food Chem. 3, 147. Wedemeyer, G . , and Dollar, A. M. 1964.The role of free and bound water in irradiation preservation: Free radical damage as a function of physical state of water. J . Food Sci. 29, 525. Weeds, A. G . , and Frank, G. 1973.Structural studies on the light chains of myosin. Cold Spring Harbor Symp. Quunt Biol. 37, 9. Weeds, A. G.,and Hartley, B. S. 1968.Selective purification of the peprides of myosin. Biochem. J . 107, 531. Weitzman, P. D. J. 1975.Evidence against the proposed interaction of thionitrobenzoate with protein disulfide bonds. Biochem. J . 149, 281, Weitzman, P. D. J., and Tyler, H. J. 1971.Sensitive polarographic estimation of thiol groups with N-ethylrnaleimide. Anal. Biochem. 43, 321. Whitcher, S. L.. Rotherham, M., and Todd, N . 1953.Radiation chemistry of cysteine solutions. Nucleonics 11, 30. White, F. H., Jr. 1960.Regeneration of enzymatic activity by air oxidation of reduced ribonuclease with observations on thiolation during reduction with thioglycolate. J . B i d . Chem. 235, 383. Wick, E. L. 1965.Chemical and sensory aspects of the identification of odor constituents in foods. A review. Food Techno/. 19, 145. Wilkinson, J . M., Perry, S . V., Cole, H. A,. and Tryer, J. P. 1972.The regulatory proteins of the myofibril: Separatin and biological activity of the components of inhibitory factor preparation. Biochem. J . 127, 215. Wilson, R. A., Mussinan, C. J., and Sanderson, A. 1973.Isolation and identification of some sulfur chemicals present in pressure-cooked beef. J . Agric. Food Chem. 21, 873. Wirth, F. 1967.Optimum temperature ranges for the heating of various canned meats and sausage products using the rotation sterilisation method. Fleischwirrschuj 47, 569. Wirth, F., and Leistner, L. 1970. Redoxpotentiale in Fleischkonserven. Fleischwirtschafi 50, 491. Witter, A., and Tuppy, H. 1960. N-(4-dimethylamino-3,5-dinitrophenyl) maleimide: A coloured sulfhydryl reagent. Isolation and investigation of cysteine-containing peptides from human and bovine serum albumin. Binchim. Biophys. Acta 45, 429. Woods, A. G., and Hartley, B. S. 1967.A chemical approach to the substructure of myosin. J . Mol. Biol. 24, 307. Woods, E. F. 1968. Dissociation of tropomyosin by urea. J . Mol. Biol. 16, 1533. Wronski. M. 1963. The determination of cysteine, thioglycolic acid. cyanide, and dithioglycolic acid. Analyst 88, 562. [Abstr.: Chem. Zentralbl. 135, 1769 (1964).] Wronski, M. 1965. Analytical methods in the chemistry of sulphur compounds based on using of mercury compounds. Chem. Listy 59, 1079. Wronski, M. 1967.Submicrodeterminations of thiols, disulfides, and rhiol esters in serum by using o-hydroxymercuribenzoic acid and dithiofluorescein. Biochem. J . 104, 978. Yamamoto, A., Omori. D.. and Yasui, H. 1970. Food seasonings. Jpn. Patent 7020,942(CI. 34 K3). [Chem. Abstr. 74, 75377g (1971).] Yasui, B., Fuchs, F., and Briggs, F. N. 1968.The role of the sulfhydryl groups of tropomyosin and troponin in the calcium control of actomyosin contractility. J . B i d . Chem. 243, 735. Yasui, T.. and Hashimoto, Y. 1966.Effect of freeze-drying on denaturation of myosin from rabbit skeletal muscle. J . Food Sci. 31, 293. Young, M. 1969.The molecular basis of the muscle contraction. Annu. Rev. Biochem. 38, 913. Yuan, H.-Y. 1970.Estimation of sulfhydryl groups in myosin of shrimp. Bull. Inst. Chem., Acud. Sin. 17, 29. Yueh, N. M.. and Strong, F. M. 1960.Some volatile constituents of canned beef. J . Agric. Food Chem. 8, 491.

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111

Zahn. H.. Gerthsen. T.. and Meichelbeck, H. 1962. Eine einfache Methude zur Analyse des Thiolgruppengehaltes von W o k und einige Anwendungen. Mrlliand 43, 1 179. Zima. 0.. and Williams, R. R. 1940. Uber ein antineuritisch wirksames Oxydationsprodukt des Aneurins. Ber. Dtsch. Chern. Ges. 13, 941. Zsolnai. T. 1970. Die antirnikrobielle Wirkung von potentiellen Thiol-Reagentien. Zmtrulhl. Brrkteriol., Parasitenkd. tnfektionskr. H y g . , Aht. I : Orig. 214, 507.

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4 D V A N C t S IN F W U H F \ I . A R L H .

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. 24

HISTAMINE (?) TOXICITY FROM FISH PRODUCTS SALLY HUDSON ARNOLD AND W . DUANE BROWN Institute of M d n i . Rrsourw Depurtmritt of Food Scieitcx*und Trchnology Uniwsity of CnliJt~rnin.Duvis. Cul$miu

I . Nature of the Problcm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Symptomology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Cases of Histamine Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . Earlier Reviews . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Mechanisms of Fornlation of Histamine in Fish . . . . . . . . . . . . . . . . . . . . . . . . A . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Histamine Fonnation by “Autolyric” Enzymes . . . . . . . . . . . . . . . . . . . . . C . Detection of Bacterial Histidine Dccarboxylases . . . . . . . . . . . . . . . . . . . . D . Bacteria Responsible for Histanline Formation . . . . . . . . . . . . . . . . . . . . . . E . Occurrence of Histamine Formers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F . Free Histidine as a Histamine Prccursor . . . . . . . . . . . . . . . . . . . . . . . . . . . G . Histidine Decarboxylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H . Bacterial Destruction of Histaniinc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. “Saurinc” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ill . Detection and Determination of Levels of Histamine in Fish . . . . . . . . . . . . . A . Guinea Pig Ileum Contraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Other Bioassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Fluoromrtric Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . Gas-Liquid Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Colorimetric Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F . Enzymatic Isotopic Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . Thin-Layer Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Relationship of Spoilage to Histarnine Forniation . . . . . . . . . . . . . . . . . . . . . . V . Unresolved Probleiiis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Is Scombroid Toxicity due to Histamine? . . . . . . . . . . . . . . . . . . . . . . . . . B . Improved Analytical Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . The Anserine and Carnosine Question . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . Possiblc Synergists or Potentiators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Allowable Levels of Histamine in Fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Copyright i@ 1978 hy Academic Press . Inr All riphrs of repnduciion in any form reserved . ISBN 0-12-016424-8

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SALLY HUDSON ARNOLD AND W . DUANE BROWN

I.

NATURE OF THE PROBLEM A.

INTRODUCTION

Histamine toxicity from fish products, often called “scombroid poisoning,” generally involves the ingestion of scombroid fish from the families Scomberesocidae and Scombridae. [Note: Because of inconsistencies in the literature with respect to the scientific nomenclature of genera and species of scombroid fishes, we are following the guidelines suggested by Klawe (1976). In some cases, this has necessitated our changing the description given in a cited paper to that used by Klawe.] Scombroid fish include saury, tuna, bonito, seerfish, butterfly kingfish, and mackerel. These fish normally contain large amounts of free histidine in their muscle tissue. The free histidine can, under certain conditions, be decarboxylated by some bacteria to produce high levels of histamine. Such levels of histamine may be reached before the fish appears spoiled or is organoleptically unacceptable. The consumption of both “fresh” and processed fish having significant levels of histamine has resulted in histamine toxicity and clinical illness.

B. SYMPTOMOLOGY Early reports suggested that histamine poisoning was caused by food allergy or idiosyncrasy (Kawabata ef al., I955a). These theories were readily disproven because people were not affected by wholesome products of the same type. Furthermore, several histamine outbreaks occurred in which all of the people eating the food subsequently showed symptoms of histamine toxicity (note references in Section 1,C). A relationship between histamine levels and fish spoilage was recognized by Geiger (1953, who observed that the skin irritation experienced after handling spoiled fish was similar to the skin irritation produced by handling pure histamine. Scombroid fish poisoning clinically resembles that of histamine poisoning intoxication, although controversy still exists as to whether histamine ingested orally is actually toxic (Halstead, 1967; Granerus, 1968; Douglas, 1970). Histamine is believed to be detoxified by bacterial enzymes during its passage through the intestinal wall (Aiso e? al., 1958a). Some workers (Geiger, 1955; Ienistea, 1973) have suggested the possibility that ingestion of large amounts of histamine may overcome the intestinal barrier, with histamine thereby gaining access to the blood; see Section V,A for a more detailed discussion. Whether histamine is the sole toxic factor or not, it is generally found at high concentrations in foods causing scombroid poisoning. Levels of histamine in excess of 100 mg% (100 mg free base per 100 gm of fish flesh) have often been associated with clinical illness (Legroux et al., 1946; Van Veen and Latuasan,

HISTAMINE

(?)

TOXICITY FROM FISH PRODUCTS

I15

1950; Kawabata et a / . , 195%). Furthermore, 100 mg% is often considered the critical concentration for histamine poisoning (Simidu and Hibiki, 1955b; Ferencik, 1970; Anon., 1973d). The onset of symptoms usually occurs several minutes to three hours after ingestion of toxic food (Sapin-Jaloustre, 1957). Typical incubation periods are less than one hour, although a wide variation can occur from individual to individual. For example, victims of an outbreak in Japan experienced incubation periods ranging from five minutes to five hours after consuming seasoned mackerel (Scomber juponicus) (Kawabata et ( I / . , 1955a). The symptoms of histamine toxicity from fish products are characteristic and vary little from outbreak to outbreak. Some victims complain that the toxic food had a characteristic sharp or peppery taste (Halstead, 1967). The most consistently noted symptom is a flushing of the facial and neck area, causing a feeling of intense heat and general discomfort (Sapin-Jaloustre, 1957). The facial flush is principally caused by the dilating action of histamine on the small blood vessels, capillaries, and venules. The flush is followed by an intense, throbbing headache which becomes a continuous dull ache deep in the head, often centered in the frontal and temporal regions (Douglas, 1975). Cardiac palpitation occurs in many instances since the heart beats forcefully but ineffectively (Douglas, 1975). Dizziness, faintness, itching, burning of the mouth and throat, rapid and weak pulse, and inability to swallow are also common characteristics (Anon., 1 9 7 5 ~ ) . Many victims develop a rash on the face and neck, accompanied by severe itching (Halstead, 1967; Anon., 1 9 7 3 ~ ) . Secondary symptoms, experienced by less than 25% of the victims, are gastrointestinal in nature. These usually include abdominal cramps, nausea without vomiting, and diarrhea. Although uncommon, some victims report gastrointestinal symptoms without the common vasomotor symptoms (Sapin-Jaloustre, 1957). In severe cases, shock, brochospasms, suffocation, and severe respiratory distress have occurred (Halstead, 1967). Morbidity values vary from 0.07% to 100% in the literature. Such figures are misleading, as different lots of canned fish may differ greatly in their histamine content. The level of histamine in a whole tuna varies with location, i.e. histamine levels are much higher in the caudal fin area than in the ventral zones (Sapin-Jaloustre, 1957). It is therefore possible for some people to eat fish toxic to others and not be poisoned. C.

CASES O F HISTAMINE TOXICITY

Many cases of scombroid poisoning are thought to be undiagnosed and unreported (Anon., 1973b). Small outbreaks remain undetected even today, as the symptoms are not particularly severe nor long lasting. In mild cases of short duration, medical consultation may not be sought (Foo, 197%). Cases of his-

116

SALLY HUDSON ARNOLD AND W . DUANE. BROWN

tamine poisoning have been reported since the late 1930s and early 1940s. More recently the Japanese performed many comprehensive studies on histamine toxicity and histamine formation in fishery products. The Center for Disease Control (CDC) in Atlanta, Georgia began its food-borne surveillance program in 1966 and most scombroid poisoning events in the United States are now reported through this Center. CDC acknowledges only thirty outbreaks of scombroid type fish poisoning in the United States from 1966 to 1975 (Anon., 1975~).A representative review of some scombroid poisoning cases follows. Atypical cases are also discussed, such as histamine type poisoning from nonscombroid fish and cases in which no histamine could be detected in the incriminated food. Legroux er ul. (1946) described two histamine outbreaks from albacore (Thunnus afulunga)and performed tests which incriminated histamine as the causative factor. The first case occurred in August, 1941 in which 22 of 28 individuals who ate "fresh" tuna showed the usual histamine poisoning symptoms. Guinea pigs could be killed by injections of the tuna homogenate, sterile filtered homogenate, and the heated filtrate. The animals showed symptoms resembling that of histamine shock. The suspension remained toxic after thirty minutes of boiling and after passing on a Chamberland candle, presumably eliminating the possibility of microbial toxins (Sapin-Jaloustre, 1957). Death of the guinea pigs could be prevented by simultaneous injection of neo-antergan, an antihistamine drug. The histamine level was estimated as 1 to 5 gm/kg of flesh (100 to 500 mg%) by assay with the guinea pig ileum test. Legroux er al. (1946) concluded that the cause was not bacterial and that the amount of histidine in the meat could not account for the amount of histamine formed. Strom and Lindberg (1945) described several cases of histaminelike poisonings in Norway. Fresh tuna (Thunnusrhynnus) and canned tuna were implicated. Symptoms included severe headache, reddening of the body, slight shivering, subnormal temperature, and cardiac palpitations. A toxic substance was isolated from a suspect tuna and identified as histamine. Intravenous injection of the toxic substance killed guinea pigs. Stram and Lindberg proposed that the histidine in the protein of tuna had been decarboxylated to form histamine. Van Veen and Latuasan (1 950) discussed the problem of histamine poisoning associated with the consumption of skipjack tuna (Katsuwonus pelamis) in Indonesia. The fish was harmless if caught and eaten immediately. Tropical temperatures appeared to enhance the poisonous characteristics, although the fish did not appear spoiled. Salted and dried skipjack were more prone to cause poisoning than were the manufactured canned product, possibly due to bacterial growth and subsequent decarboxylation of the histidine. Most toxic samples contained 500 to 700 mg% histamine. Causative bacteria were isolated, but not identified. Black skipjack (Euthynnus linearus) was responsible for a more neurotoxic type of fish poisoning outbreak on Johnston Island (situated near the Hawaiian Islands) in 1950 (Halstead, 1954). The fish was said to have been eaten on

HIS1 A M I N t (''1 TOXICITY F R O M FISH PRODUCTS

I I7

previous occasions without producing any toxic symptoms. Five out of five people consuming the fish developed nausea. comiting. tingling. intestinal cramps. cold clammy skin, and mild diarrhea a few hours after ingestion of the meal. Symptoms subsided after 35 hours with a convalescent period of several weeks, during which time weakness and muscular pains were the typical syniptoms. The Johnston Island outbreak is significant because the ingestion of black skipjack presumably caused symptoms typical of the ciguatera-type (more coninionly produced by the reef fishes) rather than symptoms of scombroid poisoning. Kawabata ef 01. (195Sa) studied the Japanese episodes of allergylike food poisoning caused by dried seasoned saury (Cololuhis suira) and other products. At that time, seafood poisoning of unknown origin accounted for 60 to 70% of all Japanese food poisonings. They differentiated these unknown poisonings from bacterial food poisoning, chemical poisoning, naturally formed toxic substanccs in plants and animals, and allergy or idiosyncrasy. Fourteen outbreaks, involving more than 1000 people were discussed by Kawabala et ul. (l955a); see Table I . Four of the outbreaks were studied epidemiologically. In all cases studied, other vagus stimulants, e.g., saurine, were believed to be present in addition to histamine (saurine is now believed to be identical to histamine; see Section 11,I). "Samma sakuraboshi" was incriminated in many of the Japanese histamine and histaminelike fish poisonings. It is made by pickling raw mackerel pike (Cololubis xrira) for twenty to thirty hours in a wheat gluten syrup, then sun dried (Aiso rt al., 1958a). The product is either broiled before consumption or eaten uncooked. The first outbreak studied by Kawabata ('1 t r l . (1955a) occurred in Hiroshima in I953 after the consumption o f sanima sakuraboshi (Cololubis srriru). All who atc the product became i l l . The incubation period ranged from 30 minutes to 2% hours. Typical symptoms included reddening of the face and upper body, palpitation, severe headache. and nausea. Diarrhea, abdominal pain, and vomiting were absent. All victims recovered. Inorganic poisons. alkaloids and other preservatives were not found in the fond. About 400 to 500 mg% histamine was detected in samples by chemical methods, as opposed to 0.3 to 1 . 1 mg% in the controls. The second outbreak occurred in 1953 in Kumamoto. Japan. Of 850 consumers of a lunch. 85 complained of burning sensations, reddening. flush, palpitation, and headache. Some vomited. others had diarrhea; all body teniperatures were normal. The incubation period ranged from 30 minutes to 1 % hours. Canned seasoned mackerel (Scotnbcr .jtrponicws) was incriminated a s thc toxic food. The bacterial count in the cans was negative. color of the meat subnormal with a pH of 6.0 to 6.2 and the meat had a slightly irritative tastc. The low morbidity (10%) was explained because the cans were from different companies and only one sample contained the toxic substance. Another outbreak occurrcd in 1954 in Kanagawa, Japan involving elcvcn people who ate frigate tuna ( A ~ r i s

TABLE I A LIST OF REPORTED SCOMBROID TOXICITY OUTBREAKS IN JAPAN, 1951-1954 BASED ON KAWABATA ET AL. (1955a).

Date

Cases

Oct. 1951

700

June 1952 Oct. 1952

17

Oct. 1952

22

Oct. 1952

94

Nov. 1952

6

Feb. 1953

II

July 1953

85

Oct. 1953

72

Oct. 1953

6

Nov. 1953

3

Dec. 1953

13

Aug. 1954

II

Oct. 1954

90

25

1215 (Total cases)

Source samma sakuraboshi (Cololabis suira) iwashi sakuraboshi samma sakuraboshi (Cololubis saira) samma sakuraboshi (Cololabis saira) samma sakuraboshi (Cololubis saira) samma sakuraboshi (Cololabis suira) samma sakuraboshi (Cololubis suiru) canned mackerel (Scomberjaponicus) samma sakuraboshi (Cololubis saira) samma sakuraboshi (Cololubis saira) samma sakuraboshi (Cololubis saira) samma sakuraboshi (Cololabis saira) frigate tuna (Auxis thaiard) samma sakuraboshi (Cololubis suiru)

Symptoms Flushing of the body and itchiness. Some diarrhea and vomiting Headache, chills, flushing, rash, and fever Facial flush, headache, nausea, and vomiting. Some diarrhea Headache, chills, and facial flush. Some diarrhea and vomiting Headache, chills. and facial flush. No diarrhea or vomiting Headache and chills Facial flush, cardiac palpitation, headache, and nausea No vomiting or diarrhea Flushing, cardiac palpitation, and headache. Some vomiting and diarrhea Headache, flushing, rash and vomiting No diarrhea Headache, chills, and reddening Diarrhea and vomiting Rash Flushing of the body and facial rash. No headache, fever, diarrhea, or vomiting Flushing, rash, and headache. Some vomiting and nausea. No diarrhea

HISTAMINE

(‘?) TOXICITY

FROM FISH PRODUCTS

1 I9

thazard) which had been caught the previous day. They experienced typical symptoms. Histamine was detected in the fish by paper chromatography, Bacteriological examination was negative. Histamine levels were 97 to 128 mg%. Volatile basic nitrogen and trimethylamine nitrogen values were normal. Boyer et al. (1956) studied a large intoxication from four tons of “fresh” tuna (Thunnus thymus). Nearly 500 people were affected, with a morbidity rate of 13%. Nine batches were analyzed with histamine values ranging from 28 to 4000 mg%; various bacteria were present in significant amounts. It was concluded that the amount of available histidine in the tuna could account for the elevated histamine levels and that the histamine was produced by bacterial decarboxylation. Initial contamination presumably occurred because the fish were not properly chilled, nor were the guts removed. The authors proposed close surveillance of sea practices of fisherman, rather than onshore inspection, which was considered useless. In 1968, eight of nine people became ill 30 minutes after eating “fresh” tuna fish (Anon., 1968). Typical scombroid toxicity symptoms with associated gastrointestinal symptoms lasted from 1 to 4 hours. Patients were given antihistamines with subsequent marked improvement. The fish contained 425.5 mg% histamine and was negative for Proteus species. In Vermont in 1972, four people experienced nausea, diarrhea, and prostration fifteen minutes to one hour after consumption of “fresh” tuna steak purchased from a local market (Anon., 1972). Histamine formation was attributed to the lengthy unrefrigerated storage (5 1-55 hours) of the tuna at the market. An employee eating 1 Vi pounds of the tuna 3 hours after storage did not get ill. Citrobacter, Proteus, Enterobacter, and Streptococcus were isolated from the remaining tuna in the market. Fresh tuna was again incriminated in New York in 1975 (Anon., 1975c,d). A family of four experienced headache, nausea, and diarrhea two hours after eating commercially smoked albacore (Thunnus alalunga) in 1972 (Anon., 1973b). Recovery occurred in 3 to 5 hours, except for the father who was hospitalized for shock. Intravenous fluids and antihistamines rapidly improved his condition and he was discharged two days later. No fish could be obtained for chemical analysis, but the outbreak was presumed to be caused by bacterial degradation of histidine to histamine. In 1973, the Center for Disease Control reported the first recorded outbreak of scombroid fish poisoning from a commercially canned food product in the United States since CDC began food-borne surveillance in 1966 (Anon., 1973d). Canned tuna caused 254 clinical cases in eight states (Anon., 1973a). The symptoms included immediate oral burning and blistering, followed in 30 to 45 minutes by headache, abdominal cramps, diarrhea, and flushing (Merson etal., 1974). No cases required hospitalization. Nine assays for histamine produced values ranging from 76 to 280 mg% in the incriminated lots, with controls at the 2.7 mg%

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level (Anon., 1973d). These lots were later recalled by the Food and Drug Administration. A case of histamine poisoning was reported in 1973 in which no histamine could be detected in the incriminated food (Anon., 1 9 7 3 ~ )Thirty . o f 4 8 children experienced a sudden onset of a rash associated with intense itching 15 minutes after beginning lunch. Symptoms lasted 15 minutes to 2% hours. A tuna casserole, made from commercially baked tuna, was incriminated as the source of the illness. The casserole was negative for bacterial histamine formers and contained no detectable histamine. Cans in the remaining cases of the lot appeared normal. Despite the negative findings, the incubation and symptoms of the outbreak were thought to be consistent with those of scombroid fish poisoning. A number of cases of histaminelike poisoning have been reported in which the incriminated food was not of the scombroid type. Scombroid poisoning was reported as the cause of three incidents in New Zealand in 1973 (Foo, 1975a). Two cases involved a canned mackerel product but the third involved smoked kahawai. This is presumably the first report of scombroid poisoning from kahawai, a nonscombroid fish, although it was assumed that other cases involving kahawai had occurred in the past. A level of 800 mg% histamine was detected in the kahawai samples. The kahawai had been allowed to dry for 3 days before smoking. It was presumed that bacterial action formed toxins during the drying and that these toxins (histamine and histaminelike compounds) survived the smoking process. Kingfish was later implicated in a histaminelike outbreak in New Zealand (Foo, 1975b). Scombroid toxicity from mahi-mahi (dolphin fish) was reported in 1973 (Anon., 1973e). The first case involved two women after eating mahi-mahi (Corypharna hippuuus) at a restaurant. Dolphin fish belong to the family Coryphaenidae which is unlike the Scombridae. However, the flesh of dolphin fish does contain large amounts of free histidine (Hibiki and Simidu, 1959). Two subsequent mahi-mahi outbreaks occurred in the state of California later in the year. It was determined that there was improper refrigeration at the time the fish was caught, and also improper handling at the retail level which could result in scombroid poisoning (Anon., 1973e). A subsequent histamine poisoning linked to mahi-mahi occurred in 1975 (Anon., 1975a). It can be concluded that most histaminelike outbreaks occur after consumption of scombroid fish or other fish normally containing large amounts of free histidine in muscle tissue. The incriminated fish generally contains histamine levels in excess of 100 mg%.

D. EARLIER REVIEWS Earlier reviews of histamine toxicity from fish products include SapinJaloustre (1957), Kimata (1961). Halstead (1967) and Ienistea (1971, 1973).

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Douglas (1970. 1975) provides an excellent review of the pharmacology of histamine.

II. MECHANISMS OF FORMATION OF HISTAMINE IN FISH A.

INTRODUCTION

Suzuki et ul. (1909) were among the first to report the presence of histamine in tuna extracts. Geiger et ul. (1944) investigated the content and formation of histamine in mackerel (Scornheromorus concolor), sardines (Surdinops sugux) and albacore (Thunnus ululungu) extracts. Early studies concentrated on histamine formation from autolysis (Geiger et al., 1944; Kimata and Kawai, 19S3e; Hayashi, 1954). although it was concluded that histamine was formed post mortem from bacterial contamination (Geiger ef ul., 1944). Histamine forming bacteria were later isolated and further characterized. Extensive studies of bacterial histidine decarboxylases were subsequently performed.

B . HISTAMINE FORMATION B Y “AUTOLYTIC” ENZYMES Geiger et al. (1944) studied the production of histamine by autolytic enzymes and concluded that bacterial action was responsible for the formation of histamine. A sample of mackerel muscle paste incubated with 3% chloroform and 5% toluene produced an insignificant amount of histamine in 24 hours, whereas a sample without these preservatives produced a considerable amount of histamine. Kimata and Kawai (1953a) arrived at similar conclusions with red meat fish (mackerel and tuna). Small amounts of histamine were thought to be produced by enzymes inherent in the meat. Under optimal autolytic conditions of pH (pH 3 . 5 4 . 5 ) and temperature (40°4S”C) only 10 to IS mg% histamine was formed from frigate tuna (Auxis thuzard) and chub mackerel (Scomberjuponicus) (Kimata and Kawai, 1953d,e). White meat fish produced substantially lower levels of histamine by autolysis (Kimata and Kawai, 1953d,f). It is most likely that the histamine produced by “autolysis” was due to previous bacterial contamination or unsterile conditions during experimentation, thus enabling histamine production by the contaminating microorganisms. Kimata and Kawai (1 953a) acknowledged that their results could have been influenced by the action of enzymes formed from bacteria which had already grown on the fish before the initial experiments. Ferencik ( 1 970) studied strains of Hufniu, Proteus morgunii, and E . c d i that were able to form high levels of histamine in sterile tuna and skipjack flesh. Histamine formation depended on the free histidine content of the fish and the histidine decarboxylase activity of the bacteria. Fish flesh experimentally infected with bacteria having no active his-

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tidine decarboxylase did not contain histamine at any stage of decomposition. Flesh kept under sterile conditions also failed to form any histamine. C.

DETECTION O F BACTERIAL HISTIDINE DECARBOXYLASES

It is not the purpose of this review to provide a comprehensive discussion of the detection of bacterial histidine decarboxylases, but a brief overview has been included. Histamine has often been detected in bacterial cultures and fish infusions by the conventional chemical and biological methods described in Section 111. However, some methods have been developed specifically for detecting, measuring, and characterizing bacterial amino acid decarboxylases. Eggerth et al. (1939) developed an improved extraction method for measuring small amounts of histamine in bacterial culture media. Gale (1940, 1946) studied bacterial amino acid decarboxylases in detail by measuring the evolution of C 0 2 with a Warburg manometer. Epps (1945) extracted and purified L-histidine decarboxylase from Clostridium welchii (now called C. perfringens) and examined its properties. Several cell free amino acid decarboxylases were made and characterized by Gale ( 1 946). Histidine decarboxylase from Lactobacillus 30a was purified by Rosenthaler et al. (1965); its substrate specificity, sterospecificity, composition, and subunit structure were studied by Chang and Snell (1968a,b). Mpller (1954a,b) developed several methods for activity determination and distribution of amino acid decarboxylases in enteric bacteria. A method for measuring the total amino acid decarboxylase contents of bacteria was devised (Mpller, 1954a). Mpller (1954b, 1955) also developed a color test for measuring various amino acid decarboxylases which was subsequently modified by Shaw and Clarke (1955). The color test for decarboxylases was later shown to be an indicator of pH change in the medium and not necessarily specific for the presence of histidine decarboxylase enzymes (Havelka, 1969). Levine and Watts (1 966) developed a radioactive method for measuring histidine decarboxylase activity. Histidine was labeled with 14C in the carboxyl carbon and the evolved [14C]C02was trapped and measured. Schayer (1971) provides a recent review on the determination of histidine decarboxylase activity.

D. BACTERIA RESPONSIBLE FOR HISTAMINE FORMATION Early microbial studies indicated that many bacteria were able to form amino acid decarboxylases. Eggerth (1939) studied the histidine decarboxylase activity of Escherichia, Aerobacter (now called Klebsiella), Salmonella, and Shigella. Gale (1946) detected histidine decarboxylase activity in Escherichia, Clostridium, and Klebsiella but not in Proteus, Bacillus, or Streptococcus. Ehrismann and Werle (1948) found that Mbrio, Proteus, and other gram-negative bacteria were able to form histamine.

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Van Veen and Latuasan (1950) isolated two strains of anaerobic salt tolerant bacteria able to produce 35 mg of histamine per 10 gm of fish flesh (350 mg%) from skipjack tuna (Karsuwonus pelarnis) indicated in a histaminelike outbreak of poisoning. They were identified only as a small gram-positive coccus and a gram-positive rod. Kimata and Kawai (1953b) isolated a “new” species of bacteria able to produce large amounts of histamine from spoiled fish. It was given the name “Achromobacter histarnineurn, although it was later shown to be identical to Proreus rnorganii (Kimata et al., 1958; Kimata and Kawai, 1958). Production of histamine by “ A . histarnineurn” corresponded to the growth of the organism when incubated at different temperatures (Kimata and Kawai, 1 9 5 3 ~ )When . a frigate tuna (Auxis thazard) extract was inoculated with the organism, more than 200 mg% of histamine was formed at 27°C in three days. Kimata and Akamatsu (1955a) later reported that two strains of “ A . histarnineurn,” known as Type 1 and Type 2, existed. Both types were able to produce large amounts of histamine from histidine and only small amounts of ammonia from amino acids. It is most probable that the two “types” were merely different strains of Proteus rnorganii, or perhaps different Proteus species. Growth and histamine formation of the two types were extensively studied (Kimata and Akamatsu, 1955b). Type 2 had a growth optimum of 30°C; Type 1 had an optimum of 20 to 25°C. Production of histamine at various temperatures varied with the growth rate of Type 1, but not with Type 2. The pH optimum for histamine formation for Type 1 was pH 5 and that of Type 2 was pH 6 . At a pH of 7, Type 1 produced very little histamine, whereas Type 2 was able to produce a significant amount. The optimum sodium chloride concentration for growth was 1% and for histamine production was 2 to 3% in both types. Proteus organisms continued to be implicated as the responsible histamine formers in scombroid and other fish. Kawabata et al. (195613) isolated 78 strains of bacteria from “sashimi” (sliced raw fish made from Parathunnus rnebachi) that had previously caused a histamine outbreak. Eleven of the 78 strains isolated from the sashimi were able to produce histamine. Five were identified as Proteus vulgaris, three as Proteus rnirabilis and three as Proteus morganii. They suggested a mode of putrefaction in which the organism could produce large amounts of the toxic substances without producing signs of deterioration in the food. Kawabata and Suzuki (1959b) reported that histidine decarboxylase activity was highest when cell division ceased, although slight activity was still detectable when the bacteria reached the death phase. Aiso et al. (1958b) studied the histidine decarboxylase activity in 84 strains of Morganella (now called Proteus morganii). Histidine decarboxylase activity was measured by the quantitative determination of histamine formed in the culture media and by estimation of the QCOz by the Warburg method. All Proreus morganii strains, irrespective of source, were able to produce histamine. A ”

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maximum of 400 mg% histamine was formed in fish meat infusion broth within 76 hours. Escherichia, Shigella, Protrus vulgaris, and Clostridium perfringens also exhibited slight histamine forming activity. Similarly, others (Kimataer al., 1960) tested 88 Proteus strains. All Proteus morganii strains could produce histamine; P . vulgaris, P . mirabilis, and P . rettgeri produced either insignificant amounts of histamine or none. The presence of high levels of Proteus organisms is currently used as a criterion of scombroid poisoning (Anon., 1972, 1975c,d). Other organisms such as Hafnia and E . coli are able to produce significant amounts of histamine, but at much slower rates (Ferencik, 1970). Proteus morganii is especially incriminated as the responsible histamine former because of its ability to rapidly produce histamine in excess of 100 mg%. E.

OCCURRENCE OF HISTAMINE FORMERS

Studies of the bacteria involved with fish spoilage indicated that only a small percentage were able to produce large amounts of histamine. Kawai (1962) indicated that histamine-forming bacteria isolated from fresh fish were found much less frequently than those organisms able to produce other amines. Kimata and Kawai (1 953g) isolated one strain out of twenty-five that was able to produce histamine in significant quantities. The responsible organism was identified as “Achromobacter histamineum” (Proteus morganii). When fresh mackerel (Scomber japonicus) were spoiled at 20°C, 5 to 30% of the total number of bacteria were histamine formers (Kimata and Tanaka, 1954a). Production of histamine was again concluded to be the result of a certain microorganism ( P . morganii) and not due to the action of all kinds of bacteria causing spoilage. Histamine formers have been detected as part of the normal surface microflora of fresh fish. However, Cantoni et a / . (1976a) reported that bacteria such as Proteus, Escherichia, and Clostridium were contaminants and not part of the normal microflora of fish. Kimata and Tanaka (1954b) found that 0.1 to I % of the total surface bacteria were usually histamine formers, whereas ammonia formers accounted for 10%. Live fish generally contained higher percentage levels of histamine formers than “fresh” (marketed) fish. The actual number of histamine formers did not decrease; the nonhistamine formers grew more rapidly and consequently the percentage level of histamine formers was lower in the marketed fish.

F. FREE HISTIDINE AS A HISTAMINE PRECURSOR Geiger (1944b) observed that the ability of E . coli to decarboxylate histidine was highly specific since histamine formation was inhibited by acylation of the amino group of histidine. Geiger (1948) later studied histamine formation with a Clostridium welchii (now called C. perfringens) enzyme preparation, a strain of

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E . coli. and a strain of marine bacteria. All were able to form histamine from free L-histidine, but none was able to form histamine from aspartyl-histidine o r histidyl-histidine. It was concluded that peptide linkages to either the -COOH or -NH, group of histidine prevented bacterial decarboxylation. It remains well documented that free histidine is required for histamine formation. Ferencik (1970) has recently concluded that the main condition for histamine synthesis was an adequate concentration of free histidine in the fish, as only free histidine can be decarboxylated. Toxic levels of histamine are generally limited to the red meat of free-swimming species because these species contain a large amount of free histidine in the muscle (Aiso et al., 1958a; Lukton and Olcott, 1958) while crustacea and white meat fish contain very low levels of histidine (Hibiki and Simidu, 1959; Lukton and Olcott, 1958).

G. HISTIDlNE DECARBOXYLASE I.

Effect of Trmperature

Gale ( 1940) observed that bacterial amino acid decarboxylases were generally most active at temperatures below 30°C. Kimata and Kawai (1953a.c) reported that Achromobacter histarnitieum (now called Proteus morganii) had an optimum growth temperature of 20" to 25"C, but produced the highest levels of histamine at 20°C. Negligible amounts of histamine were formed at 35°C. No histamine was produced by the organism at 40°C (Kimata and Akamatsu, 1955b) since growth could not occur at that temperature. Hibiki and Simidu (1959) later found that the responsible histamine-forming bacteria were killed at 60°C. Wide variation in histamine formation can commonly be found in storage trials at ambient temperatures. Edmunds and Eitenmiller (1975) recently observed that histamine production varied significantly between storage trials performed at ambient temperatures. Omura ( 1976) detected one hundredfold variations in histamine concentrations from skipjack tuna that were allowed to spoil under similar conditions. Kimata and Kawai (1953a) allowed mackerel fillets to undergo bacterial spoilage at various temperatures. The amounts of histamine present at the time in which the fish were judged organoleptically unacceptable as food was as t'ollows: Storage temperature

35°C 17°C 6-7°C

Histamine 14 mp% 354 mgL% 50-70 mg%

Time fish judged unacceptable 20 hr 75 h r 150-200 hr

A similar study with whole fish (Kimata and Kawai, 1953a) indicated that at the time in which the fish were judged unacceptable (46 hours), 826 mg% histamine had been produced at 23°C. In contrast, others (Anon., 1975b) studied the

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formation of histamine in mackerel under various storage conditions and concluded that the fish were unfit for commercial use by the time the histamine concentration reached 100 mg%. Numerous studies have been made on histamine production at near-freezing and freezing temperatures. Shewan and Liston (1955) reported that histidine was easily decarboxylated at 0°C. It has been suggested (Ota and Kaneko, 1958; Kalyani and Bai, 1965; Edmunds and Eitenmiller, 1975) that histamine-forming bacteria were killed or unable to produce histamine at this temperature. Kalyani and Bai (1965) observed that histamine production was slowed at 10°C and nearly terminated at 5°C due to the destruction of histamine-producing bacteria. Edmunds and Eitenmiller (1975) found that little histamine was formed at 4°C in storage trials of Spanish mackerel and other fish. They concluded that the psychrophilic microorganisms did not readily decarboxylate free histidine. Others (Anon., 1975b) reported that small amounts of histamine were formed at 6"C, depending on the humidity of the environment and that no histamine was formed at -20°C in two months. Current work in progress has shown that certain strains of P . morganii and P . vulgaris are able to produce levels of histamine in excess of 120 mg% when allowed to grow in tuna infusion broth for six days at 7°C. No histamine was formed by P . morganii, P . vulgaris, or Hafnia strains after one months incubation at 1°C. It is evident that other factors besides temperature and time can drastically influence histamine formation. Possible factors may be related to the original bacterial flora of the fish, prior bacterial contamination from catching and handling the fish, environmental factors, etc. However, it can be concluded that one of the best methods for retarding bacterial histamine formation in fish is to store the fish at temperatures below the freezing point of fish muscle. 2 . Effect of pH Early studies of amino acid decarboxylases by Gale (1940, 1946) and Epps (1945) demonstrated that the pH optimum was acidic, ranging from pH 2.5 to 6.5. Alin (1950) concluded that during bacterial growth an acid medium stimulated the formation of decarboxylases. Aiso et al. (1958b) found that Morganella (now called Proteus morganii) was able to form histidine decarboxylase in both neutral and acidic environments. Thirteen Proteus strains, including P . morganii, P . mirabilis, P . rettgeri, and P . vulgaris were shown to have a histidine decarboxylase pH optimum of 6.0 to 6.5 (Kawabata and Suzuki, 1959a). Kawabata and Suzuki (1959b) concluded that the maximum production of histamine in Proreus morganii occurred at pH 5.1, the lowest value supporting growth. Organisms grown at pH 5 . 5 to 7.3 possessed about 50% of the maximum activity and those grown at pH 7.6 to 8.7 possessed 10% of the maximum activity. Simidu and Hibiki (1954b,c,d) found that the pH of fresh scombroid

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fish flesh generally ranged from pH 5.5 to 6.5. The slightly acidic pH of the fish thereby enhances the production of histamine by the responsible histidine decarboxylating bacteria. 3.

Effect of Carbohydrates

Early workers (Eggerth, 1939; Gale, 1940) observed that glucose and other fermentable sugars intensified bacterial amino acid decarboxylase activity. Aiso et al. (1955) noted that the addition of glucose to diluted fish meat extracts caused a substantial increase in histamine formation. Kimata and Kawai (1958) found that carbohydrates such as glucose and fructose enhanced the formation of histamine in diluted fish extracts but not in synthetic media. The synthetic media contained sufficient amounts of carbohydrate whereas the diluted fish extract was slightly deficient. They concluded that the bacteria required a certain amount of carbohydrate as an energy source for biosynthesis and enzyme production. Others (Kawabata and Suzuki, I959b) observed that the presence of a fermentable carbohydrate, such as glucose, enhanced both growth and histidine decarboxylase activity in Proteus morganii. Glucose concentrations of 0.5 to 2% were most effective in mackerel infusion media. Levels in excess of 3% inhibited enzyme formation despite the low pH produced during bacterial growth on glucose. 4.

Effect of Vitamins and Coenzymes

Several studies of bacterial histidine decarboxylase have indicated that the addition of vitamins and coenzymes did not enhance histamine formation. Epps (1945) reported that codecarboxylase was absent in L-histidine decarboxylase extracted from Clostridium welchii (now called Clostridium perfringens). Gale ( I 946) observed that the addition of cofactors such as pyridoxine (vitamin Be) and nicotinic acid (vitamin B3) in excess of simple growth requirements sometimes promoted tyrosine, lysine, and ornithine decarboxylase but not histidine decarboxylase. Rosenthaler et al. (1965) determined that histidine decarboxylase from Lacrobacillus 30a did not require pyridoxal-5'-phosphate as a coenzyme. They concluded that no known coenzyme was required. Work with Proteus morganii by Kimata and Kawai (1958) showed that none of the vitamin B complex promoted histamine formation in synthetic or fish infusion media.

5 . Effect of 0,yygen Tension Kimata and Kawai (1958) found that the addition of cysteine, cysteine, and methionine stimulated histamine formation in P . morganii. Other amino acids (except histidine, of course) had no such effect. They suggested that cysteine,

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cystine, and methionine effectively reduced the redox potential of the media, thereby stimulating histamine production. Sodium-thioglycollate and ascorbic acid (redox potential reducers) also stimulated histamine production. Kimita and Kawai (1959) later observed that histamine formation in washed cell suspensions of P . morganii were affected by the oxygen tension. Histamine formation in aerated cultures, achieved by the addition of bubbled gas, was much less than that from anaerobic and aerobic cultures. They (Kimata and Kawai, 1959) concluded that the histidine decarboxylase activity of P . morganii was destroyed or inactivated in the presence of oxygen. Ferencik (1970) reported that anaerobic conditions caused slower histamine formation than aerobic conditions in Hafnia.

H.

BACTERIAL DESTRUCTION OF HISTAMINE

Gale (1 942) reported that any study of bacterial amine production should also take into account any further breakdown of these substances by other microorganisms. Ienistea (1971) suggested that bacterial histaminase could play an important role in foods containing high concentrations of histamine. It was proposed that an equilibrium between histamine production and destruction occurred in these foods. [Note: Histaminase refers to the enzyme capable of oxidative deamination of histamine and is frequently called diaminoxidase (Douglas, 1975)~ Bacterial histaminase (diaminoxidase) activity has been detected in several types of bacteria including some species of Pseudomanas (Werle, 1940. 194 1 ; Gale, 1942; A h , 1950), Profeus (Werle, 1940), Escherichia (Werle, 1941; A h , 1950), Vibrio (Ehrismann and Werle, 1948), Clostridium (Ehrismann and Werle, 1948). and Klebsiella (Ehrismann and Werle, 1948). Gale (1942) reported that the bacterial enzymes capable of histamine oxidation were inducible. Bacterial histaminases were best produced under somewhat alkaline conditions (pH 7.5-8), although moderate histaminase activity was detected under slightly acidic conditions (Gale, 1942; A h , 1950). Ferencik (1970) reported that a strain of Proteus morganii, after being inoculated into a sterile tuna flesh homogenate, produced large amounts of histamine. However, a significant amount of the histamine was soon decomposed by the organism. A subsequent sterile addition of histidine to the inoculated homogenate again resulted in histamine formation, then histamine destruction. Ferencik (1970) concluded that the histamine production and destruction by the P. morganii strain was determined by the concentration of free histidine in the fish homogenate. The minimum histidine concentration required for histidine decarboxylase activity appeared to be 100 to 200 mg%. Work in progress (Arnold, 1976) deals with a similar observation in a P. morganii strain and a P . vulgaris strain previously isolated from intentionally spoiled skipjack tuna.

HISTAMINE

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129

“SAURINE”

Kawabataet ul. (1955a) suspected that an additional toxin or a compound able to act synergistically with histamine was present in foods causing histamine poisoning. Chemical methods often produced histamine values ten times less than that found by the guinea pig ileum test, suggesting that an unknown vagus stimulant was present. [Note: Vagus refers to the tenth pair of cranial nerves. Among other functions, these nerves innervate the muscles of the abdominal viscera and conduct the impulses to the brain.] A newly isolated vagus stimulant was detected by one- and two-dimensional chromatography (Kawabata et ul., 1955b). It was named “saurine.” referring to the fish saury (Cololabis suira) that had often been implicated in histamine poisonings in Japan. Proteus rnorganii was later incriminated as the source of saurine (Kawabata rt d., 1956b). as it appeared to be able to produce large amounts of both histamine and saurine. Saurine was differentiated from histamine by its lower Rf value, its negative reaction with diazo reagent, and its possible insolubility in alcohol (Kawabata et al., 1955b). Saurine was characterized as a heat stable, basic, low molecular weight substance (Kawabata e f ul., 195Sb). Its chemical and biological effects were similar to those of histamine; saurine exhibited an additive and not a synergistic effect to that of histamine (Kawabata et nl., 1 9 5 5 ~ )The . effects of antihistamines on saurine were proportional to their antihistaminic activity (Kawabata et ul., 1955d). Olcott and Lukton (1961) suggested that “saurine” could be an artifact. A culture of Proteus morguriii previously able to produce saurine was obtained from Kawabata. A tuna extract was inoculated with the organism. Two spots were obtained by two-dimensional chromatography, both able to exhibit vagusstimulating activity on the guinea pig ileum. When histamine alone was added to the original tuna extract, a similar pattern was obtained, suggesting that histamine was able to produce both spots under these conditions. Kawabata subsequently informed Olcott and Lukton that the culture had lost its ability to form saurine (Olcott and Lukton, 1961). Work in the 1950’s demonstrated that amines could produce more than one chromatographic spot in some situations. Waldron-Edward (1954) observed that amines were able to produce multiple spots on paper chromatograms under certain conditions and that typical color reactions (e.g. the ninhydrin reaction) could be adversely affected. West and Riley (1954) reported that two sharply defined spots corresponding to histamine appeared on chromatograms of tissue histamine. Both spots produced vagus-stimulating activity on the isolated guinea pig ileum. There is little doubt that ”saurine” is identical to histamine. Unfortunately, the presence of saurine has been discussed in the literature since its “discovery” and it continues to be mentioned (Anon.. 1973d; Foo. 1975a).

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J.

CONCLUSION

Bacterial histamine formation is dependent on (1) an adequate concentration of histidine in the free form, (2) the presence of microorganisms able to produce histidine decarboxylase, and (3) conditions conducive to histidine decarboxylase synthesis and subsequent decarboxylation. Scombroid fish contain both a high level of histidine in the free form and a significant number of histamine-forming bacteria as part of their natural microflora. It is therefore necessary to maintain conditions that adequately supresses histamine formation; the most practical method is to keep the fish at temperatures below freezing.

111.

DETECTION AND DETERMINATION OF LEVELS OF HISTAMINE IN FISH A.

GUINEA PIG ILEUM CONTRACTION

The classical method for the determination of histamine is based on the fact that it will cause contraction of guinea pig ileum. Such methods currently employed are based on the procedures suggested by Barsoum and Gaddum (1935). These authors actually proposed that response of two tissues, guinea pig ileum and rectal cecum of a fowl, be employed. In their opinion, if a test material applied to both tissues caused contraction in both instances it could be considered to be histamine without further confirmation. Subsequently, however, most workers have employed the guinea pig tissue alone. The first application of this method to the analysis of histamine in fish tissue appears to be that reported by Geiger (1944a). He and fellow workers had earlier identified a biologically active substance in marine fish as histamine (Geiger et al., 1944). He detailed how the method could be applied to fish and reported findings on histamine levels in raw and canned sardines and mackerel. He pointed out that canning of these fish did not interfere with subsequent analysis for histamine, and demonstrated the practical application of histamine determination in canned tuna. Hillig (1956) subsequently applied Geiger’s method to several species of tuna at varying stages of decomposition while Sager and Horwiti (1957) compared the bioassay with a chemical method based on coupling histamine, in a chromatographically purified extract from fish, with a diazonium compound. Formation of the latter substance was measured spectrophotometrically. They found that values with the bioassay were usually higher than those obtained by the chemical method. However, when they slightly modified the extraction procedure used in the bioassay, they were able to obtain results with the modified bioassay that checked well with those obtained by the chemical method.

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Following the description of the use of guinea pig ileum contraction as a bioassay for histamine, there was considerable related work, much of it aimed at improvement in the extraction procedure. One early suggestion was the use of butanol extraction of an aqueous histamine-containing extract, followed by removal of histamine from the butanol by cotton acid succinate (McIntire ef al., 1947). Kadota and Inoue (1953) subsequently applied McIntire's method to the determination of histamine found in decomposed fish. They found the method suitable for rapid determination of histamine in a large number of samples. Other suggestions have included the utilization of various cation exchange resins to purify histamine from tissue extracts (Roberts and Adam, 1950; Michaelson and Coffman, 1969). Work to I956 is well summarized in a review by Code and McIntire (1 956) dealing with the quantitative determination of histamine; this review includes a detailed description of the guinea pig intestine assay as well as a summary of chemical methods developed at that time. B . OTHER BIOASSAYS The use of the guinea pig ileum contraction technique is far and away the most commonly applied bioassay. However, others have been suggested. De Waart et al. (1972) evaluated a total of 32 biological test systems, including 6 protozoa, 4 fish species, 5 insects, bull spermatozoa, 4 cell lines, 9 microorganisms, embryonated hen's eggs. Daphnia and Artemia for sensitivity to a variety of microbial toxins and histamine. Some of the protozoa, fish, Artemia, and particularly Daphnia were found to be sensitive to histamine. The latter organism, a small fresh-water crustacean, is being used presently by an investigator at the University of California at Davis (Blonz, 1976). He has found that the addition of histamine, or extracts from tuna known to have caused human illness, to water housing Daphnia causes the death of 90 to 100% of the animals in 20 to 50 minutes, compared to a 0 4 % effect in animals receiving an extract from good tuna. Daphnia are readily available and easy to house, with algae being a suitable food source. Potential significance of their use at this time awaits additional experimental findings. C.

FLUOROMETRIC ASSAY

In spite of the fact that accurate measurements are known to be possible with the commonly used guinea pig ileum bioassay, such methods have numerous disadvantages. Provisions must be made for the care and handling of guinea pigs and there is the frequently noted individual variability in ileum response. For these and other reasons, there has been increased interest in the development of suitable chemical means for histamine determination. The use of fluorometry has evolved as a major tool for the assay of histamine.

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A relatively simple fluorometric method, said to be precise and sensitive for histamine, was reported by Shore er al. (1959). It involves extraction of histamine from alkalinized perchloric acid tissue extracts with n-butanol, and its ultimate condensation with o-phthalaldehyde to yield a stable and strongly fluorescing product. The authors noted that levels of histamine as low as 0.005 pg/ml could be assayed. However methods with extraordinary sensitivity are not usually required for assay of histamine in fish, inasmuch as histamine levels in decomposed fish will be much greater than those found in ordinary tissues. Fluorometric assays have found broad application for the determination of histamine in a variety of biological materials (Noah and Brand, 1963; von Redlick and Click, 1965; Huffrt al., 1966) and wine (Ough, 1971). There is detailed description of the use of the fluorometric method for the measurement of histamine in a review by Shore (1971), which includes coverage of means of extraction of histamine from tissues together with a description of special purification procedures. More recent developments in this field include development of the use of fluorescamine to react with amino acids, peptides, proteins, and primary amines (Udenfriend el at., 1972). This reagent reacts rapidly at room temperature in aqueous media and yields highly fluorescent products. The reagent and its degradation products are nonfluorescent. It can detect arnines in the picomole range; again, however, as a practical matter, such limit of detection is not critical for assay of histamine iii fish, where levels are much higher. Hdkanson and Ronnberg (1974) have suggested an improvement in the fluorometric histamine assay involving condensation with o-phthalaldehyde at -20°C. Among other advantages claimed by reaction in the frozen state is that spermidine, which may interfere in the conventional assay, gives no fluorescence under these conditions. Still another recent modification of an o-phthalaldehyde fluorometric procedure has been proposed, this one involving the use of an IRC-50 column for purifying histamine from biological materials (Cantoni et al., 1976b). Yamada and Wakabayashi (1974) compared colorimetry, fluorometry , and bioassay for measuring the increase in blood histamine following administration of certain antibiotics. They report that fluorometry and bioassay were superior to the colorimetric determination and that the fluorometric procedure was the most simple of the three. At the time of this writing, the U.S. Food and Drug Administration is working on the development of a simpler, but still accurate, fluorometric assay for histamine in fish. The method has not yet been published, although an abstract has appeared (Staruszkiewicz et al., 1975); this method has been adopted as an official first action method by the AOAC (Staruszkiewicz, 1977). Another new method for the determination of histamine in tuna fish by

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fluoronietry has been developed (Lerke and Bell, 1976). Histamine is recovered from fish extracts by ion exchange chromatography and then derivatizecl with o-phthalaldehyde. From filtered crude extracts, 12 samples can be determined per man hour including reconditioning of the ion exchange columns. Recoveries of histamine added to extracts from acceptable quality fish ranged from 98103%. and recoveries of 94-101% were demonstrated for histamine added to extracts of decomposed fish. The method is claimed to be as accurate BS the official AOAC procedure (see Section 1II.E) and much simpler. D.

GAS-LIQUID CHROMATOGRAPHY

Histamine can be determined by gas-liquid chromatography. For example, Fales and Pisano (1962) described a procedure employing a column containing 4% SE-30 siloxane polymer as the liquid phase. Navert (1975) has described gas-liquid chromatography procedure which is said to separate adequately histidine, histamine, and the naturally occurring methyl histamines. In spite of the possibilities for application of these techniques. they have not achieved widespread use among investigators studying the histamine toxicity problem. E.

COLORIMETRIC ASSAY

Early chemical methods used for the quantitative determination of histamine involved either coupling of histamine with a diazotized aromatic amine to produce an azo-dye, or the reaction of histamine with 2.4-dinitrofluorobenzene (DNFB). Code and Mclntire (1956) discuss both methods in detail, based on knowledge at hand at the time of their review. They conclude that DNFB is the better reagent of the two in that it yields a much more stable colored histamine derivative, is more sensitive. and is potentially more specific than the azo-dye method. Subsequent to the report of Code and Mclntire. there have been additional studies on colorimetric assays. We have previously cited work in which chemical assay of histamine by diazonium reaction was compared with guinea pig ileum bioassay (Sager and Horwitz, 1957). Ota (1958a.b) has suggested a modification of the azo-dye method based on extraction o f the histamine azo compound with various organic solvents. The procedure resulted in the removal of interfering substances, including histidine. Tsuda et al. (1959) later questioned Ota's procedure and suggested that there was better removal of interfering substances with ion-exchange chromatography. Weissback et al. (1958) reports a method designed specifically for measuring both histamine and serotonin in the same extract. After protein precipitation, both compounds were extracted with butanol, and the butanol was then passed

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through a cotton-acid succinate column, which separated histamine from serotonin. The latter substance was assayed fluorometrically, while the histamine was reacted with DNFB for colorimetric measurement. Kawabata and associates (1960) reported a simple and rapid diazo method for the determination of histamine, doing their work with fish samples. They used a cationic exchange resin for separating histamine from interfering substances, including histidine, in a trichloracetic acid extract of fish flesh. Aliquots of column eluates were coupled with Pauly’s diazo reagent and the absorbance at 5 10 nm determined by spectrophotometry. They obtained recoveries of added histamine of 99 to 101%. Their column had a very high exchange capacity for histamine, and the presence of histidine, tyrosine, or tyramine in trichloractic acid extracts did not interfere with histamine values. This method, or modifications of it, appears to have been the one most frequently employed by investigators and industry personnel concerned with recent outbreak of toxicity from tuna. The “official” method for histamine determination, i.e., that detailed in the “Official Methods of Analysis of the Association of Official Analytical Chemists” also employs reaction with a diazonium compound (Horowitz, 1975). However, in the hands of workers in our laboratory and elsewhere, it is tedious and time consuming and could not be considered practical for routine analysis of large numbers of samples. For example, the time required for a single determination, not including sample extraction, may range up to 2 hours. Obviously, then, there is need for better methodology; hence the work in this direction cited earlier (Staruszkiewicz et al., 1975; Lerke and Bell, 1976) and by others to be cited later in this review.

F. ENZYMATIC ISOTOPIC ASSAY A somewhat novel means for the determination of histamine has been proposed by Snyder et d. (1966). Tissue samples were incubated with tracer in the presence of the amounts of [3H] histamine and [14C]-S-adenosylmethionine enzyme histamine methyl transferase. This enzyme methylates histamine, and [3H] [14C] methylhistamine is the only labeled product extracted. The ratio of [ 14C]/[1H]is directly proportional to the amount of unlabeled histamine present in the incubation mixture. The method is claimed to be sensitive, specific, and relatively simple to perform. G.

THIN-LAYER CHROMATOGRAPHY

Schwartzman (1973) described a thin-layer chromatographic method for the separation and quantitat ion of histamine, methy lhistarnine, acetylhistamine, methylimidazoleacetic acid, and imidazoleacetic acid. He used a butanol-glacial

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acetic acid-water system, and produced fluorescent compounds of the five amines which were quantiated by absorption or fluorescence. Schwartzman and Halliwell (1975) later described application of a thin-layer chromatographic assay to determination of histamine and its metabolities in urine. More recently, workers interested in the problem of scombroid toxicity have developed two methods that also utilize thin-layer chromatography. Both are claimed to be considerably more rapid to accomplish than those suggested by Schwartzman (1973) and Schwartzman and Halliwell(l975). In one of these new methods (Lin et uf., 1976) ground and mixed fish is extracted with methanol, the extract is filtered and subjected to thin-layer chromatography. Plates are developed with MeOH:NH40H for 70 minutes, dried for 8 minutes, and the histamine spot is developed with ninhydrin. The amount of histamine present is determined by densitometry . This method does not require preliminary column purification of the extract and is a quick and simple method for determination of histamine in fish. In the second new method, collaborators working on the same problem have employed a technique in which samples of press juice or fish flesh are applied directly to thin-layer chromatography plates (Schutz et al., 1976). The plates are then developed with an acetone-ammonium hydroxide solution, and spots are visualized with ninhydrin or Pauly’s reagent. Chromatographic separation of histamine from other fish components, including histidine, is said to be readily achieved. The method is semiquantitative, but should be quite suitable for routine screening of large numbers of samples. The authors report that histamine levels as low as 2.5 mg% in fish samples and standard solutions are readily detected.

IV. RELATIONSHIP OF SPOILAGE TO HISTAMINE FORMATION It appears to be generally accepted that the production of histamine is in itself of bacterial origin and therefore does represent a criterion of spoilage or deterioration in fishery products. However, the production of histamine per se would not be expected to be detectable by simple means, e.g., change in appearance or production of off-odors. Fish containing large amounts of histamine may have a normal appearance and odor (Sapin-Jaloustre, 1957). Thus, it is of interest to note any correlation of histamine production with other more commonly used indicators of spoilage or loss of quality in fish. While a great many methods for measuring quality deterioration in fish have been employed over the past few decades, few, if any, have received any sort of widespread acceptance. Some type of sensory evaluation may well be the method of choice, e.g., detection of off-odor, appearance of surface slime, and changes in appearance of gill tissue or eyes. Objective methods frequently suggested include the determination of total

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volatile bases or acids, volatile reducing substances, the increase in concentration of hypoxanthine associated with postmortem breakdown of tissue nucleotides (enzymatic or bacterial in origin), the production of ammonia, and, at least in several species, the production of trimethylamine (a substance with a pronounced fishy odor) by bacterial reduction of trimethylamine oxide. The last problem is of less importance with tuna than with many other species of fish. This is not the appropriate place to review the application of these and other methods for evaluation of fish spoilage. A recent book by Connell (1975) offers a useful summary of this topic and of related issues. In the 1950s, Kimata and associates published a series of papers dealing with the freshness of fish and the amount of histamine present. Their early work dealt with factors such as the effects of pH and temperature on the amount of histamine produced in “red meat” fish such as mackerel (Kimata and Kawai, 1953a,d). They later reported that histamine was produced after the production of ammonia and amino nitrogen had begun during spoilage of “white meat” fish, e.g. rockfish (Kimataet ul., 1954a). They pointed out in the paperjust cited that more histamine was produced during spoilage of mackerel species than that of rockfish. They reported similar findings with spoilage of squid and octopus, i.e. ammonia production preceded that of histamine (Kimata er al., 1954b.c). As would be expected, in every instance they noted that spoilage, ammonia production, and histamine production were enhanced at elevated storage temperatures. [Note: Some commonly used terms can prove to be confusing in this discussion. The terms “white meat” or “red meat” fish are based on the general surface appearance of the fish, i.e., the degree of redness. Thus, for example, cod species would all be “white meat” fish, while mackerel species would be “redmeat” fish. On the other hand, the terms “red” (or “blood”) and “white” muscle refer to muscle types within an individual fish, “red” muscles being those containing predominantly red fiber types and “white” muscles being those containing predominantly white fiber types. All scombroid fish contain both muscle types in varying amounts, depending on species. In the commercial processing of tuna, only white muscle is canned for human consumption, the red muscle being used primarily for pet food.] Other Japanese workers have investigated different aspects of the relationship of spoilage to histamine production. Many scombroid fish contain both red (“blood”) and white muscle. In studies of three such species (mackerel, yellowtail, and bonito) Simidu and Hibiki (1954a) found that the decrease of histidine and the consequent increase in histamine was far lower in “blood” muscle than in white muscle, They also reported that trimethylamine was produced before ammonia, and that the amounts of volatile base and trimethylamine were much larger in “blood” muscle than in white muscle. In other work they found that histamine was produced during early stages of spoilage in mackerel and suggest that spoilage at that state would be difficult to discern (Simidu and Hibiki,

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1954b). In agreement with others. they found very little histamine produced during spoilage of ‘*white” fish, squid and shrimp although large amounts of volatile base are produced (Simidu et a/., 1955). Similar results were obtained with crab (Simidu and Hibiki, 1 9 5 4 ~ )It. was subsequently proposed by Simidu and Hibiki (1955a) that volatile bases and their precursors, such as triinethylamine oxide. urea. and free monoamino acids. inhibit the formation of histamine from histidine. Kalyani and Bai (1965) reported on histamine formation during spoilage of several fresh water, estuarine. and marine fish from South India. Histamine was absent from fresh meat in all species. After 72 hours of storage at 30”C, significant quantities of histamine were formed, particularly in the marine species. However, levels were not greater than 15 mg/100 gm tissue which is considerably less than levels generally associated with histamine toxicity. Ferencik and Havelka ( 1 962), having examined a large number of samples of tuna muscle of different types held at varying temperaturcs. concluded that the histamine level was a good indicator of freshness. Hillig (1956) has reported the results of a very detailed study in which several species of tuna were analyzed as they were allowed to reach different stages of decomposition and were subsequently precooked and canned in the usual manner. Different sections of individual fish were evaluated as were fish from different boats. A number of chemical analyses were employed, together with organoleptic evaluation. It was found that canned tuna prepared from good fish normally contain a small amount of acetic acid. and that as decomposition progresses there is an increase in acetic acid content as well as that of formic acid. After further decomposition, propionic and butyric acids may appear. Hillig suggests that individual volatile acids in canned tuna are a good index of the stage of decomposition of the corresponding raw material. Succinic acid and histamine were also formed during decomposition, with histamine reaching levels of well over 1000 mgll00 gm, dry weight basis. Some of the indices were partially lost during canning but the losses were considered not to be sufficient to enable a decomposed fish to become passable when canned. In another in-depth study. Takagi ef L I / . (1969) examined the amounts of histidine and histamine in 21 species of aquatic animals during spoilage. Their findings were in agreement with those of others already summarized here in that more histamine was produced in the “red muscle” fishes such as mackerel species than in ”white muscle” fishes such as rockfish. They also found little or no histamine produced in several molluscs and crustaceans during storage. Within the same species of fish, more histidine was found in white than in red muscle, and the resulting histamine formation followed this pattern with regard to muscle type, i.e., more histamine in white muscle. Of interest is their observation that while the degree of histamine formation is governed by histidine con.tent. it is not proportional to the loss of histidine. Recently, Cantoni o r a/.

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SALLY HUDSON ARNOLD AND W. DUANE BROWN

(19764 compared histamine production with that of volatile acids and volatile bases in tuna stored at 18”-20°C. They report that toxic levels of histamine were noted after 4 days; this level was apparently defined as greater than 100 mg histamine per 100-gm fish. During the same 4-day period the development of high levels of volatile acids and bases was also noted. Thus, there is considerable evidence associating several of the objective measurements of fish deterioration with histamine levels. Clearly, however, no objective measurement, short of determination of histamine itself, emerges as an effective indicator. As discussed in the section on analytical determination of histamine levels, efforts are now being made to reduce the measurement of histamine to a rapid, routine analysis. In the tuna industry, routine quality control consists of inspection of the raw fish. Organoleptic grade classification has been employed. Such grades are based on evaluation of physical characteristics, including appearance of gills, eyes, and skin, smell, and degree of physical damage to the tuna (Lassen, 1965). Such inspection would not, of course, reveal anything about histamine levels. All commercially canned tuna is precooked. Following the precook, skin and bones are removed and the loins are cleaned. At this point a phenomenon known as “honeycombing” may be observed. This condition consists of areas of pitted, spongy-looking muscle tissue (Finch, 1963). It is apparently due to gas production accompanying microbial growth. and is the basis for rejecting for human consumption the fish in which it is found. While this problem is widely known, if infrequently encountered in industry, it has been the object of little research, or at least little published research. Otsu (1957) reported that honeycombing developed in Hawaiian skipjack held without refrigeration, independent of sexual maturity or size of fish. He noted that honeycombing resulted from delayed rcfrigeration in experiments performed at different times of the year, although the rate of honeycombing was more rapid at higher seawater temperatures. He suggested that since the months of highest water temperatures in Hawaii coincide with the peak occurrence of honeycombing noted by fishermen, there might be a close relationship between water temperature and honeycombing. In a study by Williams (1954) in which several species of fish, including yellowfin and skipjack tuna, were allowed to reach varying stages of decomposition, much higher histamine values were found in honeycombed fish, regardless of species, than in similar fish without honeycomb. Merson et al. (l974), reporting on the incident of scombroid fish poisoning traced to commercially canned tuna in 1974, stated that fish from incriminated lots showed evidence of honeycombing. This is somewhat perplexing in that, under ordinary circumstances, tuna showing signs of honeycombing would have been rejected, as indicated previously. Researchers in the field, as well as industry representatives, unanimously agree that canned tuna with high levels of histamine imparts a “peppery” feel to the mouth when chewed. However tasting per se on a routine basis does not seetn

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a feasible means of quality assurance. At present, individual lots of tuna are analyzed for histamine content. This is, at best, a tedious procedure. As mentioned earlier, the relationship between detectable olfactory changes and histamine content is not always evident (Fucker ef al., 1974). Thus, there remains a need for an efficient objective measure of decomposition or a more rapid histamine analysis. Kimata ( I96 I ) has suggested that freshness of fish kept at temperatures as high as 35°C may be judged by determining the content of ammonia, since production of histamine under these circumstances is negligible as compared to that of ammonia. However, the use of ammonia levels as a freshness indicator becomes unreliable when fish are kept at room temperatures (20°C). He includes data showing that production of histamine in certain fish predominates over ammonia production in the range of temperature from 6 to 20°C. This is unfortunate since electrodes specific for ammonia are available and conceivably could be used on a routine basis. In that connection, in a recent report by Chang et al. (1976) there appears the description of the use of a trimethylamine-specific electrode for fish quality control. The electrode can be used for measurement of trimethylamine in aqueous solutions as well as in homogenates of fish muscle. It certainly offers a much simpler means of analysis than other methods currently used for determining trimethylamine. Some readers may be familiar with the Torrymeter, a device developed by research at the Torry Research Station in Aberdeen, Scotland. It is a compact portable meter which measures changes in dielectric properties of raw fish muscle and skin which occur as freshness is lost. The sensing head of the meter, containing the electrodes, is pressed against the fish skin, and a reading appears. The actual numbers read decrease with deterioration in quality. The device is simple to use and has practical application. Unfortunately, there seems little potential for comparing quality measured by the Torrymeter with histamine production, inasmuch as it cannot be used on fish that have been frozen (as are most tuna, for example) and it is said to be unreliable on fatty or oily fish such as tuna and mackerel (Davis, 1976).

V. A.

UNRESOLVED PROBLEMS

IS SCOMBROID TOXICITY DUE T O HISTAMINE?

In our considered judgement, the answer to this question is no. At least it seems extremely unlikely that histamine, acting alone, is the sole factor responsible for scombroid poisoning. A number of reports indicate that histamine taken orally by human subjects is not toxic. Douglas (1970) states “Very large amounts of histamine can be given orally, however, without causing ef-

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fects. . . .” He attributes this to the conversion of histamine to inactive N-acetylhistamine by intestinal bacteria. Granerus ( 1968) gave human subjects up to 67.5 mg histamine orally and reported that “The subjects did not get any subjective or objective symptoms which might have been caused by histamine.” A particularly convincing report is that of Weiss et al. (1932). They fed normal volunteer subjects gradually increasing amounts of histamine phosphate from 200 to 500 mg (500 mg histamine phosphate is equivalent to about 180 mg of histamine base). They noted no subjective or objective changes. Pulse rate and arterial blood pressure were unaltered. Readers are cautioned to note carefully the form in which histamine is given in such studies. In this case the phosphate salt was used, as noted. The dihydrochloride salt is sometimes used; 100 mg of histamine dihydrochloride is equivalent to about 60 mg histamine base. Hardy and Smith ( I 976) point out that Hughes ( 1 959) found higher values of histamine during postmortem storage of herring than they found in mackerel. Of interest is their comment “yet in this country only the latter is alleged to cause illness.” Typical values in the two reports were about 22 vs 2.6 at 80 hours storage. These are both below histamine levels usually regarded as toxic. Although unreported to date, it is also known that several individuals currently investigating the histamine problem have themselves consumed sizable quantities of pure histamine as well as good tuna “spiked” with histamine (up to 100 mg) with no apparent effect. There are few papers in which a response to oral histamine has been noted. In one such report (Sjaastad, 1966), subjects given doses of histamine of 36 mg or more experienced nausea, belching, heartburn, borborygmia, and diarrhea. We are at a loss to explain this apparent contradiction except to note that these symptoms are generally inconsistent with those normally associated with socalled histamine toxicity. Thus, it is difficult to conclude that histamine per se is the causative agent. This is not to say by any means that histamine is not involved. It seems beyond question that there is a direct correlation between high levels of histamine in fish and the resulting scombroid toxicity when such fish are consumed by humans. It is equally obvious that histamine alone is not responsible and, therefore, there must be some accompanying synergistic substance(s) or potentiating condition. With regard to the role of histamine, it should be noted that much of the histamine normally stored in tissues is found in mast cells or in basophils in the blood. In these cells, histamine is stored as a complex with heparin in secretory granules. While the turnover rate is slow, histamine may be caused to be released by a variety of factors, including allergy, various drugs, physical or chemical insult. Such release could account for some, if not all, of the symptoms associated with scombroid toxicity. For more detailed information concerning the mode of action of histamine see Douglas (1975) and Beaven (1976a,b).

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IMPROVED ANALYTICAL PROCEDURES

Recent research in this area, including some not yct completed, has been quite responsive to the needs. Several new methods have been developed as mentioned in Section 111. If these methods prove to be as promising a s they appear. this may no longer be highly critical rescarch. A triore rapid “official” method is being developed (see Section 1LI.C).

On the other hand, the need for an improved bioassay is critical indeed. It is scarcely feasible to employ human subjects. but other animals tested to date have responded at best in a highly variable fashion. Geiger (1955) fed samples of tuna which had been purposely spoiled. and which had levels of histamine ranging from 190 to 210 mg per 100 grn, to dogs. cats, rats, and mice, and by stomach tube to guinea pigs. No symptoms of poisoning were observed. Work in progress confirms the lack of toxicity of tuna known to cause illness in humans when fed to dogs and cats. The same worker has noted growth impairment in Japanese quail fed toxic tuna or histamine (Blonz, 1976). It has been reported earlier that toxic tuna meal or pure histamine fed to chicks resulted in growth inhibition (Shifrine rt d., 1959). The fact that histamine alone inhibits growth may mitigate against the use of quail or chicks as a bioassay inasmuch, as previously indicated. histamine alone taken orally does not appear to be the sole toxic factor in scombroid poisoning. This same reservation applies to use of the guinea pig ileum assay inasmuch as that muscle contracts in response to pure histamine. It will be of interest to see if the work cited earlier (Blonz. 1976) with Daphnia, a fresh water crustacean. yields a new bioassay for the toxic substance(s). Blonz is also presently testing pigs as experimental animals. In a preliminary trial. one pig weighing about 73 kg was fed 900 gin of toxic canned tuna. The animal was observed for several hours. but no signs of distress were noted. C . THE ANSERINE AND CARNOSINE QUESTION Carnosine (P-alanylhistidine), and anserine (N-P-alanyl-1 -methyl-histidine) are present in muscle tissue of a number of animals, including tuna and other fish. Structures are shown in Fig. I , together with those of histidine and histamine. Analyses for levels of these two dipeptides in a variety of fish have been reported by Lukton and Olcott (1958). Yellowfin tuna light meat contained small amounts of carnosine, averaging about 1 pmole/grn. with some samples showing

142

SALLY HUDSON ARNOLD AND W . DUANE BROWN COOH H H2N-CH-CH2fi

H HpN-CH2-CHzfi

Histidine

51

YOOH H2N-CH2-CH2-C-NH-CH-CH2

Carnosine ( N -8- A Ian y I hist id i ne 1

Histamine

f] H

0 7OOH II H2N-CH2-CH2-C-NH-CH-CH2

7H3

(1

Anse r ine (N-8-Alanyl-1 -methyl histidine)

FIG. 1. Structures of histidine, histamine, carnosine, and anserine.

zero levels. Anserine, on the other hand, was present in significant amounts, averaging about 29 pmole/gm. The significance of the potential contribution of anserine to histamine formation (if this, in fact, can occur) can be seen from the fact that histidine levels in these same loins averaged about 39 pmole/gm. In light meat of albacore tuna, typical values were: carnosine, 3, anserine 25, and histidine 45 pmolelgm, respectively. In skipjack tuna light meat, corresponding values were: carnosine, 4, anserine, 16, and histidine, 53 pmolelgm, respectively. Assays were also made for L-methylhistidine, but detectable levels were not found in any of the tuna samples. It was shown many years ago that E . coli can hydrolyze carnosine and decarboxylate the histidine liberated (Nash, 1952). Under the conditions employed by Nash, the pH range was critical, the organisms accomplishing the decarboxylation at pH 4.0. Hanson and Smith (1949) had earlier described a carnosinase found in swine kidney. Other work has been done with these dipeptides, but to our knowledge none has been directed to the question of the possible contribution of either to the production of histaminelike substances in scombroid fish. Two questions suggest themselves, one being whether enzymatic systems present in fish or in microflora commonly associated with fish spoilage can hydrolyze these peptides and decarboxylate histidine to produce histamine. A second is whether decarboxylated carnosine and anserine have any histaminelike activity.

D. POSSIBLE SYNERGISTS OR POTENTIATORS If, as seems clear, histamine itself is not toxic when taken orally, but, also, if histamine is found in large amounts in fish and other foods known to cause illness when consumed, then it is evident that there must be synergistic or potentiating substances or conditions involved. A number have been suggested, as outlined below. However none has been clearly implicated, and the evidence available at

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presetit, when taken as a whole, is largely negative. Thus, there is a pronounced need for additional research in this important area.

I.

Other Amines

It has been suggested by a number of investigators that other amines, including certain diamines, may work synergistically with histamine to produce toxicity. Miyaki and Hayashi (1954) reported finding a “factor” in a dried fish product which worked cooperatively with histamine to cause food poisoning. Hayashi ( 1954) subsequently reported that trimethylamine oxide, trimethylamine, agmatine (decarboxylated arginine), and choline worked synergistically with histamine in causing contraction of guinea pig uterus. However, Kawabata et al. (1 9S6a) reported that the addition of either trimethylamine or trimethylamine oxide was without effect on the action of histamine on guinea pig uterus and concluded that they could not be involved as cooperating factors in “allergy-like food poisoning.” Aipo et al. (1967) inoculated heat-sterilized Pacific saury (Cololubis saira) with Proteus morganii and, following incubation for 48 hours, chromatographed alcoholic extracts of the inoculated material. While they found that more than 90% of the histidine in controls had been converted to histamine during the incubation, they could not detect the presence of agmatine, cadaverine, methylhistamine, or tyramine. Furthermore, of the eleven fractions they isolated, only the one containing histamine resulted in any activity in guinea pig intestine. There is some evidence that certain amines may influence the absorption of histamine. This material is covered in Section V,D,2, immediately following.

2 . Alterations in Absorption Since large amounts (180 mg) of histamine can be taken orally by man without effect while microgram quantities in the blood may cause systemic effects, there must be some inactivation before histamine reaches general circulation. Weiss et al. ( I 932) suggested that either histamine is inactivated in the intestines before it enters the portal circulation or is destroyed by the liver before it enters from the portal to general circulation. They favored the former conclusion. More recently, Douglas (1970) reported that histamine given orally is converted by intestinal bacteria, particularly Escherichia coli, to inactive N-acetylhistamine, and that any free histamine remaining is inactivated when it traverses the intestinal wall or passes through the liver. Sjaastad ( 1 966) had shown previously thal almost 80% of N-acetylhistamine administered orally to humans was excreted as such in the urine. Thus, the acetylated derivative apparently is absorbed with no difficulty. There is, however, some evidence that other materials, if present with his-

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tamine, may alter its absorption. Parrot and Nicot (1 965) reported that histamine is normally fixed by much in the gut, but that certain diamines, such as putrescine, may overcome the fixation. In their study, the administration of putrescine before that of histamine enhanced the toxicity of the latter. Ienistea (1973), in reviewing the work of Parrot and Nicot just cited and that of earlier workers, concluded that diamines may enhance the toxic action of histamine by facilitating its passage through the intestinal barrier. He further suggested that the association of several diamines, such as putrescine, cadaverine, and spermine, with histamine may exert deleterious effects on animals. Geiger ( 1 955) noting the work of others and his own earlier findings suggested that alterations of conditions of the intestinal tract might cause histamine to be absorbed at an increased rate such that detoxification could not keep up with the entry of histamine into the circulation. Two such conditions he postulated were the consumption of highly seasoned hot dishes prepared from spoiled fish or the simultaneous consumption of alcoholic beverages. Mitchell and Code (1954) reported that, when healthy adult humans were fed 60 mg of histamine, large amounts of conjugated histamine were found in the urine during the third hour after administration. None of the ingested histamine appeared in the urine as free histamine. However, they did note that when histamine was added to a meal (consisting of bread, butter, and milk) that there was an increase in excretion of the conjugate comparable to that following consumption of histamine alone, but there was also a sharp increase in the output of free histamine in the urine starting about 2 hours after the meal containing histamine. The authors suggested the hypothesis that the absorption of free histamine is increased in the presence of the meal. However no direct evidence on this point was presented. It was earlier noted herein that Weisset al. (1932) had fed up to the equivalent of 180 mg histamine base to human subjects without noticeable effect. The same investigators showed that the minimal single intravenous injection of histamine that would elicit changes in facial blood vessels and cardiac rate was 0.007 mg of histamine base. Thus, whatever the mechanism preventing intestinal absorption and/or subsequent entry into the general blood circulation, it must be extraordinarily efficient. 3 . Histaminase Widmann ( I 950) obtained a patent on the invention of a method for increasing the absorption of histamine by supressing its inactivation by histaminase. Materials he suggested using for histaminase inactivation included histidine. pyridoxine, cysteine, ascorbic acid, ethylenediamine and its hydrochloride salts, and tetramethylenediamine and its hydrochloride salts. He indicated obtaining successful clinical results with these materials, based on showing a vasodepres-

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sor effect on hypertensive individuals. He was defining histaminase as a “diaminooxidase.” In this connection. Douglas (1970) noted that, while the most important pathway of histamine metabolism involves ring N-methylation by histamine-N-methyltransferase, there is another pathway in which histamine undergoes oxidative deamination by enzymes known as histaminase or diamine oxidase. For additional coverage of histaminase, see Section 1I.H. 4 . Monoamine Oxidase Inhibitors

There is at present widespread use of monoamine oxidase inhibitors as antidepressant drugs. There have been frequent reports of problems (hypertensive disturbances, headache, palpitation. and flushing) encountered by patients using such drugs following consumption of food products known to contain certain amines such as tyramine (decarboxylated tyrosine). A brief review of this problem is available (Anon., 1965). Sen ( 1 969) surveyed a variety of food products for their tyramine levels. Significant levels of tyramine were found in a variety of cheeses and yeast extracts, with lesser amounts being found in meat extracts and salted fish. More recently, Voigt ef al. (1974) and Rice rt al. (1975) have reported results of analyses of a number of cheeses and meat products, respectfully. for content of histamine and tyramine. Histamine concentrations in cheese ranged from nondetectable amounts to 2.6 mglgm in a sample of Sap-Sago. Histamine was detected in semi-dry sausage products and country-cured hams, but amounts were very low, in the order of 0.002 to 0.003 mg/gm. These and other reports have triggered speculation that monoamine oxidase inhibitors might potentiate the effects of ingested histamine. While this cannot be ruled out on the basis of evidence at hand, it does seem unlikely to be responsible in a majority of the cases of scombroid toxicity. For example, Boyer et a/. (1 956) reported one incidence in which the proportion of victims becoming ill following consumption of tuna with high histamine levels was 400 out of 2500 people eating the product. It is clearly unlikely that all 400 individuals were taking monoamine oxidase inhibitors.

5 . Bacterial Endotoxins Bacterial endotoxins are known to be widespread. These complex lipopolysaccharide materials are produced primarily by gram-negative bacteria and are known to be relatively heat stable. These factors have led t o speculation that such endotoxins might act synergistically with histamine in scombroid fish which had been subjected to significant microbiological spoilage. It should also be noted that endotoxin is known to be capable of inducing histamine release in animals (sometimes called endotoxin shock) similar to that seen in anaphylaxis. It also causes hypersensitivity to histamine in some animals. However, these

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phenomena are observed when endotoxin is given intravenously, and when this is followed by intraperitoneal injection of histamine. No effort will be made here to review this literature. The following citations offer typical experimental findings (Hinshaw et al., 1961; Kuratsuka and Homma, 1975; Kuratsuka et al., 1975). We have found no reports in which endotoxin and histamine were fed to animals to determine their combined toxicity. In preliminary experiments, Baronowski (1976) has found only extremely low levels of endotoxin in both good tuna and in that known to have caused illness when fed to humans. These findings need to be confirmed and extended to rule out endotoxin involvement. For detailed coverage of endotoxin see Weinbaum et al. ( I 97 1) and Kadis ef ul. ( I 97 1). 6. Conclusions Of the suggested routes by which synergistic materials may act, or by which histamine toxicity may be potentiated, those deserving of additional study seem to be (1) the effects of other amines, such as putrescine, and (2) various factors that may influence histamine absorption. Since such widely disparate food products as canned tuna fish and cheese (Douglas et al., 1967) may induce histamine toxicity, it seems not unreasonable to assume that the additional factor(s) may be microbial in origin. Clearly, there may well be other contributory substances or conditions which are yet to receive consideration. Identification of synergists and/or potentiators may well result in solution of the problem of “histamine” toxicity. E.

ALLOWABLE LEVELS OF HISTAMINE IN FISH

At the present time, the U.S. Food and Drug Administration is known to be considering establishing maximum allowable levels of histamine in canned tuna fish. Since histidine is a normal constituent of tuna muscle, and since there will always be some microbial flora associated with fish regardless of the care with which they are handled, it is inevitable that some histamine will be produced. Thus, there arises the question of what levels may be accepted in good quality fish. At the time of this writing, no number has yet been officially proposed. However, the maximum allowable level most frequently discussed unofficially is around 10 mg histamine per 1 0 gm fish. It is of interest to note that Geiger (1944a) long ago said that “We do not feel ready to set standards for the freshness of the fish in terms of the histamine content, but on the basis of our experimental data we assume that fish containing more than 10 mg ‘histaminelike’ substances per 100 gm tissue should not be regarded as ‘fresh’.” It may well be that Dr. Geiger’s educated guess will prove to be prophetic.

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ACKNOWLEDGMENTS Much of the work mentioned herein as being in progress is supported by the Tuna Research Foundation and NOAA Office of Sea Grant. Department of Commerce. under Grant No. 04-6-15844021. Ms. Arnold is a Sea Grant Trainee.

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Gale. E. F. 1940. The production of amincs by bacteria (1.) The decarboxylation of amino-acids by $trains of Bacwrium c d i . Biorhcm. J . 34, 3 9 2 4 1 3. Gale. E. F. 1942. The oxidation of amines by bacteria. Birichcm. J. 36, 64-75. Gale. E. F. 1946. The bacterial amino acid dccarboxylases. Ad\*. Enzymol. R d u r . Sihj. Biochvn. 6, 1-32. Geiger. E. 1944a. Histamine content of unprocessed and canned fish. A tentative method for quantitative determination of spoilage. Food Res. 9, 293-297. Geiger. E 1944b. On the specificity of bacterium-decarboxylase. Pro(.. Soc. Exp. B i d . Med. 55, 11-13, Geiger. E. 1948. On the mechanism of histamine formation. Arch. Binchem. 17, 391-395. Geiger. E. 1955. Role of histamine in poisoning with spoiled fish. Science 121, 865-866. Geiger, E.. Courtney. G.. and Schnakenbcrg. G . 1944. The content and formation of histamine in fish muscle. Arch. Biochem. 3, 31 1-319. Granerus, G . 1968. Effects of oral histamine, histidine. and diet on urinary excretion of histamine. inethylhistamine and I-methyl-4-imidazolcaceti~acid in man. Srttnd. J. Clin. Lab. /~7vc,st., Suppl. 104, 49-58. Hikanson. R., and Riinnberg. A,-L. 1974. Improved fluommetric assay of histamine: condensation with o-phthalaldehyde at -20°C. Anal. BkJ('hW77. 60, 560-567. Halstead. B. 1954. A note regarding the toxicity of the fishes of the skipjack family. Katsuwonidae. Cali$ Fish Gamc 40, 6 1-63 Halstead. B. 1967. Class Osteichthyes: Scombrotoxic fishes. In "Venemous Marine Animals of the World." Vol. 2. pp. 639468. U.S. Gov. Print. Off.. Washington, D.C. Hanson. T . H., and Smith, E. L. 1949. Carnosinme: An enzyme of swine kidney. J . Biol. Chrm. 179, 789-801. Hardy. R.. and Smith. J. G. M. 1976. The storage of mackerel (S(.ombrr.sc.ombrus).Development of histamine and rancidity. J . S(,i. Food Agric.. 27, 595-599. Havelka. B. 1969. Contribution on the use of Mpllcr's medium for the demonstration of histidine decarboxylase. J . H y g . . Epidemiol., Mb"ibio/ , Immunol. 13, 4 6 3 4 6 7 . Hayashi. M. 1954. Investigations o n food poisoning cauaed by ordinary putrefaction. IV. Synergism between histamine and several biogenetic (sic) aniines. Yakugaku Zasshi 74, 1148-1 151. Hibiki. S . . and Simidu. W. 1959. Studies on putrefaction of aquatic products-27. Inhibition of histamine formation in spoiling of cooked fish and histidine content in various fishes. Nipport Suiscm Gukkaishi 24, 916-919. Hillig. F. 1956. Volatile acids, succinic acid and histamine as indices of decomposition in tuna. J. Assoc. Of. Agrir-. Chrm. 39, 773-800. Hinshnw. L. B., Jordan, M. M.. and Vick, J . A. 1961. Mechanism of histamine release in endotoxin shock. A m . J . Phvsiol. 200, 987-989. Horowitz. W. 1975. Fish and other marine products. In "Official Methods of Analysis of the Association of Official Analytical Chemist.\" (W. Horowitz, ed.). pp. 305-327. Assoc. Off. Anal. Chem.. Washington, D.C. Huff, J . A,. Davis. V. E.. and Brown. H. 1966. A simplified technique for the estimation of histamine in blood and urine. J. Lab. Clin. Mod. 67, 1044-1047. Hughes. R. B. 1959. Chemical studies on the hrning (Cluprtr hurengu3). 11. The free amino acids of herring flesh and their behavior during post-mortem spoilage. J. Sc i. Food Agric,. 10, 558-564. Ienisten. C. 1971. Bacterial production and destruction of histamine in foods. and food poisoning caused by histamine. Nahrung 15, 109-1 13. Ienistea. C. 1973. Significance and detection of histumine in food. In "The Microbial Safety of Food" (B. C. Hobbs and J. H. B. Christian. cda.). pp. 327-343. Academic Press. New York. Kadis. S.. Weinbauni. G.. and Ail. S. J., eds. 1971. "Microbial Toxins V . Bacterial Endotoxins." Academic Press, New York.

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Food Sci.. Kyoro Univ. 6, 3-1 I . Kimata. M., and Kawai, A. 1953d. The freshness of fish and the amount of histamine presented in the meat. 11. Mern. Res. Insr. Food Sci., Kyoto Univ. 6, 12-22. Kimata, M.. and Kawai. A. 1953e. The freshness of fish and the amount of histamine presented in the meat. V. On the poduction of histamine during the Autolysis, 3. Kyctro Daigaku Shokuryo Kagaku Kenkyusho Hokoku 11, 88-95. Kimata, M., and Kawai. A. 1953f. The freshness of fish and the amount of histamine presented in the meat. VI. On the production of histamine during the Autolysis. 4. Kyoto Daigaku Shokuryo Kagaku Kenkyusho Hokoku 12, 24-28. Kimata, M., and Kawai, A. 39538. The production of histamine by the action of bacteria causing the spoilage of fresh fish. I. K y d o Daigaku Shokuryo Kagaku Kenkyusho Hokoku 12, 29-33. Kimata, M.. and Kawai, A. 1958. Studies on the histamine formation of Proreus morganii. Mem. Coll. Agric., Kyoro Univ.. Fish. Ser. Spec. No. (June). pp. 92-99. Kimata, M.. and Kawai, A. 1959. Studies on the histamine-formation of Proreus morganii (continued). Mern. Res. Inst. Food Sci., Kyoro Univ. 18, 1-7. Kimata, M., and Tanaka. M. 1954a. On the bacteria causing spoilage of fresh fish, especially on thie activity which can produce histamine. Mern. Res. Insr. Food Sci., Kyoro Univ. 7 , 12-17. Kimata, M.. and Tanaka, M. 1954b. A study where the bacteria having an activity which can produce a large amount of histamine, so called histamine-former, are present or not, on the surface of fresh fish. Mem. Res. Insr. Food Sci.. Kyoto Univ. 8, 7-16. Kimata, M., Kawai. A,, and Tanaka, M. 1954a. The freshness of fish and the amount of histamine presented in the meat. 111. Mern. Res. Insr. Food Sci., Kyoto Univ. 7 , 6-1 I . Kimata, M.. Kawai. A., and Tanaka, M. 1954b. The freshness of fish and the amount of histamine presented in the meat. IV. Mern. Res. Inst. Food Sci., Kyoto Univ. 8, 14. Kimata, M., Kawai, A., andTanaka, M. 1954c. Freshness of fish and amount of histamine presented in meat. VUI. On the production of histamine during the bacterial decomposition of fish meat, 4. K V O ~Daigaku ~J Shokuryo Kagaku Kenkyusho Hokoku 14, 25-29. Kimata. M., Kawai, A,, and Akamatsu. M. 1958. Classification and identification of the bacteria having an activity which can produce a large amount of histamine. Mern. Res. Znsr. Food Sci., Kyoto Univ. 14, 3 3 4 I . Kimata, M., Akamatsu, M., and lshida, Y. 1960. Studies on the classification of the genus Proreus. 1. On the taxonomical situation of Proreus morganii. Mem. Res. Insr. Food Sci., Kyoto Univ. 20, 1-7. Klawe, W. L. 1976. Personal communication to H. S. Olcott. Kuratsuka. K.. and Homma, R. 1975. Comparison between histamine-sensitizing factor of Borderella pertussis and bacterial endotoxin in respect to histamine-sensitizing activity. Jpn. J . Med. Sci. Biol. 28, 316-320. Kuratsuka, K.. Homma, R.. Shimazaki. Y., and Funasaka. I. 1975. Rapid appearance of histamine hypersensitivity in mice by minute dose of endotoxins. Experienria 31, 206-208. Lassen. S. 1965. Tuna canning and the preservation of the raw material through brine refrigeration. I n “Fish as Food” (G. Borgstrom, ed.), Vol. 4, pp. 222-225. Academic Press. New York. Legroux, R., Levaditi, J.-C.. Boudin. G., and Bovet, D. 1946. Intoxications histaminiques collectives. Presse Med. 54, 545-546. Lerke. P. A , . and Bell, L. D. 1976. A rapid fluorometric method for the determination of histamine in canned tuna fish. J . Food Sci. 41, 1282-1284. Levine. R. J., and Watts, D. E. 1966. A sensitive and specific assay for histidine decarboxylase activity. Biochem. Pharmacol. 15, 841 -849. Lin, J. S.. Baranowski, J. D., and Olcott, H. S. 1977. Rapid thin-layer chromatographydensitometry determination of histamine in tuna. J . Chromatogr. 130, 426-430. Lukton. A , . and Olcott, H. S. 1958. Content of free imidazole compounds in the muscle tissue of

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aquatic animals. Foud Res. 23, 61 1-618. Mclntire, F. C., Roth, L. W., and Shaw, J . L. 1947. The purification of histamine for bioassay. J . Biol. Chem. 170, 537-544. Merson, M. H., Baine, W. B.. Gangarosa. E. J . , and Swanson, R. C. 1974. Scombroid fish ,. 1268poisoning. Outbreak traced to commercially canned tuna fish. J . Am. Med. A s s ~ ~ c228, 1269. Michaelson, A,, and Coffman, P. Z. 1969. An improved ion-exchange purification procedure for the fluorometric assay of histamine. Anal. Biochem. 27, 257-261. Mitchell, R. G . , and Code, C. F. 1954. Effect of diet on urinary excretion of histamine. J . Appl. Physiol. 6, 387-392. Miyaki. K., and Hayashi, M. 1954. Investigations on food poisoning caused by ordinary putrefaction. 111. Detection of histamine and its synergistic factor in poisoned “samma sakuraboshi.” Yakugaku Zasshi 74, 1145-1 148. Mpller. V. 1954a. Activity determination of amino acid decarboxylases in Enterobacteriaceae. Acta Pathol. Microbid. Scand. 34, 102-1 14. Moiler, V. 1954b. Distribution of amino acid decarboxylases in Enterobacteriaceae. Acta Patho/. Microbiol. Scand. 35, 259-271. Mpller, V . 1955. Simplified tests for some amino acid decarboxylases and for the arginine dihydrolase system. Acta Pathol. Microbiol. Scand. 36, 158-172. Nash, J . B. 1952. Formation of histamine from carnosine and histidine by E . coli. Trx. Rep. B i d . Med. 10, 639-646. Navert, H. 1975. New approach to the separation and identification of some methylated histamine derivatives by gas chromatography. J . Chromutogr. 106, 21 8-224. Noah, J . , and Brand, A. 1963. Simplified micromethod for measuring histamine in human plasma. J . Lab. Clin. Med. 62, 506-510. Olcott, H. S . . and Lukton, A . 1961. Is “saurine” an artifact?Nippon Suisan Gakkuishi 27,451452. Omura, Y. 1976. Personal communication. Ota. F. 1958a. On the formation of amine in fish muscle, 111. Simple method for the detection of histamine in fish muscle. Nippon Suisan Gakkaishi 24, 37-40. Ota, F. 1958b. On the formation of amine in fish muscle. IV. Rapid method for the estimation of histamine in fish muscle. Nippori Suisan Gnkkaishi 24, 4 1 4 . Ota. F . , and Kaneko, K . 1958. On the formation of amine in fish muscle. VII. Effect of freezing on the histarnine formation in the thawed fish muscle. Nippon Suisan Gakkaishi 24, 140-143. Otsu. T. 1957. Development of “honeycombing” in Hawaiian skipjack tuna. Commiv. Fish. Rev. 19, 1-8. Ough. C. S. 1971. Measurement of histamine in California wines. J . Agric. Food C h m . 19, 24 1-244. Parrot, J.. and Nicot, G . 1965. Le role de I’histamine dans I’intoxication alimentaire par le Poisson. Soc. Sri. H y g . Aliment. Aliment. Rationn. Homme, Paris 53, 16-82. Rice, S.. Eitenmiller, R . R . , and Koehler. P. E. 1975. Histamine and tyramine content of meat products. J . Milk Food Techno/. 38, 256-258. Roberts, M., and Adam, H. M. 1950. New methods for the quantitative estimation of free and conjugated histamine in body fluids. 5 r . J . Pharmacol. 5 , 526-541. Rosenthaler. J . B., Guirard, B. M . , Chang. G . W., and Snell. E. E. 1965. Purification and properties of histidine decarboxylase from Lactubarillus 30a. Pruc. Natl. Acud. Sci. U.S.A. 54, 152-158. Sager, 0 . S., and Horwitz, W . 1957. A chemical method for the determination of histamine in canned tuna fish. J . Assoc. Off. Agric. Chem. 40, 892-904. Sapin-Jaloustre, H. and J . 1957. Une toxi-infection alimentaire peu connue: L’intoxication histaminique par le thon. [A little-known food poisoning: Histamine poisoning from tuna.] Conc o w s Medical. Puris 79, 2705-2708.

HISTAMINE ('9 TOXICITY FROM FISH PRODUCTS

153

Schayer. R . W. 1971. Determination of histidine decarboxylase activity. Methods Bioc.hem. Aiiol. SUPPI. V o l . . pp. 99-1 17. Schutz. D. E., Chang, G. W., and Bjeldanes. L. F. 1976. Rapid thin-layer chromatographic method for detection of histamine in fish products. J . Assoc,. of. Anal. Chem. 59, 1224-1225. Schwartzman, R . M. 1973. Quantitative thin-layer chromatography of histamine and its metabolites. J . Chromatogr. 86, 263-268. Schwartzman. R. M . , and Halliwell, R. E. W . 1975. Thin-layer chromatographic assays of histamine and its metabolities in urine of man and dog. J. Chromatogr. 115, 129-138. Sen. N. P. 1969. Analysis and significance of tyraniine in foods. J . Food Sc.i. 34, 22-26. Shaw. C.. and Clarke, P. 1955. Biochemical classification of Proteus and Providence cultures. J . Gen. Microbiol. 13, 155-161. Shewan. J. M.. and Liston, J. 1955. A review of food poisoning caused by fish and fishery products. J . Appl. Barteriol. 18, 522-534. Shifrine. M., Ousterhout. L. E.. GtdU, C. R.. and Vaughn. R. H. 1959. Toxicity to chicks o f histamine formed during microbial spoilage of tuna. Appl. Microhiol. 7, 45-50. Shore. P. A. 1971. The chemical determination of histamine. Methods Biochem. A ~ ~ l . S u p pVol.. l. pp. 89-97. Shore. P. A.. Burkhalter, A , , and Cohn. V . H.. Jr. 1959. A method for the fluorometric assay of histamine in tissues. J. Pharmacol. E.rp. Thrr. 127, 182-186. Simidu, W.. and Hibiki, S. 1954a. Studies on putrefaction of aquatic products. X11. On putrefaction of bloody muscle. Nippon Suiscm Gakkaishi 20, 206-208. Simidu. W.. and Hibiki, S. 1954b. Studies on putrefaction of aquatic products. XIII. Comparison putrefaction of different kinds of fish ( 1 ). Nippon Suisun Gakkaishi 20, 298-301. Simidu. W . , and Hibiki, S. 1 9 5 4 ~Studies . on putrefaction of aquatic products. XIV. Comparison on putrefaction of different kinds of fish (2). Nippon Suisaji Gakkuishi 20, 302-304. Simidu. W., and Hibiki, S. 19543. Studies on putrefaction of aquatic products. XV. Comparison of putrefaction for round. fillet, minced and denatured fishes. Nippon Suisari Gukkaishi 20, 388391. Simidu, W . . and Hibiki. S . 1955a. Studies on putrefaction of aquatic products. XIX. Influence of certain substances upon histamine formation. Nippon Suisan Gakkaishi 20, 808-8 10. Simidu. W.. and Hibiki, S. 1955b. Studies on putrefaction of aquatic products. X X l l l . On thc critical concentration of poisoning for histamine. Nippon S u i s m Gakkaishi 21, 365-367. Siniidu. W . , Hibiki. S . , and Nagasaki. S. 1955. Studies on putrefaction of aquatic products. XVIII. On putrefaction of some white-muscle fishes. niollusca and shrimp. Nippon Suisuti Gukkoishi 20, 804-807. Sjaastad, 0 . 1966. Fate of histamine and N-acetylhistamine administered into the human gut. Adcr fharmucol. Toxicol. 24, 189-202. Snyder. S. H., Baldessarini, R. J.. and Axelrod. J . 1966. A sensitive and specific enzymatic isotopic assay for tissue histamine. J. fhurmc?co/.E r p . Ther. 153, 544-549. Staruszkiewicz, W. F.. Jr. 1977. Personal communication. Staruszkiewicz. W . F.. Jr.. Waldron, E . , and Bond. J. F. 1975. Quantitative determination of histamine in tuna fish by fluorometry. Ahstr.. 89th Annu. Meet.. Assoc. of/'.A U d . Chem. pp. 5-21. Strpm, A , . and Lindberg, W . 1945. Forgiftung fremkalt ved nytelse a v niakrellstflrje. Nord. Med. 26, 903-906. Suzuki. U . . Yoshimura, K . . Yamakawa, M.. and Irie, Y . 1Y09. Uber die Extraktivstoffe des Fischfleisches und der Muscheln. Hoppe-Seyler's Z . Physiol. Chem. 62, 1-35, Takagi. M.. lida. A , , Murayama. H.. and S h a , S . 1969. On the formation of histamine during loss of freshness and putrefaction of various marine products. Hokkuirlo Duigaku Suisnn Gukuhu Kenkyu Iho 20, 227-234.

154

SALLY HUDSON ARNOLD AND W. DUANE BROWN

Tsuda, A , , Mori. K . , and Tomiyama, T. 1959. Studies on the method for testing the spoilage of food. X. Errors involved in Ota’s method for determination of histamine. Nippon Suisun Gakkuishi 25, 361-367. Udenfriend, S.. Stein, S., Bohlen, P., and Dairman, W. 1972. Fuorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science 178, 87 1 -872. Van Veen, A. G., and Latuasan, H. E. 1950. Fish poisoning caused by histamine in Indonesia. Doc. Need. Indones. Morbis Trop. 2, 18-20. Voigt, M. N.. Eitenmiller, R. R., Koehler. P. E., and Hamdy, M. K . 1974. Tyramine, histamine. and tryptamine content of cheese. J . Milk Food Techno/. 37, 377-381. Von Redlick. D., and Glick, D. 196.5. Studies in histochemistry LXXVI. Fluorometric determination of histamine in microgram samples of tissues or microliter volumes of body fluids. Anal. Eiochem. 10, 4.59467. Waldron-Edward, D. M. 1954. The intereference of inorganic salts in the chromatography on paper of amino alcohols, diamines, and diamino acids. Chem. Ind. (DusseLdog) 4, 104-105. Weinbaum. G.. Kadis, S . , and Ajl, S. J . , eds. 1971. “Microbial Toxins, Vol. 4, Bacterial Endotoxins.” Academic Press, New York. Weiss, S . , Robb, G . P., and Ellis, L. B. 1932. The systemic effects of histamine in man. Arch. Intern. Med. 49, 360-396. Weissback, H., Waalkes, T., and Udenfriend. S. 1958. A simplified method for measuring serotonin in tissues; simultaneous assay of both serotonin and histamine J . B i d . Chern. 230, 865-871. Werle, E. 1940. Uber die Histaminzerstorende Fahigkeit von Bacterien. Eiochern. 2. 306, 264-268. Werle, E. 1941. Uber das Vorkommen von Diaminoxydase und Histidin-Decarboxylase in Mikroorganismen. Eiochem. Z . 309, 61-76. West. G . B., and Riley. J . F. 1954. Chromatography of tissue histamine. Nature (London) 174, 882-883. Widmann, R . R. 1950. Composition to increase the absorption by the body of histamine. U.S. Patent No. 2,498,778. Williams. D. W. 1954. Report on chemical indices of decomposition in fish (histamine). J . Assor. Off.Agric. Chem. 37, 567-512. Yamada, S . , and Wakabayashi, K . 1974. A fluorometric determination of histamine upon the administration of macrolide antibiotics. J . Antiobior. 27, 788-792.

AUVANCLS

IN FOOD R F X A K < H. VOL. 24

FOOD IRRADlATlON WALTER M . URBAIN* Michigan State University Eart Lansing. Michigan

I . Introduction-Historical Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Radiation and Radiation Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ill. General Effects of Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Action on Major Food Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Applications of Food Irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction . . , , . . , . ..................................... B. High-Dose Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Low-Dose Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Economics of Food Irradiation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Wholesomeness of Irradiated Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . VI1. The Future of Food Irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. INTRODUCTION-HISTORICAL

1

I63 I68 I68 I69 I74 I74 I74

I88 205 209 213 216

BACKGROUND

The form of energy on which the process of food irradiation is based, namely ionizing radiation, was discovered just before thc start of the twentieth century. While there were early interests in the biological effects of ionizing radiation (Goldblith, 1966). meaningful research directed toward food preservation did not start until the 1940s. Proctor ez al. (1943) probably were the first to demonstrate that ;I food (hamburger) could be preservccl by irradiation with X rays. In addition to the work at the Massachusetts Institute of Technology. a f e u years later there were the effort5 of industrial firma. namely the joint program of Electronized Chemicals Corporation and Swift Br Conipany and of the General Electric Company. These efforts subsequently were joined by programs of the United States government. The first federal agency significantly interested in foc~Iir" h ) f e c \ o r Emc.ntu\. Pre.~cnlAddress: 1064.5 Wclh Drive. S u n Cit? , , 4 r j ~ t > 18535 >~ I

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ISBN O-I?-illh4?4-8

156

WALTER M . URBAIN

radiation was the Atomic Energy Commission (A.E.C.), which had a program starting in 1950. The U.S. Army, having had difficulties with the troop acceptance of canned meats in two wars (corned ‘‘Willy” in World War I and “Spam” in World War 11) began its program in 1953. The objective of the Army work was to obtain shelf-stable meats of eating quality superior to what could be obtained by thermal processing. The A.E.C. program was discontinued after a few years, but was reinstated in 1960. Starting in 1960, by agreement, the Army program was limited to high-dose irradiation aimed at sterilized foods with unlimited shelf stability, whereas the A.E.C. program was concerned only with low-dose applications . Initially, the government program was carried out largely by contract work done in universities, commercial organizations, and existing government laboratories. While the A.E.C. program continued in this way, in 1963 the Army established a research facility at Natick, Massachusetts. About 1971, the A. E. C. terminated its program and the Army became essentially the sole United States Agency with a food irradiation activity. Since the Army program is largely an “in-house” activity, and with the termination of the A.E.C. program, funds to support university and other outside laboratory work in food irradiation have not been available. As a consequence, what had been a very large academic activity virtually has disappeared. Over the years, the American food industry had participated in the development of food irradiation to varying degrees. Initially the interest was strong, but, as problems were uncovered and it became apparent that there would be little opportunity for early commercial use of the process, this participation diminished and today industrial activity in the development essentially is nonexistent. Other countries joined in the work. England undertook an extensive program starting in the late 1940s. Programs were started also in Canada and Japan in 1956, in Argentina and the U.S.S.R. in 1957, in Poland in 1958, in India in 1959, and in Israel in 1960. In 1968, the U. S . Department of Commerce (Anonymous, 1968a) listed 76 countries which had food irradiation programs. Quite naturally groups of countries joined together. In Europe, the Commission of European Communities and the Organization for Economic Cooperation and Development both sponsored research and assisted in information transfer. In 1964, a joint activity in food irradiation of the Food and Agriculture Organization and the International Atomic Energy Agency was established. It has greatly assisted in making information on food irradiation known on a world basis, especially among the less developed countries. Among other activities, this joint FAOlIAEA division has set up regional projects of research in Asia and South America. The Council for Mutual Economic Assistance brought together the Eastern bloc countries of Europe. In 1970, approximately 25 nations banded together under the “International Project in the Field of Food Irradiation” to carry out work to establish the safety of irradiated foods for human consumption.

FOOD IRRADIATION

157

A number of countries have given approval for the irradiation of certain foods. Table I lists the approvals by food and by country which now exist or which had been given at one time. Despite these approvals, there has been little commercial use of food irradiation. Actually, only the irradiation of white potatoes to prevent sprouting has been practiced. In 1965, nearly one million pounds of potatoes were irradiated in Canada. Since 1973, potatoes grown in Japan have been irradiated. While other countries appear to be taking steps also to irradiate potatoes (e.g., Chile), other kinds of applications, despite regulatory approval, have not occurred. Unsatisfactory economics as, for example, with the case of wheat, have been a major factor in most instances. Many countries have established requirements that there must be satisfactory evidence of safety for human consumption of irradiated foods before they can be made available to the public. This need was recognized early and for over 25 years the obtaining of such evidence has occupied a very great proportion of the toial research effort. Other food processes have gained acceptance largely as a result of long-term usage which pragmatically has demonstrated safety for use with human foods. In general, they have not been subjected to definitive scientific studies to evaluate their safety. From almost the start, food irradiation, however, was considered by many as “suspect,” probably due to an unavoidable, but mistaken, association with the atom bomb and with the lethal effects of ionizing radiation on living organisms. Food irradiation also has become involved in the current general concern for the safety of processed foods and of food additives. As a consequence, the process of food irradiation and irradiated foods have been studied in terms of possible health hazards for consumers in a manner and to a degree that has not occurred with any other food process. In the United States radiation is classified as a food additive and thereby subject to regulations of the Food and Drug Administration. The United States has had a position of leadership in establishing the requirements for acceptable evidence of safety of irradiated foods. The issue of safety came to a climax in 1968 when a petition of the Army for the radiation sterilization of ham was regarded as not providing sufficient evidence of safety. A previously issued regulation permitting the irradiation of sliced bacon was withdrawn at that time. The effects of these actions by the FDA were manifold both in this country and elsewhere. Commercial organizations in the United States lost interest in food irradiation. Undoubtedly this situation was a major factor in the termination of the A.E.C. program in 1971. Other countries retrenched in their activities. Some continued, but with increased difficulty. Some attacked the actions of the FDA and, in particular, opposed the designation of radiation as a food additive and the requirements for the separate evaluation of each and every irradiated food. They sought to treat food irradiation just as any other food process.

TABLE I GENERAL SURVEY OF IRRADIATED FOOD PRODUCTS CLEARED FOR HUMAN CONSUMPTION IN DIFFERENT COUNTRIES"

Type and source of radiation Country (organization) Bulgaria

Canada

Product Potatoesh Potatoesh Onionsh Garlich Grainh Dry food concentrates') Dried fruitsh Fresh fruits" (tomatoes, peaches. apricot. cherry. raspberry, grapes) Potatoes' Onions Wheat, flour, whole wheat flour Poultry"

Cod and haddock fillets" Chile Pot atoes"." Denmark Potutoes France PotatoesY Federal Republic Deep-frozen mealsbJ of Germany Potatoesb

Purpose of irradiation Sprout inhibition Sprout inhibition Sprout inhibition Sprout inhibition Insect disinfestation Insect disinfestation Insect disinfestation

finCo

ixcS

Electrons

Dose (bad)

Date of approval

+

+ +

10

100 100

30 April 9 November 14 June 25 March

1972 1960

25 February

1969

20 June 2 October 3 1 October 27 February 8 November 24 March 26 September

1973 1973 1974 1970 1972 1972 1974

10 10

+ + + +

30

Radurization Sprout inhibition

+

Sprout inhibition

+

250 10 max. IS max. 15 max.

Insect disinfestation Radicidation (Salmonella) Radurization Sprout inhibition Sprout inhibition Sprout inhibition Radappertization Sprout inhibition

+

75 max.

+ +

700 max. I50 max.

+

+ + +

+

10 MeV

1971 1972 1972 1972 1972 1972 1972

30April 30 April 30 April 30 April 30 April 30April

15 max. 7.5-15 25004500 15 max.

1963

1965

Hungary

Israel

Potatoes'' Potatoes" Potatoes" Onions" Onions" Strawberries" Mixed spices" (black pepper, cumin paprika, dried garlic: for use in sausages) Poraroes Onions

Italy

Japan Netherlands

Poruroes Onions Garlic, Poraroes Asparagus" Cocoabeans" Strawberries" Mushrooms Deep-frozen meals' Potaroes Shrimps* Onions" Onion.\ Spices and condiments" Poultry. eviscerated (in plastic bags) Chicken

Fresh, tinned and liquid foodstuffs' \o

Sprout inhibition Sprout inhibition Sprout inhibition Sprout inhibition Sprout inhibition Radurization

Radicidation Sprout inhibition Sprout inhibition Sprout inhibition Sprout inhibition Sprout inhibition Sprout inhibition Radurization Insect diainfestation Radurization Growth inhibition Radappertization Sprout inhibition Radurization Sprout inhibit ion Sprout inhibition Radicidation Radurization Radurization, Radicidation Radappenization

+

10

+

15 max. 15max.

+

+ +

6

+

+

500

+

15 max. 10 max.

+

+

+

+ + + +

+

+

4 MeV 4 MeV 4 MeV 4 MeV 4 MeV

4 MeV

7.5-15 7.5-1s 7.5-15 15 max. 200 max. 70 max. 250 max. 250 max. 2500 min. 15 max. 50-100 15 mdX. 5 max. 800-1000

+ +

300 max. 300max.

+

2500 min.

23 December 10 January 5 March 5 March 6 August 5 March

1969 1972 1973 1973 1975 1973

2 April 5 July 25 July 30 August 30 August 30 August 30 August 7 May 7 May 7 May 23 October 27 November 23 March I3 November 5 February 9 June 13 September

1974 1967 1963 1973 1973 1973 1972 1969 I %9 1969 1969 1%9 I970 1970 1971 1975 1971

31 December 1971 10 May 1976

8 March

1972

(continued)

s

TABLE I - (continued)

Type and source of radiation Country (organization)

Product

Spices'.P Vegetable fillingh," Powdered batter-

Philippines South Africa Spain

Endive"," (prepared, cut) Potatoes" Mangoes" Potatoes Potatoes

Onions Thailand Union of Soviet Socialist Republics

Onions

Potatoes

Poraroes Grain Fresh fruits and vegetables" Semiprepared raw beef. pork and rabbit products (in plastic bags)*

Purpose of irradiation Radicidation Radicidation Radicidation Radicidation Radurization

'To

'"Cs

+ +

Electrons 4 MeV 3 MeV

+

+ +

Sprout inhibition Control of ripening Sprout inhibition Sprout inhibition Sprout inhibition Sprout inhibition

+ + + +

Sprout inhibition Sprout inhibition Insect disinfestation

+

+

Radurization

+

Radurization

+

Dose (krad)

loo0 75

4 October 26 June 4 October

1974 1975 1974

I50 100

4 October 14 January

1974 1975

lo00

15 max. 75-125 12-24

t

5-15 8 max. 10max.

+

+

10 I MeV

Date of approval

13 September 1972 6 September 1976 19 January 1977 4 November 1969 1971 20March 1973

14 March 17 July

1958 1973 1959

200400

1 I July

1964

600-800

I 1 July

I964

30 30

Dried fruits Dry food concenrrures (buckwheat mush, gruel, rice pudding) Poultry, eviscerated (in plastic bags) Culinary prepared meat products (fried meat. entrecote) (in plastic bags)* Onions" Oniuns United Kingdom

United States of America

Uruguay

-

0'

Any food for consumption by patient5 who require a atenle dietSI . an essential factor in their treatment

Insect disinfestation

100

Insect disinfestation

70

6 June

I966

600

4 July

I966

Radurization

Radurization Sprout inhibition Sprout inhibition

+

800

+

6 6

+

Radappenization

I February 25 February 17 July

I966

1967 1967 I973

1 December I969

Wheui mid urhetii ,flour (changed on 4 March 1966 from wheat and wheat Insect disinfestation product)

+

Whire poturoes

Sprout inhibition

+

Sprout inhibition

+ +

Potntoes

IS February

+ 5 MeV

+ +

20-so 20-50 20-so 5-10 5-10 5-15

21 August 2 October 26 February 30 June 2 October I November 23 June

I963 I964 I966 I964 I964 I965 1970

(continued)

TABLE I - (continued) Type and source of radiation Country (organization) World Health Organization

Purpose of irradiation

Product

Potatoes' Potatoes Onions" Papaya Stra wberries Wheat and ground wheat productsp Wheat and ground wheat products Riceg Chicken Cod and redfish'

Sprout inhibition Sprout inhibition Sprout inhibition Insect disinfestation Radurization

+

l"Cs

Electrons

+

Dose (had)

15 max.

Date of approval

12April 7 September 7 September 7 September 7 September

1%9 1976 1976 1976 1976

12 April

1969

+ +

+ +

+ +

10 MeV 10 MeV 10 MeV 10 MeV

Insect disinfestation

+

+

10 MeV max.

Insect disinfestation Insect disinfestation Radurization Radicidation Radurization Radicidation

+ +

+ +

10 MeV max. 10 MeV max.

15-100 10-100

7 September 1976 7 September 1976

+

+

10 MeV max.

200-700

7 September 1976

+

+

10 MeV max.

200-220

7 September 1976

" Compiled by K. Vas, International Atomic Energy Agency, Vienna Experimental batches. Italicized products indicate unlimited clearance. Test-marketing. Temporary acceptance. Hospital patients. Provisional.

'

T o

+ +

maxy max max. max.

3-15 2-15 50-100 100-300 75 max.

FOOD IRRADIATION

I63

The safety evaluation requirements relied heavily o n animal feeding studies and had become large and complex. As timc progressed, a knowledge of much of the radiation chemistry of irradiated foods had been acquired, and, with this knowledge, the claim was advanced that animal feeding studies were of less importance than was generally regarded and that similar foods responded t o radiation in similar ways. In fact it was stated that the radiolytic changes in foods were so small as to be undetectable by the required animal feeding studies. These viewpoints were advanced in both the United States and elsewhere and reached a focus when, in 1976, the question was referred t o a joint expert committee of the Food and Agriculture Organization, the Intcrnational Atomic Energy Agency, and the World Health Organization. As proposed by the International Project in the Field of Food Irradiation, this committee accepted the concept that food irradiation is a process (as opposed to the concept that radiation is a food additive). The same FAO/IAEA/WHO expert committee approved as “unconditionally safe” for human consumption irradiated potatoes, wheat, chicken. papaya, and strawberries. “Provisional” approval was given to rice, fish, and onions (see Table I). In the United States at least two foods, papaya and beef, shortly will be the subjects of petitions to the FDA. In the Netherlands and Denmark as a result of an animal feeding study on a totally irradiated diet, determination of acceptance o f radiation as a food process will be made. All the recent and impending actions are likely to result in a resolution of the question of the safety of irradiated foods for human consumption. The favorable FAO/IAEA/WHO action, both in accepting food irradiation as a process and in approving the indicated foods, encourages hope that other favorable actions will follow. It appears probable, therefore, that the major hold-up to the use of food irradiation will be overcome in the next few years. Once the process is available to commercial interests, its use will be determined by the conventional factors appropriate to food processes generally, namely, utility in fulfilling needs and opportunities. and economics.

II. RADIATION AND RADIATION SOURCES Chemical change in vital parts of living organisms such as food spoilage bacteria may result in their death. It is the capability of ionizing radiation to accomplish chemical changc that is the key t o most of thc particular applications of irradiation to foods. In order to break chcmical bonds, the energy level of the radiation must be sufficiently great. Typical covalent chcniical bond energies lic in the range of I to 8 electron volts.*: As thcse energies are less than the energy ‘:011rclrctron volt. e V . equals I .6 x 1 0 ~

L ‘ I ~

164

WALTER M . URBAIN

of ionization of an orbital electron, all types of ionizing radiation can break covalent bonds. Particles such as electrons can be accelerated to energies sufficient to break bonds and can be used alternatively to electromagnetic radiation. Since only chemical change is desired, it is necessary to limit the energy level of the radiation employed so as to be less than that which will accomplish nuclear change in the elements in the food and cause it to become radioactive. This limitation of energy level is accomplished by setting limits of 5 MeV for electromagnetic (gamma o r X-ray) radiation and 10 MeV for electrons (Anonymous, 1973). For this reason, also, other particles such as alpha particles or neutrons are not employed in food irradiation. Avoidance of induced radioactivity in irradiated foods is a basic requirement of the process. An important characteristic of the radiation used is its penetrability into the food. If it is to accomplish its purpose, it must reach those molecules which need to be affected. Thus for a sterilized food, all parts of it must absorb sufficient radiation to kill all spoilage microorganisms present. The penetration of X-rays and of gamma rays is a function of their energy levels. The maximum energy limit of 5 MeV for these rays provides adequate penetration for practical applications. The maximum of 10 MeV for electrons, however, restricts applications to foods less than about 5 cm thick (two-sided irradiation). For gamma ray sources, two radionuclides have been used in food irradiation: (1) Cobalt-60 (“Co) produced by neutron irradiation of 59C0and (2) Cesium-] 37 (137Cs)produced by separation from fission products. ‘j0Cohas a half-life of 5.27 years and gives gamma rays of 1.17 and 1.33 MeV. 13’Cs has a half-life of 30 years and gives a gamma ray of 0.66 MeV. For electron beam sources, the linear accelerator has proved most useful. Energies up to the 10-MeV limit can be obtained without difficulty. Irradiation facilities provide for treatment of foods under controlled conditions. In any given application, the amount of radiation is controlled by knowing the rate of energy output of the source, by controlling the physical relationship (mainly distance) between the source and target material, and by controlling the time of treatment. The amount of energy absorbed is termed the “dose” and usually has been measured in rads.* Dosimeters are devices to measure the dose. A number of kinds have been devised. The basic one is the Fricke dosimeter. It utilizes the oxidation of Fe 2+ to Fe3+ in a standardized aqueous solution. There are other chemical dosimeters, some of which are available as convenient plastic materials which change color on exposure. Some dosimeters are electrical devices. A calorimeter of appropriate design may be used, especially in cases where the energy output rate is large, as with the linear accelerator. *One rad equals 100 ergs absorbed per gram of absorber. The International System of Units (SI) replaces the rad with the Gray (Gy). One Gy equals I 0 0 rad equals one joule/kg.

FOOD IRRADIATION

165

The irradiation facility generally has three essential components: ( 1 ) the radiation source, (2) a cave or similar structure to confine the radiation within a given space in order to afford protection to personnel during irradiation, and (3) equipment to take the material to bk irradiated to the source. In the case of radionuclide sources, some arrangement usually is provided to permit storage of the source in a safe manner when it is not in use. This usually takes the form of a “pool” of water into which the radionuclide source is lowered and which has sufficient depth to reduce the radiation at the surface to a safe level. Since machine sources can be turned off, a comparable storage facility is not needed for them. The equipment to carry the food to the source usually consists of a conveyor on which the food may be placed outside the cave, and which carries the food to the source area for exposure, and then outside the cave where it is removed from the conveyor. Suitable controls are provided outside the cave for the needed oper-

FIG. I, Plan of Food Irradiation Research Laboratory, U . S. Army Natick Research and Development Command, Natick. Massachusetts.

166

WALTER M . URBAlN

FIG. 2. Irradiation area of linear accelerator. Food Irradiation Research Laboratory, U. S . Army Natick Research and Development Command, Natick. Massachusetts.

FIG. 3 . A typical food irradiator utilizing "Co. Courtesy o f Atomic Energy of Canada, Limited.

FOOD IRRADlAT10N

167

FIG. 4. Mobile irradiator. The boxed food niovcs through 3 positions on each side of the T o plaque. The entire unit is self-contained. Courtesy of Atomic Energy of Canada, Limited.

FIG. 5 . Plan of commercial potato irradiation facility in Hokkaido, Japan. Courtesy Kawasaki Heavy Industries, Ltd.

I68

WALTER M . URBAIN

FIG. 6. Wobalt potato irradiation facility in Hokkaido, Japan. Capacity 15,000 tons per month. Courtesy of Kawasaki Heavy Industries, Ltd.

ations, which can be made essentially automatic. In the area of the radiation source, the target material may be conveyed in a complex manner in order to obtain a relatively uniform dose distribution throughout it and to improve source efficiency. This kind of movement is appropriate to radionuclide sources since gamma rays cannot be directed or focused. Electron beams are easily directed. Because of the ability to direct electron beams, these sources are more efficient in terms of usefully absorbing their output of ionizing radiation than are radionuclide sources. An efficiency of 50% is considered extremely good for a radionuclide source. In order to gain a practical level of source efficiency with gamma rays especially, it is necessary to accept some latitude in dose distribution within a target material. It is virtually impossible to obtain a narrow range (e.g., 5%) between minimum and maximum dose and have source efficiencies that are realistic in terms of commercial requirements. In at least some cases, a 50% variation of dose should be considered acceptable. Costs, especially capital costs, are directly related to source efficiency. Figures 1 through 6 illustrate experimental and production irradiation facilities .

111.

GENERAL EFFECTS OF RADIATION A.

FOODS

As indicated earlier, ionizing radiation can cause chemical change. If a chemical change occurs in molecules important to the life processes of an organism,

FOOD IRRADIATION

169

there may be biological consequences that will manifest themselves in various ways. depending upon the nature of the organism, the degree and location of the damage, and environmental factors. For example, if the chemical changes involve DNA molecules in the cells of the organism, normal functioning may not be possible. Thus, ionizing radiation affects all forms of life. In food irradiation, advantage is taken of this action. Foods contain various organisms as contaminants: bacteria, yeasts, molds, helminths. and insects. These organisms can change a food, and in many cases, we term these changes as “spoilage.” Many preservation processes have as their objective the control of spoilage microorganisms. Some foods, for example, fresh fruits and vegetables, are themselves living organisms. Radiation can also affect their life processes. In some cases, the changes are useful in extending the period before senescence deteriorates the food. The general ways in which irradiation can be useful in treating foods may be listed as follows: Control of spoilage microorganisms Complete sterilization for unlimited product life Reduction of numbers to delay microbial spoilage Control of food-borne pathogenic microorganisms Control of helminths and other food-borne parasites Control of insects Delay of senescence Product improvement Of these, only the last does not involve affecting a life process, and it is not normally involved in the preservation of food. It generally is concerned with improving a functional property of a food or foodstuff. There is a large variation in the dose needed in connection with the above individual effects and part of the development of particular applications is the determination of dose requirement.

B. ACTION ON MAJOR FOOD COMPONENTS 1. Proteins

Reviews on the radiation chemistry of proteins and related compounds are available (Garrison, 1972; Urbain, 1977). In general, the effect of radiation on proteins is not great at the doses employed in food irradiation. Regardless of origin, protein molecules tend to respond to radiation similarly. To some degree, the nature of the change is related to the particular structure of a protein (namely, fibrous, globular), whether native or denatured, its composition, to the presence of other substances, and its state (wet, dry, in solution, or whether liquid or frozen).

170

WALTER M . URBAIN

Because they are large molecules, proteins generally provide a number of loci for action by radiation. Although the energy may be absorbed at one location, it can transfer to another within the molecule, where, at what might be termed a “sensitive site,” bond breaking occurs. Atoms, or groups of atoms, may split off and form free radicals. In this way, radiation acts upon large complex molecules, such as proteins, in a characteristic rather than random manner. The free radicals formed ultimately disappear. At lower temperatures, diffusion is limited and recombination of radicals is more likely. At higher temperatures, reaction with different species is more probable. The condition of a protein before irradiation affects the end results. Irradiation of a denatured protein leads to a higher level of free radical formation since its disrupted structure has less capability for recombination. Indirect action of radiation plays a very important role when water is present, unless it is “bound” or frozen. In the presence of free liquid water, there may be rupture of hydrogen bonds with consequent unfolding of the molecule, or there may be aggregation or dissociation into smaller units, or there may be fragmentation. In certain of these changes, chemically active groups may be made more available or they may be altered so as to be essentially nonexistent. Changes such as have been indicated above may alter the normal properties of a protein. It may, for example, become denatured. Enzymes no longer may be active. Chromoproteins may be changed in color. Functional properties (as in foods) may be altered. Nucleoproteins may lose their function in biological processes, which may affect the organism of which they are a part. As nutrients, proteins serve primarily as sources of amino acids. Radiation can cause amino acid destruction. At doses employed in food irradiation, however, amino acid values are virtually unchanged, and, as a consequence, proteins suffer no measurable nutritional losses.

2. Lipids Changes resulting from the irradiation of lipids may be grouped as (a) gross changes in physical and chemical properties, (b) autoxidative changes, and (c) nonoxidative radiolytic changes. a. Gross Changes. Below 5 Mrad, there are only very slight changes in the usual indexes for fat quality. At doses between 10 and 100 Mrad, there are significant increases in acid number, trans-fatty acid content, peroxide values, melting point, refractometric and dielectric constants, viscosity and density. Shifts in double bond position occur. Flavor changes in meat fat occur with doses as low as 2 Mrad. In milk fat, a “chalky” or “candle-like” flavor develops. An off odor in fish lipids has been ascribed to oxidative rancidity of unsaturated fatty acids.

171

FOOD I R R A D I A T I O N

b. Autoxidative Changes. As evidenced by electron-spin resonance (ESR) measurements, irradiation produces free radicals in fats. The types of free radicals formed and their decay rates are influenced by temperature. They are more stable at lower temperatures. If exposed to 02, they can react and form new free radicals, such as peroxide radicals (Farmer et al., 1942). The reaction with O2 can occur over an extended period of time after irradiation. The radiation-induced autoxidation process follows the same path as the usual one for fats and, through a free radical chain mechanism, yields hydroperoxides which decompose into a variety of products such as aldehydes, aldehyde esters, oxoacids, hydrocarbons, alcohols, ketones, hydroxy and ketoacids, lactones, and dimeric compounds. Irradiation accelerates the autoxidative process.

c . Nonoxidative Rudiolytic Chunges. The major compounds formed when a saturated fat is irradiated in the absence of 0, are H,, CO,, CO, a series of hydrocarbons (n-alkanes and alkenes) and an aldehyde. In general a similar pattern is obtained with unsaturated fatty acids. However, the presence of one or more double bonds causes the formation of other radiolytic unsaturated compounds. Also, some hydrogenation occurs and produces a saturated fatty acid. Significant amounts of dimers are formed. A general radiolytic mechanism has been given by Nawar (1972, 1977). In a triglyceride molecule a

L O C

d I

I

I

I

c-0-0-c,

I

c-0-0-c,,

where n is the number of carbon atoms in the component fatty acid, cleavage occurs preferentially at five locations (a, b, c, d, and e) and randomly at all the remaining carbon-carbon bonds of the component fatty acids. The resulting free radicals are terminated principally by hydrogen abstraction (from other molecules) and, to a lesser extent, by hydrogen loss or by combination with other free radicals. As a consequence, a number of radiolytic compounds are formed. With due allowance for initial composition, natural fats yield essentially the same compounds as do model systems (e.g., fatty acids, pure triglycerides. and esters). The volatile substances identified in beef fat irradiated with various doses at 25°C are given in Table 11. The same substances are formed at all doses, but the amounts are in proportion to dose. Whether a fat is irradiated in the solid or liquid state affects the relative amounts of radiolytic products formed. The compounds produced by heating a fat are quite similar to those obtained by irradiation. There are, however, both qualitative and quantitative differences.

172

WALTER M . URBAIN

TABLE

n

QUANTITATIVE ANALYSES (mg/kg) OF THE VOLATILES FORMED IN BEEF FAT BY

IRRADIATION"."

Concentration of volatiles (mglkg) at Mrad Compound n-Propane I-Propene n-Butane I-Butene n-Pentane I-Pentene n-Hexane I-Hexene n -Heptane I-Heptene n-Octane I-Octene n-Nonane I-Nonene n-Decane I-Decene n-Undecane I-Undecene n-Dodecane I-Dodecene n-Tridecane I-Tridecene Tridecadiene n -Tetradecane I-Tetradecene Tetradecadiene n-Pentadecane Pentadecene (int.)c I -Pentadecene Pentadecadiene n-Hexadecane Hexadecene (int.)r I-Hexadecene Hexadecadiene n -Heptadecane Heptadecene (int.)' I-Heptadecene Heptadecadiene "

0.5

0.40 0.07 0.34 0.18 0.50 0.02 0.06 0.01 0.26 0.07 0.30 0.05 0.81 0.12 0.49 0.22 0.40 0.16 0.37 0.67 0.95 0.67 trace 0.55

4.23 0.26 2.85 0.48

1

2

3

0.82 0.22 0.88 0.I6 0.89 0.04 0.28 0.05 0.58 0.22 0.71 0.29 0.54 0.28 0.69 0.38 0.62 0.23 0.53 1.08 1.60 0.87

1.04

I .72

0.38 I .72 0.50 0.96 0.39 1.27 0.70 1.08 0.54 0.87 1.74 2.93 1.41

1.76 0.56 1.48 0.49 1.66 0.91 1.32 0.67 1.22 2.49 3.79

trace

trace

0.90 6.43

1.31 14.00 0.86 10.40

0.50

4 1.68 0.21 I .40 0.16 1.14 0.07 0.86 0.29 2.20 0.65 2.44 0.75 1.99 0.74 2.30 1.24 1.97 0.88 1.67 3.61 6.02 3.12

5

6

2.35 4.62 8.27 3.24

2.98 0.26 2.52 0.17 2.23 0.15 1.74 0.36 4.02 1.14 5.30 1.54 3.74 1.01 4.39 I .92 3.26 I .30 2.96 6.18 1.31 4.38

2.84 0.68 3.51 0.81 2.76 0.83 3.11 I .60 2.57 1

.oo

1.51 trace

trace

trace

trace

1.58 15.40 1.22 14.80 I .60 2.42 1.06 0.96

2.53 24.20 I .66 24.50 2.11 3.50 1.68 1.82

3.37 31.10 2.02 29.40 2.59 4.28 1.93 2.30

4.75 37.80 3.07 36.25 3.88 7.29 2.67 4.50

0.26 0.60

5.04 0.48 0.97 0.41 0.78

trace

trace

trace

trace

trace

trice

trace

4.18

5.84 7.39 5.00 5.44 1.12 1.33

7.78 12.60 8.22 5.94 1.72 2.63

14.36 18.54 12.55 15.62 2.22 3.10

23.50 29.10 21.40 25.90 1.98 4.07

27.70 35.20 21.90 27.00 4.32 6.35

31.05 40.40 26.90 32.90

0.64

5.64

4.18 3.78 trace

0.96

0.5 to 6.0Mrad at 25°C. From Nawar (1977). in1 = Internally unsaturated.

1.01

1.95 0.73 1.02

5.10

8.20

FOOD IRRADIATION

173

For example, in the irradiation of triolein, hexadecadiene is formed. Heating triolein results in ethyl-, propyl-, pentyl-, and hexylcyclohexenes, which are absent in irradiated triolein. The amount of pentane produced by irradiation of tricaproin is nearly twice that formed by heat. 3. Carbohydrates

Carbohydrates are components of many foods. They also are available as isolates. The response to radiation varies with the circumstances in which the carbohydrate exists. Pure carbohydrates are very sensitive to radiation when in the crystalline state, and give a response which is dependent upon the particular crystalline form irradiated. Imperfections of the crystalline lattice, as for example, produced by freeze drying, reduce the effectiveness of energy transport in a crystal and probably account for variations related to crystalline form. Depending upon the carbohydrate irradiated, a great many substances have been identified including H,, COB,aldehydes, ketones, acids, and other carbohydrates (Dauphin and SaintLebe, 1977). In aqueous solution, irradiation of carbohydrates causes oxidative degradation. The changes are due both to direct action of the radiation and to indirect action mainly by OH. radicals produced by radiolysis of the water. For the lower saccharides, oxidation at the ends of the molecule produces acids. Ring scission forms aldehydes. D-mannose, for example, in the absence of oxygen forms D-mannonic acid, D-glucose, and two, three, and four carbon aldehyde fragments. In the presence of oxygen, secondary reactions occur leading to D-erythrose, glyoxal, oxalic acid, mannuronic acid, D-xylose, mannonic acid, D-arabinose, and formaldehyde. For aldohexoses in solution, it is clear that the effect of radiation is not confined to any particular part of the molecule and all bonds are affected. For higher saccharides, cleavage of the glycosidic link is part of the radiation effect. This results in fragmentation into smaller molecules. Corn starch, for example, yields glucose, maltose, erythrose, ribose, and mannose (Berger et af., 1973). All carbohydrate solutions produce malonaldehyde and deoxycompounds. In the normal neutral pH of foods, the yields of malonaldehyde are minimal (Phillips, 1972). The amount of deoxysugars produced in starch at 100 krad is less than 0.3 pg/gm (Diehl et al., 1978). Many substances provide protection against radiation degradation of carbohydrates (Phillips, 1972). Among these are amino acids and proteins (Diehl et al., 1978). These observations point to the influence that compounds associated with carbohydrates in a food can exert on the end results, and care must be exercised in extrapolation of findings obtained for pure substances to the complex systems that exist in foods.

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WALTER M . URBAIN

IV. APPLICATIONS OF FOOD IRRADIATION A.

INTRODUCTION

Applications of food irradiation can be divided into two categories according to the dose employed, high and low dose. This division is somewhat arbitrary but is useful. Applications requiring less than I Mrad are considered to be low dose uses; above 1 Mrad they are high dose (Anonymous, 1970a). High-dose applications generally are concerned with sterilization. All other uses fall into the low-dose category. A nomenclature has been devised to identify the objective of a particular radiation treatment in terms of its affect o n microorganisms (Goresline rt ul., 1964): Ruduppertizution-to produce a condition of “commercial sterility,” the same as with thermal processing (appertization). Rudicidutinn-to reduce the number of viable specific nonspore forming pathogenic microorganisms (other than viruses) so that none is detectable in the treated food by any standard method. Radurizution-to enhance the keeping quality of a food by means of radiation. This is interpreted to mean reduction o f t h e initial population of viable specific spoilage microorganisms to such a level that outgrowth to a spoiled condition is delayed. In addition, the term disinfestation is applied to radiation treatments whose objective is to kill o r inactivate insects or parasites contaminating a food. Other more specific terms are used for particular treatments. “Delayed senescence” applies to the irradiation of raw (living) fruits and vegetables whose usual ultimate spoilage is a kind of overripening, which can be delayed by radiation. “Sprout inhibition” refers to treatments to prevent or delay sprouting of foods such as potatoes or onions.

B.

HIGH-DOSE APPLICATIONS

I.

Meats and Seafood

As stated. the objective of high-dose applications is to achieve sterility, or perhaps, more precisely, to obtain indefinite shelf stability without refrigeration. Radiation alone cannot achieve this, but it does provide a key part of the requirements, namely destruction of spoilage microorganisms, including any that would affect the safety of the food. Irradiation must be combined with (a) suitable packaging to prevent microbial recontamination and also to isolate the product from the atmosphere and (b) inactivation of enzymes native to the food. whose action could cause undesirable changes, such as alteration of flavor or

I75

FOOD IRRADIATION

texture. Metal containers, such as presently used in thermal canning have been found satisfactory (Killoran et al., 1974). Glass containers are functionally suitable but are discolored by the radiation. Flexible film containers have been developed which meet performance requirements. A laminated flexible package consisting of chemically bonded Mylar and medium density polyethylene as the food contactant layer, aluminum foil (middle layer), and Nylon 6 (outside layer) was found to be satisfactory (Killoran, 1972; Wierbicki e t a / . , 1975). Containers are filled with product and closed prior to irradiation. The dose requirement for radappertization is determined by the microorganism associated with the food that has the greatest radiation resistance. For nonacid low-salt foods. not containing critical minimum levels of nitrite, such as many meats, this organism is the spore of Clastridium botulinum. The radiation resistance of this organism is different for the different strains and varies with the food. Table 111 shows the comparative resistance of representative strains of CI. botulinum Types A and B. The determination of the sterilization dose is not simple. While the knowledge gained with thermal sterilization is useful, radappertization is not exactly a parallel process. Most importantly, CI. botulinurn spores are not the most heat-resistant organisms found in foods. As a consequence, while there is greatest concern for the destruction of this organism, due to the potential hazard of botulism

TABLE I l l RESISTANCE TO GAMMA RAYS OF REPRESENTATIVE STRAINS OF Chslridiurn hotdinurn TYPES A A N D B 1N

PHOSPHATE BUFFER. pH 7"

Type

Strain number

D-value" (Mrads)

A A B B B A A A B B

33 36 40 41 53 62 77 1288s 9 51

0.334 0.336 0.317 0.318 0.329 0.224 0.253 0.241 0.227 0.129

From Anellis and Koch (1962). D-value is the dose necessary to accomplish a 90% destruction of the organisms present. (I

176

WALTER M . URBAIN

should outgrowth and toxin formation occur, thermal processes usually are set for organisms other than Cl. botulinum which are more heat resistant and whose outgrowth could cause spoilage. This circumstance provides an insurance factor for the safety of thermally sterilized foods. In the case of irradiation, the most radiation-resistant organism of concern is Cl. botulinum. There are other organisms whose radiation resistance is greater but they are not a factor in the production of radappertized foods (Welch and Maxcy, 1975; Maxcy et al., 1976; Anellis et al., 1977). Although the asporogenous Acinetobacter and Moraxella bacteria have a high radiation resistance, they are easily killed by heat. The preirradiation heat treatment (67"-75"C) for autolytic enzyme inactivation of radappertized meats is sufficient to be lethal to them. The safety of the thermal sterilization process is based on a 12-D reduction in the count of the most heat-resistant strain of Cl. botulinum. Early work (Hannan, 1955) had suggested that about 2 Mrad was a sufficient dose for radappertization, but Hannan (1955, p. 67) considering the 12-D concept applied to thermal processing and using the data of Morgan and Reed (1954) for the radiation resistance of CI. botulinum spores, suggested that a dose approaching 5 X lo6 rep* or 4.65 Mrad. This concept was taken up by Schmidt (1961) and, after much debate, was accepted in principle. Thus, both thermal and radiation processing are placed on the same basis as far as safety with respect to botulism is concerned. Since no other food spoilage microorganism has a greater radiation resistance, irradiation lacks the added safety factor that organisms more heat resistant than Cl. botulinum provide the thermal sterilization process. Partly because of this, the minimum radiation sterilization dose must be known accurately and the process must be carefully designed to assure its delivery. The conventional practice of estimating the 12-D dose (Schmidt and Nank, 1960) is based on the assumption that the rate of spore kill in an inoculated pack is exponential (no initial shoulder). An improved method which replaces the conventional one has been developed (Anellis et al., 1975, 1977; Ross, 1974). This method employs two interrelated functions which operate simultaneously in foods undergoing irradiation: (1) A spore inactivation rate that is not necessarily a simple exponential one; and (2) a can sterilization rate which is dependent upon inactivation of the most resistant spore in the can. The conventional method uses only the first function and assumes it to be a simple exponential function. The second function arises as the extreme or largest value derived from the first. The new method yields the largest dose complying with inoculated pack data and which also is a 12-D dose. It is, therefore, a conservative dose in the sense that it may be greater than is actually needed (Ross, in press). The inoculated pack data are obtained by inoculating the specific product in *One rep (old unit) equals 93 ergs per gram, or 0.93 rad.

177

FOOD IRRADIATION

TABLE IV INOCULATED PACK EXPERIMENTAL DESIGN FOR BEEFn

Prototype food CI. hotdinurn strains Spore inoculum Containers Foodicontainer Cansidose Vacuum seal Radiation source Radiation doses (Mrad) Radiation temperature ("C) Incubation Analysis

~~~~~~

Beef formulated with 0.75% NaCI, 0.38% TPP A mixture of 33A, 36A, 62A, 77A 12885A. 9B,40B, 419, 53B. 679 !@/strain; 107/can 21 1 x 101.5 (epoxy enamel) metal cans 40 t 5 gm 100 replicate 16 kPa T o gamma rays 1.4, 1.8, 2.2, 2.6, 3.0. 3.4, 3.8,4.2. 4.6, 5.0

-30 t 10 6 months at 30 t 2°C Swelling: daily-1st month weekly-2nd thru 6th month Botulinal toxin: 7th month Recoverable CI. hotulinum: 7th month ~

~~

~

~

From Anellis el ol. (1976). First Int. Congr. Eng. Food, as sponsored by the American Society of Agricultural Engineers. "

question with a spore level of about lo7 per unit (can). Determination of the minimum radiation dose (MRD) is based upon (a) the presence or absence of viable botulinal cells in the cans, regardless of their ability to outgrow and produce toxin and/or can swelling; (b) a single most resistant strain of Cl. botulinum, and (c) a shifted exponential (an initial shoulder followed by a semilog decline) rate of spore death. The irradiation conditions employed are identical (particularly with respect to irradiation temperature) as those of the commercial process. The experimental design of the inoculated pack of a beef prototype food is shown in Table IV. A sophisticated statistical treatment (Ross, 1976) provides a margin of safety and yields the maximum (most conservative) dose. Determination of the MRD on the basis of surviving organisms rather than on swelling or toxin formation provides an additional margin of safety. The MRDs for a number of radappertized meats and codfish cakes are given in Table V. The variation in values reflects the differences in composition and in irradiation temperatures and the impact of these differences on the resistance of CI. botulinum. Irradiation at lower temperatures reduces the lethal effect of radiation and increases the dose requirement for radappertization (Rowley et ul., 1968; Grecz ef a / . , 1971; Maxcy et af., 1976). The presence of NaCl and NaNO, lowers the dose requirement. The dose requirement for radappertization of low-acid low-salt foods has now been established. The problems related to the high MRD values have been the subject of much research. Virtually all the recent research and development in the high dose category has

178

WALTER M . URBAIN TABLE V MINIMUM RADIATION DOSE (MRD) FOR RADAPPERTIZED MEATS AND CODFISH" ____

~~

~

MRD

Food

Irradiation temperature ("C)

(Mrad)

Bacon Beef" Ham" Ham" Pork Codfish cakes Corned beef Pork sausage

5 to 25 -30 10 5 to 25 -30 2 10 5 to 25 -30 5 10 -30 2 10 - 3 0 2 10

2.5 4.1' 3.1 3.3 4.3 3.2 2.4 2.7

"

*

From Wierbicki et a / . (1975).

* With additives: 0.75%NaCI, 0.375% Na tripolyphosphate. Anellis et a / . (1977). " High NaN02/NaN03( I 56/700 mg/kg)--regular. "Reduced NaN02/NaN03(25/100 mglkg).

been concerned with meats and seafood and has been done only at one place, the U. S . Army Natick Research and Development Command. The Army program began in 1953 and, as noted, has the objective to provide foods of greater consumer acceptability, improved nutritive quality, and better storage characteristics to be used as military rations. The military program is expected to have a spin-off of benefits to the civilian sector. Among the products that have been developed are radappertized bacon, ham, pork, chicken, beef, hamburger, corned beef, pork sausage, codfish cakes, and shrimp. In the raw state, all of these foods contain indigenous enzymes, which must be inactivated for long-term preservation. At the doses employed in radappertization, radiation does not effect sufficient enzyme inactivation, as shown by the data of Table VI. In order to obtain the degree of enzyme inactivation needed for product stability, the use of heat has been found to be the only practical and effective method. Heating of meats to 7Oo-75"C prior to irradiation is sufficient (Shults and Wierbicki, 1974a). The high-dose requirement for radappertized food results in some undesired side effects, namely the formation of unpleasant and characteristic odor and flavor, texture changes, and, in meats and seafood containing myoglobin pigments, color changes. Of these, from the standpoint of consumer acceptance, the flavor change is the most important. A considerable effort over many years has been expended to find the cause of the off-flavor. Little real progress was made until gas chromatography and mass

179

FOOD IRRADIATION TABLE VI

EFFECTS OF IRRADIATION DOSE AND TEMPERATURE ON THE PROTLOLYTIC ENZYME ACTIVITY OF BEEF MUSCLE"

Irradiation temperature ("C)

Dose (Mrad)

+21 7c reduction

0 0 reduction

2 4 6 8

57

44

33

0

65 19 86

65 12 82

45 40

40

I'

- 30

- 80

%' reduction

% reduction

73

18 60

From Shults et cd. (1975)

spectrometry provided the analytical techniques needed to identify the radiolytic products present. No positive identification of the substances responsible for the flavor, however, has been secured. Wick rt af. (1967) concluded that methonal, 1-nonanal, and phenylacetaldehyde are the principal substances responsible for the flavor. The sensitivity of protein foods to off-flavor development by radiation varies with the species of animal from which the food is derived, as shown by the data of Table VII (Sudarmadji and Urbain, 1972). It was observed that irradiation in the frozen state significantly lessened the off-flavor in meat (Brasch and Huber, 1948; Coleby et al., 1961). Lean meat is of the order of two-thirds water. Irradiation of water can produce a variety of substances including free radicals such as OH., the aqueous electron e&, hydrogen atoms, and active compounds such as H,O, (Draganic and Draganic, 1971; Hart, 1972; Swallow, 1977). These radiolytic products from the water present in meat can cause an indirect action of radiation. Freezing prior to irradiation lessens the effect o f this indirect action and results in less off-flavor development. Table VIII shows data on the change of flavor and textural characteristics of beef with irradiation temperature. Lowering the temperature clearly leads to improvement of sensory properties. Harlan rt af. (1967) found a similar relationship. The beneficial effect of low-temperature irradiation, as measured by subjective criteria, has been confirmed by measurement of the amount of radiolytic products formed. Figure 7 shows the change with irradiation temperature of flavor intensity score and amounts of detected volatile radiolytic products. Correlation of flavor intensity with amount of volatiles formed seems clear. The simultaneous lowering of both with lowered irradiation temperatures points to their origin with the indirect action of radiation through the radiolytic substances produced in the water. As noted earlier. the lethal effect of radiation o n microorganisms also is reduced by lowering the irradiation temperature. In this manner the dose required

180

WALTER M. URBAIN TABLE VII THRESHOLD DOSE FOR DETECTABLE OFF-FLAVOR FOR PROTEIN FOODS FROM VARIOUS ANIMALS IRRADIATED AT 5" TO 10"C'

Threshold dose (krad)

Animal food

I50

Turkey Pork Beef Chicken Lobster Shrimp Rabbit Frog Whale Trout Turtle Halibut Opossum Hippopotamus Beaver Lamb Venison Elephant Horse Bear

175 250 250 250 250 350 400 450 450 450 500 500 525 550 625 625 650 650 875

* From Sudarmadji and Urbain (1972). Reprinted from Food TechnologylJournal of Food Science 37, 671-672, 1972. Copyright @ by Institute of Food Technologists. TABLE VlIl EFFECT OF IRRADIATION TEMPERATURES ON FLAVOR AND TEXTURAL CHARACTERISTICS OF UNITED STATES COMMERCIAL BEEF LOIN",b

Irradiation temperature ("C)

Irradiation flavorr

Mushiness"

Friability'

4.1 3.3 2.9 2. I 1.5

5.3 3.4 2.5 2.0 2.0

5.0 3.0 1.9 1.8 1.9

+60 t21 +40 - 80 - I85 "

From Shults and Wierbicki (1974b).

* Dose 4.5 to 5.6 Mrad. I'

Intensity scale of 1-9 ( I denoting "none" and 9 "extreme").

FOOD IRRADIATION

181

*Oo0[ 1500

TEMPERATURE (“C)

FIG. 7. Change with irradiation temperature of flavor intensity score and amounts of detected volatile radiolytic products of beef irradiated at 5.6 Mrad. Arrow denotes value for nonirradiated control. Flavor intensity scale of 1-9 (1 denoting “none” and 9 “extreme”) (Memtt et al., 1975). Reprinted with permission from Journal qfAgriculrural and Food Chemisrry 23, 1037-1041. Nov.1 Dec. 1975. Copyright by the American Chemical Society.

for sterility is increased. The amount of radiolytic products is a function of dose, as may be seen from the graph of Fig. 8. As a consequence, the value of low-temperature irradiation may be questioned. Is the gain in sensory quality improvement offset by higher dose requirements to gain sterility? It appears not. For its products, the Natick laboratory has selected an irradiation temperature of -30” 2 10°C. This temperature was selected as producing an adequate acceptance improvement for beef which was not significantly bettered by using lower temperatures (Shults and Wierbicki, I974b). While lower irradiation temperatures have been considered, it was concluded that -30” 2 10°C provides the

IRRAOLATION DOSE (rnrads)

FIG. 8. Graph showing relative amounts of detected volatile radiolytic products produced as a function of dose in beef irradiated at - 185°C (Merritt F I a / . . 1975). Reprinted with permission from Jourriul of Agricultural and Foad Chemisrn 23. 1037-1041, Nov./Dec. 1975. Copyright by the American Chemical Society.

182

WALTER M . URBAIN

most favorable balance of quality, cost, and required irradiation dose (Wierbicki er ul., 1975). While the use of subfreezing irradiation temperatures unquestionably yields improvement in the sensory properties of radappertized foods, trained expert evaluators can note some “irradiation flavor” in the products listed on page 178. Such persons have expressed reservations about the acceptance of these foods by ordinary consumers. The Army has compared the acceptance by volunteer troops of radappertized meats, poultry, and seafoods with nonirradiated controls. In these tests literally thousands of testers were employed. Table IX shows such consumer acceptance data obtained with Army and Air Force personnel. Army experience indicates that products scoring 5 or higher on the 9-point hedonic rating scale are acceptable as rations. Because the ratings for radappertized foods developed by the Army have exceeded the value of 5 , the views of the product experts relating to a detectable irradiation flavor have been set aside. It appears that the final test for acceptability can be made only when the products become available to consumers, both military and civilian, on a basis that allows the open competition of the market place. In this connection it should be noted that radappertized meats have obviously superior texture characteristics and do not undergo moisture release, as is the case with thermal sterilization. It is also significant that many common foods undergo flavor changes as a result of processing and yet obtain a high degree of consumer acceptance. The technological development of the products considered by Natick and listed earlier largely has been recorded in a number of publications. Bibliographies on this work are available (Wierbicki, 1974; Cohen and Mason, 1976).

TABLE IX ACCEPTANCE OF RADAPPERTIZED MEATS, POULTRY. AND SEAFOODS“

Irradiated

Nonirradiated control

Item

Number of evaluators

Rating”

Number of evaluators

Rating”

Ham Chicken Pork Beef Bacon Shrimp Codtish cakes

1,651 583 39 1 589 25,656 539 53 1

5.87 6.07 5.71 5.99 6. I6 6.09 5.40

1.437 548 458

6.66 6.36 6x5 6.61

“

644 849 578

-

6.43 6.30

From Urbain (1970).

’ Based on a 9-point hedonic scale, 9 like extremely. 5 neither like nor dislike.

1 dislikeextremely.

FOOD IRRADIATION

I83

2 . Fruits and Vegetubles The success with the development of radappertized meats and seafood must be correlated with the sustained intensive research effort of the United States Army. There has been no comparable program with other radappertized foods. In the early years of research on food irradiation many foods were examined. Apparently sensory changes discouraged further work with fruits and vegetables. Irradiation in the frozen state had a protective effect on flavor and color, but did not prevent texture damage (Hannan, 1955). Dipping fruit in a calcium chloride solution reduces the softening caused by irradiation (Al-Jasim et al., 1968). The preparation of shelf-stable fruit juices was attempted in Europe, but without definitive results (Kaindl, 1966; Anonymous, 1967; Kiss and Farkas, 1968). Just as with animal products, enzyme inactivation is required for long storage of fruits and vegetables and is best done by heating (blanching).

3 . Spices There was early interest in the radiation sterilization of spices (Proctor et al., 1950) and it has continued into the present. In this application, the objective is not preservation but rather the reduction or elimination of a bacterial population normally indigenous to these materials as used and whose presence constitutes a problem when incorporated in foods (Hansen. 1966; Farkas, 1973; Inal el al., 1975; Farkas and Beczner, 1973). Because preservation is not the objective. the minimum dose requirements, such as those for radappertized meats, are not necessary and adequate effects can be obtained with 1 to 2 Mrad. Vajdi and Pereira (1973) showed that gamma radiation was more effective than ethylene oxide in reducing the bacterial population of spices. While ethylene oxide reduced the oil content of certain spices and affected the color of paprika, gamma irradiation caused insignificant changes. Tables X and XI provide data on bacterial reduction and oil content of selected spices. Bachman and Gieszczynska (1973) obtained similar results. 4 . Diets for Speciul Patients Similar to the treatment of spices, irradiation with high doses is used to remove microbial populations of other products without having preservation as a purpose. In a few countries (United States, United Kingdom. the Netherlands, and West Germany), government approvals have been granted to permit irradiation of diets of hospital patients whose circumstances require extraordinary protection from infection. Generally these patients, as part of their therapy, have been treated to reduce their immuno response. Patients receiving organ transplants and

TABLE X THE COMPARATTVEEFFECT OF ETHYLENE OXIDE AND GAMMA IRRADIATTON ON THE BACTERlAL FLORA OF SELECTED RAW SPICES"

Treatments (Number of organisms per gram) Raw

Spices

Total count

B. pepper Paprika Oregano Allspice Celery seeds Garlic

4 . 0 X lo6 9.86 X lo6 3.26 x 104 1.74 x I@ 3.7 X lo" 4.65 x 104

Ethylene oxide

Thermophilic 1.58 X 3.24 X 1.8 x 1.5 x 1.3 X 9.0 x

lo6

lo5 I@ loR lo" 18

Aerobic spores

6.34 X 3.0 X 1.0 x 1.05 x 3.94 X 0.0

104 10' 18 I@ 10'

Total count

1.48 X 16 0.0 0.0 4.25 x 10 0.8 x 10 1.45 X I 0 4

Thermophilic

4.3

X I 8 0.0 0.0

0.0 0.0

3.5

X

l@

Gamma irradiation Aerobic spores

Total count

Thermophilic

Aerobic spores

0.0 0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0 0.0

" From Vajdi and Pereira (1973). Reprinted from Food TechnologylJournul of Food Science 38, 8 9 3 4 9 5 . 1973. Copyright @ by Institute of Food Technologists.

185

FOOD IRRADIATION

TABLE XI THE COMPARATIVE EFFECT OF ETHYLENE OXIDE AND GAMMA IRRADIATION ON THE VOLATILE A N D NONVOLATILt OIL CONTENT OF SIX RAW SPICES"

Treatments (Volatile and nonvolatile oils. a) Raw

Spices

B. pepper Paprika Oregano Allspice Celery seeds Garlic

Ethylene oxide

Volatile oil

Nonvolatile oil

Volatile

3.6 3.3 6. I6 1.73

10.436 14.345 10.401 10.910 23.420 1.340

I .6 3.2 I.6 I .73 -

-

oil

Gamma irradiation

Nonvolatile oil

Volatile 011

9.210

3.6

II ,380

-

8.060 6.130

3.33 6. I 6 I .73 -

21.360

0.607

Nonvolatile oil

9.363 15.010 10.248 9.650 23.100 1.040

" From Vajdi and Pereira (1973). Reprinted from Food TechnologylJournd of Food Science 38, 893-895, 1973. Copyright @ by Institute of Food Te
certain kinds of cancer patients are those usually involved. The value of irradiation compared with alternative food sterilizing measures, e. g., heat, lies in its ability to provide a more varied diet with greater patient acceptance. Doses of about 2.5 Mrad are used and foods generally are irradiated in the frozen state. This application is being practiced on a very limited scale.

5 . Laboratory Animul Diets A similar kind of use of irradiation is made in the preparation of diets of laboratory animals intended to be specified-pathogen-free or germ-free (gnotobiotic). Irradiation competes with heat or with ethylene oxide. Steam sterilization destroys a variable percentage of certain nutrients, while ethylene oxide destroys both vitamins and amino acids (Mickelsen, 1952). Irradiation also causes certain vitamin losses (Coates et al., 1969). Based on growth, reproduction, and general health, irradiated diets appear to be nutritionally satisfactory (Ley el al., 1969; Sickel r1 ai., 1969). Doses on the order of 2.5 Mrad are employed. It is claimed that irradiated diets are more convenient for the user and are more palatable. It has been estimated that 700 tons of feed are currently irradiated in eight countries (Adamiker, 1975). 6 . Milk Milk and dairy products are extremely sensitive to radiation and produce a disagreeable flavor, even at doses as low as 50 krad (Goresline. 1966). Irradia-

186

WALTER M . URBAIN

tion of milk in the fluid state at 4.5 Mrad caused browning and caramelization. Irradiation at -80” and - 185°C resulted in an extremely bitter flavor (Scanlan and Lindsay, 1968). Earlier work had employed conciirrent irradiation and vacuum distillation and was successful in controlling off-flavor development, but such milk was not stable on storage at 30°C due to browning and gelation (Hoff et a f . , 1958). A comprehensive literature review on preservation of milk by irradiation (Gerrard, 1969) contains the conclusion that it is not practicable to preserve milk with irradiation.

7 . Food-Borne Viruses It is assumed that many foods contain viruses. Although precise information is lacking, foods, however, are seldom considered as carriers of viruses pathogenic to man. There are, however, a number of viruses which cause disease in meat animals, among which is the foot-and-mouth (FMD) virus. Presently certain areas of the world are free of this virus and rigid controls are exercised, mainly through embargos on fresh meat to prevent its spread. Irradiation has been considered as a means of inactivating viruses (Levinson, 1957), but the small size of viruses suggested that very high doses might be needed (Desrosier and Rosenstock, 1960). In the case of the FMD virus, the size is especially small, 10-12 nm. Massa (1966) and Baldelli (1967) have investigated the inactivation of this virus. The D-value for the liquid FMD virus was 0.481 Mrad and for the dried virus 0.626 Mrad. Reduction of the number of virus particles from 10’ to 1 required 3 and 4 Mrad, respectively. These doses applied to raw fresh meats, not frozen, would produce off-flavors and possibly other sensory changes. They would also involve significant costs. Because of these negative factors, there has not been a great deal of interest in the use of radiation as a quarantine-control measure for the FMD virus.

8 . Bacterial and Mycotoxins The possibility of using radiation to inactivate bacterial and myco-toxins has been investigated (Miura et al., 1967; Miyaki et al., 1967; Roberts, 1967; Skulberg, 1970; Sakaguchi, 1970; Aibara and Miyaki, 1970). D-values as high as 4 Mrad have been obtained for botulinal toxin in complex media, such as exist in foods. Such high D-values are to be anticipated in view of the small size of the toxin entity which makes detoxifying by direct action of the radiation difficult plus the “protective” effect of the complex medium (food) constituents. Based on the limited work done, most of which was with botulinal toxin, it must be concluded that, due to the very high dose requirements, irradiation is not a useful agent for inactivation of bacterial and mycotoxins.

187

FOOD IRRADIATION

9 . Combination Processes Because radappertization employs high doses, and because of the resultant problems with sensory changes in foods, there has been an incentive to find ways to reduce the dose and yet achieve sterilization. Combination of radiation with other agents has been the principal approach for this objective. The use of heat and radiation to effect sterilization was studied (Huber er al., 1953; Morgan and Reed, 1954; Kempe, 1955). Heating was done before or after irradiation. The microbial requirements for sterilization that exist when heat or radiation is employed singly are lessened for both when they are used in combination (Kempe, 1959). Hansen (1966) reported that canned ham could be sterilized by the combination of heating to a center temperature of 65”-70”C and irradiation with 0.5 Mrad. However, both expert and consumer panels noted an “irradiated” flavor in the ham. Dharkar and Sreenivasan (1966) reported treatment of fruits with 70°C heat and 400 krad radiation produced sterile products of improved quality. Peas irradiated with 800 krad and heated to 100°C also were sterile and quality was improved. Orange juice could be made sterile with 50°C heating and 400 krad. A heat-radiation process was developed for shrimp (Savagaon ef al., 1972). This process, however, employed heating the shrimp to 121°C for 8 minutes, which alone was sufficient to inactivate CI. hotulinum spores. Irradiation served to control microbial contamination during a final packing operation. A variant of this process involved the step of drying the shrimp to 40% moisture, heating to 80°C for 5 minutes, and irradiating with 0.25 Mrad (Gore et a / . , 1970). A theoretical evaluation of combined radiation and thermal processes in food

I

- 2 0 0 -150

I

-100

I

-50

I

I

0

50

I 100

IRRADIATION TEMPERATURE ( C )

FIG. 9. Change in D-values of spore5 ol’Clostridiitnr horulimtm 33A in ground beef with change in irradiation temperature from - 196 10 100°C (Grecz (’I d . , 1971). Reproduced by perniiasion of the 17, 135-142, 197 1 . National Research Council of Canada from the Concrdim Jour/ilr/f,fMic.ro/~io/og?.

188

WALTER M. URBAIN

sterilization demonstrated no synergism for the heat-radiation sequence, but did so for the radiation-heat sequence (Purohit et al., 1971). Grecz et al. (1971) showed that the radiation D-value of C1. botulinum 33A spores rapidly approached zero in the range 65" to 100°C; Fig. 9 shows this. Unlike other work with sequential heating and irradiation (or the reverse), this involves simultaneous use of heat and radiation. This approach has been studied also with dry spores of Bacillus subtilis (Reynolds et a l . , 1970; Reynolds and Brannen, 1973). A radiation dose of about one-third of that with radiation alone was secured at 95°C. A synergistic effect also was observed. Navanugraha (1973) applied a similar technique to spores of C1. sporogenes PA3679 in raw ground beef. D-values progressively decreased with increasing temperature in the range 80" to 95°C. The D-value decreased from 0.210 Mrad at 20°C to 0.087 at 95°C. Some indication of synergism was obtained. These and other observations suggest that a trade-off can be made between heat and radiation, with the result that a combination process would produce a totality of effect of sterilization, and yield products of better sensory quality than is obtainable by heat or radiation alone. Simultaneous heating and irradiation may be basically different from sequential heating and irradiation. I n addition to heat, radiation has been used in combination with other agents. Wills et al. (1973) found that compression increased the lethality of radiation. Chemical additives also have been investigated (Lee e l al., 1965). Sodium chloride, calcium chloride, mustard oil, nutmeg, sodium citrate, Versene, sodium nitrate, and sodium nitrite reduced the radiation resistance of Cl. botulinum in ground beef (Anderson et al., 1967). Farkas et al. (1967) found that the dose requirement to insure microbiological stability of a pea preparation was reduced to one-quarter of that of control samples by the addition of 100 ppm nisaplin or 1 ppm tylosin lactate. Kim et al. (1973) showed that the preservative 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamideand potassium sorbate could reduce the dose requirements for preservation of pork sausage.

C. LOW-DOSE APPLICATIONS As has been indicated earlier, low-dose applications use doses below 1 Mrad. There are several objectives of these low-dose applications and each will be treated separately.

I . Radurization The objective here is to extend product life by reducing the microbial population initially present in a food. The dose may be adjusted to what is needed to attain the desired product life. Upper-dose limits may be set by undesired product changes, such as off-flavor. The nature of the food may be such that radiation may be combined with another preservation measure, as for example, refrigera-

FOOD IRRADIATION

I89

tion, in order to attain the desired product life. Radurization has been considered to be useful with fresh meats and poultry, seafood, fruits and vegetables, and baked goods. Radurization can affect only spoilage of foods due to microbial action. In some cases this may be not all that is needed. Chemical change due to the action of atmospheric oxygen or to indigenous enzymes may also need control. This kind of limitation must be recognized and, if possible counteracted. a. Fresh Meats and Poultry. The surface of meats becomes contaminated during dressing, cutting, and preparation. Refrigerated fresh meats keep better when in large pieces, that is, when the ratio of surface to mass is small. This fact is utilized in their distribution in that sides of beef or wholesale cuts, for example, and not retail cuts are shipped from the packing plant to the retail store, where final cutting and packaging are done. Even with good refrigeration, retail cuts of fresh meat are quite perishable. The store life of a retail cut of beef, for example, is about 3 days. The first indication of deterioration usually is discoloration, generally due to pigment oxidation by atmospheric oxygen. Pseudomonad bacteria and other types increase in numbers fairly rapidly, and in time result in other indications of spoilage. mainly odors and flavors. Fat oxidation also occurs. The cut surface of meat, especially beef, exudes a serum (drip) which creates an unsightly and generally unacceptable appearance. A considerable amount of early work was done with irradiation with the objective of delaying the spoilage of various refrigerated meats (Wolin et a l . , 1957; Ingram and Thornley, 1959; Coleby, 1959; Coleby et al., 1960; Urbain, 1965a; Rhodes and Shepherd, 1966). This early work demonstrated that radiation could delay microbial spoilage. It also showed that other deterioration changes of meat were not controlled. In fact, there was evidence that radiation itself caused discoloration and promoted lipid oxidation. Irradiation alone did not provide a procedure that was useful commercially. As has been noted, movement of fresh meat from packer to retail store requires the delay of final cutting and packaging until the product reaches the store. Efforts to centralize the final operations have not been successful. The total requirements for an acceptable procedure have been manifold and, so far, have not permitted certain technically feasible approaches to the needed preservation such as freezing. An effort to provide a solution that accomplishes what is needed was undertaken (Urbain, 1973). In this procedure, low-dose irradiation was the key agent to control microbial spoilage. Drip control and color preservation were obtained by treating the meats with a polyphosphate, such as sodium tripolyphosphate (TPP). A double packaging system was employed to provide, sequentially, both anaerobic and aerobic packaging. Vacuum packaging protects the product against oxidation during the distribution period. Aerobic packaging provides the

190

WALTER M . URBAIN

access of oxygen needed for normal red color during the period of retail sale. The steps of the process are as follows:

I. 2. 3.

4. 5.

6. 7.

Retail cuts are treated with phosphate, e.g., TPP (0.5%). Each cut is wrapped in oxygen-permeable moisture-impermeable film. A number of individual cuts are placed in a bulk container which is vacuumized. The vacuum bulk package is irradiated with doses in the range of 100 to 200 had. The bulk package is stored and transported at temperatures below 5°C. The bulk package is opened at the retail store about one-half hour before display for sale. The retail cuts are displayed at temperature below 5°C and are to be sold within 3 days.

This process provides a product stability sufficient to maintain satisfactory quality of beef for periods up to 21 days. Table XI1 gives data on the bacterial total plate count of stored beefsteaks. Figure 10 shows the color stabilizing action of TPP. Metmyoglobin percentage is an index of meat pigment oxidation. The phosphate also controls "drip." Addition of 0.5% TPP reduces drip loss from a control value of over 5% to about 0.25%. Figure I I shows lipid oxidation changes as measured by TBA values. The vacuum package protects the meat fat against oxidation. All indicated TBA values are below those which affect the taste of the meat. A product stability of 21 days is more than sufficient to permit central cutting and packaging. TABLE XI1 EFFECT OF VARIOUS DOSES OF GAMMA RADIATION ON THE TOTAL PLATE COUNT (TPC)

OF VACUUM-PACKED STORED BEEFSTEAKS"

Days storage at 4°C Dose (bad) 0

50 100 250 500 1000 "

TPClgm

7 TPClgm

1.6 X lofi 1.8 x 104 8.0 x 10'' 1.0 x IP 60
6.2 X 10'' 3.3 x 105 2.8 x lo2 I . 0 X 102
0

I . OX 107

From Urbain (1973).

14 TPClgrn

5.6 x 7.7 x 3.0 x 8.6 x 2.0 x
107 10s

10' 16

I02

21 TPC/grn 7.0 X 3.0 x 9.0 x 9.8 x 2.2 x <10

lon 107

10'

lo3 102

191

FOOD IRRADIATION 7060 -

50

m I

t-

-

0

40-

NO TI'?

'I

Ohrod

"100

- I N vnruuy

w

I

"

---IN A I R

$ 3020

-

10 -

14

7

0

21

24

DAYS AT 3OC

FIG. 10. Effect of phosphate pretreatment on metmyoglobin formation in irradiated and nonirradiated fresh beef during storage for 21 days in vacuum followed by additional 3 days in air at 5°C (Urbain. 1973).

Irradiation of meats and poultry effectively destroys the very radiationsensitive gram negative rods, including Pseudomonas, Achromobacter. and Flavobacterium. In the case of the above described process, the outgrowth was composed almost exclusively of gram positive organisms, either anaerobes or facultative anaerobes, and primarily lactobacilli (Groesbeck, 1973). Bacteria resistant to radiation have been isolated from beef. Micrococcus radiadurans was found to be more resistant than spore forming bacteria (Duggan 2 5-

. . o

NO TPP. Omrod I'

"100

a TPPMP,

2 0-

I'

IN

--- IN

0

"

0 100

'I

" "

j

VbCUUH

;:

blR

1;

2 15-

p

a

m +

3

IO-

11 I, ,I

DAYS AT 3OC

FIG. I I. Lipid oxidation changes in irradiated and nonirradiated fresh beef as measured by TBA values during storage in vacuum for 21 days plus three days in air at 5°C (Urbain, 1973).

192

WALTER M . URBAIN

et al., 1963). Tiwari and Maxcy (1972) isolated Moraxella and Acinetobacter from beef. D values from 273 to 2039 krad were observed (Welch and Maxcy, 1975). These radiation-resistant organisms are part of the normal flora of beef, but their significance in spoilage and food-borne disease is not known. b. Fish and Seafood. The perishability of fish and seafood, due primarily to microbial spoilage, causes problems in the distribution of these products. Radurization with doses not to exceed 0.3 to 0.5 Mrad is effective in significantly delaying microbial spoilage of fish and seafood when stored at refrigerator temperatures (Hannesson, 1972). Figure 12 shows the scores of an expert panel in judging the acceptability of stored haddock fillets. At least a 2 log cycle reduction of total bacterial numbers was obtained by 200-krad irradiation and was maintained during refrigerated (ice packed) shipment under commercial conditions for up to 6 days. Marketable product lives of 7, 15, and 18 days were obtained for radiation levels of 0, 100, and 200 krad, respectively (Ronsivalli et al., 1970). Irradiation alters the microflora. In temperate latitudes, pseudomonads are replaced by the more radiation resistant Achrornobacter (Laycock and Regier, 1970). In tropical regions, gram negative species other than Pseudomonas and Achromobacter and gram positive species are part of the microflora of both irradiated and nonirradiated seafoods. Gram positive cocci constituted the major portion of the flora of Bombay duck (Kumta and Sreenivasan, 1970). The elimination of pseudomonads contributes significantly to the sensory acceptability of irradiated fish.

-Not irradiated ---_100 blomds ........200 hilorods

1)

0

I

5

I

I

10 15 STORAGE TIME (days)

I

20

I 25

FIG. 12. Shelf life of irradiated and nonirradiated haddock fillets held at 1 to 4°C (Ronsivalli ct a/.. 1970). (Organoleptic score scale: 9 = excellent, 5 = borderline, I = inedible.)

FOOD IRRADIATION

193

It appears that best results are obtained with the irradiation of freshly caught fish (Hannesson, 1972). A dose of 50 krad applied to eviscerated fish as soon as possible after catching at sea was as effective in extending product life of haddock as were doses of 100 to 250 krad when the irradiation was carried out 1 to 9 days out of water (Carver et al., 1968). The spoilage microflora of vacuumpacked petrale sole fillets was predominantly Lacrobacillus, but fillets stored in oxygen-permeable films were spoiled by Achromobacrer and trichosporon yeasts (Miyauchi, 1972). Fatty species, such as mackerel, salmon, herring, and trout are not well suited to irradiation because of subsequent deterioration of fat and pigments. Low-fat marine products are more successfully treated (Spinelli er a/., 1965). Irradiation suppresses the growth of certain potential health hazard microorganisms (fecal coliforms, coagulase positive Staphylococcus aureus and enterococci) (Pelroy and Seman, 1968). However, the selective reduction in the microflora of fish and seafood may be favorable to the development of Cl. botulinum. Jay (1970) indicates the distribution of this organism. Of those types pathogenic to man, types B, E, and F are found in fish and seafood. While a number of outbreaks of type E botulism have been ascribed to fish, no fish product has been identified with type F. Segner and Schmidt (1966) determined the radiation resistance of type E spores and concluded that radurization doses are inadequate to inactivate them. Of great importance is the fact that types B, E, and F can grow and produce toxin at the low temperature of 3°C (Schmidt et af., 1961; Eklund and Poysky, 1970). Radurization permits extension of the storage period of fish and seafood and could, therefore, enhance the opportunity for toxin production. The low critical temperature for toxin formation makes difficult the maintenance of a safe condition during commercial handling. Concern for this situation has led to a great deal of work in the United States and the United Kingdom. Eklund and Poysky (1 970) concluded that irradiated haddock provided no greater health hazard from type E botulism than nonirradiated haddock, provided the storage temperature is below 5.6"C. With 100 krad and storage at 7.8"C, there appeared to be a safety factor in that sensory spoilage occurred before toxin production. a circumstance which normally would cause rejection of the product by the consumer. Increasing the dose to 200 krad reduced this safety factor due to the increased potential storage time before sensory spoilage occurred. Storage at 10°C precludes a condition of safety. At storage temperatures above the critical value of 5.6"C, consumer safety to a considerable degree depends upon his ability to identify spoilage and to reject the fish for consumption. Learson er a/. (1970). using consumer panels, to identify spoilage by odor of the raw fish, determined that 10 to 15% of a population could not make an accurate judgment of spoilage, and, consequently, could incur a hazard. Certain species of fish favor toxin formation (Cann et al., 1966). Eklund and

194

WALTER M.URBAIN

Poysky (1970) attributed enhanced toxin formation to the presence of the sugars ribose and glucose, which occur in petrale, Dover, and English sole. Hannesson (1 972) summed up the situation with respect to the botulism hazard with irradiated fish and seafoods as follows: At our present state of knowledge concerning Cl. botulinum it is concluded that a possibility exists that radurization could increase the botulism hazard. The extent of such hazard depends upon the initial level of contamination, the dose level of radiation used andlor synergistic effects of physical and chemical agents, the temperature of storage and distribution. and possibly also on the temperature at time of irradiation. Of the three dose levels most commonly referred to for radurization of fish and fishery products, i t . , 100, 200 and 300 krad, the last mentioned could lead to the most hazard as the spoilage flora would be eliminated to the largest extent.

A more optimistic view was expressed by Shewan and Hobbs (1973) with reference to fish landed in the United Kingdom. Their conclusion is as follows: If good commercial practice is followed in the processing line, during distribution until consumption, and, in particular, if the temperature of the produce is never allowed to exceed 3”C, there should be no danger from botulism poisoning, even if irradiation were employed. Even if some toxin were to be formed by malpractice at any stage during the distribution chain, including the consumer’s kitchen, subsequent cooking would almost certainly destroy any toxin present. Only when products such as smoked herring fillets or smoked salmon are eaten raw would the potential hazard become real.

Depending upon the origin of fish and seafood geographically, other organisms pathogenic to man may occur. Loaharanu (1973a) has reported the presence of Vibrioparahemolyficus in marine fish and shellfish caught in waters of Thailand. D-values for this organism were found to range from 3 to 16 krad, depending upon the serotype and salt content of the media. The microbiological situation in fish and seafood can be improved by combination processes. Mention has been made of the two shrimp processes developed in India (see p. 187). In Thailand, a native fish product is the boiled chub mackerel. This fish is boiled in saturated salt brine for 5 to 10 minutes, and can be kept for 2 or 3 days at room temperature. Greater product life can be obtained by adding irradiation. A dose of 0.1 Mrad extended the product life 7 additional days and 0.2 and 0.3 Mrad 12 and 14 days, respectively. These longer product lives will enlarge the marketing possibilities for this product (Loaharanu, 1973b). The mean salt content of boiled chub mackerel is 3.9%. Inoculated pack studies with CI. bofulinum type E spores have been made (Navanugraha, 1977, personal communication). It is probable that the salt content of this product will provide adequate protection against C1. botulinum type E. c . Fruits and Vegetables. Fresh fruits and vegetables may be irradiated for various purposes. This section deals only with radurization. The term “post

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harvest diseases” has been applied to spoilage of fresh fruits and vegetables caused by microorganisms. Fruit spoilage results principally from infections of filamentous fungi; bacteria are of minor importance in fruit spoilage. The bacterium Erwiniu c‘arofnvortris the primary cause of soft rot of vegetables. Radurization of fresh fruits and vegetables is aimed at control of these spoilage microorgan isms. Contamination of fresh fruits and vegetables with fungi and bacteria can occur in several ways. Field infections may occur during blossoming. and post harvest growth may occur within the food tissue. The epidermis of the fruit or vegetable protects against invasion, but wounds and cuts incurred in harvesting and handling may serve as entry ports. Contact infections can occur in storage and handling and spread infection from infected t o noninfected product. A great many studies have been made in the radurization of fresh fruits and vegetables. There are a number of review-type articles on early work: Morgan and Siu ( 1957); Brownell ( 1 961 ); Salunke ( I96 1 ); Bramlage and Couey ( I 965); Bramlage and Lipton (1965); Maxie and Somrner (1965); Mercier and MacQueen ( I 965); Sommer and Fortlage ( 1966); Sommer and Maxie (I 966); Vidal (I 966); Staden ( I 966); Dennison and Ahnied ( I 966); Dharkar and Sreenivasan (1966); Maxieetal. (1971a.b); Dennison and Ahmed (1971); Moy (1973). These papers indicate that most of the fruits and vegetables of importance have been examined. The findings may be summarized as follows: (1) There is a tight relationship between dose needed to control spoilage and dose that causes damage to the food. In many cases radiation damage is too great to permit application t o a particular food. (2) Irradiation is not always effective in controlling spoilage. (3) Product life extension is frequently of marginal value. Quality damage has involved texture loss or softening, pitting of citrus fruit skin, color changes, and flavor loss or change. Lethal doses of various species of yeasts and fungi associated with fruit spoilage were determined in culture media by Saravacos ct ul. (1962). The radiation resistance of yeasts varied from 0.4 to 2.0 Mrad. Fungi were inactivated with doses varying between 0.25 and 0 . 6 Mrad. These doses are seemingly very high in terms of radiation damage. especially texture loss, to fresh fruits and vegetables. Maxie et (11. ( 1 9 7 1 ~ have ) noted this. Reduction of dose may be accomplished by combining irradiation with a heat treatment, as for example. in the case of papayas and mangoes. Based on this carly work, only strawberries and fresh figs emerg’C d5 * reasonable candidates for radurization. More rclcent work. however, has indicated that the cmntrol of mthracnosc caused by the fungus, Coll~.rotric,hiuin glocosporoidirk~s,and soft brown rot, Hmdorsoiiitr cwberimtnu Syd. and Butl. nov. spec.. is accomplished by irradiation of mangoes (50 krad) and hot-water dip (55°C for 5 minutes). I n somc varieties of mango, doses LIPto 150 krad may be applied (Thomas. 1975).

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Sommer and Fortlage (1 966) criticized the work on radurization of fruits and vegetables. According to them, little study has been made on the radiation biology of postharvest pathogens in relation to the disease they cause. Much of the work concerned reduction or delay of decay but did not deal with the particular organism involved. In most cases experiments were based on natural infections, a circumstance which can result in misleading conclusions. To these criticisms may be added the view that too often there was a lack of understanding of the physiological processes of the living food and the immediate and subsequent effect of radiation on these. d . Cereal Grains and Baked Products. Only a moderate amount of research has been carried out on the radurization of cereal grains or products derived from them. It is known, however, that such products contain microorganisms that can cause problems either in their use in the preparation of uncooked foods or upon storage. Poisson et al. (1967) found that 150 krad are sufficient to make wheat flour “hygienically clean,” although 500 krad are not enough to eliminate all microorganisms. Lee et al. (1973) reported that 250 krad retarded the growth of molds in polished wheat having moisture contents greater than 14.8%. Saint-Lebe et al. ( I 973) found that 300 krad virtually eliminated all microorganisms present in maize starch, particularly molds and sulfite-reducing Clostridium spores. Maize and milo of United States origin were found to contain molds of the Aspergillus restrictus group and A . glaucus group and also Bacillus bacteria. A dose of 0.54 Mrad sterilized milo and of 1.55 Mrad sterilized maize (Watanabe et al., 1973a). A dose of 0.6 Mrad was effective in preventing mold growth in both grains (Watanabe et al., 1973b). Kim and Choi (1969) reported that, for polished and unpolished Korean rice, 400 krad were not sufficient to kill all yeasts and molds. Japanese polished and unpolished rice contained chromogenic and fluorescent Pseudomonas. Spanish rice, in addition, contained molds (Iizuka and Ito, 1973). Polished Thai rice contained Bacillus megaterium, B . cereus, B . Subtilis, Brevibacterium, Micrococcus, and Actinomycetes (Ito et al., 1973a). Doses of 0.2 to 0.3 Mrad are generally sufficient to “sterilize” all rices studied. The storage life at 30°C of rice containing 14 to 15% moisture was extended 3 to 4 times by irradiation at 0.2 Mrad (Ito et al., 1973b). Pseudomonas radiora was isolated from unpolished rice and found to be 10 to 40 times more radiation resistant than ordinary species of the genus Pseudomonas. A D-value of approximately 0.14 Mrad was determined, which is similar to that of Micrococcus radiodurans (Ito and Iizuka, 1973). Morgan and Siu ( I 957) reviewed work done up to that date on the irradiation of baked goods. Most of this work was at doses in excess of 1 Mrad, and difficulties with sensory changes were reported. Ehrenberg and Steding (1960)

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found that spiced bread and currant loaf irradiated with 600 krad would keep free of mold for more than one month of storage at 22°C. No noticeable flavor change occurred and the irradiated bread staled less. Irradiation of brown bread with 300 h a d inhibited molding for 6 weeks or more. Packaged, sliced blended-bread (70% rye and 30% white flour) was irradiated with doses up t o 0.5 Mrad. With 0.2 Mrad, several days product life extension can be had. At 0.5 Mrad, a complete suppression o f mold growth occurred for at least 1 1 weeks. The dose could be reduced to 50 krad by irradiation at 65°C (Stehlik and Kaindl. 1968). Chapaties (Indian unleavened bread) do not mold when irradiated at 1 Mrad and stored for more than 6 months. Acceptance was high up to 2 months (Savagaon et al., 1970). 2 . Radicidation

The principal interest in eliminating pathogenic bacteria in nonsterilized foods has been in connection with Salmonellae. Granville (1963) and Hobbs (1963) reviewed the epidemiology of salmonellosis in relation to its transmission by food and feed. Salmonellae are contaminants of many foods, including eggs, poultry and red meats, dairy products, and dried coconut. Salmonellae also are present in animal feeds which contain products of animal origin (fish meal and meat by-products). Proctor et al. (1953) irradiated liquid egg prior to drying. Thornley (1963) reviewed the use of radiation for the elimination of Salmonellae from various foods and feeds. A dose range of 0.2 to 0.65 Mrad was proposed for reduction of bacteria present by a factor of lo7. There is a variation of radiation sensitivity with the strain ofSalmonellae and also with the nature of the medium in which it occurs. Oxygen content. physical state (frozen or not), moisture content, and chemical composition all are factors. Table XI11 lists the doses for a lo7 reduction of count of Salmonellae in various products. In the determination of the listed doses, in most cases the resistant strains S. typhimurium and S. senfrenberg were employed. Licciardello et al. (1968) suggested a dose of 0.475 Mrad for treatment of poultry meat to obtain a 7 log cycle reduction. Quinn et al. (1967) considered organisms in addition to Salmonella, whose presence in a food can lead to a health hazard. Doses of 0.5 to 0.8 Mrad were suggested as adequate in most cases to effect a 7 log cycle reduction of most infectious or intoxicating nonspore-forming microorganisms occurring in seafoods. Organisms studied were Salmonella, Shigella, Neisseria, Mycobacterium, Estherichia, Pmteus, Streptococcus, and Staphylococcus. Radicidation falls within the area of public health measures, and some investigators place high the value of this use of radiation. It is probable, however, that few food processors would voluntarily treat foods for this purpose, since payment for the associated costs would be difficult to secure. On the other hand,

198

WALTER M. URBAIN TABLE XI11 IRRADIATION DOSES FOR THE INACTIVATION

OF SALMONELLAE IN VARIOUS PRODUCTS TO OBTAIN A

lo' REDUCTION OF COUNT"

Product Whole egg (liquid) (frozen) (dried) Egg yolk (frozen) (dried) Egg white (liquid) (frozen) (dried) Sugared egg white (dried) Horse meat (frozen) Bone meal Desiccated c o c o r ~ t " ')

Dose (Mrad) 0.2004.442 0.476-0.540 0.370 0.320 0.570 0.260 0.2 12 0.585 0.840 0.640'' 0.640 1.100

From Thornley ( I 963). lo5 reduction.

government regulations requiring radicidation of appropriate foods would overcome this difficulty by eliminating the factor of market-place competition in this action. There are, however, genuine concerns for the consequences of the doses required for radicidation on product quality. Also, to be effective, all units of a product produced would have to be irradiated and this might lead to costs which may be unreasonable as measured by the benefit to the consumer. At the present time, there seems to be no strong interest in radicidation in either the commercial or government areas. 3 . Disinfestation-l'arasites

Interest in the irradiation of foods containing worms capable of causing disease in man is very old. Wharton (1957) reviewed the very early work and listed references as early as 1904. Gould et al. (1953) reported on the irradiation of pork to control Trichinella spiralis. They reported 750,000 roentgens (630 krad) cause death of the organism, and indicated lesser amounts prevent reproduction and maturation. Gibbs et al. (1964) reported 20 to 30 krad suppressed maturation. Van Kooy and Robijns (1968) indicated that the lethal dose for Cysticercus bovis is in excess of 300 krad. Storage of beef irradiated with 300 krad, for 7 days at 2"C, however, results in death of the organism. Pawel (1968) reported

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doses of 400-500 krad for the inactivation of Cysticvrcus hovis, C. cellulosuc~, and C . pisiformis. Van Mameren and Houwing (1968) found that 600 krad were insufficient to inactivate Anisakis larvae in salted raw herring. It is clear that these food-borne parasites have relatively high dose requirements for inactivation. Considering the foods which carry them, it is probable that objectionable sensory changes will be induced at the required doses. In fact, Van Mameren and Houwing (1968) stated that 300 krad is sufficient to cause slight changes in taste and color of salted ripened herring and that 600 krad is the maximum dose for acceptable 5-day-old lightly salted herring. The approach to controlling trichinosis in man by use of the dose for prevention of reproduction and maturation of Trichinellu spiralis (20 to 30 krad) may not be adequate, since it may not prevent the initial phase of the disease associated with the release of the ingested organisms in the intestine.

4 . DisinfL.stution~nsrcts The amount of food lost after harvest to insects has been difficult to ascertain, but generally it has been judged to be large. Insects are by far the greatest cause of losses in cereal grains and cereal products (Pape, 1973). Fruits and vegetables also can become infested with insects. While such infestations do not always produce spoilage of a harvested food. they can be the basis of trade barriers in order to prevent the spread of insects. The most common approach to insect disinfestation is the use of chemical pesticides. There is a growing concern about health hazards associated with residues of pesticides in foods. Irradiation offers a way to avoid them. In some cases, chemical pesticides are not effective because the insect is within the food and is not reached by the pesticide. Penetrating radiation does not have this problem. Insects have demonstrated an ability to develop a resistance to chemicals. Insect sensitivity to radiation apparently does not change with exposure of successive generations (Brower, 1974). These are the reasons for a widespread interest in disinfestation by irradiation. Most of the research has been concerned with determination of the dose required to inactivate insects. In some foods, the infestation may involve one insect species; in others, a large number may be present. In the latter situation. the disinfestation process must operate to inactivate the most radiation resistant one. This requires investigation of all species involved in a given food. Radiation may have a short-term or long-term effect on insect development. A larva may fail to develop from an irradiated egg. An adult may not develop from a pupa. Long-term effects may appear in a development stage succeeding the one irradiated. A larva, for example, from an irradiated egg may die, shortly after emergence. Radiation may prevent reproduction. Tilton and Brower (1973) re-

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ported the doses required to kill each metamorphic stage or to sterilize adults of 26 species of insects that infest stored foods. The Lepidoptera (moths), as a group, are more resistant than are the Coleoptera (beetles). The grain mite Acarus siro has intermediate resistance. These authors conclude that a dose of 50 krad will inactivate all beetles and the immature stages of all moths. Surviving adult moths would not reproduce effectively. They indicate that 50 krad controlled insects in bulk wheat, wheat flour, cornmeal, peanuts (shelled and in shell), tree nuts, dried fruits, dried beans, rice, and a wide variety of packaged products. The results of other investigators in some instances suggest a lower value for some foods, but apparently consideration was not always given to a sufficient number of insect species in the determination of an effective dose. The disinfestation of fruits and vegetables tends to involve concern for a specific insect or for, at most, a few insects. Thus the disinfestation of mangoes grown in South Africa is directed to the mango weevil, Sfernochetus mang$erae, for which the dose completely to prevent emergence is 75 krad (Thomas, 1975). For the Hawaiian papaya, it is the oriental fruit fly, Dams dorsalis. A dose of 26 krad achieves a 99.9968% lethality when applied to infested papaya (Balock er al., 1966). In addition to plant products, some animal products, such as dried fish, in certain areas of the world are subject to insect infestation. Boisot and Gauzit ( 1 966) reported on the irradiation of African dried and/or smoked fish. Doses of 15 to 30 krad were estimated to be effective. Many factors can influence the effectiveness of radiation in disinfestation. It seems logical to determine a specific dose for a food that is appropriate to local situations and needs. The doses commonly reported do not cause immediate lethality. This may or may not be important. Delayed lethality could be of little value in short-term distribution of a food. It also could fail to meet current government regulations. In some cases, however, such as in the use of radiation as a quarantine control measure, only ultimate lethality is significant. It is probable that immediate lethality will require doses of about 500 krad, which in most cases will prove to be too high to be practical. In some cases, for example, cereal grain disinfestation, there will be the need for pre- and postirradiation treatments. The weakness of irradiation lies in its inability to protect against reinfestation. For bulk grains this can be exceedingly important and call for measures such as anoxic storage after irradiation (Willis et al., 1973). If one were to express a judgment on the uses of disinfestation by radiation, it would seem that its greatest use is likely to be as a quarantine control measure to international trade in fresh fruits and vegetables. The banning of chemical pesticides, however, could bring about widespread use of radiation disinfestation of cereal grains, leguminous seeds, etc., and derived products.

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20 1

5 . Inhibition of Sprouting and Senescence

An examination of Table I will reveal the widespread interest in sprout inhibition of white potatoes. Of all uses of irradiation. this application clearly is the one that is most advanced and that has almost universal appeal. In fact, as of this writing, it is the only commercial use of irradiation. At Hokkaido, Japan, since 1973, white potatoes have been irradiated commercially to inhibit sprouting. Dose requirements are among the lowest for any food irradiation process and fall in the range of 5 to 15 krad. A dose-variety interaction has been observed which makes i t necessary to select an appropriate dose for the variety being irradiated. Unlike chemical sprout control, irradiation is irreversible unless the potato is treated with indole acetic acid or gibberellic acid (Nair et a / . , 1973). Irradiation interferes with suberization (wound healing). Since harvesting and handling usually cause injuries, it is necessary to wait until such injuries have healed before irradiation. Otherwise irradiation can lead to losses due to rotting. Excessive dosage also causes rotting. A variety-dose interaction also occurs with black spot incidence. The dose necessary for sprout inhibition and the dose at which black spot incidence increases overlap with most varieties. This points to the need to use irradiation at the lowest doses practical for sprout control. Irradiation increases the amount of after-cooking darkening. This occurs at the dosage level necessary for sprout control and is somewhat variety-related. The postirradiation storage temperature has a significant role in the quality and quantity of satisfactory potatoes. Figure I3 shows the percentage of marketable potatoes as a function of time and storage temperatures as determined in one study (Nair et a/., 1973). It is indicated that high temperature storage such as encountered in warm countries (25"40"C) would lead to large losses. A high temperature limit of 15°C was suggested. It is these losses and not sprouting that limit the length of the storage period. As with other stored potatoes, lowtemperature storage causes a buildup of reducing sugars. This can be lowered by storage at 20°C for 2 weeks or at higher temperatures for shorter times (Takano et a/., 1973a). Nair et a / . (1973) reported a secondary benefit of irradiating potatoes for sprout inhibition. Irradiation at 10 h a d completely prevented adult emergence of the tuber moth Phthorirnaea operculella (Zeller) when the infestation is with eggs or early larval instars. Twenty krad are needed if the infestation is with late larvae. There is an extensive literature on sprout inhibition of potatoes. Early work was reviewed by Morgan and Siu (1957). Other reports of interest are by Dallyn and Sawyer (1959); Kahan and Temkin-Gorodeiski (1968); Kwiat (1968); McKinney (1970); Sandret (1973a,b); Nair et al. (1 973); Griinewald (1973); Umeda ( 1975).

WALTER M . URBAIN

..__.

FUNJAB SELECTED

.. .. PUNJAB 'AS IS'

TALEGAON SELECTED .------ATALEGAON 'AS IS'

FIG. 13. Marketable percentage of irradiated potatoes as a function of storage time and temperature (Nair et al., 1973).

Close behind potatoes in interest is the irradiation of onions. The dose for sprout inhibition of onions is roughly the same as that for potatoes, with an indication that it may be somewhat lower. Reported figures fall in the range of 3 to 15 krad. There appears to be less variety-dose interaction. Irradiation should be done as quickly after harvest as possible. Storage at warm temperatures (27"-30°C) leads to smaller losses from sprouting than storage at 20°C or lower. Spoilage losses due to rotting occur with long storage periods. A discoloration of the growth center occurs with storage time. This can cause internal rotting and is aggravated by higher doses. Delay of irradiation after harvest apparently allows the start of internal sprouting. It has been suggested that a minimal dose of 6 b a d be used and that irradiation be done promptly after harvest. Both fungi and bacteria are associated with the internal rotting (Nair et af., 1973). In addition to the article of Nair, other articles may be of interest: Nuttall et al. (1961); Kahan and Temkin-Gorodeiski (1968); Takano et al. (1973b); Bandyopadhyay et al. (1973); Loaharanu (1974). Bramlage and Lipton (1965) reported that doses as low as 8.25 krad inhibited sprouting of sweet potatoes. Park et al. (1969) indicated that 50 krad prevented sprouting of sweet potatoes during 7 months of storage. Nigerian yams did not sprout in 6 months of storage when irradiated with 7.5 to 15 krad (Adesuyi and Mackenzie, 1973). Other root foods, such as carrots and ginger, likewise can be inhibited from sprouting by irradiation. A use of irradiation related to sprout inhibition is delay of senescence. This

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also involves changes in the metabolic processes of living foods. To use senescence inhibition effectively. it is very helpful t o have an understanding of the physiological condition of the food at the time of irradiation and of postirradiation metabolic processes. For reasons not presently understood. however. radiation appears to affect various foods differently. Undoubtedly there are specific consequences of irradiation for certain foods. which are different for others. Radiation dose is critical. A minimum exists for beneficial effects. Excess radiation usually causes deleterious action, either by direct chemical change, as for example, by softening the food, or by damaging essential metabolic processes. Delay of senescence can be translated into extention of product life and can effect reduction of spoilage loss and enlargement o f market. Here, also, there is a real value potential for international trade. One important aspect of fruit physiology is the class of fruit-whether it is of the climacteric class or not. Climacteric fruits undergo marked increases in rates of respiration and ethylene production as they ripen. If they are irradiated before the onset of the climacteric, the effect may be very pronounced. If irradiation is done at later stages as the fruit ripens, the effect is smaller and becomes essentially nil in fully ripened fruits. Once the ripening process of a climacteric fruit has been initiated, a normal radiation dose does not inhibit it. Delay of ripening, however, can be obtained by irradiating before the climacteric is reached. Nonclimacteric fruits have a diminishing rate of respiration after harvest. Due probably to radiation-induced ethylene, irradiation stimulates ripening of such fruits. However, a similar kind of stimulation can occur with climacteric fruits. In the postirradiation period there can be other consequences of irradiation. Activation of enzyme systems can cause changes such as browning of the skin. Sensitivity of a fruit to chilling may be intensified. The banana was the subject of early study in connection with senescence inhibition. It is a climacteric fruit and usually is picked green. Ethylene, normally produced in the banana just before the onset of ripening, causes ripening. Maxie et al. (1965) found that the Gros michel variety irradiated with 25 to 35 krad underwent a delayed ethylene production and also a delayed ripening. Khan and Muhammed ( 1 969), on the other hand, reported that the Busri variety did not undergo either phenomenon and concluded that irradiation could not extend its life, Sreenivasan et a!. (1971) determined that for Fill Busket and Red bananas the extent of delay of respiration increase varied with the maturity of the fruit at the time of irradiation. They attributed radiation-induced inhibition of ripening of bananas to a shift in metabolic pathways from the glycolytic to the pentose phosphate pathway. Irradiation at sufficiently high doses, such as 50 krad, causes browning of the skin (Kao, 1971). Sreenivasan et al. (197 1 ) observed an increase in polyphenol oxidase in the skin and pulp, probably the result of radiationinduced enzyme activation. They postulated that radiation damage to cell membranes permitted contact between enzyme and substrate and formation of dark

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WALTER M . U R B A l N

brown pigments. These same investigators determined that ripening stimulants such as ethylene or 2,4-dichlorophenoxy acetic acid can offset radiation-induced delay in ripening. It appears that irradiation in the right dose (approximately 35 krad) and applied to appropriate varieties can effect a ripening delay of the order of a week, and yet permit bananas otherwise to be handled normally. Mangoes and papayas also are amenable to delay in ripening (Mumtaz et al., I 968; Loaharanu, 1971 ; Moy et al., 1 97 1; Pablo el a/. , 1 97 1;Sreenivasan et a/., 1971; Thomas, 1975). Both fruits have insect problems, which can be solved by irradiation. They are examples, therefore, of a dual benefit from irradiation. Both are receiving attention currently. Other fruits that have been reported to undergo a delay in ripening through irradiation are guavas, pears, avocado pears, and sapotas (sapodillas). Vegetables also can experience a delay in ripening (other than sprouting) upon irradiation. It is possible, for example, to delay the opening of the cap of fresh mushrooms (Bramlage and Lipton, 1965; Staden, 1966; Campbell et a/., 1968; Gill et a/., 1969; Acki e t a ! . , 1976). The postharvest growth of asparagus can be inhibited. 6 . Changes Related to Uses of Foods

The principal objective in using food irradiation is preservation. There are other ob.jectives, however, one of which is quality improvement. In some cases, a quality improvement is obtained more or less unintentionally while seeking another objective such as preservation or disinfestation. Finally, there are changes which are unavoidable and which set limits on process parameters such as dose because they are changes which downgrade quality. Some of these various changes will be identified. Wheat and wheat flour would be irradiated for insect disinfestation with a maximum dose of 50 krad. This dose produces measurable changes in the baking qualities of the flour (Deschreider, 1966; Pape, 1973; Sreenivasan, 1974; Rao et a/., 1975; Lorenz, 1975). Molecular degradation of the starch and protein affects the rheclogical properties, including gelatinization viscosity of starch, dough development and stability, and elasticity of gluten. These changes improve the baking quality of the flour and yield increased loaf volume. Doses greater than 50 krad can induce changes which impair baking qualities and consumer acceptance. The action of radiation on certain foods which involves molecular degradation of protein and carbohydrate components results in a tendering action. Thus beef is tendered by radappertization doses (Bailey and Rhodes, 1962; Kauffman and Harlan, 1969). The cooking time of the dry legume red gram (Cujanus cajan) is shortened (Sreenivasan, 1974). Dehydrated vegetables, such as onion flakes,

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peas, lima beans, and green beans, when irradiated with critical doses up to 4 Mrad, undergo shortened rehydration time. By adjusting the dose, a uniform tenderness for all treated vegetables can be secured within a given cooking and rehydration time period (Schroeder, 1962; Kiss et ul., 1974). This effect could be of value in dehydrated soup mixes. Doses up to 0.4 Mrad were found to increase the drying rate of blanched prunes (Emerson et al., 1965). The juice yield of grapes increased 2 to 28% in proportion to dose in the range 0.05 to 1.6 Mrad (Kiss et uf., 1974). Hasegawah and Moy (1973) reported reduction of oligosaccharide content of soybesns by irradiation and controlled germination. Doses of 250 krad were applicd to beans steeped in water for 2 hours. Malting losses may be reduced one to 2% through irradiation of dried barley with doses of 50 to 800 krad. Irradiation reduced the growth rate of barley rootlets (Farkas et al., 1963).

V. ECONOMICS OF FOOD IRRADIATION Intzrest in the economics of food irradiation has been strong. Many studies to definc costs have been made. The economic parameters of food irradiation are basicrtlly the same as those of other processes and methods used for cost analysis can be the same. Because there have been few actual experiences with food irradiation, there have been problems in estimating costs. In some estimates, there has been inadequate recognition of the fact that commercialization usually involves business activities carried out for profit. Therefore, the return on investec‘ capital must be adequate not only in absolute terms, but also in order to compzte effectively with alternate uses of capital. The significance of return on capital can vary with a great many circumstances. including the economic system of a country. Except for situations where cost is a secondary factor, as with a health hazard, advantageous economics constitute the principal incentive to use food irradiation commercially. The areas of costs for food irradiation have been identified by various studies: Pomerantz and Siu (1957); Brownell (1961); Kuckacka and Manowitz (1965); Urbain (1 965b); Baines and Mosely (1966); Killam et al. ( I 966); Rindorf (1966); Urbain ( I 966); Anonymous (1968b); Hovart et al. ( 1 972); Brouqui et ul. (1 973); Brynjolfsson (1 973); Deitch (1973); Gard and Warland ( 1 973); MacQueen ( 1 973); Oosterheert (1973); Val Cobb and Cruz Castillo (1973); Farkas (1975). The basic cost items that have been identified are listed in Table XIV. In general these are little different from those of other food processes. There are a few, however, that, to a degree, are special for food irradiation. Periodically, new radionuclide is required to make up for decay, and “source replenishment” refers to this. This cost is composed of at least four parts: radionuclide cost, radionuclide transportation, installation, and dosimetry.

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WALTER M . URBAIN TABLE XIV IDENTIFIED COST iTEMS FOR FOOD IRRADIATION

Fixed costs Plant depreciation Source depreciation Source replenishment V U r i U b l t ! C0Sf.S

Labor-operating Overhead Maintenance Operating supplieh Utilities Taxes Insurance Third party liability Interest on borrowed capital Contingency Return on equity capital Working capital

The two commonly considered radionuclides are "CO and ''j7Cs. Current costs are about 40$/Ci for 60Co and 20&i for '"Cs. These costs include a 2-year replacement schedule for both radionuclides and a 10% per year depreciation provision. They also include provision for differences between 'Wo and 1'37Csin energy per fission and self-absorption of gamma rays (Remini et al., 1977). At the present time 6oCo is much more available than In7Cs. Design of the irradiation facility affects costs (Huff, 1969). To a considerable degree, source efficiency can be improved by a greater allowable dose range within the target material. The compromise between source efficiency and dose range is governed by practical considerations and product requirements. A considerable amount of experience applicable to food irradiation is available from industrial irradiation operations, especially those concerned with the sterilization of hospital supplies (Crawford, 1973). Canadian experiences with potato irradiation demonstrated the need for more than seasonal use of an irradiation facility with one food in order to obtain an adequate return on invested capital. Holm and Christensen (1973) proposed a multipurpose irradiator as a means of improving the economics of such situations. A transportable irradiator or transport of the radiation source material to different locations could better the economics of short-term uses (Lapidot, 1973). The literature contains many cost estimates for various applications of food irradiation. These estimates are based on factors applicable to particular circumstances. Current worldwide inflation and floating rates of exchange tend to invalidate such estimates shortly after they are made. In order to provide some

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FOOD IRRADIATION

Radurirotion 0.0 L

0.5

.

I 0

$.S

2,o

2.5

3,O

1.I

4., 0

lrrodiotion costs, cents/pound

FIG. 14. Cost of inhibition, radurization. and radappertization as related to plant capacity (Anonymous, 1968b).

basis of understanding of costs, however, Fig. 14 is shown. It is adapted from the U.S. Department of Commerce estimates made in 1968 for the United States usage of food irradiation (Anonymous, 1968b). Apart from indicating the estimated costs per pound for irradiation, this figure brings out the two dominant cost factors: dose and plant capacity. The capital cost of an irradiator is tied to source size, which in turn is related to capacity and dose requirement. Throughput, determined by hourly capacity and hours used, affects unit cost of product. As can be seen from Fig. 14, radappertization is most expensive, with ripening-inhibition least and radurization intermediate in cost. The dominance of dose requirement in cost is clear. Another highly significant cost factor is source efficiency, reference to which was made above.

208

WALTER M . URBAIN TABLE XV ESTIMATED CAPITAL COST FOR FOOD IRRADIATORS"

Estimated cost (thousand U.S. dollars) Item

""Co

Land and land site improvements Ruilding Source installed Source pool and elevator Cell and shielding Conveyor Ventilation and cooling system Instrumentation and tools Spare parts Refrigeration equipment Engineering Working capital" Contingency

30 250 1,295" 42 66 60

Total capital requirements:

Electron accelerator

25 250

800" 70

35

22

20 27

29 102 560 55

80 24 15 240 50

2529

I696

18

From Deitch (1973). 3,238,000Ci 6"Coat 40p/Ci. Throughput 2000 Ib/hr at 5 Mrad. Source efficiency 30%. ' Based on 5-10 MeV 50-kw linear accelerator. Throughput 3400 Ib/hr at 5 Mrad. Source efficiency 45%. To finance receivables, inventories, prepayments, and current operations. 'I

Costs not always given adequate attention are transportation costs of product to and from the irradiator. In some cases these can severely limit usage. There appears also to have been a lack of understanding about assignment, as a cost, of the return on investment. In the United States and in other countries with comparable economic systems, return on investment is the incentive for using irradiation commercially. In the early phases of commercialization, it is likely that management will regard irradiation as a venture of high risk and consequently will require a high rate of return on invested capital. The assignment of a cost only for depreciation for this item is unrealistic. Although estimates for capital costs are apt to be accurate only at the time they are made. in order to indicate the kind of capital cost involved in food irradiation, there are given in Table XV the estimated costs for "Co and linear accelerator irradiators. These costs are based on a 5 Mrad dose and plant throughputs likely to be secured in commercial operations. A smaller dose requirement can significantly alter the capital costs. Total costs (capital and operating) are likely to be smaller for machine sources than for radionuclide sources (Brynjolfsson, 1973).

FOOD IRRADIATION

209

VI. WHOLESOMENESS OF IRRADIATED FOODS The term “wholesomeness” has been given specific meaning. A wholesome food has satisfactory nutritional quality and is toxicologically and microbiologically safe for human consumption. Because radiation is legally defined as a food additive in the United States, it is necessary to provide suitable evidence of the wholesomeness of irradiated foods. Similar requirements exist in other countries. Within the meaning of wholesomeness, particular evidence of safety is obtained in the following areas: general toxicology, carcinogenicity, teratology , mutagenicity, hepatic microsomal enzyme function, induced radioactivity, microbiology, packaging, and nutrition. Protocols for evaluation studies in the areas of concern have evolved (Anonymous, 1965; Pace, 1968; Wierbicki et ul., 1975) and still are evolving as new knowledge and techniques become available. In these protocols, the principal evidence for safety is obtained through animal feeding studies. These are multigeneration, multispecies studies in which various indexes of animal health and performance are observed, such as: growth (food consumption and efficiency), reproduction, longevity, gross pathology, microscopic pathology, urology, hematology, and enzyme function. Sufficient numbers of test animals are employed in order to yield data which permit judgment of the statistical significance of the findings. Evidence regarding teratology is obtained by mating test animals which are fed the irradiated food and examining uteri and fetuses for malformations. Because responses in experimental animals are not necessarily the same as in humans, the interpretation of results obtained by this method is open to question (Chauhan, 1974). The evaluation of mutagenic effects by means of animal studies would require very large numbers of animals and extended time periods in order to obtain meaningful data. For this reason short-temi methods which use organisms such as bacteria, Drosophila, and plant and animal cell cultures have been employed. These methods, however, are also open to question because they involve extrapolation from relatively simple organisms to man. For this reason, several tests which can be performed in vivo in mammals have been employed. The dominant lethal test reveals chromosomal mutations of the type responsible for death at the stage of the zygote or during embryogenesis. The host-mediated assay involves the use of an indicator bacterium such asSalmonella typhimurium or conidia such as Nrurospora. The host mammalian animal is fed the test food and the indicator microorganism is injected into the peritoneum. Later the microorganism is withdrawn and the number of mutants determined. Cytogenetic tests in mammals involve chromosomal analyses of cells of specific tissues, such as peripheral lymphocytes and bone marrow cells. They detect gross chromosomal damage, but not effects at the gene level.

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WALTER M. URBAIN

A number of substances including carcinogens have been found to increase the

activity of hepatic microsomal enzymes of many mammalian species. Because irradiation of foods leads to radiolytic products, measurement of the activity of hepatic chromosomal enzymes is included in some test protocols. Hickman (1973) and Chauhan (1974) have pointed out the problems of evaluating the toxicological safety of irradiated foods by use of classical pharmacological and toxicological procedures. Of the many problems, the inability to feed the test animal a level of the irradiated food that exaggerates the normal intake level, as would be done in evaluating an isolated substance, constitutes the most difficult problem in adapting the conventional techniques. In irradiated food studies, the approach to this had been to feed the maximum level of the food that can be tolerated by the test animal and which will not cause nutritional problems. Feeding foods irradiated at doses greater than contemplated in practice meets with the objection that excessive radiation can alter a food in ways to reduce its acceptance in terms of sensory characteristics and thereby affect the test. The value of animal feeding tests for evaluating the toxicological safety of irradiated foods lies in the fact that a toxic substance present in sufficient amount will provoke a response in an animal which can be recognized. It is not necessary to know what that substance is nor even the mechanism by which it produces the effect. Only identification of the effect is important in evaluating the safety of the food. If all the substances present in an irradiated food were known, it probably would not be necessary to evaluate food per se. Each substance present could be evaluated separately and standard toxicological methods would be useful. For substances as complex as irradiated foods it is not possible to have a complete knowledge of all compounds present. For this reason, animal feeding studies, which do not require this knowledge, are needed. Newer knowledge of the radiation chemistry of irradiated foods, however, has provided information that suggests that less animal testing than is currently required may be needed (Diehl, 1974; Elias and Cohen, 1977). Radiation chemistry suggests that the response of the various food components to radiation is quite similar, regardless of the food in which they occur. A knowledge of the basic composition of a food permits prediction of the radiolytic products that will be formed when it is irradiated. Hence, the need for individual testing of each and every food is reduced. In addition, different doses applied to a given food yield only quantitative differences in the radiolytic products (Merritt et af., 1975, 1978). This indicates that evaluation studies performed at one dose can be used in considering treatment of a food with another dose (Taub er al., 1976). The quantitative data on rddiolytic volatiles also demonstrate that, even with the high doses of radappertization, the amount of any given substance is small. In beef, for example, about 65 radiolytic products have been identified. Most are hydrocarbons, derived from the meat fat. Table XVI shows the amounts found in beef containing about 15% fat. The total amount is about 30 ppm and the low level of concentration of any single substance suggests that the sensitivity of

21 1

FOOD IRRADIATION

TABLE XVL ABUNDANCE OF VOLATILE COMPOUNDS

(BY CLASS) IN RADAPPERTIZED BEEF"

Volatile compound Alkanes Alkene< Alkandlh Sulfur compound., Alcohols Alkanonm Alkyl benzene5 Esters

PPm

12 14

I .s I .0

1 .o 0.5 0.1 0.1

From Merritt et al. (1975). Reprinted with permission from Journcrl qf Agriculrurul and Food Chemisrry 23, 1037-1041, Nov./Dec. 1975. Copyright by the American Chemical Society.

animal feeding studies is not adequate for the detection of a toxic effect, Advantage can be taken of the knowledge of radiolytic products by assessing the significance of their presence in terms of a health hazard. This can supplment information derived from animal studies. Because certain kinds of ionizing radiation are capable of inducing radioactivity, and because radiation is a carcinogen, evidence is required to demonstrate the absence of added radioactivity in irradiated foods. As indicated earlier, limitation of the energy and type of the radiation employed prevents induced radioactivity in irradiated foods. For radappertized meats and seafoods, microbiological hazards have been eliminated by the use of experimentally determined minimum radiation doses sufficient to kill the most radiation resistant strains of CZ. botulinum. The doses to accomplish this and other processing applied to the foods also take care of other microbiological hazards (Anellis et a/., 1977). For low-dose treatments there has been concern for possible hazards associated with surviving microorganisms. These concerns have been of two types: ( 1 ) the increased incidence of mutants due to exposure to radiation, and (2) the alteration of the outgrowth pattern to favor pathogenic organisms. Investigations of the pathogenic organisms of public health significance subjected to single or multiple irradiations gave no evidence of a microbiological health hazard associated with food irradiation (Idziak, 1973; Ingram, 1975). In foods containing CI. horulitium type E, and possibly other types, the alteration of the outgrowth pattern by radurization appears to lead to a potential health hazard; this was discussed in Section IV,C, 1,b. In other cases, as with the radurization of meat, no comparable hazard has been identified. Apart from the need to provide a package that maintains integrity under pro-

212

WALTER M . URBAIN

cessing and usage conditions, there is the need to provide evidence that the food-contacting packaging material, when irradiated, does not impart a toxic substance to the food (Wierbicki and Killoran, 1966). A number of regularly used food-contacting packaging materials have been approved by the FDA for use with irradiated foods. The normal nutritive values of the macronutrients of irradiated foods, protein, fat, and carbohydrate, are maintained, even at radappertization doses (Anonymous, 1964; Raica et al., 1972). Certain micronutrients, however, are labile to radiation. Vitamin E (alpha-tocopherol) can be lost, probably through the mechanism of peroxide oxidation (Kraybill, in press). Significant thiamine losses occur if radappertization of meats is carried out at temperatures above freezing. At subfreezing temperatures, radappertization occasions losses of thiamine and other vitamins that are no greater than those due to other preservation processes. Table XVII gives data on the effects of different processing methods for enzyme inactivated beef on four vitamins. Wheat irradiated at 20 or 200 krad retained about 90% of its thiamine, riboflavin, and niacin content (Vakil et al., 1973). The ascorbic acid content of irradiated fresh fruits is slightly reduced (Josephson et al., in press). A great many studies to evaluate the safety of irradiated foods for human consumption have been carried out. At the present time a number of foods have been given regulatory approval for consumption by the general public (Table I). TABLE XVII EFFECT OF DIFFERENT PROCESSING METHODS ON THE CONTENT OF THIAMINE, RIBOFLAVIN, NIACIN, AND VITAMIN Bo IN ENZYMEINACTIVATED BEEF (rng/kg)"

Treatment

Thiamine Riboflavin Niacin Vit. B,

Storage (months) 0 15 0 15 0 15 0 15

Freezing

Thermal canning

Cobalt Gamma* (4.7-7. I Mrad)

0.97 0.68 2.80 1.69 48.6 57.2 2.50 0.97

0.63 0. I 4 2.63 2.60 48.1 54.9 2.13 0.57

0.83 0.21 2.83 2.60 48.9 50.1 3.93 0.35

a From Josephson ef al. (1976). First Int. Congr. E n g . Food, as spon sored by the American Society of Agricultural Engineers. Irradiated at -30°C ? 10".

FOOD IRRADIATION

213

In the United States, wheat and wheat products are approved for insect disinfestation by irradiation and potatoes for sprout inhibition. In 1976, WHO gave “unconditional acceptance” as safe for human consumption of irradiated potatoes, wheat, chicken, papayas, and strawberries (Anonymous, 1970b, 1977). Japan is irradiating potatoes commercially with the approval of its government. Apparently, for all these approvals there has been provided acceptable evidence of safety for human consumption of the foods in question. Other foods, in addition to those now approved, have been the subject of evaluation studies. These studies apparently have failed to provide acceptable evidence. On the other hand, except for a small number of studies, which on repetition were not verified, no positive evidence of a toxicological hazard associated with an irradiated food has been identified in these studies. The inadequacies of the rejected evaluations appears to be an insufficiency of convincing evidence of safety. As indicated earlier, a number of new evaluations are in progress. Some, such as those being done by the U. S . Army, represent the totality of design and execution that can be applied to an evaluation study. One represents a new approach in that the evaluation is of the process of irradiation and not just an irradiated food (de Zeeuw and van Kooij, 1973). The outcome of these current studies will define the future of food irradiation.

VII. THE FUTURE OF FOOD IRRADIATION Among the reasons for reviewing a subject is a desire to judge what the future might hold. A reviewer who undertakes the task of projecting into the future is likely to use much more of his personal judgment in this than he does in covering historical aspects. He may reflect the opinions of others, but in essence he must take the major responsibility for what he writes, as does this present reviewer. It is now over 30 years since the beginnings of organized research on the development of food irradiation were undertaken. As of now, there is a considerable knowledge at hand. Despite this knowledge, the use of food irradiation has been delayed mainly by concerns for the safety of irradiated foods for human consumption. It seems probable that this situation will be resolved within the next few years. Some may regard the information now available sufficient to establish the safety of irradiated foods, but, on the whole, governments have not taken action to permit their distribution to the public. At least part of the task to be done in the next few years is to obtain regulatory approval, and there is good reason to be optimistic about the outcome. Certainly no information obtained by accepted studies has demonstrated health hazards associated with the consumption of irradiated foods. If one anticipates that government approvals for irradiated foods will be given on a broad basis, one needs then to consider what areas of usage are likely. To some extent this kind of appraisal has been made in

2 14

WALTER M . URBAIN

the various individual sections of this review, but additional thoughts can be expressed. In the case of food irradiation, generalizations are difficult and perhaps meaningless. One must deal with specific kinds of uses of food irradiation. Radappertized foods developed so far have been limited to meats, poultry, and seafoods. While there are important differences, they resemble comparable thermally processed products and presumably would serve similar market requirements. Because of ease of table preparation and because they are shelfstable, in the civilian market they must be regarded as convenience foods. The percentage of the market now held by the similar thermally processed meats, poultry, and seafood, as compared with refrigerated or frozen distribution of them, is small. An important reason for this is the higher cost of such products. Irradiation, even with substantial product quality improvement, is not likely to enlarge this market percentage. In fact, additional costs resulting from irradiation may restrict the market potential. A very fruitful area for research on radappertization would be reduction of the dose requirement, which is a principal factor of cost in the present process. If radappertized fruits and vegetables could be prepared with a quality superior to what the thermal process now produces, a very large market potential can be envisaged. A sufficient quality improvement could result in competition with frozen foods. Further research on radappertized foods aimed at quality improvement and cost reduction clearly is indicated. Combination processes, possibly simultaneous heating and irradiation, seem worth investigating in this connection. Of the other high-dose applications, sterilization of spices by irradiation appears most likely to be used. Irradiation in this case is effective, causes virtually no quality loss, and leaves no questionable chemical residue. The lower costs associated with low-dose applications offer greater opportunities for use than exist with radappertization. Also, the smaller doses are less apt to cause loss of product quality and reduce concerns for safety for human consumption. Although there are serious problems in some cases, it seems reasonable to anticipate fairly widespread use of radurization. In warm countries, for example, radurization should be useful in enlarging the distribution of salted and cooked fish without the use of refrigeration. Radurization combined with refrigeration can improve the distribution of fresh and processed meats, poultry, and seafoods, but clearly cannot be used in all situations. In certain areas such as the United States, there is need to resolve the problem with Cl. botulinum type E in marine and fresh water products. Irradiation can render important help in furthering export trade of many plant products. Radurization to prevent spoilage of fruits such as mangoes, citrus foods, and perhaps berries such as strawberries could reduce spoilage and enlarge markets. Opportunities to obtain several benefits simultaneously would favor the use of irradiation of fruits. Insect disinfestation, delay of senescence, and micro-

FOOD IRRADIATION

215

bial spoilage control can occur as a result of a single treatment. Because insects carried by the fruits of some countries are barriers to their export, disinfestation by irradiation could be the greatest use of irradiation. Its effectiveness for disinfestation has been demonstrated, even under conditions in which chemicals fail (e.g., the mango weevil inside the fruit). Continued concern for the safety of pesticides now used could lead to their replacement with radiation. The disinfestation of cereal grains and of leguminous seeds seems of uncertain future as long as chemicals can be used. Higher costs for irradiation, the need for protection against reinfestation after treatment, and the fact of a time delay in the insect killing action, all are against the use of irradiation presently. Radicidation will come only if imposed by government regulation. The same is true of parasite disinfestation. The inhibition of sprouting of white potatoes already is in use (Japan) and is likely to be extended, especially in warm countries. It could be an aid in the export of this food. A similar picture exists for onions and possibly for other root crops such as sweet potatoes, and ginger. The delay of senescence of other living foods as a means of preservation, either for distribution or temporarily holding prior to other processing, would seem to be a likely area of usage. More basic research is needed, however, to understand how to use irradiation effectively for this purpose. Changes in foods or food ingredients induced by irradiation which affect their functionality have been studied very little. Radiation could be yet another agent to accomplish desirable improvements. This area of usage might receive more attention once the food industry becomes seriously involved in food irradiation. Part of the future is the irradiation facility. A good deal of applicable experience can be secured from the current wide use of radiation to sterilize hospital supplies. It is not likely that the engineering, construction, and operation of irradiation plants will involve any serious problems. There is adequate knowledge for these available. Choices exist for the kind of radiation source, primarily whether machine or radionuclide. Technical requirements (e.g., depth of penetration needed) to a considerable degree will determine which will be best for a particular use. Economics have a role in this also, but appear to be of secondary importance. In some cases, X-rays from machines rather than electron beams may be the best form of ionizing energy. Fission by-product gamma ray sources, mainly 137Cs,presently seem limited to quantities now available in view of the U. S. Government action to defer further processing of spent reactor fuel. "Co, on the other hand, can be made available as the need develops. In the long term, it seems certain that all types of sources can be considered for food irradiation. In any particular application, a determination of the best source for it needs to be made with due consideration for technical aspects and costs. In the near term, "Co seems to be the radiation source of choice. Another part of the future is the reaction of the consumer to irradiated foods. In

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WALTER M . URBAIN

the history of food irradiation, there is little to suggest that the consumer would not eat irradiated foods. Test-marketing in a number of countries proved generally successful. Japan has been marketing irradiated potatoes since 1973. It would seem, therefore, that, with assurance from their government on the safety of irradiated foods and with suitable information provided to them, consumers will accept irradiated foods. In view of the current concerns for processed foods generally, and for food additives, it would seem prudent, however, to introduce irradiated foods to consumers with careful attention to their views. Once usage of food irradiation is established, support for research to find new applications and to improve those already identified should become available. Most importantly, this new effort will be in the private sector. The lack of industry support so far has been one of the greatest difficulties with the advancement of food irradiation. When it comes, it will have a very large impact.

REFERENCES Adamiker, D. 1975. A comparison of various methods for treating feedstuffs for laboratory animals. In “Food Irradiation Information,”No. 5. pp. 1 9 4 2 . Int. Project Field Food Irradiation., Karlsruhe. Adesuyi, S . A , , and Mackenzie, 3. A. 1973. The inhibition of sprouting in stored yams, Dioscorea roiundatu Poir, by gamma radiation and chemicals. Proc. Bombay Symp., Radiar. Preserv. Food, IAEA, Vienna pp. 127-136. Aibara, K., and Miyaki, K. 1970. Aflatoxin and its radiosensitivity. Radiat. Sensitiv. Toxitzs Anim. Poisons, IAEA. Vienna pp. 41-62. Al-Jasim, H., Markakis, P., and Nicholas, R. C. 1968. Role of calcium in softening and refirming irradiated plant tissues. Prescrv. Fruir Veg. Irradiai., IAEA, Vienna pp. 125-127. Anderson, A. W., Corlett, D. A,, Jr., and Krabben Hoft, K. L. 1967. The effects of additives on radiation-resistance of Cl. botulinum in meat. Microbiol. Probl. Food Preserv. Irradiat., IAEA, Vienna pp. 87-97. Anellis. A,, and Koch, R. B. 1962. Comparative resistance of strains of Closrridium borulinum to gamma rays. Appl. Microbiol. 10, 326-330. Anellis, A,, Shattuck, E., Rowley, D. B.. Ross, E. W., Jr., Whaley. D. N.. and Dowell, V. R., Jr. 1975. Low-temperature irradiation of beef and methods for evaluation of a radappenization process. Appl. Microbiol. 30, 81 1-820. Anellis, A,, Rowley, D. B., and Ross, E. W., Jr. 1976. Microbiological safety of radappertized beef. First Int. Congr. Eng. Food. Boston. Anonymous. 1964. “Report of the Working Party on Irradiation of Food,” Min. Health. HM Stationery Office, London. Anonymous. 1965. “The Technical Basis for Legislation on Irradiated Food,” FAO, Rome. Anonymous. 1967. “Seibersdorf Project on Food Irradiation Research,” Int. Programme Irradiat. Fruit Fruit Juices, First Activ. Rep. Organ. E o n . Coop. Dev., Eur. Nucl. Energy Agency. Anonymous. 1968a. “Food Irradiation Activities throughout the World,” U.S. Dep. Commer. U.S. Gov. Print. Office, Washington, D.C. Anonymous. 1968b. “The Commercial Prospects for Selected Irradiated Foods,” TID-24058. U.S. Dep. Commer.. Washington, D.C.

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Anonymous. 1970a. “Training Manual on Food Irradiation Technology and Techniques,” p. 59. IAEA. Vienna. Anonymous. 1970b. “Wholesomeness of Irradiated Foods with Special Reference to Wheat. Potatoes and Onions.” Tech. Rep. Ser. No. 45 I . WHO. Geneva. Anonymous. 1973. “Radionuclides in Foods.” pp. 95-97. Natl. Acad. Sci., Washington. D.C. Anonymous. 1977. “Wholesomeness of Irradiated Food,” Rep. Joint FAOiIAEAlWHO Expert Comm., Tech. Rep. Ser. No. 604 (ISBN 92 4 120604 7). WHO, Geneva. Aoki. S . , Watanabe. H., and Sato, T . 1976. Extending the storage life of mushroom “Matsutake” by y-irradiation. In “Food Irradiation in the Takasaki Radiation Chemistry Research Establishment,” JAERI-M 6548, No. 2, pp. 50-54. Jpn. At. Energy Res. Inst., Tokyo. Bachman. S., and Gieszczynska. J. 1973. Studies of some microbiological and chemical aspects of irradiated spices. Aspects Introduction Food Irradiat. Dev. Counrries. IAEA, Vienna pp. 3 3 4 1 . Bailey, A. J . , and Rhodes, D. N. 1962. Treatment of meats with ionizing radiations. X l A h a n g e s in the texture of meat. J . Sci. Food Agric. 15, 504-508. Baines, B. D., and Mosely, J. 1966. Economics of grain irradiation. Pro(... Karlsruhe Symp., Food Irradiar. IAEA, Vienna pp. 813-831. Baldelli, B. 1967. Gamma radiation for sterilizing the carcasses of foot-and-mouth disease virus infected animals. Microbiol. Probl. Food Preserv. Irradiat.. IAEA, Vienna pp. 77-86. Balock, J. W.. Burditt, A. K . , Seo, S . T . , and Akamine. E. K . 1966. Radiation as a quarantine treatment for Hawaiian fruit flies. J. Econ. Enromol. 59, 202-204. Bandyopadhyay, C.. Tewari, G. M.. and Sreenivasan, A. 1973. Studies on some chemical aspects of gamma irradiated onions. Proc., Bombay Symp., Radiut. Preserv. Foods. IAEA, Vienna pp. 11-19. Berger, G . . Agnel, J. P.. and Saint-Lebe, L. 1973. Sugars formed during irradiation of maize starch. Determination and identification. Sraerke 25, 203-210. Boisot, M. H.. and Gauzit, M. 1966. Disinsectization of African dried and smoked fish by means of irradiation. Appl. Food Irradiat. Dev. Countries, IAEA, Vienna pp. 85-94. Bramlage, W. J . , and Couey, H. N. 1965. “Gamma Radiation of Fruits to Extend Market Life,” Marketing Res. Rep. No. 717. U . S . Dep. Agric.. Washington, D.C. Branilage. W. J., and Lipton, W. J. 1965. “Gamma Radiation of Vegetables to Extend Market Life.” Marketing Res. Rep. No. 703. U.S. Dep. Agric.. Washington, D.C. Brasch. A.. and Huber, W. 1948. Reduction of undesirable by-effects in products treated by radiation. Science 108, 536-537. Brouqui, M., Eymery. R., and Saint-Lebe. L. 1973. Irradiation of foodstuffs in bags. Proc., Bombuy Symp., Radial. Preserv. Food. IAEA, Vienna pp. 577-591. Brower. J . H. 1974. Radioresistance of the red flour beetle, Triholium casfaneum (Coleopetra: tenebriondae). exposed to sublethal doses of gamma irradiation for 25 generations. Can. Enromol. 106, 24 1 - 2 4 . Brownell. L. E. 1961. “Radiation Uses in Industry and Science.” pp. 298-301. USAEC, Washington. D.C. Brynjolfsson, A. 1973. Factors influencing economic evaluation of irradiation processing. Factors Influencing Eron. Appl. Food Irradiat., IAEA, Vienna pp. 13-35. Campbell, J . D . , Stothers, S . , Vaisey, M.. and Berck, B. 1968. Gamma irradiation influence o n the storage and nutritional quality of mushrooms. J . Food Sci. 33, 540-542. Cann. D. C.. Wilson. B. B.. Shewan, J . M . , Roberts, T. A , , and Rhodes. D. N . 1966. A comparison of toxin production by Clostridium botulinum type E in irradiated and unirradiated vacuum packed fish. J . Appl. Bacteriol. 29, 540-548. Carver. J. H.. Connors, T. J . , Ronsivalli, L. J.. and Holston, J. A. 1968. “Shipboard Irradiator Studies, ” TID-2Y 332. USAEC, Washington, D. C. Chauhan. P. 1974. Assessment of irradiated foods for toxicological safety-newer methods. In

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Pawel, 0. 1968. Devitalization of cysticerci by gamma radiation. Elimination Harmful Organisms Food Feed Irradiat., IAEA. Vienna pp. 91-94. Pelroy, G. A , , and Seman, J. P. 1968. Effect of storage temperature on the microflora of irradiated and nonirradiated petrale sole fillets. J . Milk Food Technol. 31, 231-236. Phillips, G. 0. 1972. Effects of ionizing radiations on carbohydrate systems. Radiat. Res. Rev. 3, 335-35 I . Poisson, J . , Jemmali, M.. Cahagner, B., and Leclerc, J. 1967. The effect of gamma irradiation of wheat flour on its microflora and viramin 9, content. Food Irradiat., Q. Int. Newsl. 8, 2-1 I . Pomerantz, R., and Siu, R. G. H. 1957. Economics of radiation processing. In “Radiation Preservation of Food,” pp. 4 1 0 4 3 2 . Quartermaster Corps, U.S. Army, Washington, D.C. Proctor, 9. E., Van de Graaff, R. J.. and Fram, H. 1943. “Reports on Quartermaster Contract Projects by Food Technology Laboratories,” p. 217. Mass. Inst. Technol., Cambridge, Massachusetts. Proctor, 9. E., Goldblith, S. A., and Fram, H. 1950. Effect of supervoltage cathode rayson bacterial flora of spices and other dry materials. Food Res. IS, 490493. Proctor, 9. E., Joslyn, R. P., Nickerson, J . T. R., and Lockhart, E. E. 1953. Elimination of Salmonella in whole egg powder by cathode ray irradiation of egg magma prior to drying. Food Technol. 7 , 29 1-296. Purohit, K. S., Manson, J. E.. and Zahradi, J. W. 1971. Theoretical evaluation of combined radiation and thermal processes in cylindrical containers with gamma sources. J . Food Sci. 36, 750-75 I . Quinn, D. J . , Anderson, A. W.. and Dyer. J. F. 1967. The inactivation of infection and intoxication microorganisms by irradiation in seafood. Microbiol. Probl. Food Preserv. Irradiat., IAEA, Vienna pp. 1-13. Raica, N., Jr., Scott, J., and Nielsen, W. 1972. The nutritional quality of irradiated foods. Radiat. Res. Rev. 3, 447-451. Rao, S . , Hoseney, R. C., Finney, K. F., and Shogren, M. D. 1975. Effect of gamma irradiation of wheat on bread making properties. Cereal Chem. 52, 506-512. Remini, W. C., Wahlquist, E. J., and Sivinski, H. D. 1977. “Beneficial Use of Waste Nuclear Isotopess,” ERDA 77-17. U.S. Energy Res. Dev. Admin., Washington, D.C. Reynolds, M. C., and Brannen, J. P. 1973. Thermal enhancement of radiosterilization. Proc., Bombay Symp., Radiar. Preserv. Food, IAEA, Vienna pp. 165-176. Reynolds, M. C., Lindell. K. F., and Laible, N. 1970. “A Study of the Effectiveness of Thermoradiation Sterilization,” Res. Rep. SC-RR-70-423. Sandia Lab.. Albuquerque, New Mexico. Rhodes. D. N., and Shepherd, H. J. 1966. The treatment of meats with ionizing radiations. Xnl. Pasteurization of beef and lamb. J . Sci. Food Agric. 17, 287-297. Rindorf, H. 1966. Economics of food irradiation. Proc., Karlsruhe Symp., Food Irradiar,, IAEA, Vienna pp. 865-877. Roberts, T. A. 1967. Radiation resistance of botulinal toxins. Microbiol. Probl. Food Preserv. Irradiat.. IAEA, Vienna pp. 55-56. Ronsivalli, L. J., Kaylor, J. D., Murphy. E. J., Learson, R. J., and Schwartz, M. S. 1970. Studies in petition-oriented aspects of radiation pasteurization of fishery products. Preservation Fish Irradiat., IAEA, Vienna pp. 1-1 I . Ross, E. W., Jr. 1974. Statistical estimation of 12D for radappertized foods. 1. Food Sci. 39, 800-806. Ross, E. W . , Jr. 1976. Estimating 12D from one partial spoilage data point. Firs? Int. Congr. Eng. Food, Boston. Rowley, D. B., El-Bisi, H., Anellis, A., and Snyder, 0. P. 1968. Resistance of Clostridium borulinum spores to ionizing radiation as related to radappertization of foods. Proc. U.S. Jpn. Conf. Toxic Microorganisms,” Isr, Honolulu pp, 459467.

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Saint-Lebe. L.. Mucchiella. A.. Leroy. P., and Beerens. H. 1973. Preliminary studies on the microflora of maize starch before and after irradiation. Proc. , Bombay Svmp., Radial. Prfserv. Foods, IAEA. Vienna pp. 155-164. Sakaguchi, G. 1970. Radiosensitivities of Cl. borulinum type-E toxins. Radiar. Sensiriv. Toxins Anim. Poisons. IAEA, Vienna pp. 31-10, Salunke, D. K. 1961. Gamma radiation effects on fruits and vegetablcs. Econ. Eor. 15, 28-56. Sandret. F. 1973a. “Technology of Radiation Inhihition of Sprouting in Potatoes,” Ser. Proc. 19. Eurisotop Off., Brussels. Sandret, F. 1973b. “Technology of Radiation Inhibition of Sprouting in Potatoes.” 83 Ser.. Proc. 25. Eurisotop Off., Brussels. Saravacos, G. D., Hatzipctrou. L. P.. and Georgiadou. E. 1962. Lethal doses of gamnia radiation of some fruit spoilage microorganisms. Food Irrudiut., Q . lnr. N e w s / . 3, A6-A9. Savagaon. K . A,. Dharkar. S. D.. and Sreenivasan. A . 1970. Radiation preservation of chapaties (Indian unleavened bread). Food Techno/. 24, 1158-1 160. Savagaon, K . A,, Venugopal. V.. Kamat. S. V.. Kunita. U. S . , and Srecnivasan. A. 1972. Radiation preservation of tropical shrimp for ambient temperaturc storage. l . Development of a heat-radiation combination process. J . Food Sc.i. 37, 148- 150. Scanlan. R. A., and Lindsay, R. C. 1968. Observations on thc low temperature irradiation of milk. J . D a i v Sci. 51, 1967-1968. Schmidt. C. F. 1961. “Report of the European Meeting on the Microbiology of Irradiated Foods.” Paris, Appendix 11. FAO, Rome. Schmidt, C. F.. and Nank, W. K. 1960. Radiation sterilization of food. I . Procedures for the evaluation of the radiation resistance of spores of Closrridium bofulinum in food products. Food Res. 25, 32 1-327. Schmidt. C . F., Lechowich. R . V.. and Folinazzo. J . F. 1961, Growth and Toxin production of type E Closrridium botulinum below 40°F. J . Food S c i . 26, 626-630. Schroeder. C. W. 1962. Dehydrating vegetables. U.S. Patent 3.025.171. Segner. W. P.. and Schmidt. C. F. 1966. Radiation resistance of spores of Closrridiurn botulinum type E. Proc.. Karlsruhe Symp.. Food Irradiar.. IAEA. Vienna pp. 287-298. Shewan, J . M . . and Hobbs. G. 1973. The botulism hazard in the proposed use of irradiation of fish and fishery products in the United Kingdom. Preserv. Fish Irradiut.. IAEA. Vienna pp. 117124. Shults. G. W.. and Wierbicki, E. 1974a. “Changes in Nonprotein Nitrogen Content and the Sensory Characteristics of Beef Steaks as Affected by the Heat Trcatment,” Tech. Rep. 75-8-FEL. U.S. Army Natick Lab., Natick. Massachusetts. Shults. G. W . . and Wierbicki, E. 1974b. Development of Irradiated Beef. I . Acceptance of Beef Loin Irradiated at Cryogenic Temperatures.” Tech. Rep. 74-57-FI. U . S . Army Natick Lab.. Natick. Massachusetts. Shults. G. W.. Cohen, J . S.. and Wicrbicki. E. 1975. “Radiation-inactivation of Proteases as Determined by a ‘‘C-Labelled Hemoglobin Method.” Tech. Rep. TR-76-33 FEL. U.S. Army Natick Res. Dev. Command. Natick, Massachusettq. Sickel. V . E.. Diehl. J . F.. and Griinewald. T. 1969. Comparison of the suitability of heat and radiation-sterilized presrarter feed for the young pig for SPF status. 2. Tierphysiol.. 7ierernurhr. Futtermittelkd. 25, 258-269. Skulberg. A . 1970. Toxins produced by Cl. botulinum and their radiosensitivity. Radiar. Sensiriv. Toxins Anim. Poisons. IAEA, Vienno pp. 19-30. Sommer. N . F.. and Fortlage. R. J . 1966. Ionizing radiation for control of postharvest diseases of fruits and vegetables. Adv. Food Res. 15, 147-193. Sommer. N . F.. and Maxie, E. C. 1966. Recent researches on the irradiation of fruits and vegetables. Proc. Karlsruhe Symp.. Food Irradiat., IAEA. Vienna pp. 571 -587.

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Spinelli, J., Eklund, M.. Stoll, N., and Miyauchi, D. 1965. Irradiation preservation of Pacific Coast fish and shellfish. 111. Storage life of petrale sole fillets at 33” and 42°F. Food Technol. 19, 1016- 1020. Sreenivasan, A. 1974. Compositional and quality changes in some irradiated foods. Improve. Food Quai. Irradiat., IAEA. Vienna pp. 129-155. Sreenivasan. A,. Thomas, P., and Dharkar, S. D. 1971. Physiological effects of gamma radiation on some tropical fruits. Disinfestafion Fruit Irradiat.. IAEA, Viennu pp, 65-91, Staden, 0. L. 1966. Experiences with the irradiation of vegetables in the Netherlands. Proc.. Karlsruhe Symp., Food Irradiar.. IAEA, Vienna pp. 609-617. Stehlik, G., and Kaindl. K. 1968. “Preservation of Bread by Means of Gamma Radiation,” ORNLIIC-5. Oak Ridge Natl. Lab., Oak Ridge, Tennessee. Sudarmadji, S . , and Urbain, W. M. 1972. Flavor sensitivity of selected raw animal protein foods to gamma radiation. J. Food Sci. 37, 671-672. Swallow, A. J. 1977. Chemical effects of irradiation. In “Radiation Chemistry of Major Food Components” (P. S. Elias and A. J. Cohen, eds.). pp. 5-20. Elsevier, Amsterdam. Takano, H ., Tanaka, Y.. Umeda, K., and Sato, T. 1973a. Sprout inhibition of potatoes by ionizing radiation (Part 3). Dose to inhibit sprout and the change of sugar content during low temperature storage. In “Food Irradiation in the Takasaki Radiation Chemistry Research Establishment,’’ JAERI-M 5458, No. I , pp. 79-84. Jpn. At. Energy Res. Inst., Tokyo. Takano, H., Tanaka, Y.,Umeda, K., and Sato, T. 1973b. Sprout inhibition of onions by ionizing radiation (Part 2). Effects of radiation dose and storage conditions on the sprout of var. “Senshuki.” In “Food Irradiation in the Takasaki Radiation Chemistry Research Establishment,” JAERI-M 5458, No. I , pp. 83-87. Jpn. At. Energy Res. Inst., Tokyo. Taub, 1. A.. Angelini, P., and Merritt, C., Jr. 1976. Irradiated Food: Validity of extrapolating wholesomeness data. J. Food Sci. 41, 942-944. Thomas, A. C. 1975. Gamma irradiation-an answer to the mango growers prayer. In “Food Irradiation Information,” No. 5, pp. 9-18, Int. Project Field Food Irradiat., Karlsruhe. Thornley, M. J. 1963. Microbiological aspects of the use of radiation for the elimination of salmonellae from foods and feeding stuffs. In “Radiation Control of Salmonellae in Food and Feed Products,” Tech. Rep. Ser. No. 22, pp. 81-106. IAEA, Vienna. Tilton, E. W., and Brower, J. H. 1973. Status of the U.S. Department of Agriculture Research on irradiation disinfestation of grain and grain products. Proc., Bombay Symp., Radiar. Preserv. Food, IAEA, Vienna pp. 295-309. Tiwari, N. P., and Maxcy, R. B. 1972. Moraxella Acinetobacter as contaminants of beef and occurrence in radurized product. J. Food Sci. 37, 901-903. Umeda, K. 1975. Background to the establishment of the first food irradiation plant in Japan. Require. Irradiar. Food Commerc. Scale, IAEA, Vienna pp. 113-131. Urbain, W. M. 1965a. Radiation preservation of fresh meat and poultry. In “Radiation Preservation of Foods,” Publ. No. 1273, pp. 87-98. Natl. Acad. Sci., Washington, D.C. Urbain, W. M. 196%. A look toward commercial use of radiation preservation. In “Radiation Preservation of Foods,” CONF-650552, pp. 75-98. USAEC, Washington, D.C. Urbain, W. M. 1966. Technical and economic considerations in the preservation of meats and poultry. Proc., Karlsruhe Symp. Food Irradiat. IAEA, Vienna pp. 397410. Urbain, W.M., 1970. Radiation update. Proc. Symp. Feeding Mil. Man, U . S . Army Natick Lab., Narick, Mass. pp 105-1 13. Urbain, W. M. 1973. The low-dose radiation preservation of retail cuts of meat. Proc., Bombay Symp., Radiat. Preserv. Food, IAEA. Vienna pp. 505-521. Urbain, W. M. 1977. Radiation chemistry of proteins. In “Radiation Chemistry of Major Food Components” (P. S . Elias and A. J. Cohen, eds.), pp. 63-130. Elsevier. Amsterdam. Vajdi, M., and Pereira, R. R. 1973. Comparative effects of ethylene oxide gamma irradiation and microwave treatments on selected spices. J . Food Sci. 38, 893-896.

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Vakil, U. K.. Aravindakshan, M.. Srinivas, H., Chauhan. P. S.. and Sreenivasan. A. 1973. Nutritional and wholesomeness studies with irradiated foods: India’s program. Proc.. Bornboy Symp., Radiat. Preserv. Food. I A E A , Vienna pp. 673-702. Val Cob. Del M.. and Cruz Castillo. de la F. 1973. The effect of technological parameters on the economic design of food-irradiation units. Fuctors Influencing Econ. Appl. Food lrrudiut., IAEA. Vienna pp. 37-55. Van Kooy. J. G., and Robijns. K. G. 1968. Gamma irradiation elimination of Cysficercus bovis. Elim. Harmful Organisms Food Feed Irradiut., I A E A . Viennu pp. 8 1-89, Van Mameren. J . . and Houwing, H. 1968. Effect of irradiation on Anasakis larvae in salted herring. Elim. Harmful Organisms Food Feed Irradiat. I A E A , Vienna pp. 73-80. Vidal, P. 1966. Irradiation of fruits and vegetables in France. Proc. Karlsruhe Symp. Foodlrradiut. IAEA. Vienna pp. 589-599. Watanabe, H.. Ito. H.,Shibabe, S . . and lizuka. H . 1973a. Effect of gamma-irradiation on the microflora of maize and milo. I n “Food Irradiation in the Takasaki Radiation Chemistry Research Establishment.” JAERI-M5458. No. I , pp, 21-24. Jpn. At. Energy Res. Inst., Tokyo. Watanabe. H., Ito, H., Shibabe, S . , and Iizuka. H. 1973b. Effect of gamma-irradiation on the storage of maize and milo. I n “Food Irradiation in the Takasaki Radiation Chemistry Research Establishment,” JAERI-M5458. No. 1, pp. 25-20. Jpn. At. Energy Res. Inst.. Tokyo. Welch. C. B . , and Maxcy. R. B . 1975. Characterization of radiation resistant vegetative bacteria in beef. Appl. Microbial. 30,242-250. Wharton. D. R. A. 1957. Action of ionizing radiations on helminths. I n “Radiation Preservation of Food.” pp. 235-239. Quartermaster Corps, U . S . Army, Washington, D.C. Wick. E. L., Murray, E.. Mizutani, J . , and Koshika, M. 1967. Irradiation flavor and the volatile components of beef. In “Advances in Chemistry Series.” Vol. 65. pp. 12-25. Amer. Chem. Soc.. Washington, D.C. Wierbicki. E. 1974. “Radappertization (Radiation Sterilization) of Foods,” Tech. Rep. NaticWTR74-3-FL,. U.S. Army Natick Lab.. Natick, Massachusetts. Wierbicki. E., Brynjolfsson. A,. Johnson, H. J.. and Rowley, D. B . 1975. Preservationofmeats by ionizing radiation-an update. Eur. Meet. Mcat Res. Workers, 2 / s t , Rapp. Pap. No. 14. Wierbicki, E., and Killoran, J. J. 1966. Packaging for radiation-sterilized foods. Present status. Act. R c ~ 18, . 18-29. Wills. P. A., Clouston. J. G . , and Gerraty. N. L. 1973. Microbiological and entomological aspects of the food irradiation program in Australia. Pro(,., Bombuy Symp., Radiat. Preserv. Food, I A E A , Viennu pp. 231-259. Wolin. E. J., Evans. J. B.. and Niven, C. F., Jr. 1057. The microbiology of fresh and irradiated beef. Food Res. 22, 682-686. ~

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ADVANCES I N

FOOD RESEARCH. VOL. 24

TEA ROBERT L. WICKREMASINGHE Tea Research Institute of Sri Lanka, Coombs, Talawakelle. Sri Lanka

St.

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Composition of Tea ... ..... A. Chemical and Bi ... ..... B. Factors Affecting Chemical Composition . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Changes during the Processing of Tea . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . A. Black Tea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Green Tea . . . . . . . . . . . . . . . . ........... IV. Organoleptic Properties . . . . . . . . . . . . . . . . . . . . . . . . . . A. BlackTea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Green Tea ...... ........ V. Storage of Tea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Tea Leaves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. TeaBrews. . . . . . . . . . . . . . . . . . . . . C. Green Tea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Black Tea . . . . . . . . . V1. Potential By-products . . . . . . . . . . , . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . VII. Clinical Effects VIII. Host Plant-Pest Relationships. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Instant T e a . . . . . . . . . . . . . . . . . . . ... ...... .. . X. Additional Research Needs . . . . . ... ......... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

229 232 233 248 25 1 25 1 26 I 263 263 265 266 266 266 267 267 268 269 27 1 272 213 273

INTRODUCTION

Tea originated in China where its legendary history dates to as far back as 2737 although the earliest mention of tea is found in Erh Ya, an ancient Chinese dictionary of 350 B.c., and the first monograph on tea was published by Lu Yu in 780 A.D. Several centuries later, in 1559, tea was brought to Europe by Gian Battista Ramusio, the noted Venetian writer, and became a widely consumed B.C.

229 Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-016424-8

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beverage during 1784-1789. From that time on the growth of its popularity was rapid, and it is now the most widely consumed beverage in the world. The average annual production and exports of tea in 1972-1974 were about 1,213,000 and 663,000 tons, respectively, derived from the countries listed in Table I (Food and Agricultural Organization, 1976). The figures for China (including Taiwan Province) and the Democratic Republic of Wet Nam are not available. Most of the cultivated tea in the world is highly heterogeneous (KingdonWard, 1950) as a result of the large-scale dispersal of the tea plant during the long history of its cultivation, its outbreeding nature, and the free hybridization beTABLE I AVERAGE ANNUAL PRODUCTION AND EXPORTS OF TEA FROM DIFFERENT COUNTRIES FOR THE PERIOD 1972-1974''

Country NET EXPORTING COUNTRIES FAR EAST AND OCEANIA India Sri Lanka Indonesia Bangladesh Others Africa Kenya Malawi Mozambique Uganda Tanzania Others Near E m / Turkey Laiin America Argentina Others PRODUCING-IMPORTING COUNTRIES Japan South Africa Iran Malaysia U.S.S.R. WORLD TOTAL

Production

Exports

783.6 472.7 209.6 63.9 28.0 9.4 156.6 54.4 22.5 18.4 22.3 13.0 26.0 44.2 44.2 35.4 26.8 8.6

482 225 190 41 22 4 I38 48 22 18 20 10 20 17 17 26 20 6

90.9 1.8 23.0 3.4 73.7 1212.6

663

" Figures in lo00 metric tons, according to the Food and Agricultural Organization (1976).

TEA

23 1

tween geographical races, although all cultivated tea is generally assigned to one species Carnellia sinensis (L.) 0. Kuntze (Eden, I976).* In this classification, small-leaved plants are grouped as vat-. sirletisis while the large-leaved plants are included in the vur. assumica, but the bush population even in a single location shows great variation in growth habit, branching size, shape, texture, and pose of leaf, as well as in inherent yield and other characteristics (Bezbaruah, 1974). These variations are due to the majority of existing tea bushes (which have a useful life span of 60-70 years) having been derived, during the greater part of the past century of tea propagation, from seed of widely diverse genetic constitution. In more recent years however, tea growing countries have perfected the art of vegetative propagation from single-leafed cuttings (Visser and Kehl, 1958) and propagation from seed is now hardly ever practiced. Selection of mother bushes from which cuttings are taken is based on visual estimation of bush size, frame, yield, resistance to pests, diseases. and drought. characteristics of the prdcessed tea and other factors. The final assessment of these characteristics depends on the results of field trials spread over a period of 7 to 10 years, but it is possible that other methods of selection of mother bushes, e.g., a study of morphological (Wight and Barua, 1954; Wu ef al., 1968; Memedov, 1961; Venkataramani and Padmanabhan, 1964; Toyao, 1966) and anatomical features (Wu, 1968; Pochet ef al., 1974) as well as chemical and biochemical characters (Toyao, 1975) will provide a means of shortening the time required for evaluation. Free-growing tea will attain a height of about 9 m, but in commercial tea cultivation the tea bush is maintained at a height of 1-1.5 m by regular pruning (Eden, 1976). The pruning frequency varies from 3 to 6 years in different teaproducing countries, and the purposes of this practice are to encourage branching, revitalize the bush, and to facilitate plucking of the leaves (Tubbs, 1936). Plucking is done by hand or by mechanical means, such as with shears, and tea of the highest quality is made from young shoots (“flush”), consisting of the tender bud and first two leaves. The frequency of plucking is dependent on climatic and other factors affecting regeneration of the flush, but is generally at intervals of 6 to 9 days. Four main types of tea may be processed from these young shoots: black tea, green tea. oolong tea, and instant tea, and of these black tea is quantitatively the major type produced. Comprehensive reviews of tea have been published (Stahl. 1962; Roberts, 1962; Bokuchava and Skoboleva, 1969; Sanderson, 1972a) all of which have dealt with the chemistry of tea and tea manufacture. The purpose of the present *In Japan, the tea plant is assigned to the genus Then, which is distinguished from Camellia on the basis of chemotaxonomical characteristics. particularly the occurrence of eugenol glycoside in the essential oil of Camellia but not Thea (Fujita el a / . . 1973) and the presence of L-pipecolic acid (Ozawa et al., 1969) in the unripe fruit of Thea species only.

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ROBERT L. WICKREMASINGHE

review is to update and extend the coverage to include some of the other aspects of tea.

II. COMPOSITION OF TEA Published data have, in the past, been generally confined to analysis of the flush (Kursanov, 1956; Vuataz et af., 1959; Millin and Rustidge, 1967) because it is this portion of the tea bush which is of economic importance. A more general picture of the chemical composition of the tea bush is given in Table I1 which describes the distribution of various constituents in mature leaves, green and mature stem, roots, and seeds, in addition to the flush. Flush contains high levels of polyphenols, amino acids, and caffeine, while mature leaves are rich in carotenoids. The distribution of saponins, nucleotides, nonsaponifiable material, lipids, and carotenoids has been the subject of recent research, which is described in some detail in the following pages of this review.

TABLE I1 DISTRIBUTION OF CHEMICAL COMPOUNDS IN THE TEA BUSH"

Compound Polyphenols Amino acids Nucleotides Phosphate esters Caffeine, theobromine Carbohydrates Lipids Organic Acids Chlorophyll Carotenoids Unsaponifiables Saponin Minerals Volatile compounds

Flush

Mature leaf

Green stem

++ ++ +

+ +

+ +

+

+

ND"

++ + + f + + + +

+ + + + + ++ + +

+

+

+

+

+

+

+

Mature stem

Root

Seed

+ + +

+ ++ +

ND

ND

+ +

+ +

ND

ND

+

+ + +

-

ND

+ + + + + + +

+ +++ + +

Signs denote relative distribution of each compound; from Wickremaqinghe (1978). ND = not determined.

-

+ + ND -

+ +++ + +

TEA

A.

233

CHEMICAL AND BIOCHEMICAL CONSTITUENTS

I. Enzymes a . Tea Leaf Polyphenol Oxidasr. Tea leaf polyphenol oxidase (0-diphenol : o2 oxidoreductase I . 10.3.1) plays an important role in black tea manufacture (see

Section 111) and has been the subject of intensive research by several investigators. Sreerangachar (1943a,b) established that polyphenol oxidase was a copper-containing protein, and some years later the enzyme was purified and found to consist of at least four isoenzymes (Bendall and Gregory, 1963; Gregory and Bendall, 1966), of which the major component had a molecular weight of 144,000 5 16,000 and contained 0.32% (wlw) of copper. The enzyme was considered to be insoluble by these and previous workers, but Sanderson (1964a, 1965) isolated polyphenol oxidase in a soluble state by the inclusion of polyphenol adsorbents in the extraction medium, showing that the previously assumed insolubility of the enzyme was due to a binding effect of polyphenols and their oxidation products on the enzyme protein. Using an improved method of extraction (Coggon et al., 1973), which included cryomilling the leaf in liquid nitrogen, adjustment of the extraction medium to pH 7.0. addition of Tween 80 according to Takeo (1965a). and passage through Sephadex G 50, a highly active, crude soluble polyphenol oxidase preparation was obtained. This crude soluble preparation was purified over 200-fold, using isoelectric focusing, and shown to have optimum activity at a pH near 5.7 and a K , of 2.3 X IO-’M (at 25°C) for (-)epigallocatchin gallate. The presence of isoenzymes was confirmed in agreement with the previous findings of Bendall and Gregory (1963) and Gregory and Bendall (1966), as well as of Takeo and Uritani (1966), Buzun et af. (1970). Perera and Wickremasinghe (1972), and Takeo and Baker (1973). The last named investigators also found 70% less polyphenol oxidase activity in mature leaves as compared to young tea leaves, possibly due to variation in the relative amounts of the individual isoenzymes. Seasonal variation of polyphenol oxidase activity was reported by Takeo (1966a) who also found a marked increase (2-3 times) of enzyme activity during the earlier stages of black tea processing (i.e., withering, rolling) followed by a decrease during fermentation. Evidence was presented in support of the view that the initial increase was due to enzyme synthesis, and that the decrease of enzyme activity during fermentation was a result of the formation of insoluble complexes of the polyphenol oxidation products with the enzyme protein. Location of the site of polyphenol oxidase in the tea leaf has been the subject of several studies, the majority of which used differential centrifugation to determine the particles with which the enzyme was associated. Li and Bonner ( 1 947) and Oparin and Shubert (1 950) concluded that the enzyme was bound in the chloroplasts; Bokuchava et al. (1970) also found activity in the chloroplast

234

ROBERT L. WICKREMASINGHE

fraction, but observed that there was low activity in the mitochondria as well, while Takeo (1966b) reported that the major part of activity was not in the chloroplast, but bound on the precipitable particles in the range of 1400g to 15000g. Kato et al. (1976) employed sucrose density gradient to study the position of polyphenol oxidase in the particulate fraction separated at 80,OOOg from tea leaf enzyme extracts, freed of polyphenols by the incorporation of Polyclar AT [poly-(vinylpyrrolidone)] in the extraction medium. The centrifugal pattern in the gradient indicated that the position of polyphenol oxidase coincided with those of the markers of peroxisomes, catalase, and malate dehydrogenase, but not with those of mitochondria and chloroplasts, cytochrome c oxidase and chlorophyll. Using fluorescent antibody techniques, Wickremasinghe et al. (1967) concluded that the major portion of the enzyme was localized in the epidermis of the leaf, and that young leaves contained the enzyme in both upper and lower epidermis, whereas in older leaves, the enzyme was apparent in the lower epidermis only.

b. 5-Dehydroshikimate Reductase. The presence of 5-dehydroshikimate reductase (E.C. 1.1.1.25)in tea leaf was demonstrated by Sanderson (1966), who found that specific activity was greater in the growing shoot than in mature leaves. This enzyme plays a key role in the biosynthesis of polyphenols via the phenylalanine pathway (Neish, 1960; Stafford, 1974). c . Phenylalanine Ammonia Lyase. Phenylalanine ammonia lyase activity in tea leaf was found to be related to clone, age of leaf, and shading during growth (Iwasa, 1976), being higher in clones suitable for black tea rather than green tea production, less in mature than young leaves, and reduced by shading. It was also observed that the activity of the enzyme was related to catechin content, implying that phenylalanine played an important role in polyphenol biosynthesis.

d . Tea Leaf Peroxidase. The occurrence of an insoluble form of peroxidase in tea leaf homogenates was detected in the early years of biochemical studies of tea leaves by Bokuchava (1950) and Roberts (1952). Subsequently, the enzyme was isolated in a soluble form by the addition of Tween 80 (Takeo and Kato, 1971) or Polyclar AT (Tirimanna, 1972), when it was found to exist in the form of several isoenzymes, the activity of one of which was found to increase during processing to black tea (Tirimanna, 1972). Ethylene and iodoacetate also caused increased peroxidase activity, and column chromatographic studies indicated that these two compounds enhanced the activity of different isoenzymes (Saijo and Takeo, 1974). However, although tea leaf peroxidase has been studied in some detail, its exact effect on tea processing is uncertain. e . Tea Leaf Peptidase. Peptidase activity in tea shoot tips was investigated by Sanderson and Roberts (1964) who developed a procedure for its assay in cell-

'TEA

235

free preparations. This enzyme is responsible for the breakdown of protein to amino acids during the withering stage of black tea manufacture, and was found to show marked difference in activity in the various parts of the shoot tip and in different clones of tea.

f. Tea Leaf Chlorophyllase. Studies of tea leaf chlorophyllase are limited to those of Ogura and Takamiya (1 966) and Ogura ( 1 969). The latter investigation indicated that there was a seasonal variation in enzyme activity, which was inversely related to the content of chlorophyll. This finding may play a part in determining the proportions of pheophytin and pheophorbide in processed black tea as described later. g. Acid Phosphatases of Tea Leaf. Multiple forms of acid phosphatase were detected in tea leaves (Tirimanna, 1967; Baker and Takeo, 1974) but no specific function has, as yet, been assigned to this enzyme.

h. Leucine- a-Ketoglutarate Transaminase. An enzyme which catalyzes transamination between leucine and a-ketoglutardte was detected in tea leaves by Wickremasinghe et af. (1969). and this is considered to be the first step in one of the two routes of biosynthesis of volatile compounds responsible for tea flavor (Wickremasinghe, 1974). The enzyme was not specific for leucine and caused transamination of a-alanine and valine as well.

i. Oxidation of Linolenic Acid by Tea Leaf. It has been shown (Gonzales et al., 1972; Saijo and Takeo, 1972) that linolenic acid was converted to trans-2hexenal by macerated tea leaves, and Hatanaka ( I 976) found that incubation of this fatty acid with isolated chloroplasts of tea leaves led to the formation of cis-3-hexena1, which isomerized to trans-2-hexenal. It was suggested by Hatanaka and Harada (1 973) that cis-3-hexenal was formed enzymically in the presence of oxygen; but Coggon et aI. ( 1 977) have recently proposed that nonenzymic, metalloprotein catalyzed oxidation of unsaturated fatty acids may be the mechanism of trans-2-hexenal formation in tea. The relative proportion of this aldehyde in the aroma complex of tea has an important effect on the flavor of tea (Yamanishi et a(., 1968a; Gianturco et al., 1974) and the effect of climate on its formation has been discussed by Wickremasinghe (1974). j . Alcohol Dehydrogenase of Tea Leaj and Tea Seed. The occurrence in fresh tea leaves of alcohol dehydrogenase (alcohol NAD oxidoreductase E.C. 1 . I . 1 . 1 .) which catalyzes the conversion of cis-3-hexenal and trans-2-hexenal to their respective alcohols has been discussed by Hatanaka and Harada (1973). These alcohols. especially their proportions in relation to other volatile constituents, play a role in determining tea flavor (Yamanishi et al., 1968a; Gianturco et al., 1974). Alcohol dehydrogenase was studied in tea seeds as well, from

236

ROBERT L. WICKREMASINGHE

which source it was extracted and purified by Hatanaka et al. (1976a), who determined its molecular weight as 95,000, dissociable to two homogenous subunits by o-phenanthroline. This enzyme was capable of utilizing acyclic terpenes (e.g., nerol, geraniol, citronellol) as substrates, but showed little or no activity toward cyclic terpenes.

k. Pecrin Methylesterase. Pectin methylesterase was detected in tea leaves by Ramaswamy and Lamb (1958) who suggested that the pectic acid, produced by demethylation of pectin, formed a gel which impeded oxygen diffusion, and thereby reduced the rate of oxidation reactions during the processing of tea. Pectin methyl esterase activity was found to be inhibited by polyphenols, and to a greater extent, by their oxidation products. Addition of pectin to tea leaf reduced oxygen uptake during processing (Lamb and Ramaswamy, 1958) and resulted in a tea which was considered by tea tasters as being “underfermented.” 1. Malate Dehydrogenase. The occurrence of malate dehydrogenase in tea leaves has been reported by Morchiladze et al. (1972), who obtained 3 enzyme fractions by DEAE cellulose chromatography and polyacrylamide gel electrophoresis.

m. Tea Leaf Ribonuclease. Two types of endonucleases were separated from crude enzyme preparations of tea leaves by DEAE-cellulose column chromatography (Imagawaet al., 1976), and one of them was further purified by gel filtration. Using similar techniques, Tsushida and Takeo (1976) isolated four fractions showing ribonuclease activity and studied the properties of the two major components. Of these, one was found to have a molecular weight of 13,000 and the other 16,000, the pH optimum being 4.75 and 4.9, respectively. Both were inhibited by cupric and mercuric ions, and were inactivated by temperatures above 50°C. n. Enzymes of Tea Roots. Enzymes detected in tea roots are glutamyl transferase (Roberts and Fernando, 1975), amylase, nitrate reductase, and glutamine synthetase (Roberts and Fernando, unpublished) all of which may play a role in the nutrition of the tea plant.

2. Polyphenolic Compounds Polyphenolic compounds are the quantitatively major component of young tea leaves, and are mainly responsible for the unique character of processed teas (Roberts, 1962). They have been the subject of intensive study for several years and a list and the amounts of polyphenolic compounds identified in fresh tea flush are given in Table 111. Of these, the group of flavanols are oxidized by polyphenol oxidase during processing to black tea, and make an important con-

237

TEA

tribution to the characteristics and color of tea brews (see Section 111, A , 4). In green tea production, on the other hand, the first step is destruction of the polyphenol oxidase either by steaming (Japan) or dry heat (China) in order to arrest enzymic oxidation of the flavanols. One of the fundamental differences between black and green tea is, therefore, the quantitative and qualitative differences in the polyphenols present in the finished product. The polyphenolic composition of tea leaves undergoes changes as the leaf is allowed to mature, and this is one of the reasons why the finest teas are made

TABLE 111 PHENOLIC COMPOUNDS FOUND IN FRESH TEA FLUSH"

Structure No. in text

Flavunols (-)-Epicatechin (-)-Epicatechin gallate (-)-Epicatechin digallate ( - )-Epigallocatechin (-)-Epigallocatechin gallate (- )-Epigallocatechin digallate (+)-Catechin ( +)-Gallocatechin Fluvonols and jlavonol glycosides Quercetin Kaempferol Quercetin-3-rhamnoglucoside (rutin) Kaempferol-3-rhamnoglucoside Quercetin-3-rhamnodiglucoside Myricetin-3-glucoside Flavonei Vitexin 6, 8-di-C-glucosyl apigenin Leucoanthocyanins Acids und depsides Gallic acid Chlorogenic acids (4 isomers) p-Coumarylquinic acids (4 isomers) Theogallin Ellagic acid Totdl polyphenols

Amount in flush (9% dry wt)*

I

1-3

I1

3-6 3-6 9-13 I -2 3 4

I11

IV

v V1 VII VIIl

2-3 XI XI1 Xlll XIV

xv

-5 -

-

-

25-35

Adapted from Sanderson (l972a). instances in which no figures are given quantitative data are not available. I'

* In

238

ROBERT L. WICKREMASINGHE

only from young flush. These changes have been studied by Nakagawa and Torii (1964), Bhatia and Ullah (1968), Forrest and Bendall (1969), and Wickremasinghe and Perera (1973), and the results indicated that as the leaf matured there was a progressive decline in total phenolic material accompanied by an alteration in the proportions of flavanols, relative to each other. Furthermore, those parts of tea flowers, such as sepals, petals, and pericarp, with morphological affinities to the leaf, were found to show a phenolic pattern which was dissimilar to that of organs such as stigma, carpel, anther, and testa (Bhatia and Ullah, 1968). The flavonols and tlavonol glycosides occur in small quantities and have been

I I-)- Epicolechin, R , = R2= H

I V I - I - E p i gollocatcchin;

R = R,= H

I1 1-1- Epicatechin g a l l a t e ,

V (-1-

E p l gollocatechln gallate;

R,= 3 , 4 , 5 - l r i h y d r o a y -

RI= 3,4,5-

benzoyl, R,= ti

trihydroxybenzoyl. R z = H

X I ( - ) - Eplcatechln d q o l l o l e ;

V I 1-1

Epigallocatachin dlQollale ,

R , = R,= 3 , 4 . 5 - trihydroxy-

R

banroyl

lrihydrorybenzoyl

I =

R2= 3,4,5-

OH

OH

VII

(+I-

Calechin

Vlll

1x x

Quercetin; R Koempferol ,

:

OH

R = H

(+I-

Gallocotechin

239

TEA

‘OH

I HO XI

Gallic

acid

XI1

Chlorogenic J c i d , R = O H

Xlll

p - Cournarylquinic a c i d , R = H

0

0

II no

0

HO XIV

Theogallin

XV

Ellapic acid

studied in some detail by Ul’yanova (1963) who developed a method for the quantitative estimation of kaempferol and quercetin glycosides and found that different organs of the tea plant (bud, first leaf, second leaf, third leaf, fourth to sixth leaves, flowers, and petals) contained different amounts of flavonols. It was also pointed out (Ul’yanova and Erofeyeva, 1966) that the vitamin P activity (see Section VII) of tea resided in the flavonol glycoside complex. The flavones of tea have been studied by Sakamoto (1967) who found 19 flavones in green tea infusion, four of which were identified (Sakamoto, 1970) as C-glycosyl flavones, namely, vitexin, isovitexin, and two isomers of 6. 8-di-C-glycosyl apigenin, theiferin A and theiferin B. Among the depsides, theogallin is of particular interest because of its unique occurrence in tea, its relatively high level in tea flush, and its statistical correlation to black tea quality (Biswas et al., 1971). It was first detected by Cartwright and Roberts (1954). and identified as a galloyl quinic acid by Cartwright and Roberts (1955) and Roberts and Myers (1958), while its structure as 3-galloyl quinic acid was determined by Stagg and Swaine (1971). Polyphenols were originally estimated as total oxidizable material by Lowenthal titration (Association of Official Agricultural Chemists, 1960) but this nonspecific method was later superseded by methods similar to those described by Swain and Hillis (1959). Paper chromatography (Bradfield and Bate-Smith, 1950) and column chromatography (Vuataz et al., 1959) have also been used to separate and estimate the amount of individual polyphenols, and more recently Collier and Mallows (197la) employed gas-liquid chromatmraphy of the

240

ROBERT L. WICKREMASINGHE

trimethyl silyl ethers of flavanols for this purpose. The method of choice for analysis of the polyphenols, as well as other nonvolatile constituents, of tea would, however, appear to be the reverse phase high-pressure liquid chromatography technique, which has been very recently developed by Hoefler and Coggon (1976). Studies on biosynthesis of polyphenols indicate that this may occur in the tea leaf by alternate routes, only one of which proceeds by the well-established pathway which involves phenylalanine as an intermediate (Neish, 1960; Stafford, 1974). The identification by Zaprometov (1961) of 0.035% (dry weight basis) of shikimic acid, and presence of the enzymes, 5-dehydroshikimate reductase (Sanderson, 1966) and phenylalanine ammonia lyase (Iwasa, 1974), as well as the incorporation of activity from phenylalanine-14C to flavanols (Iwasa, 1976), collectively indicated that the shikimic acid-phenylalanine-flavanolpathway (Zaprometov, 1962, 1963) does operate in tea leaves. Nevertheless, Zaprometov and Bukhlaeva (197 1) and Iwasa (1976) found that shikimic acid-I4C was more efficiently incorporated into tea leaf flavanols than phenylalanine-14C itself, suggesting the existence in tea leaves of a pathway of flavanol biosynthesis which may not include phenylalanine as an obligatory intermediate. The existence of such an alternate route had also been proposed by Hillis and Ishikura (1970) for the biosynthesis of polyphenols in eucalypt leaves and heartwood, and by Swain and Williams (1970) for sun flower leaf disks. The site in the tea leaf of biosynthesis of polyphenols is unknown, but histochemical studies (Tambiah et a/., 1966; Chalamberidze er a/., 1969) indicated that the polyphenols themselves are localized in the vacuoles of the palisade cells of the leaves, an observation which has been confirmed by electron microscopic examination (Selvendran and King, 1976).

3 . Amino Acids The amino acids present in young tea leaves have been separated by paper and column chromatography and the following amino acids identified: aspartic acid, glutamic acid, glycine, serine, glutamine, tyrosine, threonine, a-alanine, p-alanine, valine, leucine, isoleucine, phenylalanine, lysine, arginine, histidine, trytophan, asparagine, proline, and theanine (Krishnamurthy et a/., 1952; Bhatia and Deb, 1965; Roberts and Sanderson, 1966). Theanine accounted for more than 50% of the total amino acid content and comprised about 1% of the total dry weight of tea leaves. This amino acid which was first found in tea leaves by Sakato (1950) and identified as 5-N-ethyl glutamine (Sakato et al., 1950) is particularly abundant in clones of tea used for processing to green tea, and the finest variety of green tea (Gyokuro or “Pearl Dew”) is grown under conditions of shade and fertilizer application which encourage maximal formation of theanine. In fact, one of the striking differences between green tea and black tea is the much greater content of theanine in the former as compared to the latter.

IEA

24 1

Sakato (1957) believed that theanine content is of prime importance in determining the taste of green tea, and it has been suggested that this amino acid plays a role in protecting enzymes from inactivation by plyphenolic products (Wickremasinghe and Perera, 1973) and as a constituent of the colored thearubigin complex of black tea liquors (Perera, 1972). Theanine is present in highest concentration in the roots of tea plants, but is absent from tea seeds in which the principal amino acid is pipecolic acid (Ozawa et al., 1969). Studies on the biosynthesis of theanine in tea seedling homogenates (Sasaoka and Kito, 1964) provided evidence that the precursors were glutamic acid and ethylamine, and it has been recently shown (Takeo 1974a) that ethylamine could be derived from alanine. Konishi (l969), as well as Wickremasinghe and Perera (l972a), established that the site of biosynthesis of theanine in the tea plant was the root from where translocation occurred to the young leaves. In the young leaves, some degree of incorporation occured of the N-ethyl carbon of theanine into the phloroglucinol nucleus of tea polyphenols (Kito et a/., 1968; Konishi and Takahashi, 19691, but the exact role, if any, of theanine in the tea plant remains to be established. Compounds similar to theanine, namely N-ethyiamide of asparagine (Wickremasinghe and Swain, 1965) and N-methylamide of glutamic acid (Konishi and Takahashi, 1966), have also been reported to occur in small quantities in tea leaves. 4 . Phosphate Esters, Nucleotides and Caffeine

The nucleotides and phosphate esters of tea have been studied by Selvendran (1969), who found that tea shoots contained glucose-6-phosphate as the predominant phosphate ester together with small amounts of fructose-6-phosphate, glucose- 1 -phosphate, glucose- 1,6-di-phosphate and sucrose-6-phasphate. The concentrations of these esters underwent changes during the withering, fermenting, and firing stages of black tea manufacture and these changes, together with those of the tea nucleotides, which were investigated in the same study, are indicated in Table IV. It was observed that all phosphate esters decreased during the withering stage of processing but that, during fermentation, there was an increase of fructose-6-phosphate and glucose- 1 , 6-diphosphate, followed by a further increase of the last named phosphate ester, as well as of glucose-lphosphate, during the firing stage. Sucrose-6-phosphate was found to remain at a constant low level throughout processing. In the case of nucleotides, the triphosphates of cytidine, uridine, and adenosine showed a significant increase during fermentation, but the most striking change was the sharp increase of uridine-3’monophosphate during firing. The diphosphates showed little change during processing of the leaves to black tea. Takino et al. (1972) investigated the composition of nucleotides in green tea and identified the 5’ monophosphates of adenosine and uridine, as major constituents, together with uridine-5’monophosphate and adenosine diphosphate in smaller amounts. These workers also found that there was no difference in distribution pattern in several kinds of

242

ROBERT L. WICKREMASINGHE TABLE IV

PHOSPHATE ESTERS AND NUCLEOTIDES IN TEA, AND THEIR CHANGES IN CONCENTRATION DURING BLACK TEA PROCESSING“

Compound Phosphate Esters Glucose-6 phosphate Fructose-6 phosphate Glucose- 1 phosphate Glucose- I phosphate Glucose- 1, 6-diphosphate Sucrose-6 phosphate Nudeotides Uridine-5 monophosphate Uridine-3 monophosphate Adenosine monophosphate Uridine diphosphate sugars Uridine diphosphate glucose Uridine diphosphate Adenosine diphosphate Cytidine triphosphate Uridine triphosphate Adenosine triphosphate “

Fresh shoot

Withered shoot

Fermented material

Black tea

9.7 I .4 0.6

6.6 0.8 0.5

5.1 1.2 0.2

4.7 1.1 0.7

0.06 0.01

0.01 0.01

0.04 0.01

0.1 0.01

2. I 1.5 0.02 22 7.1 3.8 9.2 5.7 7.0 15.0

3.5 2.7 0.02 15.7 3.4 4.0 9.2 0.1 0.6 0.1

2.8

3.3 36.3 0.02 11.0 4.5 4.7 11.6 ?

0.02 19.6 3.6 4.0 10.2 3.6 2.8 9.4

?

4.7

Values expressed as pmole/l00 gm fresh weight.

green tea. In black tea, however, Takino and Imagawa (1973) found that the nucleotide composition was quite different to that of green tea in that the 2‘ and 3’ isomers of the monophosphates of adenosine, cytidine, and guanosine were detected in addition to the 5’ ribonucleotides, and these workers suggested that this was a consequence of enzymic degradation of ribonucleic acid during black tea processing. Subsequently Imagawa et al. (1976) and Tsushida and Takeo (1976) fractionated crude enzyme preparations from tea leaves to endonucleases, one of which released mainly purine-5’-ribonucleotides,and the other 2’,3‘cyclic nucleotides, when incubated with yeast ribonucleic acid. In this connection, Bhattacharya and Ghosh (1968) noted a decrease in ribonucleic acid during the processing of black tea, and suggested that caffeine was one of the degradation products formed. Caffeine occurs in tea to the extent of 2.5% to 5.5% (dry weight basis) together with much smaller quantities of the dimethylxanthine, theobromine, and the monomethylxanthine, theophylline (Wood et al., 1964). Caffeine plays an important part in determining the taste (Millin et al., 1969a) and “briskness” (Roberts, 1962) of tea beverages, and its mode of formation has been the subject of several studies. Using tea stem caIIus tissue, the results obtained by Ogotuga and Northcote (1970a,b) suggested that caffeine was formed by methylation of purines derived from nucleic acid catabolism. The earlier studies of the biosynthesis of caffeine in tea sprouts and tea leaves (Proisier and Serenkov, 1963) also

TEA 7-Methylxanthine

+

243

3.7-Diniethvlxanth111e + 1.3.7-Triinethylxantl11ne (theobromine) (caffe ine )

1.7-Dimethylxanthme 1-Methylxanthine

/

(paraxanthlne)

1.3-Dimethylxanthine (theophylline)

FIG. 1. Scheme of blosynthesis of caffeine. (Proposed by Suzuki and Takahashl, 197Sb.)

indicated that the mechanism involved methylation of purines, and Konishi et al. ( 1 972) found that the N-methyP4C of 14C-glutamylmethylamide was incorporated into caffeine. In a more detailed study, Suzuki and Takashashi (1975a) demonstrated the conversion in tea shoots of hypoxanthine and, to a lesser extent, xanthine, to theobromine and caffeine. They also found (Suzuki and Takahashi, 1975b, 1976) that extracts of tea leaf contained 2 methyltransferase activities catalyzing the transfer of the methyl group from S-adenosylmethionine to 7-methylxanthine to form theobromine, and to theobromine to form caffeine. On the basis of their findings these workers proposed that caffeine was not a degradation product of ribonucleic acids but the result of methylation of purine nucleotides in the nucleotide pool, as depicted in Fig. 1.

5 . Carbohydrates The distribution of carbohydrates in tea flush and tea roots has been summarized by Sanderson (1972a). Studies of the polysaccharide contents of root wood, root bark, stem wood, and stem bark (Selvendran and Selvendran, 1972) indicated that starch was the main reserve polysaccharide and that starch content (dry weight basis) was highest in root wood. The polysaccharide fraction extracted from the ethanol-insoluble material of tea flush was investigated by Selvendran and Perera (197 1) who determined the percentage composition of hot water soluble polysaccharides and proteins, ammonium oxalate-soluble pectic acid, sodium hypochlorite-soluble compounds (lignin and structural proteins), hemicelluloses A and B, and a-cellulose. It was also found (Selvendran ef al., 1972) that maturation of tea leaves was accompanied mainly by an increase in the contents of lignin, hemicelluloses, and a-cellulose. 6 . Lipids Studies (Roberts, 1974) of the polar lipids of tea leaves and seeds indicated that young leaves contained a higher concentration of phosphatidyl ethanolamine and phosphatidyl choline than mature leaves, whereas monogalactosyl diglyceride and digalactosyl glyceride were predominant in the mature leaves, and phosphatidyl choline was the major lipid in tea seeds (Table V). Tea seeds were

244

ROBERT L. WICKREMASINGHE TABLE V THE CONCENTRATION OF POLAR LIPIDS IN PARTS OF THE TEA PLANT""

CompoundC

Young shoots

Coarse leaf

Seed kernel

7.62 (34) 1.72 ( 8) 3.45 (16) 1.87 ( 9) 3.34 (IS) 2.71 (12) 1.17 ( 5)

1.39 (16) 1.66 (20) 0.97 (1 I ) 0.26 ( 3) 3.77 (45) 0 0.41 ( 5)

~~

1

2 3 4

5 6

MGG PE DGG PG PC SL PI

2.78 (18) 2.16 (14) 1.58 (10) 1.33 ( 8) 4.66 (31) 1.38 ( 9) 1.40 ( 9)

Values expressed as p n o l e lipid/gm fresh weight. Figures in parentheses represent the percentage of total polar lipid content in that tissue. According to Roberts (1974). MGG, Monogalactosyl diglyceride; PE, phosphatidyl ethanolamine; DGG, digalactosyl diglyceride; PG, phosphatidyl glycerol; PC, phosphatidyl choline; SL, sulfolipid; PI, phosphatidyl inositol.

found to also contain 18 to 33% (dry weight basis) of nonpolar triglyceride lipids, the composition of which was similar to that of olive oil (Roberts and de Silva, 1972).

7 . Triterpenoids a . Unsaponifable Fraction. In early investigations of triterpenoids in tea, Matsumoto et al. (1955) obtained 0.6 gm a-spinasterol and 2.6 gm P-amyrin from 400 gm of the wax arising as a by-product of the industrial extraction of caffeine from tea leaves. More recently, Itoh et al. (1974) made detailed studies of the unsaponifiable fraction of seed oils from three species of Theaceae, Camellia sasanqua L., Camellia sasanqua Thunb., and Thea sinensis L. They found that the unsaponifiable fraction of tea (Thea sinensis L.) seed oil consisted of triterpene alcohols (59%), less polar compounds (22%), sterols (18%), and 4-methyl sterols (I%), and that this was in contrast to the composition of the majority of vegatable oils in which sterols constituted the major fraction of the unsaponifiables. The triterpene alcohol fraction of tea seed oil was found to contain P-amyrin (36%) as the major component, together with butyrospermol (24%), lupeol (24%), and smaller quantities of other unidentified alcohols. The sterol fraction consisted exclusively of A7-sterols with a-spinasterol (59%), A7stigmastenol (33%), 24-methylcholest-7-en01 (4%), A'-avenasterol (2%), and trace amounts of other unidentified sterols. The relatively small 4-methyl sterol fraction contained citrostadienol, gramisterol, and/or cycloleucalenol, obtusifoliol, and other unidentified components. Analysis of the unsaponifiable fraction of extracts of different segments of the tea bush (Wickremasinghe et al., 1976) showed that a-spinasterol, a-spinasterol glycoside, and P-amyrin occurred in root and stem segments as well as in leaves; oleanolic acid was detected only in the aerial portion of the bush, with epi-

245

TEA

friedelinol and friedelin being confined to the brown portion of the stem. 6 . Saponins. The occurrence of saponin in green tea was reported by Machida (1938) who obtained 4 gm of a compound, mp 223°C (decomp.) from 5 kg of green tea. Saponin was also isolated from steamed tea leaves by Hashizume (1967) who designated the sapogenins formed on hydrolysis as camelliagenin A, barringtogenol C, and barringtogenol R (Hashizume, 1969). Vogel et a l . (1968) ascribed a structure similar to aescin for tea seed saponin, while Yosioka et a l . (1966) described the structure of theasapogenol A. It is evident from these diverse reports that the structure of tea saponins has not, as yet, been fully elucidated, although its triterpenoid nature is well established. The component sugars of tea seed saponin have been described by Mizuno (1968), and the structures, taste, and physiological activities of tea saponins reviewed by Hashizume ( 1 970). The distribution of saponin activity in various segments of the tea bush at periods of 6, 10, 14, and 18 months after the bush had been pruned was investigated by Wickremasinghe et a l . (1976). The results (Table VI) indicated very high saponin activity in the roots and a progressive decline toward the aerial portion of the bush, as well as variation of saponin activity at different periods after pruning. This distribution of saponin activity plays a role in conferring resistance to infestation of tea by the insect pest, Xyleborusfornicatus, as discussed later (see Section VIII).

8.

Chlorophyll and Carotenoids

a . Chlorophyll. The amount of chlorophyll in tea leaves is dependent on climatic factors and clonal characteristics (Wickremasinghe et al., 1966). The initial chlorophyll content of the leaf and the extent of production of enzymic TABLE VI SAPONIN ACIIVITY IN SEGMENTS OF TEA BUSHES AT PERIODS OF 6-18 MONTHS AFTER PRUNING"

Months after pruning Segment

6

10

14

18

Feeder root Base of root Upper root Base of stem Mid-stem Upper stem

2048 1024 512 64 32 8

1024 1024 512 32 8 4

I024 I024 512 8 8 8

2048 1024 1024 256 16 8

" Saponin activity expressed as highest dilution of aqueous extract of segment, which caused hemolysis of rabbit red blood cella.

246

ROBERT L. WICKREMASINGHE

breakdown products, such as chlorophyllide, during processing are factors which affect the desired appearance of processed green and black teas. The principal changes undergone by chlorophyll during black tea processing are depicted in Fig. 2; it was found (Wickremasinghe and Perera, 1966a) that the formation of pheophorbide detracted from the appearance of black tea, whereas pheophytin made a positive contribution. Saijo (1970) reported that chlorophyllides and pheophorbides occurred in fermented tea leaves and that pheophytins were formed on heating. Nikolaishvili and Adeishvili (1966) reported a marked decrease in chlorophyll content of green tea leaves during processing to black tea, while Blanc (1972) and Sanderson (1972a) found that this decrease of chlorophyll was due to its transformation to pheophytin. b. Carotenoids. Tirimanna and Wickremasinghe (1965) identified 14 carotenoid compounds in tea flush and observed that total carotenoid content decreased during processing to black tea. Nikolaishvili and Adeishvili (1966) also found a decrease in yellow pigments of tea leaf from 0.731 mglkg to 0.320 mg/kg (dry weight basis) in the black tea derived therefrom. Sanderson and Gonzalez (1 97 1) identified neoxanthin, violaxanthin, lutein, and @-carotene as major components, and showed that @-carotene was degraded to @-ionone and other compounds when dried in the presence of oxidized flavanols in a model system. The distribution of carotenoid pigments in tea leaves has been studied by Venkatakrishna et al. (1977) whose findings are summarized in Table VII. The results indicated that mature leaves contained a higher content than young leaves of carotenes and xantophylls, and that there was a very marked increase of @-carotene and lutein + zeaxanthin during leaf maturation. 9.

Minerals

The average total ash content of tea is around 5% of the dry matter, and it was observed (Hasselo, 1965) that the nitrogen, potassium, phosphorus, sodium, and copper contents of tea leaves decreased with increasing age of the leaves, whereas calcium, magnesium aluminum, manganese, iron, boron, and molybdenum were higher in old than young leaves. In an investigation of the aluminum status of tea leaves, Chenery (1955) found that young leaves contained 108 ppm of this element, increasing to between 5000 and 16,000 ppm (dry weight basis) in mature leaves, and that the

,

chlorophyilase+

Chlorophyllide

Pheophorbide (brown)

Chlorophyll

Pheophytin

(black)

FIG. 2. Changes undergone by chlorophyll during processing of black tea.

247

TEA TABLE VII CAROTENOID ANALYSIS OF TEA-Thcii

Carotenoids Total Hydrocarbons Xanthophylls Hydrocarbon: Xanthophyll @Carotene: Lutein zeaxanthin Phytofluene a-Carotene p-Carotene

1 E t leat

2nd leaf

3rd and 4th leaf

Mature leaves

25.42 6.91 17.51

35.8

41.3

10.72 23.80

10. I 1

30.24

I 26.08 53.68 72.20

1:2.5

1:2.22

1:2.99

1:1.34

1:2.3

I 3.28

1:3.20

1:1.37

Traces

Traces

Traces 0.I5

Trdces

0.15

(0.59) 6.24 (24.52)

/3-Zeacarotene

0.38

.$-Carotene

(1.49) 0.I4

Mutatochrorne

ui~ornii~~"

(0.55) Traces

0.24 (0.66) 6.72 (18.74) 3.52 (9.82) Traces

0.16 (0.44)

Aurochrome Cryptoxanthin

Cryptoxanthin-5,6-rnonoepoxide Cryptoxanthin-5.6-diepoxide Cryptoxanthin-5 &diepoxide Lutein and zeaxanthin Lutein epoxide Violaxanthin Luteoxanthin Neoxanthin

Traces 0.53

(2.10) 0.72 (2.83) Traces

(56.59) Traces

1.53 (6.01) 0.24 (0.94) 0.09 (0.35)

0.08 (0.22) 0.20 (0.55)

0.12 (0.33) Trace\

(64.39) 0.46 (1.28) 0.48 (0.50)

0.16 (0.44)

0.30 (0.83)

(0.36) 8.02 ( 19.40) 1.56 (3.77) Traces 0. in

(0.43) 0.20 (0.48) 1.05

(2.54) 0.I6 (0.38) 1.01

(1.08) (0.29) (62.96) 0.52 (1.25) 0.46 (1.11) 0.15

0.23

(0.in)

49.86 (39.38) 0.61 (0.48) 0.64 (0.50) 1.47 (1.16) 0.87 (0.68) 1.20 (0.94) 0.10

(0.07) 0.78 (0.61) (0.47) (53.98) 0.7 (0.55)

0.04 (0.03)

(0.36) 0.75

0.I8 (0.14) 0.26

(1.80)

(0.20)

" Value> indicate rng/100 grn dry wt; the value$ in parentheses indicate percentage of the total carotenoids. Data of Venkatakrishna ct ul. (1977).

level was also determined by the genetic structure of the plant, rainfall. altitude of growth, and soil conditions. The fluoride content of tea is also unusually high, being about 150 ppm for black tea and more than twice this level for green tea (Singer et al., 1967). The

248

ROBERT L. WICKREMASINGHE

major mineral component of tea is, however, potassium, which accounts for about 50% of the total mineral content (Sanderson et al., 1976), and deficiency of this element leads to defoliation and death of the tea bush (Portsmouth, 1953). Deficiency of zinc has been found (Tolhurst, 1962) to lead to decline of yield, and the spraying of tea with zinc is now a standard practice in tea-growing countries. Symptoms produced in tea leaves of deficiencies of nitrogen, potassium, calcium, magnesium, and sulfur have been described (Pethiyagoda and Krishnapillai, 1970) and this study was extended to include the effect on leaves of deficiencies of the minor elements, manganese and boron (Pethiyagoda and Krishnapillai, 197 1). 10. Volatile Compounds The mixture of volatile compounds present in tea constitutes only about 0.01% of its dry weight but plays an important role in determining the overall aroma of the brew. More than 300 compounds have been identified in this mixture, and the characteristic aroma of a tea depends on the correct balance between the proportions of certain key compounds (Wickremasinghe et al., 1973; Kozhin and Treiger, 1973), the nature of which is described in Section IV. Subsequent to the compilation of a list of the compounds identified as constituents of tea aroma (Yamanishi, 1975; Sanderson, 1 9 7 3 , several other compounds (Table VIII) have recently been found to be also present as constituents of the volatile fraction of black tea (Renold et al., 1974; Vitzthum et al., 1975). However, in spite of detailed analysis of the tea aroma complex, there are still a number of constituents which remain unidentified. Furthermore, tea aroma is not one single entity, but a complex characteristic which is dependent on location of growth of the tea bush (Yamanishi et al., 1968a), climate (Wickremasinghe, 1974), genetic constitution of the tea clone, rate of fertilizer application, conditions of processing, and a number of other factors. Green tea flavor is distinct from black tea flavor, and the flavor of a Darjeeling black tea is distinct from that of a black tea from the Dimbula District of Sri Lanka. Nevertheless all of these teas have a qualitatively similar profile of volatile constituents (Yamanishi et al., 1968b), and it would appear that it is the balance between volatile constituents that determines the flavor of tea. B . FACTORS AFFECTING CHEMICAL COMPOSITION 1.

Climate

Ramaswamy (1964) reported that the composition of black tea liquors was related to the weather conditions (wet or dry) prevailing at the time the tea was produced, as well as to the elevation of growth of the tea bush. In an analysis of a limited number of liquors, the level of soluble solids and nitrogenous substances was found to be higher during the dry season than the wet season; mineral

TEA

249

constituents showed the opposite trend. With respect to the effect of elevation, the level of theaflavins and oxidizable matter was higher in liquors obtained from high- than low-grown tea, whereas nitrogenous constituents and mineral content were lower. These changes in liquor composition with climate are related to the composition of the fresh tea flush, and Evans (1930) reported that there were marked seasonal variations of total soluble solids, tannin, total soluble nitrogen, and total nitrogen in fresh tea leaf; these findings were confirmed and extended by subsequent workers (Sanderson, I964b; Sanderson and Kanapathipillai, 1964; Wickremasinghe ct al., 1966) who studied the variations with season of minerals, pectin, polyphenols, oxidase activity, amino acids, and chlorophyll. Gianturco et a/. (1 974) studied the seasonal variations of the volatile constituents of black teas from three different locations over a period of fifteen months, and found a correlation between their composition and the quality of tea aroma. In a study of the effect of shade on the chemical composition of tea flush, Hilton ( I 974) found that 25% shading of clonal tea bushes increased the proportion of gallated to nongallated flavanols and also increased polypbenol oxidase activity. Anan and Nagakawa (1 974) observed that shading of developing tea shoots with several sheets of black net for 13 to 25 days had no effect on the content of (-)epicatechin and (-)epigallocatechin, which however, showed a gradual increase in the unshaded shoots. They also observed that total amino acids and caffeine increased on shading. Extensive studies have been carried out in Tanzania on the effect of irrigation on the yield of tea (Carr, 1970, 1974) and it was found that irrigation during the long dry season doubled the annual yield of tea and, to some extent, evened the yield over the year. In Malawi experiments, Ellis (1976) obtained a yield increase of 30% over unirrigated tea, but considered that this return was uneconomic in relation to the high cost of irrigation. Devanathan (1975) found a correlation between yield of tea and the product of rainfall and average daily hours of sunshine of the previous month. This correlation was subsequently refined by inclusion of factors for variations in soil and the temperature coefficient of photosynthesis (Devanathan, 1976).

2 . Clone The differing genetic constitution of the various clones of tea would predictably influence the chemical composition of the tea flush produced. Studies have been made of the clonal variations in respect of polyphenol oxidase activity, individual polyphenols, individual amino acids, and chlorophyll of green leaf (Tocklai Exp. Sm., 1974). Analyses of flush from 11 Sri Lanka clones for ash, total flavanols, caffeine, total nitrogen, and polyphenol oxidase activity (Sanderson, 1964b) indicated that there was some intercloncal variation, and that there appeared to be a relationship between the quality potential of a clone, polyphenol oxidase activity, and ash content-this relationship was not, however, perfect.

250

ROBERT L. WICKREMASINGHE T A B L E VIlI NEWLY IDENTIFIED VOLATILE CONSTITUENTS OF BLACK TEA“

1. ALDEHYDES cis-3-pentenal trans-2-heptenal trans-2-nonenal trans-2-decenal rrans-2-undecenal trans-2, cis-4-hexadienal trans-2, trans-4-octadienal trans-2, cis-4-octadienal trans-2, rrans-4-nonadienal trans-2. cis-4-nonadienal trans-2, cis-6-nonadienal rrans-2, cis-4-decadienal neral P-cyclocitral safranal 2-methylbenzaldehyde 4-methoxybenzaldehyde

4-methyl-2-phenyl-2-pentenal 5-methyl-2-phenyl-2-hexanal 4-ethyl-7, 1 1 -dimethyl-trans-2, trans-6, 10-dodecatrienal 4-ethyl-7, I I -dimethylrrans-2. cis-6, 10-dodecatrienal 2. KETONES 2-heptanone 5-isopropyl-2-heptanone 2-octanone 3-octanone trans-3, cis-5-octadien-2-one 2-nonanone 6, 10-dimethyl-2-undecanone benzyl ethyl ketone 2. 6, 6-trimethylcyclohex-2-enI -one 2, 6, 6-trimethylcyclohex-2-en1, 4-dione P-damascenone a-damascone P-damascone I , 5, 5, 9-tetramethylbicyclo [r. 3. 01 non-8-en-7-one 3. ESTERS hexyl formate rrans-2-hexenyl formate cis-3-hexenyl formate

trans-2-hexenyl acetate ethyl phenylacetate hexyl phenylacetate trans-2-hexenyl propionate trans-3-hexenyl propionate cis-3-hexenyl propionate hexyl butyrate trans-2-hexenyl butyrate trans-3-hexenyl butyrate benzyl butyrate cis-3-hexenyl 2-methylbutyrate rrans-2-hexenyl hexanoate cis-3-hexenyl trans-2-hexenoate trans-3-hexenyl cis-3-hexenoate methyl octanoate ethyl octanoate methyl trans-dihydrojasmonate 4. MISCELLANEOUS 2-ethyl- 1-hexanol 4-terpineol 4-methyl-5-hexen-4-olide carvacrol thymol 2-acetylfuran safrole 2, 6, 10, 10-tetramethyl-I-oxa-spiro [4, 51 dec-6-ene (“theasprane”) 6, 7-epoxy-2, 6, 10, 10-tetramethylI-oxa-spiro [4, 51 decane (“6, 7epoxy -dihydrotheaspirane ”) 6-hydroxyl-2, 6, 10, I O-tetramethyl1-oxa-spiro [4, 51 decane (‘ ‘6-hydroxy-dihydrotheaspirane”)

phenylacetic acid trans-geranic acid trans-2-octenoic acid 5 . PYRIDINES Pyridine 2-Methylpyridine 3-Methylpyridine 4-Methylpyridine 2-Ethy lpyridine 3-Ethy lpyridine 2.6-Dimethylpyridine (continued)

25 1

TEA

TABLE VIII-(continued) 2-Methy lbenzothiazole 8. QUINOLINES 2-Methy lquinoline 6-(or 7-)Methylquinoline 2,6-Dimethylquinoline 2,4-Dimethylquinoline 4.8-Dimethylquinoline 3-n-Propylquinoline 4-n-Butylquinoline 9. AROMATIC AMINES Aniline N-Methylaniline N-Ethylaniline o-Toluidine N.N-Dimethy lhenzylamine 10. AMIDES N-Ethy lacetamide N-Ethylpropionamide 1 1, MISCELLANEOUS Benzoxazole I .4-Diacetylhenzene I .3-Diacetylhenzene 2,4-Dimethylacetophenone p-Ethy lacetophenone 2.4-Dimethy lpropiophenone p-Ethylpropiophenone 3,4-Dimethoxyacetophenone

2SDimethylpyridine 2-Methyl-6-ethy lpyridine 2-Methyl-Sethylpyridine 3-Methoxypyridine 4-Vinylpyridine 2-Acetylpyridine 2-n-Butylpyridine 2-Phenylpyridine 3-Phenylpyridine 6. PYRAZINES Methylpyrazine 2.6-Dimethylpyrazine 2.5-Dimethylpyrazine 2.3-Dimethylpyrazine Ethylpyrazine 2-Ethyl-6-methylpyrazine 2-Ethyl-5-methylpyrazine Trimethylpyrazine Tetramethy lpyrazine 2-Ethyl-3.6-dimethylpyrazine 7. THIAZOLES 2,4-Dimethylthiazole 2,5-Dimethylthiazole 5-Methylthiazole 2,4,5-Trimethylthiazole 2,5-Dimethyl-4-ethylthiazole Benzothiazole ~

Groups 1-4 identified by Renold

cf

ti/.

(1974) and 5-1 I by Vitzthum

el a1

(1975).

Ill. CHANGES DURING THE PROCESSING OF TEA A.

BLACKTEA

The starting material of black tea processing is the young tea shoot, consisting ideally of the terminal bud and two adjacent leaves, which are generally handpicked by teams of experienced pluckers. In some countries the high cost of manual labor has led to the development of devices for mechanical plucking, but this practice suffers from the drawback that more mature leaves are included in the harvest, with a resulting loss in quality of the processed black tea. One of the reasons for this loss of quality is the progressive decline with leaf maturity of polyphenol oxidase activity, polyphenols, caffeine, and amino acids (Wickremasinghe and Perera, 1973).

252

ROBERT L. WICKREMASINGHE

The processing of tea flush to black tea comprises the following stages: withering, preconditioning, rolling, fermentation, firing, and grading.

I. Withering Withering is generally accomplished by thinly spreading the flush on tats, or the more recent practice of loading it into troughs. The loss of moisture may be hastened by blowing hot air through the “withering loft,” and withering is allowed to proceed for a period of 8 to 18 hours, during which time the moisture content of the leaf drops to between 60% (“soft” wither) and 50% (“hard” wither), and the leaf acquires a “kid glove” feel. It was believed for many years that physical conditioning of the leaf was the only purpose of withering, but it is now known that several chemical changes (Table IX) occur during this stage of processing. All of these changes with the exception of the increase in cell wall permeability are independent of moisture loss during withering (Sanderson, 1968) and play an important part in improving the character of the finished product.

2 . Preconditioning Preconditioning of withered tea leaf consists of rolling the leaf for 10 to 15 minutes without the application of any pressure-the action is similar to lightly rubbing the leaf between the palms of one’s hands, which was the traditionally used method, still employed in a few countries. The purpose of preconditioning is to impart the desired “twist” and compactness to the leaf (Keegel, 1958) and also to make available the maximal amount of polyphenol oxidase for fermentation (Wickremasinghe, 1978). This enzyme was found to be located in discrete cells of the epidermis of the tea leaf (Wickremasinghe et af., 1967) and light rolling results in disruption of the separating walls and the production of a homogenous layer of enzyme. Rupture of this layer at even one point during the subsequent state of rolling under pressure would, therefore insure full utilization of the leaf complement of polyphenol oxidase, which plays a central role in the following stages of processing.

3 . Rolling The purpose of rolling is to macerate the leaf in order that the contents of the leaf (such as enzymes and their substrates) may be intimately mixed. There are several machines which have been developed for this purpose, but the so-called conventional or orthodox roller (Keegel, 1958), the rotorvane, and the C.T.C. (Crushing, Tearing, Curling) machines, or the use of these machines in various combinations, are the most popular in present-day black tea processing. During rolling some of the changes initiated during withering and preconditioning proceed at an accelerated rate, consequent to the rise in temperature caused by frictional forces. Additionally, new reactions that are of importance

253

TEA TABLE IX CHANGES DURING THE WITHERING STAGE OF BLACK TEA PROCESSING

Importance to black tea characteristics

Nature of change Formation of amino acids

Precursors of compounds determining flavor and extent of nonenzymic browning reactions

Formation of keto acids Formation of mevaionic acid

Precursor of compounds determining flavor Precursor of compounds determining flavor

Formation of caffeine

Pharmacological activity and taste of tea Improves fermentation

Increase of polyphenol oxidase activity Breakdown of chlorophyll

Affects appearance

Increased cell wall permeability

Enhances efficient mixing of reactants during fermentation Unknown

Increase of organic acids Breakdown of polysaccharides

Unknown

Reference Roberts and Wood (1951); Bhatia and Deb (1965); Roberts and Sanderson (1966); Wickremasinghe ( I 978) Wickremasinghe (1964) Wickremasinghe and Sivapalan ( 1966) Wood and Chanda (1955); Stagg and Millin (1975) Takeo ( 1966a) Wickremasinghe and Perera ( 1966a) Sanderson ( 1968)

Sanderson and Selvendran (1965) Sanderson and Perera ( 1965)

for black tea character are themselves initiated because of more intimate mixing of the leaf constituents. These reactions continue during rolling and are allowed to proceed to the desired stage in the fermentation stage of tea processing. The nature of these reactions is described in the following section. 4.

Fermentation

The designation of this stage of black tea processing as fermentation is a misnomer, stemming from the erroneous view, held at one time, that the changes occurring at this stage were mediated by microorganisms. The procedure for fermentation is to pile the leaf, which has already been macerated by rolling, in a layer 5 to 7.5 cm thick, and let it stand at room temperature for periods of time varying from 45 minutes to 3 hours, depending on the qualities being sought for in the processed black tea. Some of the reactions occurring during fermentation are enzyme-catalyzed, and of these the more important are the oxidation of tea

254

ROBERT L. WICKREMASINGHE

flavanols by polyphenol oxidase, which leads to the development of color, strength, and quality in tea brews, and the occurrence of reactions responsible for the characteristic aroma of black tea. a . Tea Flavanol Oxidation and Development of Color, Strength, and Quality during Fermentation. Following the pioneering studies of Roberts and his coworkers (Roberts, 1962), it became clear that the formation of substances known as theaflavins and thearubigins was one of the central reactions which occurred during the fermentation stage of black tea processing. Roberts (1958) using a model tea fermentation system demonstrated that theaflavins, gallic acid, and a number of unidentified substances were formed on incubating an acetone-dried powder of fresh tea flush (Roberts and Wood, 1951) with an extract of dried flush containing a mixture of (-) epigallocatechin, (+) gallocatechin, (-) epicatechin, (+) catechin, (-) epigallocatechin gallate, and (-) epicatechin gallate together with small quantities of flavonol glycosides, leucoanthocyanins, chlorogenic acids, andp-coumarylquinic acids, but no caffeine, sugars, or amino acids. In this model system there was no detectable formation of thearubigins (as occurred during fermentation of tea flush), and this lack of thearubigin formation was ascribed to the comparatively low concentration of reagents in the system studied. Sanderson et al. (1972) also studied the oxidation, in model systems, of the major flavanols found in tea leaves by partially purified soluble polyphenol oxidase preparations (Co and Sanderson, 1970). Their results confirmed the earlier finding of Roberts and Wood (1950) that epigallocatechin and its gallate were oxidized more readily than the epicatechins, and also showed that theaflavins and thearubigins are formed only in those fermentation systems which contained the appropriate combinations of flavanols. Based on the results of this investigation, the authors proposed that theaflavins are formed according to Eqs. (1) to (4), which are consistent with the reaction proposed by Takino et al. (1964) for the formation of theaflavins. ( I ) epicatechin + epigallocatechin + i)O, + theaflavin (XVI) + CO, ( 2 ) epicatechin + epigallocatechin gallate + 0, + theaflavin gallate A (XVII) + COP (3) epicatechin gallate + epigallocatechin + +02+ theaflavin gallate B (XVIII) + COP (4) epicatechin gallate + epigallocatechin gallate + 0, theaflavin digallate (XIX) +

+

co,

+

--f

In earlier studies Bryce et a f . (1970) and Coxon et a f . (1970a) had isolated and fully characterized the three theaflavin gallates proposed in these reactions. Of the compounds formed during tea fermentation, it may be considered that theaflavins play a premier role in determining the characteristic cup quality of black tea brews (Roberts, 1962; Hilton and Ellis, 1972) and considerable attention has therefore been paid to determining the structure of theaflavins. Roberts et al. (1957) were the first to isolate theaflavin from black tea liquors, and suggested that it was derived from one molecule of (-)epigallocatechin plus one molecule of ( - ) epigallocatechin gallate. However, Dzemukhadze et al. (1957, 1964) found that all catechins decreased during processing of tea flush to black

255

TEA

OH XVI

Theofloum.

R , = R, = H

XVll

Theoflovln gallale A , R , = 3 . 4 . 5 i r i h y d r a x y b e n z a y l , R,: H

Xvlll

Theaflovin pollate B ,

XIX

R, =H

0

,

OH

R , = 3 . 4 , 5 - lrihydrorybenzoyl Theaflavin digallole. R , = R 2 = 3.4.5trihydroiybenzoyl

XX

OH

Irotheoflovin

tea, and Vuataz and Brandenberger (1961) and Bhatia and Ullah (1961, 1965) confirmed that epicatechin gallate decreased during tea fermentation. Takino et al. (1964) next demonstrated that a mixture of (-) epicatechin and (-) epigallocatechin was oxidized with either a polyphenol oxidase preparation, or potassium ferricyanide in a bicarbonate solution, to give a compound which had the same properties as theaflavin. Shortly afterward, Takino and Imagawa (1964) established that the compound formed was identical to that isolated from black tea by Roberts ( 1 958). Takino et al. ( I 964) proposed structure XVI for theaflavin, the configuration of which was determined by Takino et al. (1965) and by Brown ez al. (1966). More intensive studies on separation and characterisation of theaflavins have led to the identification of isotheaflavin ( X X ) (Coxon er al., 1970b) as well as of epitheaflavic acid (XXI) (Coxon et al., 1970c; Bryce et al., 1972) and epitheaflavic acid-3-gallate (XXII) (Bryce et al., 1972). It is evident, therefore, that the theaflavin fraction of black tea consists of a number of benzotropolone derivatives, the approximate relative proportions of which were determined (Coxon et al., 1970a) as being 8% theaflavin gallate A, 20% theaflavin gallate B, 40% theaflavin digallate, and 4% isotheaflavin, together with epitheaflavic acids. The commonly used photometric method for the quantitative estimation of total theaflavins in tea liquors (Roberts and Smith, 1961, 1963) gives values in the range 0.3 to 1.8% of the dry weight of black tea, amounting to about 1 .O to 5.9% of tea solids in a cup of brewed tea. More recently individual theaflavins have been separated from theaflavin gallate by gel filtration using Sephadex L H 20 (Lea and Crispin, 1971), and by gas-liquid chromatography of their trimethyl silyl ethers (Collier and Mallows, 1971b). Apart from the theaflavins which impart the properties of quality and brightness of color to tea liquors, (Roberts, 1962), the group of thearubigins (Roberts, 1958) also makes an important contribution to the color, strength (Roberts and

256

ROBERT L. WICKREMASINGHE

OH XXI

Epitheaflavic a c i d , R = H

XXll

Epitheaflavic acid - 3 ' - g a l l a t e ,

R = 3 , 4 5 - trihydroxybenzoyl

Smith, 1963) and mouthfeel (Millin et a l . , 1969a) of tea liquors. Thearubigins are a heterogenous group of compounds which are estimated according to the method described by Roberts and Smith (1963). They constitute about 10 to 20% of the dry weight of black tea (Roberts, 1962) and comprise 30 to 60% of the solids in brewed tea liquors. Roberts (1962) obtained thearubigin fractions (Sl, S1,, Sl,) by solvent extraction and found that their absorption spectra differed markedly from those of the theaflavins. He proposed that they were derived from theaflavins by oxidation leading to destruction of the benzotropolone nucleus. Vuataz and Brandenberger (1961), however, detected the presence of nitrogen in the thearubigins extracted with 80% ethanol-water and obtained fourteen amino acids on acid hydrolysis. Brown e f al. (1969a,b) isolated thearubigins by successive extraction of aqueous extracts of black tea with different solvents, followed by acidification and further extraction with n-butanol. This procedure yielded five main fractions, which yielded cyanidin and delphinidin, or flavan-3-01s and flavan-3-01 gallate, or gallic acid under different conditions of hydrolysis, and it was concluded on the basis of these findings that theambigins are mixtures of polymeric proanthocyanidins containing flavanoid residues. Berkowitz et al. (1971) investigated the oxidation of mixtures of epicatechin and gallic acid, as well as epicatechin gallate and gallic acid in a model tea fermentation system containing a crude soluble tea enzyme preparation, and obtained bright red phenolic compounds, which were identified as epitheaflavic acid (XXI) and its gallate (XXII). The epitheatlavic acids were found to be present in very low levels in black tea, probably because of further oxidation to thearubigins through coupled oxidation with oxidizing tea catechins (Berkowitz et a f . , 1971), as depicted in Fig. 3. From these diverse studies it is apparent that the mode of formation and nature of the theambigins has not, as yet, been satisfactorily resolved, and this is undoubtedly due to the complexity of this group of compounds.

6 . Development of Tea Aroma during Fermentation. The aroma of tea is an important parameter in the commercial valuation of tea, as is evident by the price

257

TEA Catechol oxidase

/I

Epicatechin

(02)

4

Epicatechin

Oxidized epicatechlne

7Thearubigins Epitheaflavic

acid-3'-gallate Gallic acid

Oxidized callie acid

FIG. 3 . Formation of epitheaflavic acid and epitheaflavir-3'-gallate and their transformation to theambigins. (From Berkowitz el a / . , 1971 .)

of tea with flavor being 2 to 3 times higher than that of a tea which is devoid of flavor. The available evidence suggests that black tea aroma develops during fermentation, and Yamanishi et al. (1966a) studied the changes in flavor constituents during the various stages of black tea manufacture and found an increase in almost all constituents during fermentation, especially in the contents of I-penten-3-01, cis-2-penteno1, benzylalcohol, rruns-2-hexena1, benzaldehyde, n-caproic, cis-3-hexenoic and salicylic acids. Saijo and Kuwabara (1967) also observed increases during fermentation of rruns-2-hexen- 1 -a1 and cis-3-hexenoic acid, as well as of n-capronaldehyde, together with a decrease in the amounts of n-hexyl alcohol, cis-3-hexen-1-01, and methyl salicylate. Two primary mechanisms have been suggested for the formation of volatile compounds, the first being dependent on the polyphenol oxidase mediated oxidation of tea flavanols (Sanderson, 1975) and the second on direct biosynthetic reactions (Wickremasinghe, 1974). The first of these is based on the evidence that oxidized flavanols cause oxidative degradation of compounds, notably amino acids, carotenes, and linolenic acid, during fermentation. It has been established (Bokuchava and Popov, 1954; Popov, 1956; Nakabayashi, 1958; Wickremasinghe and Swain, 1964; Saijo and Takeo, 1970a; Co and Sanderson, 1970; Saijo, 1973) that amino acids are transformed to carbonyl compounds in the presence of oxidized flavanols by Strecker degradation according to Eq. 5 . (5) RCHNH,COOH

+0

oxidized

tlavanols

RCHO

+ COO+ NH3

The bouquet of freshly brewed tea may be greatly influenced by the production of carbonyl compounds in this manner, e.g., the formation of phenylacetaldehyde from phenylalanine (Finot et ul., 1967; Saijo and Takeo, 1970b). c . Carotenes. It was suggested that the decrease in carotenes during black tea processing (Tirimanna and Wickremasinghe, 1965; Miiggler-Chavan er al., 1969) may have been due to their conversion to volatile compounds which contribute to tea flavor. This suggestion was examined by Sanderson et al.

258

ROBERT L. WICKREMASINGHE TABLE X BLACK TEA AROMA CONSTITUENTS SUPPOSED TO BE DERIVED FROM CAROTENOID COMPOUNDS',!' ~

Carotenoids found in tea leaves (Tirimanna and Wickremasinghe, 1965) p-Carotene

Secondary oxidation products

Primary oxidation products

Dih ydroactinidiole

+p-Ionone +TAKr

f 2.2-6-Trimethyl

cyclohexanone \r 5,6-Epoxy ionone 2,2,6-Trimethyl-6hydroxycyclohexanone

+

a -Carotene

Lutein

Phytoene

Theaspirone

+ +

Lycopene y-Carotene

Cryptoxanthin

+

Violaxanthin Zeaxanthin ~~

ja-lonone +p-lonone + TAK 3 (3-Hydroxy-/3 ionone)+ + (3-Hydroxya-ionone) + TAK jLinalool TAK jLinalool TAK 3 @-lonone +Linalool +TAK j/3-lonone +(3-Hydroxy-P-ionone) +TAK (3-Hydroxy-5.6-epoxy ionone + TAK 3 (3-Hydroxy-/3-ionone) + TAK

~

~

(Known reaction based on results of this investigation, + ; Highly probable reaction based on results of this investigation, 3 ; Probable reactions, + ; Supposed reactions, j; Compounds shown in brackets have not yet been identified in tea). Data of Sanderson er al. (1 97 1). TAK = Terpenoid-like aldehydes and ketones. Oxidation products of all the carotenoids listed.

(1971) and Kawashima and Yamanishi ( I 973) who obtained evidence that @carotene was converted during black tea processing to p-ionone and other compounds, which included (Reymond, 1976) damascenones, a-ionone, theas-

259

TEA

pirone, and dihydroactinidiolide. Using a model system, Sanderson et al. (1971) found that three basic ingredients, an active tea enzyme preparation, tea flavanols, and P-carotene, were necessary for the production of p-ionone, and the results further indicated that many of the important black tea aroma constituents are probably formed during black tea processing by oxidative degradation of the carotenoid compounds present in the system (Table X). d. Fatty Acids. The formation during black tea processing of trans-2hexenal from linolenic acid has been demonstrated by several workers (Saijo and Takeo, 1972; Gonzales et al., 1972; Hatanaka and Harada, 1973; Hatanaka et al., 1976b; Sekiya et al., 1976). This reaction assumes importance in view of the finding (Gianturco et al., 1974), that the proportion of trans-2-hexenal in the aroma complex is one of the factors which determine the flavor of tea. Hatanaka and his associates have proposed the mechanism indicated in Fig. 4 for the formation of trans-2-hexenal from linolenic acid. The second mechanism for the formation of volatile compounds during fermentation is direct biosynthesis. Wickremasinghe and Swain (1 965) observed that flavory teas contained less leucine than nonflavory teas and this, together with the correlation between flavor and the formation of a-ketoisocaproic acid during withering (Wickremasinghe, 1964) led to the suggestion (Wickremasinghe, 1967) that L-leucine could be a precursor of compounds responsible for tea flavor. This suggestion was supported by the identification of leucine-a-ketoglutarate transaminase in tea flush (Wickremasinghe et al., 1969). and the conversion of 14C-leucine to 14C-mevalonic acid and unidentified 14Cvolatile compounds during tea processing (Wickremasinghe and Sivapalan, 1966). In this study, it was found that I4C-acetate too could act as precursor of mevalonic acid and of volatile compounds, among which nerolidol has been

enzyme

Phospholipid

Linolenic acid

1

enzyme*/O,

IPeroxide)

t

ADH'

ris-3-Heienal

1

GZ=

cis-3-Herenol (leaf alcohol)

enzyme *

It-wi.+2-Hexenal (leaf aldehyde)

ADH' ZZ=

tmns-2-Hexenol

*Located I n chloroplast +Alcohol dehydrogenase

FIG. 4. Mechanism of formation of trans-2-hexenal from linolenic acid. [Based on Hatanaka cr a!. (1976b) and Sekiyaer al. (1976).]

260

ROBERT L. WICKREMASINGHE

identified (Saijo and Uritani, 1971). It was proposed (Wickremasinghe, 1974) that the well-established fact that flavor develops in conditions of climatic stress was related to whether leucine or acetate acted as precursor of volatile compounds. In conditions that are favorable for plant growth, normal intrachloroplastidic reactions are dominant and lead to the formation of acetate which acts as precursor of the volatile compounds; whereas in conditions of climatic stress, extrachloroplastidic biogenesis of terpenoid compounds from leucine, rather than from acetate, is operative. It was proposed that the acetate pathway favored the formation of linolenic acid which was converted to trans-2-hexenal during processing; whereas formation of this aldehyde, an excess of which detracts from tea flavor, is minimal when the leucine pathway comes into operation. Additionally, the qualitative and quantitative changes in the tea leaf carotenoid composition under conditions of climatic stress lead to the formation of compounds (such as p-ionone, theaspirone, and dihydroactinidiolide) in those proportions which were necessary for the organoleptic perception of flavor. 5.

Firing

Firing is the final stage of tea processing when the rolled and fermented leaf having a moisture content of about 45-50% is dried to produce a black tea containing 3% moisture. This is accomplished by blowing hot air through the fermented leaf as it is conveyed on an endless chain. The temperature of the hot air at the inlet is 87"-93"C, and that of the outlet is 56"-57"C, and the drying process normally takes about 20 minutes. More recently the technology of fluid bed drying has been applied to the firing of tea (Kirtisinghe, 1974), using a temperature of 125°C for 20 minutes. Important changes occur during the firing stages of tea processing and some of these are: a. loss of moisture to a level (3%) which makes the product suitable for storage; b. arrest of fermentation reactions due to destruction of polyphenol oxidase and other enzymes. There is, however, some acceleration of enzyme-mediated reactions during the initial stages of firing, and 10-15% of the theaflavin content of black tea is formed during the first 10 minutes of firing (Wickremasinghe, unpublished); c. conversion of chlorophyll to pheophytin, which imparts to black tea the desired black appearance (Wickremasinghe and Perera, 1966a). This transformation occurs at the elevated temperature of firing in the acidic conditions of the tea leaf. It has been observed, in this connection (Wickremasinghe, unpublished), that freeze drying, or raising the pH of the fermented leaf, yields a brown product which is not acceptable to the tea trade; d. reduction of astringency of fermented leaf due to combination of polyphenols with tea leaf proteins at the elevated firing temperature (Wickremasinghe and Swain, 1965). Prior to firing the taste is harsh and metallic, but this mellows on firing;

TEA

26 1

e. firing is essential for the development of black tea aroma (Bhatia and Ullah, 1965) because the loss of low boiling volatile compounds (Yamanishi et al., 1966a) is accompanied by the formation of other compounds which are considered to be important constituents of black tea aroma, e.g., p-ionone (Sanderson et a / . , 1971; Kawashima and Yamanishi, 1973), theaspirone, and dihydroactinidiolide (Ina et af., 1968). The resultant change in the relative proportions of the different volatile constituents probably has an important effect on the overall flavor of the tea (Yamanishi et al., 1968a). Additionally, the pyrazines, pyridines, and quinolines detected in the basic fraction of tea (Vitzthum et al., 1975) are probably formed during firing as a result of interaction between free sugars and amino acids (Reymond, 1976). 6 . Grading Grading is based entirely on the physical separation of the different sizes of particles of the fired tea. This is achieved by the use of mechanically oscillated sieves fitted with mesh of varying sizes, and the different grades of tea are defined by the mesh size of the sieves. Some of the tea grades which are commonly produced are known as Broken Orange Pekoe (B.O.P.), Broken Pekoe (B.P.), Broken Orange Pekoe Fannings (B.O.P.F.), Orange Pekoe (O.P.), Flowery Broken Orange Pekoe (F.B.O.P.), Fannings, and Dust. The tea is finally cleaned by winnowing to remove the fine dust and fiber and passing through a stalk extractor, which works on the principle of electrostatic attraction. Each grade of tea is then packed in plywood boxes and sealed with similar material. Further particulars regarding the grading of tea are described by Harler (1 963). B.

GREENTEA

The world production of green tea is quantitatively less than that of black tea, but it is the principal form in which tea is drunk in several countries, e.g., China, Japan, Taiwan, and Indonesia. Small-leaved tea varieties (so-called China or low jat types) are generally more suitable than the large-leaved varieties (so-called Aassam or high jat types) for green tea production, although Indonesian green tea is, in fact, produced from the large-leaved varieties. In Japan, green tea is manufactured from particular clones of tea (e.g., Yabukita, Natsumidori, Tamamidori), whereas black tea is made from other clones (e.g., Benihomare, Benifuji); the principal chemical differences between green and black teas appear to be the relatively higher content of amino acids and the lower content of polyphenols in the former (Table XI). Recognition of these differences is reflected in agricultural practices where every effort is made to increase the content of nitrogenous material in the leaf by the liberal application of fertilizer, while reduction of polyphenolic material is achieved by shading.

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ROBERT L. WICKREMASINGHE

TABLE XI COMPARISON OF VARIOUS TYPES OF GREEN TEA AND BLACK TEA“

Tea

Polyphenols

Amino acids

Finest grade (Japan) Popular grade (Japan) Popular grade (China) Black tea High grown (Sri Lanka) Low grown (Sri Lanka)

132 229 258

48 21 18

280 302

16 17

Green tea

“ Values expressed as mg/grn dty weight

Processing of Green Tea

As mentioned earlier, the main difference in processing of green tea and black tea is the prevention of fermentation in the former by initial heat inactivation of the enzymes present in the tea flush. This inactivation is achieved in Japan by steaming the flush, and in China, Taiwan, and Indonesia by a process known as “panning” where the flush is fed into a hot rotating drum. In Japan the steps of manufacture are described as follows in an undated publication of the Ministry of Agriculture and Forestry, Japan: “SteamingPrimary rolling and firing-Rolling-Secondary drying-Final rolling-Final drying. ” Steaming is the first step of green tea manufacture in which the polyphenol oxidase and other enzymes are inactivated, and the green color of the leaf maintained. Steam is introduced from a boiler into a rotating cylindrical drum containing the tea leaves and the period of exposure to steam is 15-20 seconds. The heated leaves are cooled by a fan as soon as possible after steaming; and in the next stage of primary rolling and firing, the leaves are introduced into a wooden box containing a rotating shaft fitted with sweepers and forks, through which hot air is blown. The leaves are subjected to this treatment for about 55 minutes, when the moisture content is reduced to about 50%. They are then rolled under pressure for 10 minutes, and dried again in a rotating drum for about 20 minutes, until the moisture content drops to about 30%. Final rolling is for 35 minutes in heated machines which impart a twist to the leaf, and the final drying is by hot air at about 65°C until the moisture content is reduced to 3 4 % . In the panning process of green tea manufacture, the steps followed are similar to those for the steaming process, except that the initial inactivation is effected by dry heat (Wu, 1976). An important component of green tea flavor is dimethylsulfide, the precursor of which was identified as methylmethionine sulfonium salt (Kiribuchi and

TEA

263

Yamanishi, 1963). The other volatile constituents of green tea have been extensively studied by Yamanishietul. (1956, 1957, 1963, 1965, 1966a,b. 1970) and Nose er ul. (197 1). A list of the numerous compounds identified, together with a comparison of their occurrence in unprocessed tea leaf and black tea, has been compiled by Yamanishi ( I 975). A further step in the processing of green tea commonly employed in Indonesia, and which is gaining popularity in Japan. is further roasting at 200°C. This roasted green tea (“Hoji-cha”) has an aroma which is quite distinct from green tea, and was found to contain a total of 66 compounds, including 21 pyrazines (Yarnanishi et ul., 1973). In a study of the changes in aroma components during the roasting of green tea, Hara and Kubota ( 1 973a) described the production of pyrazines, furans, and pyrroles, and it was also found (Hara and Kubota, 1973b) that the amount of carbonyl compounds doubled during roasting, presumably due to Strecker degradation of the free amino acids in green tea. In a study of the polyphenols in roasted green tea, Nakagawa ( 1 967) found that these had undergone marked epimerization, polymerization, and thermal decomposition with an accompanying decrease in the amounts of flavanol gallates.

IV. ORGANOLEPTIC PROPERTIES A.

BLACKTEA

Evaluation of tea is carried out by skilled and experienced tasters who determine the market value of a tea on a purely subjective basis. Apart from the appearance (black or brownish) of the dry tea and the color (coppery or greenish) of the leaf after infusion, it is the tea taster’s palate which assesses the characteristics of the brewed liquor. Some of the important characteristics are the “color.” “strength,” “quality,” and “briskness” of the tea liquor, as well as the formation of “cream” on cooling. These, however, are only a selected few of the terms employed in the tea trade and a fuller description is given by Harler (1963) and Eden (1976). The relationship between color and strength of tea liquors in terms of chemical compounds was studied by Roberts and his co-workers (Roberts, 1962) who concluded that apart from small contributions by flavanotropolones, triacetidein, and possibly products of nonenzymic browning, the color or a liquor is due to theaflavins and thearubigins. Strength of tea liquors was found to be directly related to oxidase activity and polyphenol content in the green leaf, and considered to be determined by the oxidation products produced during fermentation. In subsequent studies, Roberts and Smith (1963), and Nakagawa (1969), found a positive correlation between theaflavin content and the theaflavidthearubigin ratio and the tea tasters’ assessment of quality, color, and strength of tea brews. Similar results were obtained by Takeo (1974b) who observed that the optical

264

ROBERT L. WICKREMASINGHE

density values of theaflavins, thearubigins, and also theaflavins plus thearubigins showed a high positive correlation with the quality of tea infusions. In a statistical evaluation of North-East Indian Plains teas, Biswas et al. (1 97 1) found that total oxygen uptake of unprocessed tea shoots, and the theaflavin, epicatechin gallate, and theogallin contents of the processed black teas determined the cash valuation, which was itself found to be dependent on quality and/or briskness (Biswas and Biswas, 1971). From a study of the characteristics of black teas processed from the tender stem, and tea leaves of differing maturity, Wickremasinghe and Perera (1 973) concluded that the factors affecting quality, strength and color of black tea liquors were the proportions of different polyphenols, polyphenol oxidase activity, and contents of caffeine, theanine, theogallin, and the unidentified compound G 36 detected by Forrest and Bendall ( I 969). The ability of black tea infusion to “cream” and the color of the cream formed (bright or dull) is one of the yardsticks employed by tea tasters to judge the quality of tea. “Cream” is the haze or precipitate that is formed when a strong infusion of tea is allowed to cool down; the relationship between cream formation and quality was first demonstrated by Bradfield and Penny (1944). It was found by Roberts (1963) and Bhatia (1964) that the theaflavins, thearubigins, and caffeine were the main constituents of cream, and later studies (Wickremasinghe and Perera, 1966b; Smith, 1968) showed that theobromine, theaflavin gallate, epigallocatechin gallate, epicatechin gallate, triacetidin, caffeic acid, gallic acid, ellagic acid, chlorophyll, bisflavanols A and B, flavonol glycosides, and mineral matter were also components of the cream complex. A relatively high proportion of theaflavins may be expected to yield a cream which has the desirable bright color, whereas a high proportion of thearubigins results in a dull cream (Wickremasinghe and Perera, 1966b). In this empirical practice, the tea taster is therefore assessing the black tea infusion for its content of theaflavin gallate. A study of the contribution of the nonvolatile compounds of black tea to the character of the beverage was made (Millin et al., 1969a) by tasting pure compounds and various fractions isolated from black tea liquors. It was found that, with the exception of caffeine, none of the monomeric nonvolatile substances examined (flavanols, flavonols, theogallin, chlorogenic acid, p-coumarylquinic acid, caffeic acid, theanine) contributed significantly to the taste of the beverage. Among the oxidation products of flavanols, theaflavin and other oxidation products of intermediate molecular weight were astringent, and it was suggested that together with caffeine, and in the absence of protein, these compounds could influence briskness and strength of tea liquors. Oxidation products of high molecular weight were thought to be responsible for “soft,” “flat,” “thin” liquors, and overall quality depended on the correct balance of a number of substances. Sanderson et al. (1976) confirmed that the astringency of a tea beverage is largely dependent on the amount of polyphenolic compounds present, the degree of oxidation (polymerization) of the tea flavanols, and particu-

TEA

265

larly by the amount of galloyl groups present on the flavanols and their oxidation products. These workers also confirmed the beneficial effect of caffeine on the briskness of tea brews (Roberts, 1962; Wood and Roberts, 1964; Millin et ul., 1969a) and demonstrated that the addition of milk or lemon juice modified the taste of tea polyphenolics. The effect of flavor constituents on the character of black tea has been evaluated by a number of workers; and, in gas chromatographic separations of the aroma constituents, it was observed (Yamanishi et al.. 1968b) that the proportion of compounds having a R, value greater than linalool to those of lower R, value was related to the country of origin of the black tea. In an investigation of Ceylon tea, Yamanishi et al. (1968a) found that the proportion of compounds of relatively high boiling point to more volatile compounds was several times higher in black teas with quality than in those devoid of this characteristic. Similar results were obtained on comparison of flavory and nonflavory Ceylon black teas (Wickremasinghe et a / . , 1973) and in a study of the seasonal variations in the composition of volatile constituents of black teas (Gianturco et al., 1974). Using a mathematical approach to the evaluation of tea quality, Vuataz and Reymond (1 970) identified three regions, namely ionones, linalool. and dimethylsulfide of gas chromatograms as being positively related to quality. It is evident, therefore, that several parameters exist for the evaluation of the characteristics of black teas, but in this plurality lies their disadvantage. An experienced tea taster assesses several hundred of samples in a working day and it is still impractical to replace him by chemical procedures that would need several weeks of work by an experienced chemist.

B.

GREEN TEA

Green tea brews, unlike those of black tea, contain no highly colored products formed by the oxidation of polyphenolic compounds, and the desired color is greenish or lemon yellow without any trace of red or brown color. The liquor should remain clear on cooling without any turbidity, and the infused leaf should be green with no sign of discoloration due to damage. The desired greenish yellow color of the liquor is believed to be dependent on the composition and content of flavonols and their glycosides. as well as flavones (Sakamoto, 1967, 1970). Nakagawa ( 1970) studied the correlation between chemical composition and organoleptic properties by analysis and sensory evaluation of various grades of tea, that is, Sencha (common green tea) of high, medium, and low grades and Gyokuro (the finest grade of green tea). His results indicated that the multiple correlation coefficient between sensory evaluation and catechins, amino acids, caffeine, and other soluble substances was highly significant. In a subsequent study, Nakagawa and Ishima (1971) reported that the taste of green tea brews was affected by the contents of aspartic acid, glutamic acid, theanine, epigal-

266

ROBERT L. WICKREMASINGHE

locatechin gallate, epigallocatechin, other catechins, and other soluble residues. In a more detailed study, Nakagawa (1975a,b) concluded that defined compounds contributed to the four main taste sensations of tea which he described as bitter, astringent, brothy , and sweet. In this investigation, tea infusions were separated to five fractions by gel filtration and the results of chemical and organoleptic tests indicated that the bitterness and astringency of green tea brews was determined by catechins and other phenolic compounds, the brothy taste by amino acids (particularly theanine), and sweetness by sugars. It was considered that the balance between these different tastes was of importance, and that individual taste elements could be accentuated by the conditions of brewing.

V. STORAGE OF TEA A.

TEALEAVES

Changes of the chemical constituents of tea leaves during their storage for 10-21 days at 5" or 10°C in atmospheres containing varying proportions of nitrogen, oxygen, and carbon dioxide was investigated by Tsushida et al. (1976) who found that total ascorbic acid content decreased markedly after storage for two weeks in a manner which was positively related to the content of oxygen in atmosphere. The amount of tannin and caffeine were not greatly affected by storage and in the conditions used; glutamic acid, aspartic acid, and theanine decreased, while glutamine and asparagine increased. Ethylene was produced by the stored leaves, and the extent of production was directly related to the oxygen concentration in the storage atmosphere. Zarnadze (1971) found that storage of fresh leaf at O"-l"C and 96-100% relative humidity for up to 10 days did not affect its suitability for processing. In these storage conditions, there was an increase in polyphenol oxidase, peroxidase, and invertase activities, a decrease in chlorophyll and protopectin contents, but no significant change in the level of catechins. In a study of the essential oils in tea leaves stored at 5°C for 10-12 days, Takeo (1956b) reported the occurrence of quantitative changes in some of the volatile compounds, accompanied by a deterioration in the flavor of green tea and black tea made from the stored leaves.

B. TEABREWS Storage of black tea brews was observed to lead to darkening of the liquors (Roberts, 1959), and was accompanied by an increase in the amount of "nondialyzable theambigins" (Roberts, 1961). The occurrence in tea brews of substantial amounts of nondialyzable material, containing polysaccharide as the major component together with protein, nucleic acid, and polyphenols, was described by Millin et al. (1969b); it was also found (Millin el al., 1969c) that

TEA

267

darkening of tea brews was accelerated by heating and accompanied by a large increase in the quantity of nondialyzable polyphenolic material. C.

GREENTEA

Storage of processed green tea was found (Furuya, 1970) to lead to deterioration of aroma, color, liquor characteristics, taste, and ascorbic acid content. The factors responsible for this loss of quality were considered to be the moisture and oxygen content in the atmosphere and the temperature of storage.

D.

BLACKTEA

Brews of freshly fired black tea have a “raw” or “green” taste but after storage of the fired tea for some weeks this rawness is replaced by a balanced astringency and flavor. However, prolonged storage for several months, especially under unfavorable conditions of exposure to light, elevated temperatures, and high humidity, causes considerable loss of astringency and flavor and the brew is considered, in tea taster’s parlance. to be “flat” or “soft.” Hearne and Lee (19%) observed that the amount of carbon dioxide produced during the storage of black tea was in excess of that expected from “browning” reactions, and suggested that breakdown of constituents occurred during the aging of tea. It was also observed that the extent of carbon dioxide production was dependent mainly on moisture content and temperature of storage of the tea. These results were confirmed and extended by Roberts and Smith ( I 963), who found that carbon dioxide production was associated with a loss of theaflavin. Studies of the changes occurring under different conditions of storage (Wickremasinghe and Perera, 1972b) showed that, during a period of 22 weeks, moisture content of the tea rose from 4.2 to only 5% in an airtight container, whereas it increased to 9.9% in conditions when air was not excluded. Theaflavin and epicatechin gallate content decreased during storage, and levels of thearubigins, amino acids, and total polyphenols showed an undulating pattern. Cash valuations of the samples increased during the first few weeks after storage, after which they declined. In a more detailed study, Stagg (1974) found a decrease of theaflavin and creaming index and an increase of nondialyzable material with time; these trends were accelerated by moisture uptake. There was also a reduction in content of free amino acids (particularly theanine), glucose, volatile compounds (particularly aliphatic aldehydes and aocohols), whereas the levels of total lipids and total fatty acids (particularly palmitic and other free fatty acids of chain length above C 12) increased. The absorption of moisture was considered to be the most important single parameter operative during storage because all the changes described (with the exception of lipid oxidation) were accelerated by moisture contents in excess of 6.5 to 7.5%, and it was suggested that optimal conditions for storage of black tea would include maintenance of its moisture content in the range of 3 to 5% at a temperature below 30°C.

268

ROBERT L. WICKREMASINGHE

VI. POTENTIAL BY-PRODUCTS Black, green, and instant teas together with smaller amounts of oolong and paochong tea are the only products obtained from the tea bush, and, at the present time, none of the other products obtainable is being exploited on even a small scale. A list of these potential by-products has been compiled (Wickremasinghe, 1972) and it is possible that some of them may become useful in the future. Considering each of the possible by-products individually, the extraction of caffeine from waste tea (such as the residue of stalk and fiber remaining after black tea processing) was at one time a profitable industry. Tea waste contains 1.5 to 3% caffeine and there are numerous patented processes for its extraction but these have diminished in importance due to competition from synthetic caffeine. A method for the extraction of vitamin-containing food dyes is the subject of a patent (Bokuchava and Pruidze, 1970), where the starting material is any tea forming material or nonstandard tea leaf. This raw material is heated in hot air or water vapor at about 75°C for 1-10 minutes, after which it is ground, and extracted with aqueous alcohol at 50°-55"C to produce a green food dye, or with water at 65"-80"C to produce a yellow dye. For production of a brown food dye the yellow aqueous extract was heated at about 90°C at atmospheric pressure or at 130" to 200°C at 1.5-1.6 atmospheres. After extraction all dyes were filtered and either spray or freeze-dried. The use of tocopherols extracted from tea flush as antioxidants has been suggested by Tirimannaet al. (1967), who found that a-tocopherol accounted for an appreciable portion of the total tocopherol content of 0.387 mg/gm (dry weight basis). It has also been found (Lea and Svoboda, 1957) that the unoxidized polyphenols extractable from tea may be used as antioxidants, but neither of these possibilities has been placed on a commercial basis. The possibility of using waste products of the tea industry as a source of protein for cattle feed and for composting has been considered by Croyle et al. (1974) who suggested that spent (extracted) tea leaves from instant tea processing plants may be used for this purpose. It was found that this material contained about 25% crude protein, the availability of which would need to be improved by acid-heat or fermentation treatments, prior to its utilization as a feed material. Seeds of Camellia sinensis contain about 20% of oil which is remarkably similar to olive oil in composition (Chakrabarty and Chakrabarty, 1954; Roberts and de Silva, 1972). The content of oil in seeds of Camellia sasanqua is higher (about 35%) and an unconfirmed report (Reddy, 1958) states that tea seed oil production was, at one time, a commercial enterprise in China. The main difficulty at present, would appear to be the difficulty in obtaining the tea seeds themselves because the current practice of vegetative propagation of tea has reduced the availability of seed. Seed residues after extraction of oil contains about 15% (dry weight basis) triterpenoid saponins (de Silva and Roberts, 1972)

TEA

269

which may be used as detergents, or foaming agents, but the extraction of tea seeds for these products is not, at the present time, considered to be a commercially feasible proposition.

VII. CLINICAL EFFECTS A comprehensive account of the nutritional and therapeutic value of tea has been recently published by Stagg and Millin (1975), and this together with the reviews of Das et a1. (1964, 1965) cover much of the published literature on this subject. The more important constituents of tea, from a clinical point of view, are considered to be caffeine and the polyphenol fraction, because many of the beneficial effects claimed for tea may be traced to these constituents. However, as indicated by Stagg and Millin (1975), these effects of tea may be due to interaction between a number of compounds rather than to any single component or group of components. Caffeine is a vasodilator having diuretic and stimulant properties, and tea drinking has been recommended for the treatment of a variety of disorders (Krantz, 1955; Das et al., 1965) since an average cup of tea contains about 40 mg caffeine (Sanderson ef al., 1976). Furthermore, it seems that harmful effects of caffeine, such as a rise in fatty acids in the blood, are not apparent when caffeine is administered in the form of tea (Akinyaju and Yudkin, 1967), and it has been suggested (Stagg and Millin, 1975) that a possible reason for the ameliorating effect could be due to cream formation (see Section IV, A) which may influence the rate of assimilation of caffeine from tea. The other group of medically important compounds of tea are the polyphenols which constitute 48.5% of the total solids in a cup of tea (Sanderson et al., 1976). These polyphenols include the flavanols and flavanol gallates, flavonol glycosides, theaflavins, thearubigins, bisflavanols, epitheaflavic acid, gallic acid, and chlorogenic acid; a variety of pharmacological activities have been ascribed to this group of compounds. It has, for instance, been claimed for many years that tea polyphenols possess the property of strengthening the walls of blood vessels and regulating their permeability (Ul’yanova and Erofeyeva, 1966) and the substances responsible for this action were designated vitamin P. Preparations derived by extraction of green tea leaf are marketed in the U.S.S.R. for their content of this vitamin, although U.S. Food and Drug Administration in 1968 sought withdrawal of such bioflavonoid drugs from the market on the grounds that they were ineffective. Other effects of tea flavonoids discussed in the review by Stagg and Millin (1975) are their effect in increasing levels of catecholamines, the capillary strengthening action, the anti-inflammatory action, normalization of thyroid hyperfunction causing thyrotoxicosis, protection against the harmful effects of exposure to radiation, bacteriostatic effect on a number of microorganisms, and stimulation of folic acid biosynthesis. In this connection, it has been pointed out (Stagg and Millin, 1975) that the common practice of referring to tea polyphenols as “tannins” is misleading, because the strong, irreversible

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protein-binding effects associated with true tannins are not exhibited by the tea pol yphenols. Several reports have appeared on the antiatherosclerotic effect of tea (Little et al., 1966; Akinyaju and Yudkin, 1967; Young et al., 1967; Naismith et ul., 1969; Mahendra et al., 1972). The results (Table XII) of Mahendra et al. (1972) on feeding mice with an atherogenic diet containing respectively, whole and fractionated black tea extract, green tea extract, and coffee indicated that tea, but not coffee, had a beneficial effect in combating the rise of serum cholesterol, triglyceride, and total esterified fatty acids. Black tea was not very different from green tea in its effect, but the black tea fractions separated by gel filtration (Wickremasinghe, 1977) were more effective than whole black tea, and the fraction containing the polyphenols was found to be especially effective in reducing triglyceride levels. Black tea has a relatively high content of fluoride (Cheng and Chou, 1940; Zimmerman et al., 1957; Singer et al., 1967; Okada and Furuya, 1969) and it was found that a black tea brew provided 1-2 ppm fluoride (Karunanayake et al., 1972) indicating that tea drinking could make a significant contribution to the fluoride intake, which is required for the prevention of dental caries (Schwerp, 1971). All of the properties of tea mentioned above are beneficial to human health, but there are also reports that tea may be injurious. Among these is the claim (Fedrick, 1974) that there is some correlation between tea drinking among expectant mothers and the subsequent incidence of anencephalic births. However several factors cast doubt on the validity of this claim as the experimental procedure, interpretation of the statistics, incorrect assumptions, and lack of a doserelated effect do not support the conclusion drawn (Stagg and Millin, 1975). It has also been reported that the phenols of tea have the effect of promoting the TABLE XI1 EFFECT OF TEA AND COFFEE ON SERUM LIPID LEVELS OF RATS ON AN ATHEROGENIC DIET

Treatment

Total esterified fatty acids

None Coffee Green tea Black tea Black tea Fraction 1" Black tea Fraction 2"

Triglycerides

Cholesterol

8

156

8 6

102 70 64 90 48

108 111 91 92 64 67

5.4 6.5 5.8

" Contains non-polyphenolic material of high molecular weight.

* Contains polyphenols and other material of low moleculer weight (e.g., caffeine and amino acids).

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27 1

carcinogenic effect of benzpyrene painted on the necks of mice (Kaiser, 1967), but the lack of adequate controls in the experimentation procedure nullifies the inference drawn. It has also been claimed (Morton, 1972), on the basis of epidemiological study, that there may be a correlation between the incidence of esophageal cancer and the consumption of tannin rich plants, among which tea is included, but this conclusion rests entirely on evidence of a purely speculative nature.

VIII.

HOST PLANT-PEST RELATIONSHIPS

Studies of host plant-pest relationships in tea are few and comparatively recent, but there are already indications that the results obtained may afford practical means of pest control. In a study of the factors influencing the infestation of Ceylon tea by the scolytid beetle, Xyleborus fornicutus, it was found (Wickremasinghe et ul., 1976) that ambient temperature determined the distribution of this pest, while moisture content and the availability of a-spinasterol (a possible precursor of insect moulting hormones) determined the degree of infestation by the beetle. It was found that (1) the beetle preferentially attacked that portion of the tea stem which had a moisture content of 61-63%, as well as a relatively low level of saponins. the mid-portion of the stem (Table VI); (2) those clones of tea which were tolerant to infestation by X . fornicatus contained more saponin than those which were susceptible; and (3) tea saponins had the property of binding sterol, and so reducing its availability. On the basis of these findings, it has been suggested that control of X . .fornicutus could be effected by measures which increased the amount of saponin in the host plant. Another use of saponins is the improvement of laboratory methods for the recovery of nematodes from tea roots (Sivapalan, 1976) where it was found that incorporation of saponin to the extraction medium lead to a fourfold increase in the number of nematodes recoverable from the roots. In a study of the relationship between polyphenol content of tea roots and degree of susceptibility to nematode infestation, Sivapalan and Shivanandarajah (1974) reported that there was a significant increase in the total free polyphenol content in the feeder roots of nematode-tolerant clones following infestation with the root lesion nematode, Prutylenchus loosi, whereas infestation of nematode susceptible clones lead to a decrease of polyphenol content. On the basis of this finding. it was suggested that polyphenols played an important role in determining infestation of tea roots by P. loosi. A correlation between the carotenoid pigment, rhodoxanthin, and the degree of attack of tea by the red spider mite Oligonychus coffeue Nietn., was proposed by Fernando (1967) who found that the severity of mite infestation was related to the rhodoxanthin content of the tea leaf, and it was suggested that this carotenoid probably acted as a phagostimulant or reproductive stimulant.

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IX. INSTANT TEA The commercial production of instant teas began in the 194Os, and this form of tea has grown in popularity in the United States to the extent that it now comprises 42% of tea sales in that country, although on a worldwide basis instant tea accounts for only a very small proportion (less than 5%) of consumption. The demand in the United States is for instant teas soluble in cold water, because it is iced tea which is the real basis for the success of instant tea in that country. In other countries, hot tea is the preferred beverage, and it would appear that here the popularity of tea bags has been the factor responsible for the limited consumption of instant teas. The methods used for instant tea production have been protected by patents, and the patents published up to 1969 have been reviewed by Pintauro (1977). The basic steps in the preparation of instant teas are extraction of tea solids from fermented but unfired tea leaf, black tea, or green tea, followed by concentration of the extract, and drying of the concentrate to a powder. Extraction may be effected by a variety of methods among which counter current extraction and percolation methods have been widely used. Concentration of the extract is effected by evaporation of the water under reduced pressure at a moderately elevated temperature, and during this process various methods for trapping the escaping volatile compounds have been devised. These trapped volatiles are concentrated and retained for incorporation into the final dried product. The concentrated extract is turbid due to the formation of cream (see Section IV, A), and solubilization of this cream is a fundamental problem in the production of instant teas soluble in cold water. Methods for solubilization include treatment with tannase (Takino, 1971), and the use of sulfites and of oxidation as outlined by Sanderson (1 972b), who also discusses the additional problem of “dehazing” for imparting hard water stability to instant tea products. The final step of drying the concentrated tea extract is commonly achieved by spray drying, but other methods, such as freeze-drying or drum-drying are the subject of published patents. The importance of instant teas to the World Tea Industry may be gauged by the intense activity which is current in the field, and this activity is itself an indication that a truly acceptable instant tea has not been yet produced.

X.

ADDITIONAL RESEARCH NEEDS

A great deal of fundamental work has been done in research into various aspects of tea cultivation and tea processing during the last few decades. There are, however, several fields of investigation which remain open, one of which is the development of methods for the rapid selection of planting material which

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will provide bushes with a capacity for higher yields, preferably by reason of a genetic constitution which makes for more efficient absorption and utilization of the fertilizer applied. Yield increase may also be achieved by a systematic study of host plant-pest relationship in tea, which could afford a means for a biological and integrated program of pest control without placing undue reliance on the necessity for chemical pesticides and their attendant drawbacks. In the field of biochemistry, a more thorough understanding of the mechanism of biosynthesis of compounds contributing to the unique characteristics of tea may, in the future, provide a means of tailoring conditions during the cultivation and processing of tea to realize the full potential of the tea leaf. At the same time, the numerous clinical effects need to be studied in greater detail, as these show promise of providing a pleasant, inexpensive and easy method for promoting the general well-being and better health of tea consumers. Better packaging and improvements in technology for prolonging the storage life of tea are also areas which need further study, particularly in view of the rather long time lag between production and consumption, and finally, innovations leading to the presentation of tea in a convenient form will contribute to the continuance of tea as being the most popular beverage in the world.

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Wight, N., and Barua, P. K. 1954. Morphological basis of quality in tea. Narure (London) 173, 63043I . Wood, D . J.. and Chanda, N. B. 1955. Tocklui Exp. Stn. Annu. Rep. 1954, Rep. Biochem. Branch pp. 4 5 4 4 . Wood, D. J., and Roberts, E. A. H. 1964. Chemical basis of quality in tea. 111. Correlation of analytical results with tea tasters' reports and valuations. J . Sci. Food Agric. 15, 19-25. Wood, D. J . , Bhatia, I. S., Chakraborty, S., Choudhury, M. N . D., Deb, S. B.. Roberts, E. A. H . , and Ullah, M. R. 1964. The chemical basis of quality in tea. I. Analyses of freshly plucked shoots. J . Sci. Food Agric. 15, 8-14. Wu, C. T. 1968. The anatomy of tea leaves and its relation with tea leaf quality. J . Agric. Assoc. China 64, 2 9 4 . (Plant Breed. Abstr. 40, 3556.) Wu, C. T. 1976. Tea in Taiwan. Symp. Teh I , Bandung, Indonesia. Wu, C. T.. Wu, H. K . , and Fong, C. H. 1958. The distribution of plucking leaf hair of tea varieties and its correlation with yield and quality. J . Agric. Assoc. China 24, 78-82. Yamanishi. T. 1975. Tea aroma. Nippon Nogei Kugaku Kuishi 49, 1-9. Yamanishi, T., Takagaki, J., and Tsujimura, M. 1956. Studies on the flavor of green tea. Part 11. Changes in components of essential oil of tea leaves. Bull. Agric. Chem. Soc. Jpn. M ,127-130. Yamanishi, T., Takagaki, J . , Kurita, H., and Tsujimura. M. 1957. Studies on the flavor of green tea. Part nI. Fatty acids in essential oils of fresh tea leaves and green tea. Bull. Agric. Chem. Soc. Jpn. 21, 55-57. Yamanishi. T.. Kiribuchi, T., Sakai, M.. Fujita, N.. Ikeda. Y . . and Sasa, K. 1963. Studies on the flavor of green tea. Part V. Examination of the essential oil of the tea leaves by gas liquid chromatography. Agric. Biol. Chem. 27, 193-198. Yamanishi, T . , Kiribuchi. T., Mikumo, Y., Sato, H.. Ohmura, A., Mine, A., and Kurata, T. 1965. Studies on the flavor of green tea. Part VI. Neutral fraction of essential oil of tea leaves. Agric. Biol. Chem. 29, 300-306. Yamanishi. T.. Kobayashi, A , , Sato. H..Nakamura, H . . Osawa. K..Uchida, A., Mon, S., and Saijo, R. 1966a. Flavor of black tea. Part IV. Changes in flavour constituents during the manufacture of black tea. Agric. Biol. Chem. 30, 784-792. Yamanishi. T., Kobayashi, A,, Uchida, A., and Kawashima, Y. 1966b. Studies on the flavor of green tea. Part VU. Flavor components of manufactured green tea. Agric. Biol. Chem. 30, 1102-1105. Yamanishi. T., Wickremasinghe. R. L., and Perera, K . P. W. C . 1968a. Studies on the quality and flavour of tea. 3. Gas chromatographic analyses of the aroma complex. Tea Q. 39, 75-80. Yamanishi. T . , Kobayashi, A , , Nakamura, H.. Uchida. A., Mori, S., Ohsawa, K., and Sasakura, S . 1968b. Flavor of black tea. Part V. Comparison of aroma of various types of black tea. Agric. Biol. Chem. 32, 379-386. Yamanishi. T.. Nose, M.. and Nakatani, Y. 1970. Studies on the flavor of green tea. Part VIII. Further investigations of flavor constituents in manufactured green tea. Agric. Biol. Chem. 34, 599-608. Yamanishi, T.. Shimojo, S., Ukita. M., Kawashima, K . . and Nakatani, Y. 1973. Aroma of roasted tea (Hoji-cha). Agric. Biol. Chem. 37, 2147-2153. Yosioka. I., Matsuda, A , . Nishimura. T.. and Kitagawa, I . 1966. Structure of theasapogenol E. Chrrn. lnd. (London) p. 2202. Young. W.. Hotovec. R. L., and Romero, A. G . 1967. Tca and atherosclerosis. Mature (London) 216, 1015. Zaprometov, M. N. 1961. lsolation of quinic and shikimic acids from the shoots of the tea plant. Biokhimiyu 26, 373-384. Zaprometov. M. N . 1962. The mechanism of biosynthesis of catechins Biokhirniya 27, 366-367.

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Zaprometov, M . N. 1963. Catechin biosynthesis in tea shoots. Fiziol. Rust. 10, 73-78. Zaprometov, M. N . , and Bukhlaeva, V. Y. 1971. Efficiency of various carbon-I4 precursors for the biosynthesis of flavonoids in the tea plant. Biokhirniya 36, 270-276. Zarnadze, D. N . 1971. Biochemical study of the preservation of a tea leaf at a low temperature. Subtrop. Kul’t. 50, 148-153. (Chem. Abstr. 76, 44839.) Zimmerman, P. W., Hitchcock, A. E., and Gwirtsman, A. 1957. Fluoride in food with special reference to tea. Contrib. Boyce. Thompson Inst. 19, 44-53.

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ADVANCES IN F M D RESEARCH VOL .

24

JONATHAN W . WHITE. JR . Agricultural Reseurt.h Service.

U S. Department of Agriculture Philudelphiu. Pennsdvaniti

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Production and Processing . . . . . . . . . . . . . . . . . . . A . Principal Areas and Types . . . . . . . . . . . . . . . . B . Production Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Harvesting . . . . . . . ............................... D . Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Market Forms of Honey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Retail Products . . . . . . . . . . . . . . . . . . . . . . . . . B . Product for Manufacturing Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Analysis and Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Moisture Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................. C . Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................. D . Mineral Content . . . . . . . . . . . . . . . . . . . . . . . . E . Proteins and Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... F . Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . Flavor and Color . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................... H . Vitamins . . . . . . . . . . . . . . . . . . . . . .................. I . Toxic Constituents . . . . . . . . . . . . . V . Physical Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Rheology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Thermal Propetties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Hygroscopicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . Crystallization . . . . . . . . . . . . . . . . . ........................... Vl . Storage of Honey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Effects of Time and Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Fermentation . . . . . . . . . . . . . . . . . . . . . . C . Recommended Storage for Honey . . . . . . . . . . . . . . VII . Nutritive Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . As a Carbohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . €3 . Minerals and Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Folklore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

288 289 289 292 293 293 295 295 296 297

304 305 305 312 331 332 333 333 333 335 338 339 344 344 351 352 352 352 354 354 287

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Copyright 0 1978 by Academic Press Inc . All rights of reproduction in any form reserved .

ISBN 0-12-016124-8

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VIII. Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Nonfood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Standards, Specifications, and Quality Control A. United States Standards . . . . . . . . . . . . . . . . B. Codex Alimentarius . . . . . . . . . . . . . . . . . . . C. Specifications . . . . . . . . . . . . . . . . . . . . . . . . D. Quality control . . . . . . . . . . . . . . . . . . . . . . . X. Research Needs.. . . . . . . . . . . . . . . . . . . . . . . . . References ..............................

1.

357

INTRODUCTION

Honey is the only sweetening material that can be stored and used exactly as produced in nature. No refining or processing is necessary before enjoying this unique material, which can be traced through the entire span of recorded history. Honey, which was man’s first sweet, was used earliest as a ceremonial material and a medicinal ingredient. Not until the era of the Greeks and Romans did honey come to be regarded as a food also. It so remained until relatively recently displaced by cane and beet sugar during the past 100 years. Honey is the sweet, viscous substance elaborated by the honeybee from the nectar of plants. This simple definition excludes honeydew honey, which is produced by the bee from honeydew excreted by various plant-sucking insects. The bee harvests, transports, and processes the nectar to honey, and packages and stores it in the comb. Processing consists of simultaneously reducing the moisture content from the 30-60% common to nectars to the self-preserving range of 15-19%, inverting the considerable proportion of sucrose by the addition of invertase, preserving it meanwhile by adding a glucose oxidase which produces small amounts of acidity and hydrogen peroxide. Ripening takes place in open cells of the comb, which are sealed when the honey reaches full density. The combs, of course, are constructed by the bees from wax they secrete, the production of which requires about 8-10 times its weight in honey. As a unique natural product, honey produces an interesting link to earlier times, and a wealth of observations such as: a bee colony flies about 75,000 miles (12 1,000 km) to produce a pound (454 gm) of honey, but the “fuel” consumption in this (at 1 million miles per gallon or 426,000 km per liter) is only about three ounces (85 gm). The purpose of this review is to provide scientists and technologists with information on the composition, properties, processing, and uses of honey necessary to making informed decisions about its use and value in their operations.

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II. PRODUCTION AND PROCESSING A.

PRINCIPAL AREAS AND TYPES

Honey, even when processed for comniercial use, is essentially a natural product. As produced, it is highly variable, particularly in color, flavor, moisture content, and sugar composition, indeed in nearly every constituent. These attributes depend upon climate, the floral type, and individual beekeeping practices. While bees are kept in all 50 states of the United States and in every country of the world, conditions favorable to commercial beekeeping (honey production) are not as widely available. Further, as agricultural practices and crops change, the value of areas for beekeeping or the quality, type, and amount of honey produced will be influenced. Table I shows honey production, imports, and exports for the major honey-producing and consuming countries, providing a summary of world trade in the commodity.* I.

United States

About one-third of the United States honey crop is sold by the producer directly to the consumer, the remainder to packers. Nearly half of the crop is produced by about 1200 fulltime beekeepers (400 or more hives), about twofifths by parttime beekeepers (25-400 colonies), and the remainder by hobbyists (<25 colonies) (Bauer, 1960). Each of the 48 contiguous states produces at least a million pounds (454,000 kg) annually, except Delaware, Maryland, South Carolina, those in New England, and the largely desert states of Nevada and New Mexico; the primary producing areas are the intermountain area, west coast, central states, Texas, and the southeast. California and Florida vie for the greatest production, averaging 20-30 million pounds (9.07-1 3.6 1 million kg) annually. Minnesota, South Dakota, and Texas generally exceed 10 million pounds (4.54 million kg). Annual variation in a state’s output of 50-100% is not uncommon. Honey is characterized by its color and floral type. A limited number of commercially available honeys are essentially monofloral, i.e., not appreciably blended by the bees. This includes such types as clover, alfalfa, tupelo, “orange” (which is actually better called “citrus” since grapefruit is present). gallberry, and cotton. “Clover” honey may be relatively pure, or may be called “clover” but be more or less naturally blended with other sources in areas where clover pasturage is not widespread. This is generally obvious because o f the darker color. A group of naturally blended honeys is available on a consistent *More detailed statistics are available annually from the Foreign Agriculturat Service, U . S . Department of Agriculture, Washington, D.C. 20250.

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JONATHAN W. WHITE, JR. TABLE I HONEY PRODUCTION AND TRADE“,* (in millions of

Country United States Canada Mexico Cuba El Salvador Gu atema 1a Argentina Brazil Chile Austria Belgium-Luxembourg France West Germany Greece Italy Spain Netherlands Switzerland United Kingdom Bulgaria Czechoslovakia Hungary Poland Romania Yugoslavia USSR Japan Turkey Australia New Zealand Peoples Republic of China

Production

212.4 50.3 80.3 10.1 3.3 6.8 51.8 11.4 15.0 13.5 2.2 22.1 27.2 19.8 13.6 20.6 0.4 4.2 8.1 13.7 17.2 17.8 23.2 18.5 9.0 258 15.5 37.0 43.7 12.2

Imports

Exports

25.2 0.9

8.7 13.2 57.7 6.0 3.3 5.9 38.1 2.5 2.0

7.6 5.5 9.9 97.9

2.4 3.4 2.7

3.5 6.6 10.1 32.5

4.4

18.2 1.2

6.1 4.6 13.3 0.7 8.5 0.4

47.2 15.8 3.3 30.9

~~

‘’ From USDA (1975).

’ Averages for 1972-1974.

‘ 1 million pounds equals 453.59 metric tons. Missing value indicates no data available.

basis, such as fall flowers, alfalfa-sweet clover, and “mixed flowers” from various specified areas. Actually, many recognized types of honey are produced in the United States; a USDA analytical study included 83 different single-source and 93 blends of known composition (identified by the beekeepers) among the 490 samples of honey analyzed (White et a / . , 1962). Most of them are not available in sufficient

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29 1

TABLE I1 COMMERCIALLY SIGNIFICANT HONEY TYPES AND NATURAL BLENDS OF THE UNITED STATES

Honey

Area of Production

“Clover”“ Sweet clover Sweet clover-alfalfa Alfalfa Basswood Buckwheat, wild Cotton Fireweed Gall berry Goldenrod Locust, black Meaquite Orange-grapefrui t Sage Sourwood Spanish needle Star thistle Tulip tree Tupelo Vetch

Central. North-Central, EastCentral Central, North-Central, EastCentral Intermountain West Central and Mountain West, California Mid-Atlantic to Wisconsin California Southwest Oregon Southeast Northeast Mid-Atlantic, East Central Southwest Florida, California California Virginia, Carolinas Central California. North Central Mid-Atlantic to Indiana Florida California. Oregon

“Clover” may include alsike, white Dutch, crimson, ladino, and red clovers, vetch, and trefoil. ‘(

quantity to be of commercial significance. On a local or statewide basis, however, they assume some importance. Table I1 lists the commercially significant types of domestic honey usually available in the United States. Depending upon seasonal and environmental factors, some variability in color and, to some extent, flavor within any type should be expected; honey is after all a natural product. Most food manufacturing use of honey requires such large quantities that the choice is restricted to the clover blends or other natural or prepared blends. Table honey, except for the specialty markets where floral type is preserved, is blended for season-to-season uniformity of color and flavor.

2 . Other Countries Table I lists average (1972-1974) production in several countries; only those with significant (about 1 million pounds (454,000 kg) foreign trade are listed.

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JONATHAN W . WHITE, JR.

Crane (1975b) lists production estimates and other data for 26 European, 21 Western Hemisphere, 20 Asian, and 19 African countries, and Australia and New Zealand. With a few exceptions, honey in world trade is specified and priced by color. Experience with typical honey blends available from the exporting countries allows importers to provide suitable types of honey for specific manufacturing or blending uses. Specialty honeys for table use are available from certain areas: heather from the British Isles, northern and western Europe; thyme honey and pine forest honeydew from Greece; acacia from Hungary, Rumania, Yugoslavia; orange from Spain; mild-flavored light alfalfa, white clover, and thistle from Argentina; legumes and rape from Canada. Honeys of more pronounced flavor include the eucalyptus types from Australia. Mexican honeys, sold largely by color as “mixed flowers,” are (Willson, 1975): acahual (Viguiera grammatagrossa) (extra light amber), and mesquite (light amber); and from Yucatan, tah (Compositae), which is extra light amber to light amber and of pronounced flavor, and dzidzilche (Gymnopodium antigonoidcs), a fragrant light amber honey much used in baking. B.

PRODUCTION METHODS I.

Migratory Beekeeping

Commercial production of honey was made possible by the invention in the mid-nineteenth century of the movable-frame hive and the centrifugal extractor for removing stored honey without destruction of the comb, allowing its reuse. Beekeeping has changed greatly in this century, from being a craft for which every family farm had a few hives for pollination and a sweetener, to our present monocultural agricultural production methods making available vast acreages of such honey sources as orange, cotton, and legumes (for seed production, since modern forage-producing practices largely recommend cutting just before bloom). Many such areas are not suitable for permanent location, further encouraging migratory beekeeping where thousands of colonies are moved thousands of miles, following the blooming crops from south to north.

2. Permanent Locations Much of the nation’s honey comes from wild sources, encouraging much competition for locations where hives can be kept permanently. Such areas must have a succession of pollen and nectar sources as well as sufficiently moderate winters that the cost (in honey or sugar) of keeping the colony alive is not excessive. Many northern beekeepers destroy the colony each fall and begin anew with purchased queens and package bees in the spring. A major beekeeping

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industry (concentrated in the South, Texas, and California) is the production and sale of queens and package bees in the early spring. Colonies depleted by removal of bees for sale may be moved north for strengthening and honey production.

3 . Pollincition Although most of the beekeepers’ income arises from sale of honey, a significant fraction, especially in fruit-, nut-. and seed-production areas comes from planned pollination. Rental prices per colony of bees vary. depending upon such factors as strength of colony, availability, time required, and possibility of a honey crop, from $4-$15 in California (almond, prune, cherry) to $10-$36 in Michigan, Wa>.hington.New York. and other fruit-producing areas.

C.

HARVESTING

Management of honeybee colonies for maximum honey production is a blend of art and science and is beyond the scope of this review. Details are described by Cale et al. (1975). The hive bodies (”supers”) containing combs of ripened honey, largely capped over, are removed from the colony. freed of bees, and taken to a central location for extraction. Therc the cappings of the cells are removed mechanically and the honey is extracted by centrifugation. I t may be run directly into 55-gallon drums for shipment to processors or storage or may be cleaned (to a greater or lesser extent) by allowing it to stand to permit extraneous material to rise to the surface (ripe honey has a density of around 1.42) for removal. It may also be strained through coarse (23 mesh) or fine (100 mesh) screens depending upon the needs of the immediate customer. The frequency of removal of supers will depend upon the honey tlow and the need to prevent mixture of different floral types.

D.

PROCESSING

I.

Why Process Y

Honey immediately after extraction is at its best in terms of flavor and color. It is not suitable for large-scale marketing without further treatment, however, unless the producer has carried out the required processing (which qualifies him as a “producer-packer”). Most producers sell most of their honey to processors who prepare it for marketing and package it. As extracted, “raw” honey contains extraneous matter such as pollen, bits of wax, variable amounts of sugar-tolerant yeasts, and probably crystals of dextrose

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hydrate.* It is thus prone to fermentation unless the moisture content is below 17%; most honey will crystallize in time unless action is taken to prevent it. Processing of honey thus includes controlled heating to destroy yeasts and dissolve dextrose crystals, combined with fine straining or pressure filtration. 2.

Processing Methods

a. For a Liquid Product. Even though supersaturated with respect to glucose, honey will not granulate for months if correctly processed, handled, and stored. Pressure filtration, introduced by Lothrop and Paine (1 934) for honey, improved shelf life of liquid honey by eliminating seed crystals and fine particles of crystallization-inducing substances. Heat exposure, because of the great sensitivity of honey to heat resulting from its acidity, fructose content, and high viscosity, should be limited only to that necessary to accomplish the functions: ‘‘melting” (dissolution of dextrose granulation), pumping, filtration, pasteurization, and filling. Figure 1 diagrams a plant packing 12 million pounds (5.44 million kg) of honey per year. Honey is received from producers in 55-gallon (208 liter) drums (660 Ib, 300 kg), classified for color, floral type, flavor, and moisture and held for use. The melter is designed to liquify 24 drums in about 4 hours without exposure to excessive heating. Most of the liquefaction occurs in the tank beneath the oven from which honey is pumped to batch storage. From this point, it is raised to 150°F (65.6”C) by a heat exchanger, passed through a plate-type filter, and cooled to 120°F (49°C) in the heat exchanger before going to a series of holding tanks in the packing area. Total time at 150°F (65.6”C)is about 30 seconds in this operation. The filling lines for liquid pack honey are conventional; care is needed to avoid reseeding the liquid from holdout residues of honey in lines and equipment. b. For a Solid Product. A semisolid honey product results from the controlled crystallization of some of the dextrose in very fine grain, producing a fondant-like texture. The line for this product is shown on the bottom of the diagram. Not shown is the addition of about 10% of finely crystallized “seed” honey previously prepared by grinding crystallized honey and storing it at 57°F (14°C) for 5-7 days. This is added in the creamer after the batch temperature is reduced to 80°F (27°C). After thorough mixing, the material is filled into retail containers and held at 57°F (14°C) for a week to complete the fine-textured crystallization. The process as described was patented by Dyce (1935); most production is based on it. Details of equipment may be found in the article by Geddes (1964). Townsend (1975) has described several sizes of honey packing lines. *The terms “glucose” and “dextrose” are equivalent, as are “fructose” and “levulose.”

295

HONEY

AT EXCHANGE

COIL

GRILL DRUMS REST ON

HEATER FOR53-GALOPEN END DRUMS CAPACITY 24 DRUMS

I T I FEEDFROM 5 ORPlGE DOUBLE HEAT EXCHbNGER BbTCH STORAGE TANKS 111)

1II

SIOUX HONEY’S NEW PROCESSING-FACKAGING OPERATION

I I

I

I

FILLER SEAYER

I

I

PLbSTlC CAPPER 12OO.LB BATCH STORAGE

I

TWIN PLSTCU FILLER

ICE-TANK WATER CIRCULATES THROUGH COIL

CREAMED HONEV LINE-DIXIE CUP FILLING

FIG. I . Layout and flow diagram for commercial honey packing plant with an annual capacity of 12 million pounds. (From Geddes. 1964.)

Ill. MARKET FORMS OF HONEY A. RETAIL PRODUCTS 1 . Liquid

The United States retail market appears to favor liquid honey, while in many other countries a solid form is preferred. Supermarket exposure seems to require the clear, nonturbid product which results from filtration. A considerable amount of honey is sold in alternate markets such as health food stores, roadside stands, or department or specialty stores. This may not be filtered or processed for clarity and may also be partially granulated, a natural state for honey. Unfortunately, if the honey has been pasteurized, granulation may be coarse and gritty, reducing

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JONATHAN W . WHITE. JR

the appeal of the product. If the honey has not been heated, however, fermentation may take place unless moisture content is 17% or less. Containers include clear glass, translucent or opaque plastic, and, in larger sizes, metal. Dispensers such as squeeze bottles or drip-cut servers are sometimes available. 2 . Comb Honey Forms Honey in the comb is the ultimate in natural flavor and unprocessed nature. It has virtually disappeared from most urban markets, being difficult and expensive to produce and ship. A sealed comb is a real guarantee of a natural product, exactly as prepared by the honeybee. Market forms include: section comb, a 43 inch (1 1.4 cm) square frame which the bees have filled with honey; cutcomb, which is a piece cut by the beekeeper from a larger comb; and chunkcomb or bulk-comb honey, which is a piece of sealed honey comb in a container filled with liquid honey. 3 . Solidified Honey

Since most honeys are supersaturated in dextrose, the most stable form would seem to be biphasic. The truth appears to be that for nearly all honey there is actually no completely stable form, although for most marketing requirements the liquid form is sufficiently stable. As briefly noted above, honey is also sufficiently shelf-stable for sale in a semisolid form known as “creamed,” “spun,” “churned,” recrystallized, or “honey spread.” This is a fondant of fine dextrose hydrate crystals in the honey matrix. It has a “short” consistency and can be spread or handled without the difficulties of a thick syrup. Nothing extraneous is added in manufacture; the product is a result of a controlled crystallization process which follows the normal pasteurization. This is necessary because the liquid portion of the product is somewhat higher in moisture content than before crystallization and hence more liable to fermentation. Storage at temperatures over about 81°F (27°C) will lead to softening and eventually partial liquefaction, since the equilibrium between soIid and solution is temperature dependent.

B.

PRODUCT FOR MANUFACTURING USE

Honey for use in food, confectionery, and pharmaceutical products is currently available in the liquid form. Generally darker honey types or blends of more pronounced flavor are required to ensure that an identifiable flavor contribution is made to the product. Such types are also somewhat less costly. A mild, light clover type will provide honey attributes other than flavor in a product (hygro-

297

HONEY

scopicity, browning of baked goods, "doctoring" or fondants, etc.) but may not contribute its flavor significantly to that of a product with a flavor of its own. Industrial honey is generally purchased in 55-gallon drums, but tank trucks can be available. It should be purchased on sample and on specification if possible; specification should be concerned only with attributes pertinent to use. Honey must contain, at most, 18.6% moisture, and be of acceptable color and flavor for the intended use, be filtered to assure cleanliness, be processed to remain liquid, and, if needed, to inactivate enzymes (see later). Since the semisolid honey spread retail form is more easily handled at the table without drip and stickiness, it is conceivable that a similar form would be of use in certain food manufacturing operations; it could be handled similarly to solid shortenings. Such a product could be made available by honey processors if demand indicated a need.

IV. ANALYSIS AND COMPOSITION Honey as produced by honeybees from plant nectars is rather variable in its composition, reflecting contributions of the plant, climate and environmental conditions, and beekeeper skills. Table 111 summarizes the general composition of United States honey. Data available from similar studies in other countries (White, 197Sa) provide similar values. TABLE III AVERAGE COMPOSITION OF 490 SAMPLES OF HONEY AND RANGE

OF VALUES"

Characteristics measured

Average

Moisture, percentage Levulose, percentage Dextrose, percentage Sucroqe. percentage Maltose. percentage Higher sugars. percentage Undetermined, percentage PH Free acid, meq/kg Lactone, meq/kg Total acid, meqlkg Lactone/free acid Ash, percentage Nitrogen, percentage Diastase value

17.2 38. I9 31.28 1.31 7.3 1 1.50 3. I 3.91 22.03 7.11 29.12 0.335 0. I69 0.041 20.8

"

From White et ul. (1962)

Standard deviation

1.46 2.07 3.03 0.95 2.09 I .03 I .97 -

8.22 3.52 10.33 0.135 0. I5 0.026 9.76

Range

13.4-22.9 27.2544.26 22.0340.7 5 0.25-7.57 2.74- 15.98 0.13-8.49 0.0-13.2 3.42-6.10 6.7547.19 0.00-18.76 8.68-59.49 0.000-,950 0.020-1.028 O.ooO-. 133 2.1-61.2

298

JONATHAN W . WHITE, J R .

A. MOISTURE CONTENT

I.

Analysis

The amount of water in honey is of major importance to its stability against fermentation and granulation. Normally ripened honey has a moisture content below 18.6%;honey of higher content does not qualify for the USDA grading classifications. The determination of honey moisture has been reviewed extensively by White (1969); no significant developments have occurred since that time. In that review, moisture determination by direct drying, Karl Fischer reagent, measurement of viscosity, and density by weighing and hydrometry are critically discussed, as are certain errors and inconsistencies in the literature. The most accurate and convenient procedure uses the refractometer with the conversion table recalculated by Wedmore (1955), which appears as method 3 1.1 12 of the Association of Official Analytical Chemists (Honvitz, 1975). Approximations suitable for many purposes (standard error 2 0.4%) may be obtained with a hand refractometer, providing proper calibration is used, since sucrose solutions and honey of the same refractive index differ in their solids content. Table IV provides a conversion of solids (sucrose) by refractometer to honey solids. 2 . Relation of Moisture Content to Stability The principal short-term instabilities of honey are granulation and fermentation. Liability to each is related to moisture content: fermentation by osmophitic TABLE IV CONVERSION OF REFRACTOMETER CALIBRATION AT 20°C FROM SUCROSE TO HONEY SOLIDS" _____

___

Sucrose

Honey solids

Refractive index

76.00 77.00 78.00 79.00 80.00 81.00 82.00 83.00 84.00

77.56 78.56 79.60 80.64 81.68 82.76 83.76 84.80 85.80

1.4804 1.4829 1.4855 1.488 1 1.4907 1.4934 1.4960 1.4987 1.5014

Calculated from data of Wedmore (1 955) and AOAC table 52.012 (Honvitz, 1975).

299

HONEY

TABLE V LlABlLlTY OF HONEY TO FERMENTATION”

Moisture content (%)

Liability

Below 17.1 17.1-18.0 18.1-19.0 19.1-20.0 Above 20.0

None None if yeast count < 1000/gm None if yeast count < lO/gm None if yeast count < l/gm Always liable

Data of Lochhead (1933) ba\ed on 319 honey samples.

yeasts will ensue if the combination of moisture content, temperature, and yeast count is favorable (Lochhead, 1933); granulation tendency appears to be fairly predictable by the glucose/water ratio (White et a l . , 1962; Hadorn and Zurcher, 1974). Normally ripened honey with a moisture content of 17.5-18%, with a water activity of 0.58, requires a natural inoculum of about 1000/gm to ferment (Lochhead, 1933). Table V shows the general relation between moisture content and yeast count in honey liable to fermentation. USDA (optional) standards (USDA, 1951) require that honey contain no more than 18.6% water to qualify for grading. Retail honey is usually blended to 18% water or less. A survey of composition of United States honey (White et al., 1962) showed that honey from the Western and intermountain areas is lower in moisture than that from the East and West North Central areas, with other United States areas intermediate (average 17.5% for 238 samples). Exact values will be affected by seasonal factors.

B . CARBOHYDRATES The sugars of honey have been intensively reviewed recently by Siddiqui (1970), White (1975b), and Doner (1977). For that reason, the discussion here will be limited, including only a description of our present understanding of the carbohydrate composition of honey and its analyses. I.

Average Composition and Ranges

Table 111 lists the amounts of glucose, fructose, sucrose, “maltose” (reducing disaccharides), and higher sugars found in a survey of nearly 500 samples of United States honey. The variability of honey is illustrated by the ranges shown. A better conception of this is seen in Fig. 2 which illustrates the distribution of individual values within the range for these sugars. Individual analyses for these sugars (and other components) are given by White et al. (1962) for 504 honey

300

fK1

JONATHAN W WHITE, JR.

LT

#

I

40

2 20 0

25

30

35 40 40 LEVULOSE (%)

LEVULOSE / D E X T R O S E

FIG. 2. Distribution of carbohydrate contents among 490 honey samples. Arrows indicate means. (From White et al., 1962.)

and honeydew samples from 47 states, representing 83 single floral types, 93 blends of “known” composition, and 4 honeydew types, all from two crop years. The routine paper chromatography carried out as a control in the fractionation procedure indicated that all samples had the same pattern of sugars present. Also in the publication, the carbohydrate (and other) composition of 74 honey types was compared with the average values. Effects of area of production were examined for alfalfa, cotton, and orange honeys produced in widely different areas. Any differences found were not significant. It is noteworthy that only 3 of the honey samples had a 1evulose:dextrose ratio

30 1

HONEY 46

I

I

I

I

I

I

1

D E X T R O S E I%)

FIG. 3. Dispersion of monosaccharide content of 457 honey samples; line indicates L/D = 1 . (Data of White el al., 1962.)

less than 1.0. Figure 3 shows the individual values of this ratio found for 457 honey samples. The floral source has the strongest influence on carbohydrate composition; area and seasonal influences are minor.

2 . Identity of Sugars Sugars which have been unequivocally identified in honey are listed in Table Vl. Identification requires isolation and identification by sound physical or chemical methods of analyses, not simply by comparison of chromatographic mobility. Details of isolation and identification are included in the reviews of Siddiqui (1970) and Doner (1 977). Table VII provides an approximation of the amounts of the oligosaccharides found in honey by Siddiqui and Furgala (1967, 1968a). 3 . Analytical Problems

In common with other syrups, the carbohydrate analysis of honey remained empirical for many decades. Not until White and Maher (1954) applied class separation on charcoal columns was a reasonably accurate method available for determining dextrose and levulose in honey; three fractions are obtained. This method, accepted by the AOAC, remains the method of choice; it is somewhat

302

JONATHAN W. WHITE, JR. TABLE V1 SUGARS ESTABLISHED AS HONEY CONSTITUENTS'

Trivial name Glucose Fructose Sucrose Maltose

Systematic name

a-D-glucopyranosyl-/3-D-fructofuranoside O-a-D-glUCOpyranOSyl-(1+4)-~-glucopyranose

Reference

Elser (1924); van Voorst (1941)

Isomaltose Maltul ose Nigerose Turanose Kojibiose Laminaribiose a ,P-Trehalose Gentiobiose Melizi tose 3-a-Isomaltosylglucose Maltotriose 1-Kestose

Panose Isomaltotriose Erlose Theanderose Centose Isopanose ISOmdkO-

tetraose Isomaltopentaose

O-a-D-glUCOpyranoSyl-(1+6)-~-glucopyranose ~-a-D-g~ucOpyranosy~-( 1+4)-D-fructose O-a-D-glUCOpyranOSyl-(1+3)-~-glucopyranose O-a-D-glucopyranosyl-(1+3)-~-fructose O-a-D-glUCOpyranOSyl-(I+2)-~-glucopyranose O-P-~-glucopyranosyl-( I+3)-~-glucopyranose a-D-ghcopyranosyl-P-D-ghcopyranoside O-P-D-glUCOpyranOSyl-(1+6)-~-glucopyranose O-a-D-glUCOpyranOSyl-(l-+3)-0-/3-~-fructofuranosyL(2- 1)-a-D-glucopyranoside O-a-D-glUCOpyranOSyl-(1+6)-O-a-o-glucopyranosyl-( 1+3)-D-gh1COpyranOSe O-a-D-glUCOpyranoSyl-(1-+4)-O-Cr-D-ghCOpyranosyl-( 1+4)-D-glUCOpyranOSe O-a-D-giucopyranosyl-(I+2)-P-D-frUCtOfuranosyl-(1+2)-/3-~-fructofuranoside O-cY-D-glucopyranosyI-(1+6)-O-c~-~-glucopyranosyl-( 1-+4)-D-g~ucopyranose O-a-D-glUCOpyranoSyl-(1+6)-U-cY-D-gIUCOpyranosyl-( 1+6)-D-glucopyranose O-a-D-glUCOpyranOSyl-(1+4)-a-D-glUCOpyranosyl-/3-o-fructofuranoside O-a-D-glUCOpyranOSy1-(1 -+6)-a-~-glucopyranosyl-P-D-fructofuranoside O-a-D-glUCOpyranOSy1-(1+4)-O-a-D-ghCOpyranosyl-( 1-+2)-D-glUCOpyranOSe O-a-D-glucopyranosy1-( 1+4)-O-Cy-D-glUCOpyranosyl-( 1+6)-D-glucopyranose O-a-D-glUCOpyranoSy1-(I+6)-[O-a-D-gIUcOpyranosyl-( 1+6)],-~-glucopyranose O-cY-D-glUCOpyranOSyI-(1+6)-[O-a-~-glucopyranosyl-( 1+6],-~-glucopyranose

' From Doner (1977).

White and Hoban (1959) White and Hoban (1959) White and Hoban (1959) White and Hoban (1959) Watanahe and Aso (1959) Siddiqui and Furgala (1967) Siddiqui and Furgala (1967) Siddiqui and Furgala (1967) Siddiqui and Furgala (1968a)

Siddiqui and Furgala (1968a) Siddiqui and Furgala (1968a) Siddiqui and Furgala (1968a) Siddiqui and Furgala (1968a) Siddiqui and Furgala (1968a) Siddiqui and Furgala (1968a) Siddiqui and Furgala (1968b) Siddiqui and Furgala (1968a) Siddiqui and Furgala (1968a) Siddiqui and Furgala (1968a)

303

HONEY TABLE VII

YIELDS OF THE PRINCIPAL SUGARS IN THE OLIGOSACCHARIDE FRACTION (3.65%)OF HONEY”

Disaccharide

(%)

Trisaccharide

(7o)

Higher oligosaccharide

(%)

~~

Maltose Kojibiose Turanose lsomaltose Sucrose Maltulose, (and 2 unidentified ketoses) Nigerose a ,P-Trehalose Gentiobiose Laminaribiose

29.4 8.2 4.7 4.4 3.9

3. I 1.7 1.1 0.4 0.09

Erlose Theanderose Panose Maltotriose I-Kestose Isornaltotriose Melizitose lsopanose Centose 3-a-Isomaltosylglucose

56.99 a

4.5 2.1

Isomaltotetraose lsomaltopentaose

2.5

1.9 0.9 0.6 0.3 0.24 0.05

0.33 0.16 0.49

Acidic Fraction

Not investigated

6.51

trace

13.69

Data of Siddiqui and Furgala (1967, 1968a).

lengthy if many analyses are required. Recent development of high-performance liquid chromatographic (HPLC) analyses of sugar mixtures (Conrad and Palmer, 1976) promises a more rapid procedure without loss of accuracy. The several gas chromatographic (GLC) procedures are of intermediate value, suffering as they do from the need to derivatize. A large degree of empiricism remains in the charcoal column procedure, however. Sucrose can be analyzed specifically only if yeast invertase hydrolysis is used; melezitose is eluted in the disaccharide fraction and interferes if acid hydrolysis is used. The remaining mixture of reducing disaccharides is measured by reducing power and reported as “maltose.” It is too complex to allow individual sugars to be quantitated, even by HPLC. All oligosaccharides (other than melezitose) are present in the higher sugars fraction, reported as glucose after hydrolysis. Literature reports of the sugar composition of honey must be examined with a knowledge of the analytical procedures employed. Values obtained by polarimetric or saccharimetric means are only roughly approximate; those using specific methods for either glucose or fructose and calculation of the other by difference are not accurate unless a class separation has first been done. Values from GLC analysis for the monosaccharides are usually acceptable; for sucrose and other sugars they may not be since demonstration of the singular nature of peaks, considering all of the honey sugars, has not been done. Data from HPLC may be quite acceptable for monosaccharides and sucrose, but all other disaccharide peaks should be combined as “maltose,” since the

304

JONATHAN W . WHITE, JR.

chromatograms do not reflect the known complexity of the honey sugars. Improvements in columns and detectors may eventually provide analyses of more components.

C . ACIDS The characteristic flavor of honey (if such a variable commodity can be said to have a characteristic flavor) includes a contribution due to its acidity. The pH of honey (Table 111) averages 3.91, with a range for 490 samples of 3.42 to 6.10. This level of active acidity probably also contributes to the stability of honey against microbiological attack.

I.

Gluconic as the Principal Acid

Gluconic acid, in equilibrium with gluconolactone, is the principal acid of honey (Stinson et a l . , 1960). It is produced by the action of the glucose oxidase normal to honey on the glucose (White et a l . , 1963b). This reaction is extremely slow in full-density honey but rapid when honey is diluted. It has been proposed that this acid is produced from nectar glucose during the ripening of nectar to honey by the bee. The combined effect of acidity and the hydrogen peroxide concurrently produced is thought to assist in preserving nectar from spoilage during the ripening. Burgett (1974) has shown that this also occurs in nine other eusocial Hymenoptera.

2. Other Acids Ten other organic acids have been identified in honey by suitably rigorous procedures, and seven more are probably present. The former group includes acetic, butyric, lactic, and pyroglutamic (Stinson et al., 1960), citric and succinic (Nelson and Mottern, 1931), formic (Vogel, 1882, cited by Farnsteiner, 1908), maleic (Goldschmidt and Burkert, 1955), malic (Hilger, 1904), and oxalic (von Philipsborn, 1952). In the latter group are glycollic, a-ketoglutaric, and pyruvic (Maeda et al., 1962), tartaric (Heiduschka and Kaufmann, 1913), and 2- or 3-phosphoglyceric acid, a- or P-glycerophosphate, and glucose-6phosphate (Subers et a l . , 1966).

3 . Analysis The titration of total acidity of honey had been an empirical procedure because of a fading endpoint. This was shown to be caused by hydrolysis of gluconolactone (White et a l . , 1958); the present procedure measures free acid and lactone. Of 490 samples of United States honey, only two were found not to contain

HONEY

305

gluconolactone; their pH values were uncommonly high: 5.01 and 6.10 (White et al., 1962). The amount of gluconic acid in honey should be a reflection of several contributing factors, the most significant being the time between the collection of the nectar by the bee and the attainment of full density in the comb, since the action of glucose oxidase essentially stops at full density. This is governed by the sugar content of the nectar, the weather, the strength of the colony, and the quality (i.e., density and volume) of the nectar flow. A greater time needed for ripening permits production of more gluconic acid; it also results in more manipulation of the ripening honey by the bees, with addition of more enzyme. A need exists for an analytical procedure to determine total gluconic acid and gluconolactone in honey. Present lactone titration is not satisfactory because the position of the lactone-acid equilibrium, as related to honey pH, is not known.

D. MINERAL CONTENT 1 . Average Amounts of Principal Minerals The wide variability of honey composition is reflected also in the ash content. Table 111 shows an average of 0.17%, with a range from 0.02-1.03%. The predominating mineral element is potassium, which averages about one-third of the ash; sodium content is roughly one-tenth as much. Schuette and his students at Wisconsin (Schuette and Remy, 1932; Schuette and Huenink, 1937; Schuette and Triller, 1938; Schuette and Woessner, 1939) published the data summarized in Table VIII. They found honey in the two lightest color classes to have lower mineral content than the darker honey types. This was confirmed by White et ul. (1962). 2.

Trace Minerals

The literature on the content of these minerals and 13 others in honey from other areas of the world was reviewed by White (1975a). Tong et al. (1975), in an examination of the value of trace analysis of honey as an indicator of pollution. reported ranges for 41 elements in 19 New York State honey samples. Samples collected by bees in the vicinity of the New York Thruway appeared to contain elevated levels of elements known to be emitted by internal combustion engines. E.

PROTEINS AND AMINO ACIDS

The nitrogen content of honey is low and variable. Table 111 shows an average for United States honey of 0.04196, with a standard deviation of 0.026 (63%).

306

JONATHAN W. WHITE. JR. TABLE VlIl MINERAL CONTENT OF HONEY”

As parts per million of honey

Mineral element Potassium (K) Sodium (Na) Calcium (Ca) Magnesium (Mg) Iron (Fe) Copper (Cu) Manganese (Mn) Chlorine (Cl) Phosphorus (P)

Sulfur (S) Silica (Si02)

Honey color

No. samples

light dark light dark light dark light dark light dark light dark light dark light dark light dark light dark light dark

13 18

13 18 14 21 14 21 10 6 10 6 10 10 10 13 14 21 10 13 10

10

Range

Average

100-588 1154,733 6-35 9-400 23-68 5-266 11-56 7-126 1.2O-4.80 0.70-33.50 0.14-0.70 0.35-1.04 0.17-0.44 0.46-9.53 23-75 48-20 I 23-50 27-58 36- I08 56- I26 7-12 5-28

205 1676 18 76 49 51 19 35 2.40 9.40 0.29 0.56 0.30 4.09 52 I13 35 47 58 100 9 14

“ Data of Schuette and Remy (1932), Schuette and Huenink (1937), Schuette and Triller (1938). and Schuette and Woessner (1939). Paine et al. (1934) reported an average of 55% of the nitrogen lost by ultrafiltration (range 26-93%); White and Kushnir (1967b) noted that about 4 0 4 0 % of the nitrogen is lost on dialysis. Bergner and Diemair (1975) more recently reported 3345% to be removed by ultrafiltration (10,000 limit). Most of the nonprotein nitrogen is in free amino acids.

I.

Proteins

Early interest in protein content was in distinguishing honey from artificial mixtures and blends. The volume of precipitates with honey and tannin (Lund, 1909), phosphotungstic acid (Lund, 1910), or alcohol (Laxa, 1923) was used. Immunological tests were studied as early as 1903 (Langer, 1915). Thoni (1913) proposed using an antiserum to royal jelly or “beebread” for this purpose.

307

HONEY

Indeed, Langer (1 915) immunologically differentiated honey protein and proteins of hand-collected pollen, refuting Kiistenmacher’s earlier claim that protein in honey was extracted from the pollen and that Langer was in error in ascribing it to the bee. Studying the colloidal material removed from honey by ultrafiltration, Paine et al. (1934) found it to be more than half protein, isoelectric at pH 4.3, and precipitable by colloidal bentonite. Helvey ( 1 953) found three components in the colloidal material from a buckwheat honey: proteins of molecular weight of 146,000 and 73,000 and a presumed polysaccharide of 5,000 weight. White and Kushnir (1967b), using gel filtration, ion-exchange chromatography, and starch-gel electrophoresis, examined proteins of eleven floral types of honey and sugar-fed stores. From four to seven proteins were found, of which four originate with the bee. The molecular weights of two of the latter were approximately 40,000 and 240,000; those from the plant were about 98,000 and >400,000. Figure 4 shows gel filtration and starch-gel electrophoresis of a preparation from

4.9 A

B

LA) C

C * H H

-

D

1

-

20

ORIG

I

,

4 0 60 80 100 120 140 F R A C T I O N NO. L2rnl)

II I @ ,H,I,

20 4 0 60 80 100 120 140 M l G R A T l O N TOWARD ANODE ( m m l

FlG. 4. (A) Filtration of I ml dialyzed concentrate (= 10 gm honey) goldenrod-aster honey on 2.1 X 60-cm column of Sephadex (3-200 in 0.01 M phosphate pH 6.5; (B) Starch gel electrophoresis of fractions combined in pH 8.9 borate, 4.0 Vlcm as indicated in A. Pattern at bottom is from original material as applied to column. (From White and Kushnir, 1967b.)

308

JONATHAN W . WHITE. JR.

(A)

0.5 0.4

0.3

~

t A

0.2 0.1

u =

'0

05

" 01 40 60 80 F R A C T I O N NUMBER ( 2 m l )

FIG. 5 . DEAE-cellulose chromatography (bed 0.8 x 18 cm) in 0.01 M phosphate (pH 8.0) of concentrated dialyzed protein preparations. Solid lines: apparent protein by optical method. Broken lines: by Lowry method (scales left). Straight solid lines: gradient of concentration (scale right). (A) Goldenrod-aster preparation, 0.25 ml, (B) Lespedeza, from single comb, I .O ml. (From White and Kushnir, 1967b.)

a goldenrod-aster honey. In Fig. 5 is seen the greater resolution produced by ion exchange cellulose chromatography of this preparation. The chromatogram of a preparation from sugar-fed bees (no nectar components) is shown in the same figure to indicate its less complex nature. In Fig. 6 are gel filtrations of protein preparations from another honey and from stores from sugar-feeding. The larger number of components in the former is apparent. Bergner and Diemair (1975) have also examined by gel filtration protein preparations from several types of honey and from sugar-feeding. Their results have generally confirmed those of White and Kushnir. They ascribed three of the five elution peaks to the bee and two to plant components.

2. Amino Acids The formol titration, essentially a measure of total amino acid content, was applied to honey by Tillmans and Kiesgen (1927) who proposed that it be used to

HONEY

309

FIG.6 . Filtration of concentrated dialyzed honey preparations (0.5 ml) on Sephadex G-200. Protein by Lowry method. (A) cotton honey, (B) stores from sugar-fed bees. (From White and Kushnir, 1967b.)

authenticate honey. European limits for this value were shown by Schuette and Templin ( 1 930) to be inapplicable to United States honeys, which were generally lower and more variable. Lothrop and Gertler (1933) described a procedure for amino nitrogen in honey, reporting an average of 0.0033% (range 0.00240.0066%). Schuette and Baldwin (1944) reported averages of 0.0034% for light and 0.0058% amino nitrogen for dark honeys. The introduction of paper chromatography renewed interest in honey amino acids; several investigators identified up to 17 amino acids in various samples. Komamine (1960), quantitating paper chromatography, first noted that proline was the preponderant amino acid. Later the automatic amino acid analyzer was used for honey analyses; a considerable body of analyses is now available (Curti and Riganti, 1966; Mizusawa and Matsumuro, 1968; Michelotti and Margheri, 1969; Hahn, 1970; Biino, 1971; Bergner and Hahn, 1972; Petrov, 1974; Davies, 1975). Table IX shows Davies’ values for free amino acids in 98 honey samples. All agree that proline predominates, representing 50-85% of the total. Davies (1975) has reviewed the sources of honey amino acids. Since pollen contains about 1.5% amino acids, with proline predominating, Komamine proposed this as the source. Nectar contains small amounts of free amino acids but little proline. Davies calculated that far too little pollen is present in honey to account for the proline. Bergner and Hahn (1972), noting proline to comprise 80% or more of the generally lower amino acid content of sugar-fed bee stores, ascribed

W

0

TABLE IX AMINO ACID ANALYSIS OF

No. samples:

Argentina 8 Avg.

Glucosaminic acid Methionine sulphoxide Aspartic acid Unknown A "Amides" Glutamic acid Proline Unknown B Glycine Alanine Cystine Valine Methionine

SD

1.14 1.76 0.64 6.48 2.12 53.10

1.241 0.587 0.218 2.961 0.809 16.589

0.55 1.73

0.176 0.679

1.27 0.07

0.346

Australian eucalypt/clover 16 Avg.

SD

Australia unspecified 15 Avg.

SD

Canadian 16 Avg.

SD

SD

0.955 2.169

2.05

0.245

0.59 4.50

1.463

9.90

1.592

6.68 2.02 51.81

0.561 0.2% 6.954

14.82 5.73 44.96

4.532 1.831 16.170

0.71 1.85 0.30 1.32

0.115 0.482 0.125 0.48 1

30.90 6.00 83.42 13.54 1.48 3.84 0.45 6.16 0.09

4.673 0.643 23.930 3.691 0.208 0.601 0.179

7.57 1.66 93.95

1.120 0.245 16.658

50.49

3.439 1.981 25.424

0.54 2.42 0.66 1.43 0.25

0.169 0.673

0.43 1.21

0.190 0.632

0.43 1.16

0.102 0.121

0.318 0.090

0.92 0.33

0.372 0.223

1.32 0.97

0.248

1.51

Avg.

SD

1.664

1.268 0.577

1.47 0.46 5.09

Avg.

Yucatan 14

3.17 2.38 1.24

1.44

U.S. clover 13

r

1.545

AII samples

98 Avg.

SD

3.21

1.688

1.74 3.44 0.95 11.64 2.94 59.65 21.04 0.68 2.07 0.47 2.00 0.33

1.174 3.212 0.937 9.334 2.163 26.765 20.612 0.407 1.523 0.212 1.854 0.232

Isoleucine Leucine Tyrosine Phenylalanine P-Alanine y-Amino butyric acid Unknown C Unknown D Unknown E Lysine Unknown F Ornithine Histidine Tryptophan Arginine Total:

1.76 0.74

0.317 0.288 0.420 0.204 0.223

0.69 0.54 1.13 3.35 0.66

0.278 0.205 1.280 3.757 0.387

3.82 2.89 7.26 60.49 1.00

1.52 0.41

0.207 0.156

0.26 1.28

0.1 15 0.337

0.131 0.602 1.431 1.238

0.421 0.113 0.148 0.327 0.393 0.139 0.112 1.238 0.986 0.767

4.23 0.87 0.61 0.63 1.97 0.93 0.43

0.600

0.12 5.42 1.91 0.86

1.30 0.37 0.25 0.37 1.06 0.31 0.25 4.83 3.83 1.12

33.976

83.88

7.530

90.46

26.601

0.72 0.69 0.91 2.07 0.98

0.279 0.267 0.391 1.525 0.452

0.73 0.64 1.29 3.17 I .57

0. I70 0.203 0.382 1.241 0.400

0.47 0.88 2.24 5.07 1.29

0.229 0.753 1.689 4.253 0.260

1.34 0.63 0.30 0.33 I .32 0.64 0.24 6.04 2.06 0.82

0.318 0.681 0.068 0.175 0.768

2.76 1.49

0.770 0.242

0.69 0.44 0.34 0.20 2.70

0.082 0.206

1.62 1.19 0.77 0.64 0.34 0.41 0.27 1.85

0.810 0.538 0.176 0.225 0.1 18 0.191 0.191 0.398

0.437

1.30

0.690

1.12

84.10

26.484

127.96

19.857

77.25

0.064

2.305

0.106

0.084 0.728

0.75 0.59 1.04

~~

' From Davies (1975).

* mg amino acid/100 gm honey (dry wt). A blank in the SD column indicates that only one sample contained the amino acid.

0.514 2.176 13.800 0.402

1.12 1.03 2.58 14.75 1.06

1.191 0.898 2.826 24.806 0.510

2.15 0.81 0.66 0.57 0.99 0.50 0.26 3.84 3.84 1.72

1.186 0.513 0.476 0.442

6.17 6.05

0.617 0.275 0.092 0.244 0.486 0.440 0.172 0.838 0.996 3.372

0.359 0.223 1.982 3.393 2.269

252.28

41.846

118.77

69.491

5.64

0.%1

0.666

312

JONATHAN W . WHITE, J R .

05[

03

,

,

I

O""'S

0005 001

003 005 01 ASPARTIC / P R O L I N E

3

FIG. 7. Regional separation of honeys by ratios between concentration of individual amino acids. A Australian, 0 Canadian, 0 United States clover, 0 Yucatan. (From Davies, 1975.)

it to the bee. Petrov related it to the important part proline plays in aerobic muscle exchange products in all insects. Davies, using data for 98 samples of honey, has suggested that certain ratios between contents of various amino acids could be used to determine the geographic source of a honey; Fig. 7 indicates one such approach. Later (Davies, 1976) this approach was refined by using a computer-aided selection of 60 amino acid ratios. Fifteen of 16 samples not used to establish the program were correctly assigned to one of the four locations shown in Fig. 7 , showing that while there are variations in the ratios between samples of the same area, the variation between sources is much greater.

F. ENZYMES That honey contains enzymes has been known for more than a century since Erlenmeyer and Planta (1 874) reported their presence in bees, pollen, beebread, and honey. As the author has noted earlier, The enzymes are among the most interesting materials in honey, possibly have received the greatest amount of research attention over the years, and have supported the greatest burden of nonsense in the lay and even scientific press. The use of enzyme activity in some countries as a test for overheating of honey seems to support by implication the occasional supposition by food faddists that the enzymes of honey have a dietetic or nutritional significance of themselves (White, 1975a).

The greatest volume of literature reports on honey enzymes until most recently dealt with their use as indicators of honey identity and quality, largely heat

313

HONEY TABLE X ENZYME ACTIVITIES OF HONEY

Enzyme a-Glucosidase (invertase, sucrase) Diastae (a- and P-amylase) Glucose Oxidase

Catalase Phosphatase

Average activity

Number of samples

7.5-10

1468

References Duisberg and Hadorn (1966) Duisherg and Hadorn (1966)

16-24 20.8 80.8 167 210 4.97" 86.8" 13.4 5.07

263 90 24 10

2R 10 25

White er ul. (1962) White and Suhers (1963) Dubtmann (1971a) Dustmann ( 197 1h) Schepartz and Suhers (1966a) Du\tmann (1971h) Dzialoszynski and Kuik (1963) Zalewski ( I 965)

units

a-Glucosidase: Diastase: Glucose oxidase:

Catalase:

Phosphatase: "

gm sucrose hydrolyzed per 100 gm honey per hour at 40°C gm starch converted per 100 gm honey per hour at 40°C pg H,O, accumulated per gm honey in I hour under experimental conditions. Because honey contains substances oxidized by H,Oz, this is not a true measure of glucose oxidase Catalatic activity per gram, K , = I h (In x d x ) DIW where x o is initial substrate. x is substrate at time, r , D is dilution. and W is sample in grams (Schepartz and Suhers, 1966a) mg P/100 gm honey/24 hours

Includea nine values of zero

' Includes four values of zero. exposure. Most countries other than the United States require minimum values for amylase activity and proposals for use of other enzyme activities for this purpose still arise. The honey enzymes of most direct interest in food applications are amylase, invertase, and glucose oxidase. Catalase and acid phosphatase are also present. The amounts of these enzymes normally found in (unheated) honey are shown in Table X , to provide an idea of order of magnitude. I.

Invfrtase

A sucrose-splitting enzyme is added to nectar by the honeybee during its harvesting and ripening to honey. It continues its activity in extracted honey unless destroyed by heating. It is an a-glucosidase (White, 1952; White and Maher, 1953a) with inherent transglucosylase action. During its action on su-

3 14

JONATHAN W. WHITE. JR

crose, six oligosaccharides are formed, all eventually hydrolyzed to glucose and fructose by the completion of the reaction. The principal intermediate is a-maltosyl p-D fructofuranoside (White and Maher, 1953b) trivially named erlose (also termed glucosucrose, fructomaltose). It can accumulate to as much as 11% of the original sucrose (White and Maher, 1953b) during the reaction. Maltose is formed in lesser amounts. Echigo and Takenaka (1973) have studied the carbohydrates and a-glucosidase in stores produced by sucrose-fed caged bees; during the ripening they reported erlose to appear in the earlier part of the ripening period and remain throughout the ripening period. Figure 8 shows the progress of ripening of sucrose stores. The optimal conditions for the transferase reaction were found to be pH 6.0, 30°C, and 0.25 M sucrose. Examination of a-glucosidase from several honeys and from stores of sucrose-fed bees (White and Kushnir, I967a) indicates that preparations seemingly homogeneous by Sephadex gel filtration (Fig. 9) show 3-9 components by ion-exchange chromatography. The preparation from sugar-fed bees, however, appeared homogeneous with an approximate molecular weight of 5 1,000 indicated. Figure 10 illustrates DEAE cellulose chromatography of a-glucosidase from honey and of stores from sugar-fed caged bees. A highresolution starch gel electrophoresis procedure (White and Kushnir, 1966) further resolved all preparations into 7-18 isozymes. Figure l l compares the pattern of a-glucosidase isozymes from a bulk honey (i.e., extracted from combs taken from many colonies at several locations) with that of the a-glucosidase from a comb honey (i.e., produced by a single colony) and that of stores from sugar-fed bees.

-

\

50c3

0

\ \

\

24

48

72

96

HOURS

FIG. 8. Changes in sugar content during the process of honey formation. x fructose, A glucose, 0 sucrose, 0 erlose. (From Echigo and Takenaka, 1973.)

315

HONEY

8

r-----l

0

FIG. 9. Sephadex (G-200) filtration (2. I X 31 cm) in 0.014 M phosphate (pH 6.5) of enzyme preparations from honey (solid lines, scale left): a-glucosidase from (1) 0.5 ml preparation (= 1 . 1 g) goldenrod-aster honey; (2) 0.5 ml preparation ( g 5 . 5 g) clover honey; (3) 0.25 ml preparation from (= 8.9 gm) stores from sugar-fed bees. Broken line (scale right) from same clover honey. (From White and Kushnir. 1967a.)

The greater complexity of the preparation from bulk honey is probably the result of blends of honey from many colonies. The single-colony samples have equivalent numbers of isozymes. Noteworthy is the much lower migration rate of the sugar-fed samples, which have no plant components. White and Kushnir suggest that the bands may represent genetic differences among bees. Methods

0 u 2

D

0 FRACTION NUMBER 121111 1

40 60 ao FRACTION NUMBER ( 2 m I l

FIG. 10. (A) Chromatography on DEAE-cellulose of a-glucosidase preparation from cotton honey elution with 0.01 M potassium phosphate (pH 8.0); KCI gradient as shown; solid line: a-glucosidase activity (scale left); broken line: “protein” measured by optical method (scale right). ”Protein” retained and fractionated, 14.9 mg (73%); a-glucosidase retained and fractionated. 73.5 units (27%); (B) a-glucosidase preparation from stores of sugar-fed bees. (From White and Kushnir, 1967a.)

316

JONATHAN W . WHITE, JR. 401

I

I

I

1

I

I

1

I

1

M I G R A T I O N TOWARD ANODE ( m m )

FIG. 1 I . Starch-gel electrophoresis of a-glucosidase preparations from honey, borate (pH 8.9): (A) clover honey. 96 unitslml; (B) Lespcdeza honey, 38 unitslml; (C) stores from sugar-fed bees. 44 unitdml. A at 3.70 V/cm, B and C at 3.52 V/cm. (From White and Kushnir, 1967a.)

used are sufficiently sensitive to examine the a-glucosidase of single bees in this fashion. a . Origin and Kinetics of Honey Invertase. The question of the source of the sucrose-inverting enzyme of honey has intrigued scientists since its discovery in honey. No purpose is served by reviewing the earlier literature; it has been accepted for many years that the major portion is that added by the bee during the collection of nectar and the ripening process. Whether any plant enzyme from nectar or pollen is present has not been definitively shown. Gothe (1914) concluded that both plant and insect were sources, since more enzyme activity was present in honey than in stores from sugar-feeding. Schonfeld (1927), however, found the invertase activity of sugar-fed stores to be inversely related to the concentration of the feed; no information is available on the concentration fed by Gothe. The kinetic study of honey invertase by Nelson and his colleagues (Nelson and Cohn, 1924; Nelson and Sottery, 1924; Papadakis, 1929) remained the definitive work until recently. Differences from yeast invertase were found in pH optima and the initial reaction course, initial yeast invertase rates being practically

317

HONEY

constant, in contrast to a marked rate increase in the honey invertase inversion. Two kinetic studies with the objective of determining the source of honey invertase provide the only recent kinetic data on the reaction. Rinaudo et al. ( I 973) undertook to demonstrate that invertases from the other possible sources (pollen, nectar) differ from that of honey. Invertase from the hypopharyngeal gland of the bee and from honey were shown to have the same pH and temperature sensitivity, substrate and reaction products (glucose and fructose; intermediates were not mentioned), and inhibition by fructose. Comparison of reciprocal rate plots for the enzymes from the bee, honey, two pollens, and nectar showed identical Michaleis constants (0.17 M )only for the first two. However, they reported that none of the preparations showed maltase, contrary to earlier reports (White and Maher, 1953a; Gontarski, 1954; Maurizio, 1961). The definitive study of the a-glucosidase of the hypopharyngeal gland of the honeybee (and hence of honey) is that of Huber. Huber (1975) and Huber and Mathison ( 1 976) have purified two sucrases from honeybees, confirming Gontarski’s ( 1 954) earlier studies. The less soluble of these precipitated between the same values of ammonium sulfate saturation and exhibited kinetics very similar to those of honey sucrase. Final purification was by affinity chromatography. The previously reported transglycosylase activity was confirmed and a kinetic study of the hydrolytic and synthetic reactions was carried out. As seen in Fig. 12, the rate of release of fructose is rectilinear; the rate of glucose release drops at

an FRUCTOSE

40

0

ZOO

400

600

BOO

1000

Yo/ [ SU C R 0sE l

FIG. 12. Kinetic Hofstee plot of the production of glucose and fructose from sucrose. For incubation details see original. Units are micromole product per minute from 0.2 M sucrose at 30°, pH 6.5. From Huber and Mathison, 1976. Reproduced by permission of the National Research Council of Canada from the Canadian Journal of Biochemistry 54, 153-164, 1976.

318

JONATHAN W . WHITE, JR.

FIG. 13. Proposed mechanism for action of the major sucrase of honey bees; S . sucrose, G . glucose, F. fructose. G-S trisaccharide. W , water. From Huber and Mathison, 1976. Reproduced by permission of the National Research Council of Canada from the Canudion Journal ofBiochcrnisrry, 54, 153-164, 1976.

high sucrose concentrations. The scheme in Fig. 13 explains this: the reaction releasing fructose (K3) is rate limiting, and sucrose is not only initial substrate, but an acceptor for the transglucosylation to form the glucose-sucrose trisaccharide, erlose. Huber did not identify the trisaccharide with erlose. The K cat for fructose, glucose, and erlose formation according to the scheme are: Kcat (fructose) = K3

(1)

K for formation of all three products by this mechanism is K 2 / K I . (K cat values which include substrate concentrations are not, in fact, constants, since the values change with substrate concentrations.) The earlier value of 51,000 for the molecular weight (obtained by Sephadex filtration in 0.1 M phosphate pH 6.5) was confirmed by equilibrium ultracentrifugation, but Huber and Mathison found values of 82,500 by Sephadex filtration ( 0 . 2 M citrate pH 6.5) and 78,000 by SDS electrophoresis. The glycoprotein nature of the enzyme was confirmed by amino acid and amino sugar analysis. Honey a-glucosidase has been reported to have optimal maximum activity at pH 6.0 (10% hydrolysis), 5.7 (35-55% hydrolysis) (Nelson and Cohn, 1924), 5.9 (Rinaudo et af., 1973), and 5.5 (Huber and Mathison, 1976). Hadorn and Zurcher ( I 962), comparing several published procedures for determination of sucrase in honey with particular attention to the pH optimum, selected 6.3, commenting that very little difference in activity was found between pH 5.8-6.5. Huber and Mathison, who used the most highly purified enzyme, show a flat maximum between pH 5.5 and 6.0. This discrepancy in K , values reported for the major honeybee enzymes (and for honey sucrase) by Rinaudo et al. (1973) (0.17 M ) and Huber and Mathison (1976) (0.030 M ) may perhaps be resolved by a calculation of K M for honey

HONEY

319

invertase using the data published by Nelson and Cohn ( I 924). A LineweaverBurk plot of the 5-minute data from their Table 10 gives KM = 0.031 M. Considering the crude nature of their preparation, this is excellent agreement. When comparing data among the few recent reports on these a-glucosidases, the sources and extent of purification must be considered. Huber and Mathison prepared their material from whole honeybees, necessitating extensive purification. They also prepared the enzyme from an unpasteurized supermarket honey. The latter was purified by dialysis and ammonium sulfate precipitation. They stated that the kinetic properties and apparent K M values of the honey enzyme were the same as the “major honeybee sucrase.” The sucrase of the head portion of the honeybee is almost entirely of this type. Rinaudo er a / . (1973) prepared honey sucrase by dialysis, and that from excised honeybee food (hypopharyngeal) glands and pollen by extraction and centrifugation only. No estimates of purity were made. The hypopharyngeal glands are known to contain an active glucose oxidase (Gauhe, 1941) which, if not removed, can distort results by preferentially removing glucose. This glucose oxidase activity was probably eliminated in Huber’s preparation from bees, and it was probably present in those of Rinaudo. It is not clear, however, whether the less extensive purification given the honey enzyme by Huber effectively removed the glucose oxidase known to occur in honey. Huber makes no mention of glucose oxidase. There is no question that the a-glucosidase of honey acts upon maltose; no reason is apparent for the contrary report of Rinaudo et a/. (1973). Huber and Mathison (1976) report activity against maltose to be 83% that against sucrose. White and Kushnir (1967a) show exactly parallel activity for 13 isozyme peaks separated by starch-gel electrophoresis; the activity against maltose was only 30% of that against sucrose. Takenaka and Echigo (1975) purified honey a-glucosidase by DEAE cellulose chromatography and obtained K M for maltose = 0.00526M, with optima at pH 6.0 and 30°C. Sucrose was hydrolyzed at three times the rate for maltose. b. Heat Inactivation. The papers of Rinaudo et al. (1 973) and of Huber and Mathison (1976) include data on heat inactivation of the a-glucosidase in buffer. The latter report only that the activity rapidly disappears at 55’45°C; Huber and Mathison state that with 10-minute exposure destruction begins at 40°C and is essentially complete at 60°C. Trade interest in the invertase activity of honey centers about its possible use as an indicator of heating history. Most of the literature on the subject deals, therefore, with inactivation in full-density honey. White et a / . (1964) have shown that the rate constant for inactivation of honey invertase in buffer is 24 times that in full-density honey. Several proposals have been made to establish minimum values for sucrase in honey, to be used together with diastase values for estimating heat exposure

320

JONATHAN W . WHITE, JR.

(Kiermeier and Koberlein, 1954; Duisberg and Gebelein, 1958; Hadorn et a / . , 1962). The Codex Alimentarius standards, however, do not include sucrase activity. No discussion of analytical methods will be included here, beyond the observation that Dustmann (1972) pointed out that existing methods in which the reaction takes place in diluted honey (including those of the preceding three papers, above) underestimate the activity by 10-30%, presumably because of the inhibitory effect of the monosaccharides of honey. He recommended dialysis as a pretreatment. The procedure earlier used by White et al. (1 964) in their study of the effect of storage and processing on honey enzymes had used dialysis for this reason. In that study, a single expression was found adequate to describe the inactivation of invertase in full-density honey by heating and long-term storage. The first-order rate equation is log K = 26.750 -

39730 2.303 RT

(4)

which is plotted in Fig. 14. Figure 15 shows the half-life of honey invertase over the temperature range of 2Oo-80"C. 10,

00

1

I

I

'

-10 L L9

5 -20

-30

-4.0I 280

1

I

I

300

320

340

f

1

x lo5

FIG. 14. Effect of temperature on rate of heat inactivation of diastase and invertase in honey; + Schade et al. (1958). W Lampitt et a / . (1929), 0 Duisberg and Warnecke (1959), A Kiermeier and Koberlein (1954). From White er al., 1964. Reprinted from Food TechnologylJournal of Food Science 18(4), 153-156, 1964. Copyright @ by Institute of Food Technologists.

HONEY

32 I

DAYS

68

104 140 STORAGE TEMPERATURE

FIG. 15. Approximate time required at a given temperature between 20°C (68°F) and 80°C (176°F) for the diastase and invertase activities of a honey sample to be reduced to one-half of the initial value. (From White, 1967.)

2 . Amyluse For an enzyme whose occurrence in honey has been known for 100 years, with hundreds of reports written on methods of quantitation, factors affecting activity in honey, and reporting assays of many thousands of honeys, very little is known of its kinetics, mode of action, and indeed, the significance of its presence in honey. a . tsolation and Pur$cation. The importance attached to amylase assay as a quality factor in honey is indicated by its inclusion in the Codex Alimentxius standards for honey. This reflects the preference of consumers in many countries for honey with relatively minor exposure to heat. Establishment of minimum acceptable values for honey “diastase” provides a control procedure. Since a few honey types are known that are naturally deficient in diastase, special provision is made for exceptions. Kerkvliet and van der Putten (1973) have compared five methods for determining the diastatic activity of honey by measuring the loss of iodine-coloration power.

322

JONATHAN W. WHITE, JR. TABLE XI SUMMARY OF ANALYTICAL DATA ON THE PREPARATION OF AMYLASE FROM HONEY"

Preparation stage Original honey (200 gm) Dialysed solution Acetone precipitate Amm. sulph. precipitate DEAE (cellulose) bands: A B

Total diastase units

Total a-amylase units

Protein (mg)

Diastase

a-Amylase Diastase ___ mg protein mg protein a-amylase

6520 6191 4590 1853

220.0 229.0 148.0 59.0

3180.0 1663.0 731.0 134.0

2.0 4. I 6.3 13.8

0.07 0.14 0.20 0.44

29.6 29.7 31.0 31.4

292 241

31.0 8.2

2.2 66.2

130.0 3.1

13.80 0.12

9.4 30.1

-

~

" From Schepartz and Subers (1966b) Honey has both a-and P-amylase activity; both the increase in reducing sugar and loss of coloration with iodine have been used in assays. The latter is most commonly used. Lampitt er a f . (1929) reported optimal pH for the a-amylase to be about 5.0 in the 22"-30"C temperature range, and 5.3 between 45"-50"C. For P-amylase, a value of 5.3 was reported. Schepartz and Subers (1 966b) attempted to separate the a- and P-amylase activities of honey by several procedures. Used as the final isolation procedure, ion-exchange cellulose chromatography provided two principal fractions. Table XI summarizes the research. Efforts to characterize the pooled fractions were unproductive because of their instability; the presence of a-glucosidase further complicated interpretation of the results. A 200-fold purification of the a-amylase was attained. White and Kushnir (1967a) carried out Sephadex gel filtration of dialyzed honey concentrates and determined amylase activity on the fractions. As seen in Fig. 16 a single peak was obtained using maleate buffer, indicating an approximate molecular weight of 21,600. Interaction with the Sephadex is evident when phosphate buffer is used with three maxima in the elution curve. Bergner and Diemair (1975), however, obtained single elution peaks in phosphate (pH 5.3) from G-200, as seen in Fig. 17, but considerably more retarded than the glucosidase peak in maleate, similar to the highest peak in phosphate in Fig. 15.

b. Heat Inactivation. Diastase has been used for at least 75 years as an indicator of honey heating, Nearly all of the reports are therefore oriented to this aspect, as is true for the invertase. Most of this work has measured a-amylase activity. The few papers reporting P-amylase inactivation in honey must be discounted because the a-glucosidase and glucose oxidase in honey may vitiate the results by their effect on maltose or glucose from the amylolytic reaction.

323

HONEY 1

t L

= w

1

1

SEPHADEX G-200 -M A L E A T E _.. PHOSPHATE .

L

3l

2

F R A C T I O N NUMBER (2111 I

FIG. 16. Sephadex (2.1 x 31 cm) filtration of enzyme preparation from cotton honey in 0.01 M maleate (pH 6.5). and in 0.01 M phosphate (pH 6.5). (From White and Kushnir, 1967a.)

The most complete study of heat inactivation of diastase (a-amylase) in honey remains that of White et al. (1964), who showed that loss of diastase by heating and by extended storage at lower temperatures obey first-order kinetics and can be described by the equation

Iog K

=

22.764

-

35010 2.303 RT

as shown in Fig. 14, which also includes data from other investigators. Figure 15 provides an estimate of the half-life of diastase in honey within the temperature range 20"-8OoC. It should be pointed out that although the relationship between time, temperature, and invertase and diastase activity have been widely quoted by honey

GLUCOSE OXIDASE

i I I

400

V, m l

-

500

V, ml

-

FIG. 17. Elution diagram of 10 ml protein concentrate on Sephadex G-200 (2.3 x 83.5 cm) in 0.03 M phosphate (pH 5.3) after freezing (-20°C) and remelting. (From Bergner and Diemair, 1975.)

324

JONATHAN W. WHITE, JR

scientists and control officials, they are based on a study of only three United States honeys (White et a/., 1964). This work should be extended to include other representative honeys. c . Source of Honey Amylase. In contrast to the a-glucosidase, which has a clear and essential function in the conversion of nectar to honey, no such function has been assigned to the starch-digesting enzymes in honey. Nectar contains no starch or dextrins. The question of its origin, in view of this, has been examined for many years. The presence of definite amounts of diastase in stores from sugar-fed bees (diastase numbers about 10 or less) led Gothe (1914) and others to ascribe it mostly to the bee, with a contribution from pollen. Vansell and Freeborn (1929) later contended that pollen, known to have diastatic activity, was the principal source, and Lothrop and Paine (193 1 ) supported this, citing the great variation in diastase value among honeys of different floral type. On the other hand, Fiehe (1932) considered nectar to be the major source; most honey has diastase numbers considerably in excess of the lower values common to stores from sugarfeeding. Braunsdorf (1932) found diastase numbers of 17.9 in two sugar-fed samples and proposed that it originates largely from the bees, with the variability resulting from the different degree of manipulation by strong or weak colonies upon slow or heavy flows of nectar. Weishaar (1933) ascribed only 1.5-2.5% of the diastase to nectar, 0.25-0.75% to pollen, and the remainder to the bee. Rinaudo et al. (1973) considered honey amylase originating from the bee as the basis of an optimal pH of 5.6-5.9 for preparations of the enzyme from honey and from honeybee hypopharyngeal glands. Nectar amylase pH optimum was 7.2, but that from pollen at pH 5.9 did not differ appreciably from that of honey. Amylase from honey and the bee was activated by chloride ion, in contrast to that of pollen and nectar. If diastase originates largely in the food glands of the honeybee, as does a-glucosidase, it would be expected that the ratios of these two enzymes in honey be relatively constant. Since a-glucosidase is more heat (and storage) labile, values for unheated honey should be used. The writer has calculated the correlations between diastase number and sucrase number of 39 unheated samples from the literature: 30 Swiss honeys from Table 1 of Hadorn et a/. (1962), 4 United States honeys from Table 2 of the same paper (actually supplied to the authors by the writer), and 5 stores from sugar-feeding, described in Table 1 from Hadorn and Zurcher (1963). A correlation coefficient of +0.83 resulted with F = 26.2, significant at less than 0.01%. There appears to be little doubt that the major portion of the diastase in honey originates from the bee, and the variability probably reflects the specific conditions during gathering and ripening of the nectar.

HONEY

325

3 . Glucose Oxidase Honey has been thought from ancient times to have wound-healing and antiseptic properties, and within the past 40 years a distinct heat-labile antibiotic activity has been the subject of considerable interest. The activity was named "inhibine" and a biological test was devised for its measurement in honey (Dold et al., 1937). During analytical studies on honey, the writer and his colleagues found that the drifting end-point common in the determination of acidity in honey, ascribed by Cocker (19%) to an acid-producing enzyme, was actually caused by the hydrolysis of lactone material in honey (White et al., 1958). Further studies indicated gluconic acid, in equilibrium with gluconolactone. to be the principal honey acid (Stinson er af., 1960). With the knowledge that a glucose oxidase had been reported in the hypopharyngeal glands of honeybees (Gauhe, 1941), it was demonstrated that the enzyme was present in honey and its production of gluconic acid and hydrogen peroxide during the standard microbiological test for inhibine was responsible for the major part of the antibiotic effect (White et al., 1963b). A chemical assay was described (White and Subers, 1963) in which the accumulation of hydrogen peroxide in diluted honey during a I-hour incubation was measured colorimetrically. From the results on 45 samples assayed by the Dold et af. (1937) plate assay and by the chemical assay, a relation between the inhibine number and log of peroxide accumulation was found. The effect of heating honey for 10 minutes at 70°C on the inhibine number and on peroxide accumulation was investigated for 29 samples, and for 6 the half-life of the peroxide accumulation system was determined (White and Subers, 1964a). A wide range in stability was found: most samples lost 85-95% of the activity when heated 10 minutes at 70°C, but seven lost less (6-71%) and five lost more (96-100%). Figure 18 shows a comparison of the heat sensitivity of the peroxide accumulation system in six honeys with that of honey diastase and invertase. The great variability, which precludes its use an index of heating exposure, is obvious. In a study of the previously reported instability of inhibine to light, White and Subers ( I 946b) found a wide variation in this effect; some honeys lost 90% of the activity on exposure to normal laboratory fluorescent light for 1 hour, others lost only 10% in full sunlight for 10 minutes. The sensitivity is maximal at 425-525 nm and pH 3, and is negligible at pH 6-7; a heat- and light-stable. nonvolatile sensitizer was postulated. The use of inhibine number or glucose oxidase activity as a measure of honey quality on heat exposure is therefore impractical because of the wide range of activity and the wide range (70-fold) of heat sensitivity shown by authentic honeys.

326

JONATHAN W. WHITE. JR TEMPERATURE

(OF)

I00

10

Y W

-

_I

u.

0. I

A U I

0.0 I

TEMPERATURE

('CI

FIG. 18. Effect of temperature on the half-life o f the peroxide accumulation system in honey. Diastase and invertase shown for comparison. (From White and Subers, 1964a.)

Schepartz and Subers (1964) and Schepartz (1965a,b, 1966a) have examined the glucose oxidase of honey. The enzyme is a true glucose oxidase, aerobically transferring H2 directly to molecular oxygen. Its pH optimum is 6.1 and requires 0.1 M Na+ for maximal activity. Of 32 carbohydrates tested, only glucose (100%) and D-mannose (9%) were oxidized. The optimal temperature is 40°C; it is completely inactivated at 60°C. It is strongly inhibited by NaCN and semicarbazide, somewhat by EDTA, mannose, fructose, and azide. Heavy metals at 0.001 M did not inhibit. The enzyme shows a preference for P-D-glucose over a-D-glucose of about 6:l. A kinetic study (Schepartz, 1965b) revealed the unusually high optimum substrate concentration of about 2.7 M (equilibrium glucose) as indicated in Fig. 19A. The Michaelis constant is 1.49 M, shown in Fig. 19B. In terms of 0-D-glucose, the optimal substrate concentration is 1.8M. The reaction follows zero order kinetics and is stoichiometric. Further investigation of the apparent contradictory action of fructose as an inhibitor or activator under certain conditions was carried out by Schepartz (1966a). Using manometric procedures, the results in Fig. 19 were obtained using varying concentrations of glucose (0.5-2.7 M ) and fructose (0.1-2.2 M).

HONEY

0

10 SUBSTRATE

327

20 30 40 C O N C E N T R A T I O N (MI (A1

(El

FIG. 19. (A) Effect of substrate concentration in velocity of reaction of honey glucose oxidase with glucose. measured manometrically. For details see original. (B) Lineweaver-Burk plot. Points were derived from those in A; v , initial velocity, (S), substrate concentration. K,, Michaelis constant, 0, points included in statistical analysis, A points not included in statistical analysis since beyond optimum concentration, 63, points derived from statistics and used to locate line. (From Schepartz, I965b.)

The plotted data in Fig. 20A suggest a coupling or uncompetitive inhibition by fructose. The plot of the transposed data from the experiments in which both sugars were present by the method of Hunter and Downs (1945) yielded the hyperbolic curve in Fig. 20B, which is found, according to Webb (1 963), in the rare instance of coupling or uncompetitive inhibition wherever the inhibitor combines only with the enzyme-substrate complex, never with the enzyme alone.

328

JONATHAN W . WHITE, JR.

0

200

100

300

400

v /IS1

12,

I

FIG. 20. (A) Plot of velocity-substrate data. In (a) the complete system contained: 0.5to 2.7 ml 3.5 M glucose in 0 . 2 M sodium phosphate (pH 6. I ) , and enough of the same buffer to total 3.4ml in the main space; 0. I ml (419 units) enzyme preparation in a side-arm sac; 0 . 2 ml 10% KOH in the center well. In (b) the system was the same except I .7M fructose was present in the main space at the 1.7 M and 1.OM glucose levels. Blanks were run without enzyme, without substrate; v , initial velocity in p molehin, (S). substrate concentration in M , vgOI. maximum velocity, K,,,Michaelis constant. (B) Plot of transposed velocity-substrate data. Conditions same as in A, except that fructose concentration was varied from 0. I to 2 . 2 M ; the combined sugar concentrations never exceeded 3.4M.(From Schepartz, 1966a.)

HONEY

329

Webb states that this gives rise to circumstances in which the inhibitor can cause activation. There is little doubt that honey glucose oxidase originates in the bee. Gauhe (1941) has shown that the glucose oxidase of the hypopharyngeal gland of the honeybee also has a high optimal substrate (about 2 M ) and a high Michaelis constant (KM0.63), greatly in contrast to those of other reported glucose oxidases such as the KM of 0.0042 for a mold enzyme reported by Keilin and Hartree (1948). Bee and honey glucose oxidases have pH optima at 6.1 and 6-7, respectively, and are equally specific for glucose. Most of the few other glucose oxidases oxidize a number of substrates. The great variability among honeys of peroxide accumulation, related to the antibiotic effect. does not imply a corresponding variability in glucose oxidase content of honey. The peroxide accumulation assay is carried out with diluted honey so that any constituents oxidized by peroxide will depress the value found. Thus data such as those of White and Subers (1963) showing a thousandfold variability in peroxide accumulation cannot be cited as indicating that the honeybee is not the source of the enzyme. To the writer’s knowledge, no true assays of glucose oxidase in honeys have been reported. 4 . Other Enzymes Catalase and an acid phosphatase are the remaining enzymes demonstrated to occur in honey. Gontarski (1948) described a “vitamin C oxidizing enzyme” in the hypopharyngeal glands of bees and observed a similar action in honey. He proposed that it might be identical with the glucose oxidase Gauhe (1941) reported in the bee glands. It is now apparent that this enzyme is in fact glucose oxidase; Schepartz (1966a) showed ascorbic acid to be a powerful activator of honey glucose oxidase by way of product removal and not due to action on the enzyme itself.

a. Caruhse. Schepartz (1966b) has reviewed critically the eight reports of catalase in honey which have appeared since Auzinger first reported it in 1910. Because of the earlier use of inappropriate methods and inconclusive experiments, he rejected them and, using manometric and spectrophotometric procedures, has claimed the first unequivocal evidence for catalase in honey. Using a dialyzed honey solution, he found a pH optimum at 7-8.5, a Michaelis constant of 0.0154M. and an optimum substrate concentration at 0.018M H202, with the reaction being first order. Subsequently Schepartz and Subers (1966a) described a kinetic assay procedure and reported catalase values for 28 honeys. Since diastase and peroxide accumulation values were available for the same samples, correlations were calculated. A direct correlation (r = -0.76, sig. at 0.01 probability level) was found between diastase and catalase, and as expected, an inverse

330

JONATHAN W . WHITE. JR

correlation (r = -0.71, sig. 0.01) between catalatic activity and peroxide accumulation. In the latter calculation, however, 12 samples which showed little or no catalase but also had little or no peroxide accumulation were not included. Inclusion of these samples reduces the correlation to 0.11, significant at less than the 90% probability level. It is evident that catalase activity is but one of the factors contributing to variability in peroxide accumulation. Dustmann ( 1 97 1 b) provides further evidence; he assayed 11 samples for catalase, using Schepartz’s procedure, and also for peroxide accumulation. The four samples with extremely high peroxide values (380-662) were totally devoid of catalatic activity. Dustmann’s other catalase values (46.1-241) are all greatly higher than those of Schepartz (0.5-17.8); the same procedure and units appear to have been used. Calculations by the writer based on the seven samples with catalatic activity showed a correlation coefficient between catalase and peroxide accumulation of 0.023 (not significant). When all samples were included, the correlation coefficient is -0.71 (sig. at .05). Using the assay procedures that Schepartz has since declared inapplicable (Schepartz, 1966b), Gillette (1931 ) reported source of catalase to be pollen. Dustmann (1971b), in the only study using acceptable procedures, has found very high catalase activity for pollen, very little in nectar. No reports could be found of catalase assay of stores of sugar-fed bees on which acceptable assay methods were used. b. Phosphutuse. Giri (1938), on the basis of the production of inorganic phosphorus from P-glycerophosphate during a 24-hour incubation at 35°C with diluted honey, stated that honey contains an acid phosphatase. The activity was maximal at pH 4.5-6.5 and was increased by magnesium ions. His two (of eleven) most active samples were “slightly fermented”; Giri stated that fermented honey samples were characteristically high, and values for unfermented samples were decidedly low, and it is lowered somewhat by pasteurization. He suggested that it is “derived chiefly from fermentation yeast and bees and partly from the plants.” Giinther and Burckhart (1967) described an improved procedure requiring a 3-hour incubation with p-nitrophenylphosphate. Zalewski (1965) assayed honey, pollen, nectar, and bees for acid and alkaline phosphatases using disodium phenyl orthophosphate as substrate, with incubation of 2.25 hours at 37°C for honey and nectar, 18-24 hours for pollen. Chloroform was added to control microbiological action. Acid phosphatase activity in honey and nectar ranged between 30-2140 and 15-2750 pmolell00 gm dry weight, respectively. Stores from caged sugar-fed bees had about ‘/16 of the honey average. The acid phosphatase assay of pollen ranged from 1260-145,500 pmole/l00 gm. It is implied that pollen is the principal source, although it is apparent that nectar contains sufficient to account for the activity in honey. Whether the enzyme can pass through the wall of the intact pollen grain is debatable.

HONEY

33 1

G. FLAVOR AND COLOR 1.

Flavor

Table honey is attractive to the consumer for a variety of reasons, flavor possibly being the most significant. While there seems to be a characteristic “honey flavor,” the wide variety of flowers attractive to bees overlays a great multiplicity of source-specific flavors and aromas. Color is also variable and strongly influenced by source, but more susceptible to environmental factors than is flavor. Flavors are sufficiently distinctive that dozens of different floral types can be identified by flavor alone by the experienced taster. Typical flavors can range from the most delicate and desirable to some that are harsh and objectionable. Generally, though not invariably, the lighter colors are associated with the milder, more pleasant flavors. The flavor complex includes, in addition to the volatile aromatic materials dominating sweetness, contributions from the acids, traces of polyphenolics, amino acids, and in some cases specific bitter or characteristic nonvolatile notes.

2. Aroma Relatively little attention has been given to the volatile aroma constituents. Gas-liquid chromatography has been applied by several groups of investigators (Dorrscheidt and Friedrich, 1962; ten Hoopen, 1963; Cremer and Riedmann, 1964). As is true of most natural products examined in this way, the lower aliphatic aldehydes, ketones, alcohols, and esters make up the bulk of the identified components. Cremer and Riedmann identified over half of 120 compounds separated by a 1 mm X 100 m Golay column and observed, after long storage, increases in pentanol, 2-methyl- I-butanol, 3-methyl-]-butanol, and n-propanol, suggesting that these may arise from the corresponding amino acids. Sixteen of the 22 honeys examined contained phenylethyl alcohol, and 14 also had benzyl alcohol. It is noteworthy that most synthetic honey flavors contain large amounts of lower aliphatic esters of phenylacetic acid and phenylethylsalicylate or phenylacetate; Jacobs (1955) states that nearly all phenylacetic esters are characterized by a honeylike taste and odor. An example of a type-specific aromatic is methyl anthranilate (MA), reported in citrus honey by Lothrop (1932). Lavender honey also contains it (Hadorn, 1964). Knapp (1 967) has elaborated upon White’s (1966) suggestion that MA content may be a useful quality measure for citrus honey, commenting that only 1 of 1000 samples would be expected to contain less than 1.5 ppm. He proposed that additional work be done with known single-source citrus samples as needed. The 80 predominately citrus samples reported by the two investigators averaged 3.8 ppm MA, range 0.844.9. Twelve noncitrus samples analyzed by White

332

JONATHAN W . WHITE, JR

averaged 0.07 ppm (range 0-0.28). Data for Knapp's 14 noncitrus samples were not available. 3.

Color

Little is known of the specific compounds responsible for the color of honey. Of 92 honeys Browne (1908) analyzed, 25 gave a positive test for polyphenolic compounds with FeCI,; the most intense reactions were from the darkest honey. Milum ( 1 939) ascribed the increase in color of honey upon storage to reaction of iron from processing equipment and containers with polyphenols, the browning reaction of reducing sugars and amino acids, and the instability of fructose in acid solution. Von Fellenberg and Rusiecki ( 1938) found water-soluble coloring materials to increase with honey color more than did fat-soluble colors. H.

VITAMINS

Honey has measurable amounts of six vitamins but at such low levels that they have no nutritional significance. Table XI1 summarizes the significant data. Widely conflicting reports of the ascorbic acid content of honey have been ascribed to interfering materials in the chemical determination. Most honeys contain less than 5 mg/100 gm. Some reports of values as high as 390 mg/100 gm by chemical means should be discounted, but Griebel(1938) confirmed chemical values for mint honey of 160-280 mg/l00 gm with bioassays in which 1 gm honey/day protected guinea pigs, corresponding to 100-200 mg/l00 gm. High ascorbic acid content ( I 18-240 mg/l00 gm) of Iranian honey was reported by Rahmanian et al. (1970) by chemical analysis and TLC of derivatives, and also confirmed by bioassay, which indicated levels of 75-150 mg/100 gm. They TABLE XI1 VITAMIN CONTENT IN MICROGRAMS PER 100 GRAMS OF HONEY

Samples

Riboflavin

Pantothenic acid

Niacin

Thiamine

Pyridoxine

Ascorbic acid

61 63 22 26 12-54

105 96 20 54 -

36Oh 32Oh I24 108 442-978

5.5 6.0 3.5 4.4 8 -22

299 320 1.6 10.0 -

2400 2200 2000-3400

~~

29 Minnesota" 38 U.S. and Foreign" 21 U.S. 3-7 years old" 19 U.S. 1-2 years old" 4 India"

Haydak et al. (1942).

* Corrected from original data in publication as later shown (Haydak et a / . , 1943). Kitzes er a / . (1943). Kalimi and Sohonie (1965).

HONEY

333

proposed use of this specific honey type (of unknown floral source) for helping relieve marginal vitamin C deficiency often found in Iran.

I.

TOXIC CONSTITUENTS

Since tremendous numbers of organic compounds are synthesized by various plants, many with substantial physiological activity, it is inevitable that some may, on occasion, be found in honey. The remarkable aspect is that, as widely as bees forage, the instances of toxic reactions are so few. White (1973) has reviewed the subject in some detail. Perhaps the best-known toxins are those of honeys from the Ericuceue (Rhododendron, Azulra, Andromeda, Kulmiu spp.), with literature descriptions reaching back to Xenephon's description of the mass poisoning of the expedition of Cyrus in 401 B.C. in Asia Minor, presumably by honey from Rhododendron; instances still occur in that area. Other areas from which reports of intoxication from Ericucear honeys are USSR, eastern and Pacific Northwest United States, and Japan. Beekeepers are largely aware of the problem and take appropriate steps to avoid it. Other toxic honey types are those from the tree tutu of New Zealand (actually a honeydew), henbane (Darura metel), Datura stramonium and Hyoscyamus niger, yellow jasmine (Jessamine), euphorbia, and arbutus. Details of toxicology, compounds responsible, and other aspects are in the review by White ( I 973).

V.

PHYSICAL CHARACTERISTICS

The physical attributes of honey are largely conferred by the high concentrations of sugars that compose most of the solids. Viscosity, refractive index, and specific gravity are so closely related to solids content that each has been used to measure moisture (solids) content. Refractive index is the most easily used, as implied in Table IV. A complete table of refractive index-moisture content equivalents appears in the Book of Methods, AOAC (Horwitz, 1975). Specific gravity (20/20") varies regularly with moisture content, between 1.4404 at 14.0% moisture through 1.4174 at 18.0% to 1.3550 at 21 .O%. A table at 0.2% intervals is available (Wedmore, 1955). Because of the fairly wide natural range, care must be taken to mix thoroughly when blending honeys of different moisture content to avoid layering. A.

RHEOLOGY

Much early effort was expended in attempts to determine moisture of honey with such instruments as the hydrometer (Chataway, 1933) and the falling-ball viscosimeter (Chataway , 1932; Oppen and Schuette, 1939) with approximately

334

JONATHAN W. WHITE, JR

the accuracy, but without the facility, of the refractometric measurement. Using absolute viscosity values, Lothrop (1939) found a rather wide variation among honeys adjusted to equivalent moisture contents. Munro's (1943) data (Table XIII) are the most extensive available. The high viscosity of honey is most apparent when draining containers or in pumping or processing it. Although Munro stated that most of the decrease of viscosity on warming takes place from room temperature to about 30°C, his observation was based on a linear plot. Pryce-Jones' (1953) plot of Munro's data as log viscosity versus 1/T shows that rate of change is relatively constant; only the extent of heating needed to obtain the required viscosity reduction should be applied to minimize heat-induced damage to color and flavor. MacDonald (I 963) has examined the effect of temperature on the flow of honey through pipes under a constant head. Table XIV shows the results. For the average of all four pipe

TABLE XI11 VISCOSITY OF HONEY

Moisture content

Temperature

(8)

("C)

Sweet clover" (Melilotus)

16.1

Sage" (Sulviu)

18.6

White clover!' (Trifoliurn repens)

13.7 14.2 15.5 17.1 18.2 19.1 20.2 21.5 16.5 16.5 16.5

13.7 20.6 29.0 39.4 48. I 71.1 11.7 20.2 30.7 40.9 50.7 25.0

Type

Sageb Sweet clover" White clover" a

Data of Munro ( 1 943).

* Interpolated from Munro's data.

25 25 25

Viscosity (poise) 600.0 189.6 68.4 21.4 10.7 2.6 729.6 184.8 55.2 19.2 9.5 420 269 138 69.0 48.1 34.9 20.4 13.6 1 I5 87.5 94.0

335

HONEY

TABLE XIV RELATIVE FLOW OF HONEY IN PIPES”

Temperature Pipe diameter (inside)

82°F (28°C)

102°F (39°C)

122°F (50°C)

% in. (19 mm) 1 in. (25 mm) 1% in (31 mm) 1% in. (38 mm)

149 361 129 1263

400 913 1895 2609

1125 2353 5000 6192

Rate of flow (in pounds per hour) through 4-inch (10-cm) length of pipe with 4-inch head. Data of MacDonald (1963).

sizes, the rate of flow increases equally with each of the two temperature increments. The importance of pipe diameter in moving honey at the lower temperatures is shown by the eightfold increase in flow obtained when the cross-section area of the pipe is increased four times. This effect declines as the viscosity decreases with increasing temperature. Most honeys are Newtonian liquids but some have been reported to have non-Newtonian properties. Pryce-Jones (1953) has examined the rheology of heather honey, which is so thixotropic that it cannot be removed from the comb by a centrifugal extractor unless the gel-sol transformation is effected by applied vibrating rods. This property is ascribed to the properties of the proteins; if isolated heather honey protein is added to clover-honey, it exhibits thixotropic behavior. Manuka (Leptospermium scoparium) honey from New Zealand and Karvi (Carvia caflosa)from India (Deodikar et al., 1957) are markedly thixotropic. Pryce-Jones (1952) also reported that Opuntia honey from Nigeria and several Eucalyptus types exhibited dilatancy , which he ascribed to the presence of a high-molecular dextran.

B. THERMAL PROPERTIES Relatively little data are available on the physical properties of honey with respect to heat, even though honey can easily be damaged by its improper application. Processing equipment design has generally been based on data from sugar processing. The specific heat of honey at 17.4% moisture was reported by Helvey (1954) to be 0.54 cal/gm/”C at 20°C with a temperature coefficient of 0.02 caV”C. He also measured specific heat of honey solutions. Townsend (1954b) has described McNaughton’s determination of specific heat. He used a considerably larger sample and obtained somewhat higher results, as seen in Table XV.

336

JONATHAN W. WHITE, JR

TABLE XV SPECIFIC HEAT OF HONEY"

Moisture content (%)

Specific heat

20.4 19.8 18.8 17.6 15.8 14.5 Coarsely granulated Finely granulated

0.60 0.62 0.64 0.62 0.60 0.56 0.64 0.73

" Data of MacNaughton (Townsend, 1954b). Basic data necessary in designing a heat exchanger for honey processing have been obtained by Detroy (1966). Using a concentric-tube exchanger, the surface conductance or film coefficient for honey was determined at flow rates of 700975 lb/hour and two temperature ranges of interest in processing, preheater (65"68°C) and flash heater (85"-88"C). Figure 21 shows the values calculated from these data for a range of honey flow velocities. In this work honey was in laminar flow, water in turbulent flow. Detroy used Helvey's value for specific heat of 0.54. He pointed out the desirability of experimental verification of his values

I

I

I

I

0.18 0.20 0.22 0.24 0.26 V E L O C I T Y OF HONEY FLOW ( f t / s e c )

FIG.21. Change of film coefficient with velocity of honey flow in the honey-to-watertemperature difference range of each heating circuit. Upper line, flash heater water circuit; Y = 10.6 + 272x; Sv,z = 1.68. Lower line, preheater water circuit;y = 26.51 + 53.3~;SZ," = 1.7. (From Detroy, 1966.)

337

HONEY

under accurately controlled conditions, since the possibility for experimental error is considerable. The heat sensitivity and relatively low heat conductance of honey have encouraged examination of high-frequency heating of honey. Lackett and Wilson (1971) used a kitchen-type microwave oven operating at 2450 mHz to heat and liquify completely granulated honey in 1 -Ib jars. With the metallic caps removed, heating was rapid but not uniform; when a temperature of 60°C was reached in the center of the jar, a damaging 98°C was reached at the top. Difficulty was encountered in heating larger jars (2% Ib) without boiling the surface layers. Bergel and Stuwe ( 1 972) have proposed the use of dielectric heating for honey processing. They have estimated from small-scale heating experiments that a 25-kW dielectric heating installation would be required to heat I000 kg per hour from 30" to 55°C. The frequency was not specified. Normal range for dielectric heating is 2-100 mHz. The arrangement and results of the small-scale tests are y

0 5 K W HIGH FREOUENCY HE ATlN G EO U I P M E N T

60

IW

a

ELECTRODE AREA 240 I I B O m m

13

I0: 4

I

20 0

CONTAINER PETRI D I S H

wt-

WEIGHT OF HONEY m:200q

Lg 0

BETWEEN ELECTRODES ON T E F L O N S U P P O R T S

(A1

0

05 10 1 5 2 0 2 5 HEATING T I M E t I m i n )

FIG 22 (A) Dielectric heating of honey in a glass dish (From Bergel and Stuwe, 1972 ) (B) Temperature variation in high-frequency heating of honey in ajar (From Bergel and Stuwe. 1972 )

338

JONATHAN W . WHITE. JR.

shown in Fig. 22A. The authors point out that, as seen in the figure, the power absorption decreases with increasing temperature, i.e., the dielectric loss value for honey decreases with increasing temperature. This automatically provides temperature equalization. In Fig. 22B is seen the results of heating honey in a commercial jar (500 gm) with metal cap. Under the proper conditions the jar of honey is heated to 59'43°C in 2% minutes. Overheating induced boiling in a ring under the cap. Analysis of all honey samples in the latter experiment showed no change in diastase value or HMF content. Samples remained liquid for at least 6 months. By heating I kg in two jars in the 2-kW chamber at full power, a linear heating rate of 32"C/minute was found between 20" and 60°C. From this a high frequency power requirement of 25 kW was calculated for heating 1000 kg/hour from 30" to 55°C. C.

HYGROSCOPICITY

I. Equilibrium Relative Humidity The ripening of nectar to honey by the bee includes its repeated exposure in a thin film to warm air. The solids content reached is a function of the extent of moisture saturation of the air in the hive, which is related to temperature and to the external air conditions. Nearly all honey contains less glucose than fructose, the more hygroscopic carbohydrate, and is remarkably hygroscopic for a natural material. As seen in Table XVI, honey in its normal moisture range of 16.818.3% is in equilibirum with air at 55-60% RH. In general, attention must be given to hygroscopicity in handling and processing, since, as Martin (1958) has TABLE XVI APPROXIMATE EQUILIBRIUM BETWEEN RELATIVE HUMIDITY OF AIR AND THE WATER CONTENT OF A CLOVER HONEY"

Relative humidity

Water content

("/.)

(%)

50 55 60 65 70 75 80

15.9 16.8 18.3 20.9 24.2 28.3 33. I

"

Interpolated from the data of Martin (1958).

339

HONEY

shown, moisture from the air diffuses only slowly into the mass, so that aerobic yeast growth is encouraged at the surface. For example, Martin (1958) showed that a honey sample at 22.5%moisture exposed to air at 86% RH for 7 days had 26% moisture in the surface layer; 2 cm below no change was found.

2 . Comparison with Other Carbohydrates Relatively few data are available to provide for comparison of commercially available carbohydrates. Table XVII summarizes values for honey, invert syrup, fructose syrup, and commercial glucose at 20% moisture. Uncertainty among values of different investigators makes it difficult to determine if real differences exist among honey, invert syrup, and fructose syrup. Conventional corn syrup is definitely less hygroscopic. Data are not available for high fructose corn syrup.

D.

CRYSTALLIZATION 1 . Glucose

a . Cause and Prediction. As noted earlier, the stable form of most extracted honey is a matrix of glucose hydrate crystals in a syrup. This is due to a considerable extent to the lower storage temperature to which the honey is exposed after removal from the bee colony. Nearly all honey is supersaturated with respect to glucose except for a few nongranulating types that are relatively low in glucose, such as tupelo and sage. Crystallization of glucose from honey while in the

TABLE XVlI EQUlLIBRlUM RELATIVE HUMIDITY OF VARIOUS CARBOHYDRATES AT 20% MOISTURE"

Material Honey Invert sirup

Levulose sirup Commercial glucose

'I

tJ

E.R.H.

Reference

63.5%" 63.2 67 67.5 57.5 63.5 61.3 75 72

Lothrop ( 1937) Martin ( 1958) Lothrop (1937) Dittmar (1935) Money and Born (1951) Money and Born (1951) Lothrop (1937) Lothrop (1937) Money and Born (1951)

Most values interpolated from original data Average of five samples.

340

JONATHAN W . WHITE, JR.

comb, though relatively rare, may be encountered with such honey as dandelion, blue curls, and ivy. It is likely that the extraction process encourages subsequent granulation by introducing fine glucose crystals from equipment, from the air of the extracting plant, and from containers. Natural crystallization, before heating, is usually fine grained, reflecting the presence of myriads of fine seed crystals and initiators such as dust, pollen, and fine air bubbles. After honey is heated and/or filtered, seed crystals are no longer present, and when crystallization finally takes place it is usually coarse grained and slow. Proper heating and processing will delay granulation for many months. Two general approaches to predicting granulation tendency of honey have been made: study of model systems and empirical correlation of various parameters with observed behavior. Examples of the former are the work of Jackson and Silsbee of the U.S. Bureau of Standards, Lothrop of the U.S. Department of Agriculture, and Kelly of the University of Tasmania. Jackson and Silsbee (1924) examined several systems at 30°C and discussed the glucose-fructosewater system with reference to honey. In the presence of solid glucose hydrate, solubility of glucose decreased from 54.6% without fructose to 32.5% at 39.4% fructose. Their conclusion that all honey is supersaturated with respect to glucose (even never-granulating tupelo honey) was based on inadequate analytical procedures then used for honey, which overestimated glucose; in addition, their data did not extend to the higher fructose concentrations found in some honey. Lothrop (1943), in an unpublished thesis, extended their data to higher fructose concentrations. He found an abrupt increase in dextrose solubility at a fructose concentration of about 150 gm in 100 gm water. The solid phase in the region of higher solubility was anhydrous glucose. Identification was by crystal form. Lothrop felt that the increased solubility was not related to the a-p equilibrium, but rather to the extent of hydration of the glucose in solution, and concluded that this accounted for the failure of certain honeys to crystallize. Lothrop's data, replotted on a ternary diagram, are shown in Fig. 23, since they were never published. Kelly (1 954), without knowledge of Lothrop's work, published the complete diagram for the system at 30°C. He also noted an area in which anhydrous glucose is the solid phase (Fig. 24) and an invariant point at which both forms are in equilibrium. He suggested that in solutions saturated with fructose, the transition temperature of the monohydrate was reduced from >50"C to <3OoC. He noted that published analyses of honey relate to the area at which anhydrous glucose is in the solid phase at 30°C. However, honey crystallizes only below 30"C, below the transition temperature, so that only the hydrate crystallizes. Villumstad ( I 952) has described the simultaneous presence of both plates and needles of glucose in honey, without speculation on the reason. Kelly (1 957) suggested using the quaternary glucose-fructose-sucrose-water diagram (Kelly, 1955) in an effort to predict granulation. As a first approximation, all disaccharides were considered as sucrose, and a honey composition fell

341

HONEY WATER

GLUCOSE

FRUCTOSE

FIG. 23. Effect of temperature on the fructose-glucose-watersystem. (From Lothrop, 1943.)

GLUCOSE

FRUCTOSE

FIG. 24. The system water-glucose-fructose at 30°C. (From Kelly, 1954.)

342

JONATHAN W . WHITE, JR

into the area in which the solid glucose is anhydrous at 30"C, but hydrated at some point below 30°C. An additional complication may be the finding of Dean (1976) of a phase in the glucose-water system thought to be P-D-glucose monohydrate metastable at 38"-50"C, transforming to stable a-D-glucose monohydrate at 32"-38°C. The metastable phase is apparently orthorhomic, as are both anhydrous forms. A further problem in applying model systems is the presence of a variety of other sugars at > 1 % of the total. Knowledge of granulating tendency would be useful in selecting honey for both liquid and semisolid pack. Earliest attention was given to the ratio of fructose to glucose for prediction, but difficulty was encountered (Jackson and Silsbee, 1924) because of inadequate anlytical methods for these sugars on mixture. White et al. (1962) reviewed the literature and calculated several of the proposed indices for the 490 honeys they analyzed. The ratio DIW, proposed by Austin (1958), showed a highly significant relationship with granulating tendency (as defined) equivalent to theD-WIL of Jackson and Silsbee (1924). White er al., considering that the latter required more analytical work, suggested that the DIW ratio be used; values of 5 1.7 seemed to be generally associated with nongranulating honeys; values Z2.1 would predict rapid granulation to a solid. Codounis (1962) has suggested that the relationship B-DID (B = Brix, D = glucose) provides a better index. Hadorn and Zurcher (1974) found only a loose association between DlW and subsequent crystallization of honey. The calculations of White et al. (1 962) were based on observation of the extent of granulation of samples after 6 months of room temperature storage following liquefaction. A more useful index may have resulted had the samples been seeded with glucose hydrate after cooling. b. Controlled Crystullization. Since the texture of the semisolid pack depends upon a three-dimensional interlaced network of dextrose hydrate crystals in a liquid phase, it will not return to the initial hardness after stirring or softening by heat or improper storage. The initial texture is influenced by moisture and dextrose content of the honey and by the amount and quality of the seed material. Best (smoothest) texture results from growth of a network of interlaced fine crystals, induced by large numbers of very fine crystals and fragments in the seed material, The temperature of the honey at the addition of the seed must be low enough to avoid destroying the smallest crystal particles in the seed. The addition of seed at 14°C with continued blending at that temperature however does not seem to lead to the most desirable texture because too much of the crystallization takes place too early to permit an adequately interlaced network of crystals to form in the retail container. This product is not commonly packed in clear glass, but rather in waxed paperboard or plastic tubs or jars or in opaque glass jars. The reason appears to be

HONEY

343

that changes at the container-honey interface can give the appearance of spoilage, although the product is in good condition. This is an uneven separation of the contents from the wall of the container, sometimes combined with a white irregular patchy appearance termed “frosting.” Thomson (1938), in a detailed study of this, concluded that it resulted from the presence of air bubbles in the honey before setting, together with a contraction of volume accompanying granulation. Crystals in frosted areas were found to be smaller than in other areas and consequently appeared lighter in color. The volume contraction was found to be 0.500 cc per 100 gm, or about 0.60% V / V . No consideration was given by Thomson to avoiding or eliminating the defect. Green ( I 95 I ) , without reference to Thomson, ascribed frosting to the presence of dissolved air and tiny air bubbles in the honey and their segregation by rejection from the crystal. He ascribed no relevance to the composition of the container wall and proposed no remedies. Gonnet and Lavie (1963) studied the adhesion of crystallized honey to container walls. They ascribed separation to thermal shock (rapid temperature drop) and also to continued storage at 14°C after setting, with separation occurring in the third week of such storage. They eliminated it by exposure to 30°C, but noted it recurred after sharp cooling. The color change was ascribed to desiccation of the exposed glucose crystals. They examined several treatments of the glass container walls to eliminate this without marked success. McDonald (1964) also studied the factors affecting the formation of voids (i.e. space between container and product) and recommended prompt removal from the 14°C crystallizing temperature after 5 days to 20°C or higher temperatures, followed by conditioning. A conditioning for 2 days at 35°C of the particular honey used prevented subsequent void formation regardless of subsequent lower storage temperature; McDonald indicated that lower temperatures might be preferable for honey of higher moisture content, since considerable softening was evident after conditioning. Guilbault (1965) included this problem in his study of quality factors in recrystallized honey packs. He recommended conditioning in opaque packs after the 4-5 days 14°C crystallizing step by storage 7-14 days at 25”-27”C followed by holding 10 days or till shipped at 14°C. For glass packs, holding for not over 5 days at 30°C would prevent subsequent frosting. Other recommendations included control of seed quality by making crystal counts, since a 100% variation can occur in this parameter, which affects product quality.

2. Melezitose Other than glucose, melezitose is the only sugar known to crystallize from honey, or more correctly, from honeydew honey. It is relatively rare to encounter sufficiently high concentrations of melezitose for crystallization. Such crystallization usually takes place in the comb. Hudson and Sherwood (1920) described

344

J O N A T H A N W . WHITE, JR

several instances where this occurred, finding 10-25% melezitose in the honeydew honeys they described.

VI. STORAGE OF HONEY A.

EFFECTS OF TIME AND TEMPERATURE

It is not surprising that honey is susceptible to physical and chemical change during storage, considering its nature as a highly concentrated, somewhat acid solution of fructose and glucose. The changes caused by heating also take place at any temperature above about 5°C.

I. Color Development Depending upon the composition (fructose, moisture, acidity) honey darkens during storage and heat processing. Considerable variation among samples has been observed. Noting that addition of formaldehyde prevented darkening of honey on storage, Ramsey and Milum (1 933) proposed that most of the darkening resulted from the Maillard reaction between amino acids and reducing sugars. The most extensive study if that of Milum (1948). He concluded that darkening during storage is dependent in part upon the amount of previous darkening; thus discoloration during processing tends to reduce the subsequent rate. Table XVIII shows a summary of Milum's data obtained by replotting and interpolation. The values should be regarded as approximations only. Smith (1967) stored several Australian commercial-type honeys at temperatures between 43" and 80°C, all TABLE XVIII APPROXIMATE RATE OF HONEY DARKENING IN STORAGE"

Temperature of storage

"F

Darkening in mm Pfund/month

"C

Original color < 34 mm

Original color 34-50 mm

Original color > 50 mm

10.0 15.6 21.1 26.7 32.2 37.8

0.024 0.08 0.27 0.90 3.0 10.0

0.024 0. I25 0.70 4.0 7.7 14.0

0.024 0.10 0.40 1.5 5.0 11.0

~

50 60 70 80 90 100

"

Calculated from data of Milum (1948).

HONEY

345

above those given in Table XVI. One honey (Dryandra) was more unstable than the others. Smith found for the remaining five that the times required at various temperatures to produce 10 mm darkening (Pfund) were of the same magnitude as those calculated by White et a / . (1964) to produce 3 mg HMFl100 gm honey. Wootton et al. (1976a) examined the changes induced by storage of six Australian honeys at 50°C for 44 days, analyzing for color, acidity, total nitrogen, sugars, and free amino acids. Rate of increase in color varied markedly; the least stable required approximately 5 days to increase 20 mm in color, and the most stable, about 16 days. This is a wider range than found by Smith. The retarding effect of added sulfite indicated the Maillard reaction to play a major role; ascorbic acid addition had no effect, eliminating an oxidative mechanism. Changes in carbohydrates (Wootton et uf., 1976b) differed from those reported by White et al. (1962) and Kalimi and Sohonie (1964), but all experimental factors involved differed. Amino acid content decreased markedly for five honeys (26-83% decrease), and apparently increased (6%) for one, tea-tree (Leptospermum scoparum). Most of the quantitative decrease was loss of proline, which represented approximately 80% of the free amino acids, except for tea tree (about 30%). Increases were found in a few (1-3) amino acids, losses in all others; again tea tree was the exception, showing an increase in total amino acids, including 7 of the 15 present. A major increase in phenylalanine was recorded for alfalfa and tea-tree honeys. The greatest decrease reported would remove only about 0.16% of monosaccharide from the honey, less than the analytical error in the sugar analysis. Wootton ef a / . ascribed the increases to protein breakdown.

2 . Hydroxymethylfurjural Several colorimetric tests were devised many years ago to indicate the addition of acid-inverted syrup to honey. These tests, the resorcinol (Fiehe) test, and the aniline (Feder) test have been intensively studied and modified. Considerable early controversy was concerned with the interpretation of the colors produced. These reagents were known to be reacting with hydroxymethylfurfural (HMF), which is formed from fructose by action of acid and heat. Invert sugar prepared with acid contains variable amounts of HMF, depending on the conditions used. The minimum amount of added invert sugar detectable by these tests thus depends on their sensitivity and the characteristics of the invert syrup used. Much controversy in the literature revolved about this because the color tests responded differently for different investigators, thus in effect using differing standards for judging adulteration by invert sugar. It was long ago recognized (Browne, 1908) that honey if heated sufficiently would give a positive test. Such heating was said to destroy the flavor characteristics. Nothing is to be gained by reviewing earlier studies, of which there

346

JONATHAN W . WHITE, JR.

were many, which attempted to show that the Fiehe and Feder tests were, in fact, suitable to distinguish between heated and adulterated honey. For example, these included studies (Shannon, 1916; Sherwood, 1924; Greenleaf and Browne, 1929) in which collaborative testing was done, and two papers concluding that the color tests indeed were useful (Lampitt et al., 1929; Gautier et al., 196 1). de Boer (1934) suggested that extended ordinary storage could also lead to accumulation of enough HMF for a positive test, the time required being a function of the storage temperature. The publication by Winkler (1955) of two quantitative methods for HMF in honey provided the means for European countries to extend their examinations of imported honey to include the HMF levels of honey, as well as diastase content. These countries had for many years insisted that honey sold for direct consumption meet minimum standards for diastase to assure that it had not been “denatured” by overheating. They ascribe rather obscure health-giving properties to honey which they feel are vitiated by heating. Enforcement of these rules has provided a considerable volume of analytical data on honey from the major exporting countries. When the quantitative HMF method became available, limiting values for HMF content of honey were established in addition to diastase values. An extensive collection of analyses of imported honey is summarized in the paper of Duisberg and Hadorn (1966), where HMF analyses of 1554 imported samples of commercial honey received between 1960 and 1966 by laboratories in Switzerland and Germany are given. Figure 25 shows the results. Using Winkler’s methods for quantitation of HMF in honey, Schade et al.

H M F CONTENT FREQUENCY D l S T R l O U T l O N 0 1 3 0 7 Importsd Honsyr, Brsmen Lab

U I I l 247 -39

0

1

I m p o r t e d Honeys, Easel Lob SWISS

2

Honeys, Basel Lob

,

3 4 H M F (rng %)

5

6

FIG. 25. Frequency distribution of HMF content of honey. (From Duisberg and Hadorn, 1966.)

347

HONEY

(1958) demonstrated variability among honeys, of the effects of storage and heating, and of the compositional factors influencing HMF formation, and reported the rate to correlate directly with moisture content and with initial HMF content. Other unknown factors also affected rates. Lampitt et al. (1929) had earlier confirmed the positive effect of the acidity of honey in the formation of HMF; Hadorn et al. (1962) ascribed the lower rate of HMF formation in heated Swiss honeys to their higher pH (4.5-5.0) in contrast to that of most other honey (pH 3.8). Several groups of workers have reported the effect on HMF content of various heat treatments of honey. The temperature exposures ranged from those normally used in honey processing to deliberately excessive treatments, in terms of deleterious effect on general organoleptic qualities. Schade et al. (1958) showed HMF to increase in four samples during storage 13-15 months at 20°C (68°F); in one case an increase of 3.3 mg% was recorded. Three alfalfa honeys accumulated HMF at higher temperatures as shown in Fig. 26. Hadorn and Kovacs (1960) reported the effect of holding several types of imported honey at 50°C (122°F). Their results are summarized in Table XIX. Most investigators reported data from small-scale laboratory tests. An exception is the work of Hadorn and Zurcher (1962), who followed HMF content in 300-kg (660-lb) drums as they were taken through the procedure normally used in Switzerland to liquefy the contents, holding in a room at 48°C. The honey reached 48°C in 24 hours. At 120 hours, HMF content in three drums had increased from I .2 to 2.2, 2.7, and 2.4 mg/100 gm. White et al. (1964) subjected three honey samples to storage at seven temperatures ranging from -20" to 60°C (-40" to 140°F) and analyzed them for HMF 70

60

5

50

I 0

I .

40

30

E

g

20 10 0

0

2

4 6 8 WEEKS OF STORAGE

10

FIG. 26. Comparison of the rate of formation of HMF in three alfalfa honeys during storage at elevated temperatures. From Schade et uf., 1958. Reprinted from Food TechnologylFood Research 23, 446-463, 1958. Copyright @ by the Institute of Food Technologists.

348

JONATHAN W . WHITE, JR TABLE XIX HYDROXYMETHYLFUFWURAL CONTENT OF HONEY HELD AT 5O0C"* ~~

_______

~

Source

Initial value

Guatemala Central America California Mexico

2.5 0.6 1.6 0.1

~

_

_

_

_

_

100 hr

_

_

_

_

~

300 hr

5

16 8 8 26

2 4.3 0.8

" Interpolated from graph of Hadorn and Kovacs ( 1 960). In rng/100 grn of honey.

content. Figure 27 shows the approximate time required for a honey to accumulate 4 and 20 mg HMFf100 gm. The effective use of HMF levels to demonstrate addition of invert sugar to honey requires that a maximum value be established representing the combined effects of commercial storage and processing so that genuine, albeit storage or heat-abused, honey not be discriminated against. Variability in the response of honey to heat compounds the difficulty of setting an equitable level. In any event, since honey may, due to economic conditions, be stored a year or more in

0.1 I

20

I

30

I

I

40 50 TEMPERATURE ('C)

I

I

60

70

FIG. 27. Heat exposure required to develop indicated amount of HMF in honey. (Calculated from data of White et al.. 1964.)

HONEY

349

high ambient temperatures at tropical places of production, a history of storage of suspected samples may prevent unjustified allegations of adulteration. 3 . Flavor Changes As honey is heated or stored for several months at temperatures common to much of the United States, the more delicate aspects of flavor and aroma may change. These changes are relatively minor; to detect them a sample must be compared with a control kept at freezer temperatures. It is easily possible to damage flavor by excessive heating; heating that causes darkening will certainly have a deleterious effect on flavor. Fresh honey in the comb, a delicacy of yesteryear, has the maximum of volatile “top notes” and desirable flavor quality. Present-day closed-system high-temperature short-time processing does provide a better flavored product than do the batch processes that were previously used and are still used by some smaller operators.

4 . Enzyme Inactivation The effect of storage on enzyme inactivation is of importance largely in honey intended for export to countries with minimum limits for diastase (and in some cases, invertase). Difficulty has been encountered with rejection of United States shipments by European countries over many years. Many papers describe the effect of elevated temperatures on enzyme activity, particularly diastase, in honey, but it remained for Schade et al. (1958) by applying their quantitative procedure for honey diastase to record the effect of storage at 20°C on diastase. The loss which they described as “slight, but not significant in most cases” can be calculated at about 10% in 13-15 months. Later White er af. (1964) emphasized that the changes are relatively predictable over the temperature range of 10”-80°C; Table XX shows the half-life of honey diastase and invertase over this range, based on their data. 5.

Carbohydrate Composition

The most obvious change in the sugar of honey takes place during ripening, with the inversion of sucrose and the production of transglycosylation sugars, which persist in the ripened product. Enzymic inversion continues in full-density honey at a greatly reduced rate and can contribute to error in the analysis of sucrose. The most striking evidence is seen in certain floral types that on occasion produce so heavily in relatively warm, dry weather that the honey reaches full density while the sucrose content is still 10%or more. This may lead to legal difficulties, since maximum permitted values for sucrose can be as low as 5%. Unless the honey is heated, the sucrose content can be expected to decline to

350

JONATHAN W . WHITE, JR.

TABLE XX CALCULATED HALF-LIVES OF HONEY ENZYMES“

Temperature

“F

“C 10 20 25 32.2 35 40 50 62.4 71 80

“

50 68

71 90 95 104 122 145 160 176

Half-life Diastase

Invertase

12,600 days 1,480 days 540 days 126 days

9,600 days 820 days 250 days 48 days 28 days 9.6 days 1.3 days 3 hr 4 0 min 8.6 min

78 days 31 days 5.4 days 16 hr 4.5 hr 1.2 hr

From White ef a / . (1963a).

legal levels, although the time required is variable. Smith (1965) in Australia described a crop of honey fromBanksia menziesii which fell from 8-12% sucrose to about half that in a year’s storage. The acacia (locust, Robinia pseudoacacia) honey flow is at times of this nature; Borus et al. (1966) reported an instance in which locust honey of 9.6-12.7%sucrose fell to 1.7-4.3%in a year’s storage. In general, the nature of the honey flow, as described above, results in lower-thannormal levels of bee-added enzymes. Citrus honey is also one of those the Germans call “naturbelassen,” being generally low in enzymes for this reason. Three 1976 citrus honey samples with sucrose contents immediately after extraction of 5.3,7.1, and 9.3%fell during 12 weeks room temperature storage to 2.9, 3.1, and 2.2%, respectively (White, 1976). Changes in other carbohydrates during storage are not as obvious. Taufel and Muller (1 953), finding minor sugars in honey by paper chromatography, suggested that they might arise from acid or enzyme conversion of the major honey sugars. Later (Taufel and Muller, 1957) they concluded, using conventional analyses and paper chromatography, that significant changes do not occur on storage. Using more appropriate analytical procedures and statistical treatment, White er al. (1961) examined the effect of storage of honey on carbohydrate composition. In this work, honey stored at -20°C was compared with aliquots held up to 2 years at room temperature, with and without heating 30 minutes at 55°C for pasteurization, without excessive enzyme inactivation. The storage caused an increase of 69% in reducing disaccharides, a slight increase in sucrose and higher sugars at the expense of glucose and fructose, which decreased 13 and 5.5%, respectively. It is likely that this decrease in glucose is a

35 1

HONEY

major cause of texture loss and partial liquefaction of finely granulated honey during long-term storage. These changes would appear to be caused by two mechanisms: enzyme activity and acid reversion. At the low water concentration in ripe honey, the formation of disaccharides by slow a-glucosidase action should be favored over the accumulation of free hexoses by transfer to water as the acceptor. In concentrated solutions of monosaccharides in the presence of acids, appreciable reversion to disaccharides and higher sugars takes place (Pigman and Goepp, 1948).

B.

FERMENTATION

The development of commercial honey production during early decades of this century encountered major difficulties, particularly in Canada, with fermentation. Osrnophilic yeasts, which can ferment honey even at its low water activity, are nearly ubiquitous on the bodies of bees, in nectar, soil in apiaries, and extracting and storage areas. Even though a honey be in the “safe” area (Table V) the subsequent granulation will enrich the liquid phase in water and increase the risk. Many investigators in the northern United States and Canada studied this problem between 1928 and 1932, with the most definitive work that of Lochhead and his co-workers (Lochhead and Heron, 1929; Lochhead and Farrell, 1930a,b, 1931a,b; Lochhead, 1933; Marvin, 1928, 1930; Wilson and Marvin, 1929, 1931 , 1932; Marvin et ul., 1931). Table XXI lists the yeasts isolated from honey. Martin (1958), in his work on hygroscopicity, examined factors leading to yeast growth at the surface and also in the depth of the container. He found that, when TABLE XXI YEASTS ISOLATED FROM HONEY

Type

Reference

Nematospora ushbya gossypii Saccharomyces bisporus Saccharomyces torulosus Schizosaccharomyces octosporus Schwanniomyces occidentilis Torula mellis Zygosaccharomyces spp. (2) Zygosaccharomyces barkeri Zygosaccharomyces japonicus Zygosaccharomyces mellis Zygosaccharomyces mellis m i d i Zygosaccharomyces nussbaumeri Zygosaccharomyces priorianus Zygosuccharomyces richteri

Aoyagi and Oryu (1968) Aoyagi and Oryu (1968) Aoyagi and Oryu (1968) Lochhead and Farrell (193 1b) Aoyagi and Oryu (1968) Fabian and Quinet (1928) Nussbaurner (1910) Lochhead and Heron (1929) Aoyagi and Oryu (1 968) Fabian and Quinet (1928) Richter (1912) Lochhead and Heron (1929) Fabian and Quinet ( I 928) Lochhead and Heron (1929)

352

JONATHAN W . WHITE. JR.

surface moisture increased above about 22%, yeast count increased massively at the surface; although from 2 cm down, counts remained stable. Further handling then can distribute the inoculum throughout the mass with subsequent anaerobic fermentation. C.

RECOMMENDED STORAGE FOR HONEY

Problems to be considered in storage of honey are fermentation, granulation, discoloration, flavor damage, and, if intended for export, destruction of enzymes and production of HMF. The only condition in which all dangers and changes are eliminated is in freezer storage. Since this is not a practical procedure, some compromise is to be expected, depending upon the type and intended use of the honey. In any case, honey must be protected from atmospheric moisture. Cold (i.e., below 10°C) storage has been recommended for unprocessed honey to prevent fermentation (Marvin, 1928). Fermentation generally does not take place in unheated warehouse storage in northern winters, but it can be expected when temperatures become favorable, since most honeys will have granulated by then. Pasteurized honey, though not liable to ferment, will granulate (coarsely) if held in fluctuating temperatures between 11" and 15°C; this will necessitate further processing. Processed honey is best stored between 18" and 24°C; short-term exposure to higher temperature is permissible. Since heat damage is additive, care must be taken to limit heat exposure as much as possible: 10 days at 32°C are equivalent to 100-120 days at 21°C (White el al., (1963b). Reduction of storage temperature by 6"-8°C will reduce rate of deterioration to l/3-% of that at the higher temperature.

VII. NUTRITIVE VALUE A.

AS A CARBOHYDRATE

There is probably no area in which scientific opinion clashes with folklore more than in the nutritive (and medical) aspects of honey. Many articles in the lay or trade press over the years in all parts of the world attest to the superiority of honey as a nutrient. The subject was recently reviewed in the book edited by Crane (1975a) in a chapter on the biological properties of honey. There, six collaborators agreed that "the time had come to make a realistic appraisal of the position and to clear away some of the misconceptions that are published from time to time." After noting that over 2000 papers and articles have been published on the subject and referring to several books that have appeared since the second World War, several aspects are discussed that appear to be relatively well founded, including nutritive value. Honey is a nutritive sweetener, with prop-

HONEY

353

erties arising from its high content of glucose and especially of fructose and its variable content of trace minerals. As noted elsewhere, the vitamin content has no nutritional significance. Haydak (1936) described a study in which he continued normal work for a 12-week period on a diet consisting solely of milk and honey. No subjective or clinical problems were noted, except a need for ascorbic acid supplementation. Later (Haydak et a f . , 1944), five adults alternated 1-week test periods with 1-week normal diet for 4 weeks. The test diet, milk and honey, was supplemented with thiamine, ascorbic acid, and iodide. No effect on normal health and activity was noted. Haydak (1 955) reviewed the nutritional aspects in general. The rapid absorption of honey monosaccharides and the slower metabolism of the fructose content appear to be the basis of its popularity as a source of quick energy for athletes, scuba divers, and mountain climbers. Townsend (1954a) reviewed this aspect. I.

Infant and Geriatric Diet

A considerable literature, largely European, has accumulated in the past 50 years on the value of honey supplementation of milk in infant feeding. For details the reader is directed to the reviews of Haydak (1955) and White (1975b). The facile absorption of the monosaccharides, improved weight gain, relief from constipation, decrease of diarrhea, and good tolerance by infants at special risk is cited. Improved calcium retention (Knott et al., 1941) and utility in feeding prematures (Vignec and Julia, 1954) are also cited. Most of the articles conclude that honey should have a wider use in infant feeding. At the other end of the human experience, honey appears to have some particular utility in geriatric feeding. Albanese et al. (1954) pointed out that utilization of glucose is markedly decreased with aging, while that of fructose is only slightly affected. Results of his experiments suggest that levulose or levulosecontaining products are sugars of choice for the aged in that they may provide a ready source of energy and an optimal protein-sparing effect. In a later study Albanese et al. (1968) reported that glucose tolerance is not significantly altered by age in healthy subjects, but a greater loss of tolerance to glucose than to honey was seen in patients recovering from strokes. Similar differences prevailed in patients with diabetes, with hemiplegia complicated by diabetes, and in those recovering from coronary occlusion. Distinct differences in the metabolism of glucose and fructose have been reviewed recently (Pawan, 1973). 2 . Honey and Diabetes Beekeeping and lay publications at times contain suggestions that diabetics can use honey without incident. This is nonsense, since honey contains a considera-

354

JONATHAN W . WHITE, JR

ble proportion of glucose. Because on the weight basis, honey (at 80% solids) is about as sweet as sugar and provides an average of 31% as glucose, compared with sugar’s 52%, some advantage in “sweetening power” is theoretically available to the stabilized diabetic. Selection of tupelo honey, averaging about 25% glucose, would approximately double the “sweetness” intake without increasing glucose. It must be emphasized, however, that any such substitution be undertaken only upon the advice of the physician. B.

MINERALS AND VITAMINS

As already noted, honey does contain measurable amounts of several vitamins and quite variable levels of a number of minerals. The real nutritional significance of these may be assessed by examination of Table XXII, which shows order of magnitude for the more important nutrients in relation to the United States minimum daily requirements.

C. FOLKLORE Space is not available for a discussion of the folklore of honey, which dates back about 5000 years. The interested reader is referred to the excellent review by Crane, “History of Honey” (1975c), and to the less scholarly “Honey and Health” by Beck (1938).

V111. A.

USES FOOD

Most of the honey sold for food is used directly as table sweetener or spread. The most significant indirect uses are in baking, cereal, and confectionery. Use in baking has decreased in recent years because of the run-up in price and the introduction of fructose-containing syrups that approach the functional values of honey. Unduplicated, however, are the flavor advantages conferred by honey and the freedom to use the word “honey” in advertising and promotion, which carries a definite connotation of quality and ‘‘old-fashioned goodness” not conferred by any other sweetener. Over the years articles have appeared in the baking trade press describing the use of honey in various products. Griffith (1934a,b,c) produced information and recipes for crullers, sweet rolls, rye and white bread, icings and glazes, Bohemian water rolls, black walnut bread, raisin whole wheat bread, poppyseed horns, cheesebread, and challis, all using honey. Glabau (1944, 1945) described formulas for various breads, cakes, and cookies.

355

HONEY TABLE XXII NUTRIENTS IN HONEY IN RELATION TO HUMAN REQUIREMENTS‘

Nutrient Energy equivalent Vitamins: A B, (Thiamin) B2 complex: Riboflavin Nicotinic acid (niacin) BB (Pyridoxine) Pantothenic acid Folk acid B 12 C (Ascorbic acid)

Unit

Average amount in 100 gm honey

2800

kcal

i.u. mg

5000 0.004-0.006

1.5

0.024.06 0.1 14.36

1.7 20

0.0084 . 3 2 0.02-0.1 I

2.0 10 0.4 6.0 60 400 30 0.3

2.2-2.4

D E

H (Biotin) Minerals: Calcium Chlorine Copper Iodine Iron Magnesium Manganebe Phosphorus Potassium Sodium Zinc ‘I

Recommended daily intake U.S.A.

0.004-0.03 0.002-0.02 0.01-0. I 0.1-3.4 0.7-13 0.02-10 0.002-0.06 0.014 . 4 7

1 .o

2.0 0.15 18

400 1 .o

O.OOO6-0.04 0.2-0.5

15

Taken, with omissions, from Crane (1975a. p. 264)

Uses of honey in commercial baking have been rather thoroughly explored in a series of papers from Kansas State University. Advantages for honey-sweetened baked goods in moisture retention, texture, keeping quality, flavor, and the undefinable “eating quality” have been shown for white and whole wheat bread (Smith and Johnson, 1951), cakes and sweet doughs (Smith and Johnson, 1952), cake, cookie, and sweet goods production (Johnson and Smith, 19531, cookies (Smith and Johnson, 1953a). and fruit cakes (Smith and Johnson, 1953b). Further publications include a bulletin with commercial-scale recipes (Miller et al., 1960) and home recipes (Johnson et al., 1959) for the same products.

356

JONATHAN W . WHITE. JR

The recent popularity of the granola-type of breakfast cereal has provided an additional use for honey. Although the original granola recipe requires a considerable proportion of honey, formula modification and price competition has eliminated it or reduced it in some instances to only a token-literally “below the salt” in ingredient statements. Nevertheless, quality formulation and enlightened promotion should permit honey to retain a position in this area. Certain confections have been and are always properly made with honey-the nougats, halvah, torrone-but, in general, as in other areas, honey has been largely displaced by less costly (and less flavorsome) sweeteners. The possibilities and instructions for using honey in confections have been described by Barth (1952), Anderson (1958, 1963), Meineke (1967), and Watson (1968). Honey is an optimal sweetening ingredient in the FDA Standards of Identity for fruit butters, jellies, jams, and preserves, providing it either be the sole such ingredient or represent at least 20% of the solids in mixtures with certain other of the optional sweeteners. It is unlikely that any appreciable amount of honey is presently in such use. The inclusion of 2-3% honey in prune juice is permitted under the standard of identity: a honey-sweetened canned grapefruit juice has enjoyed some recent success. Attempts to prepare beverages or beverage bases containing lemon juice and honey have been retafded by formation in storage of an unsightly floc. Recent experimentation (White, 1976) has shown that this can be eliminated by removing the colloidal materials in honey by treatment with bentonite (White and Walton, 1950). A different type of fruit and honey product is a high-density honey-fruit spread. These spreads are easily made by mixing high-solids fruit juice concentrates with five to eight parts of full-density honey, followed by the controlled crystallization process described earlier. Grape, citrus, and berry flavors blend especially well with the honey flavor (White, 1950). Another process mixes comminuted dried fruits with honey, followed by controlled crystallization (Berthold and Benton, 1968). Products containing honey intended for food manufacture include several dried mixtures. A spray-dried blend of 40% honey and 60% nonfat milk has been described (Walton et al., 1951) and a tunnel-dried mix containing up to 70% honey was patented by Webb and Walton (1952). The milk solids content of these products limits their value to baking, beverage, or confectionery use. A continuous process using a wiped-film vacuum evaporator which can dry pure honey was described by Turkot et al. (1960); the product is highly hygroscopic, but suffers no flavor or color damage in the process. The addition of 35% sucrose before drying is recommended in order to improve storage capability by raising the softening point. A schematic diagram of the process is shown in Fig. 28. In spite of its hygroscopicity, limited testing has shown it not to induce caking in prepared dry baking mixes.

357

HONEY

V

FIG. 28. Flowsheet and diagram of pilot-scale apparatus used to dehydrate honey. 1, feed tank; 2 , feed pump; 3, feed preheater; 4. back pressure valve; 5 . evaporator; 6. condenser; 7, absolute pressure manometer; 8, vacuum regulator; 9, condensate receiver; 10. sightglass; 1 I , product pump; 12. check valve; 13, chilling rolls. T , thermometer or thermocouple; S, steam; C, condensate; W . water; V , vacuum source; A, ammeter; P, pressure gauge. From Turkot e/ ai.,1960. Reprinted from Food TechnologylFood Technology 14, 387-390 ( 1960). Copyright @ by Institute of Food Technologists.

Several dry honey mixtures are commercially available and produced under the patents of Straub (1954) in which gelatinized starch is used to aid drying, and Northcutt and Northcutt (1 945) which covers atmospheric drum-drying. In general, prospective users of honey in food products will seek either product or promotion advantages to offset the higher cost of the material. Its prime attribute is flavor, which cannot be discerned at very low ingredient levels but cannot be duplicated when honey is present at optimal amounts. Its nature as a somewhat acid reducing sugar or solution with distinctive flavor attributes must be kept in mind in formulation. Heat treatment to inactivate enzymes should be specified to insure product stability.

B.

NONFOOD

Probably the largest nonfood use of honey is in pharmaceuticals. In addition to home use with lemon juice for easing sore throats, honey has been compounded into a number of successful commercial cough remedies. No effort will be made to review this area in view of the subject matter of the series; articles by Rubin ef al. (1959) and Gennaro et al. (1959) may be consulted. The use of honey in medicine is a subject reported intermittently for the past 4000 years. Since this is also outside the food area, the interested reader is referred to the review by Stomfay-Stitz (1960) for the earlier aspects; for more recent documented articles on the successful use of honey for wounds to Temnov

358

JONATHAN W . WHITE, JR.

(1944), Gubin (1945), Bulman (1955); for infected wounds, to Gundel and Blattner (1934) and Zaiss (1934); for bums, to Phillips (1933), Voigtlander (1 937); and to a remarkable article by Cavanagh et al. (1 970) on the successful direct application to extensive wounds following surgery, in which undiluted honey-killed organisms cultured from the wounds of twelve patients. Willson and Crane (1975) have written an extensive review on honey uses.

IX. STANDARDS, SPECIFICATIONS, AND QUALITY CONTROL A.

UNITED STATES STANDARDS

There is no Federal Standard of Identity for honey; the general regulations for food products apply, of course. The definition from the earlier Act of 1906 is still considered a guide in what is considered honey: “Honey: The nectar and saccharine exudations of plants gathered, modified, and stored in the comb by honeybees (Apis mellifera and apis dorsata). Honey is levorotatory and contains not more than 25% of water, not more than 0.25% of ash, and not more than 8% of sucrose” (USFDA, 1936). This definition excludes honeydew honey or any honey containing honeydew sufficient to render it dextrorotatory. Criticism of the limits of moisture (too high), sucrose (too high), and ash (too low) have been expressed by White et al. (1962) and Feinberg (1951). The U.S. Department of Agriculture has established voluntary grading standards for extracted honey and comb honey.* Honey is classified into seven color categories as indicated in Table XXIII. The USDA color comparator is a device in which honey in specified 2-oz square bottles is compared to glass standards representing the established limits of each grade (Brice et al., 1956, 1965). Color is not a quality factor. Honey is assigned to one of four quality grades (U.S. Grade A or U.S. Fancy, U.S. Grade B or U.S. Choice, U.S. Grade C or U.S. Standard, and U.S. Grade D or Substandard) by evaluating solids content, flavor, absence of defects (particles of comb, propolis, or other material in suspension or deposited as sediment), and clarity. Moisture minima are 18.6% for the two upper grades, 20% for Grade C (honey for reprocessing), and unspecified for Grade D. B. CODEX ALIMENTARIUS The European Economic Community has directed the member countries to incorporate the FA0 European Regional Standard for Honey (Codex Alimen*Copies of standards may be obtained from Chief, Processed Products Standardization and Inspection Branch, Fruit and Vegetable Division, Agricultural Marketing Service, USDA, Washington, DC 20250.

359

HONEY TABLE XXlIl STANDARD COLOR DESIGNATION OF HONEY AND RANGE FOR EACH COLOR‘

USDA color standards Water White Extra White White Extra Light Amber

Light Amber

Amber Dark Amber

Color range USDA color standards Honey that is Water White or lighter in color than Water White Color Standard. Honey that is darker than Water White but not darker than Extra White Color Standard. Honey that is darker than Extra White but not darker than White Color Standard. Honey that is darker than White but not darker than Extra Light Amber or Golden Color Standard. Honey that is darker than Extra Light Amber but not darker than Light Amber Color Standard. Honey that is darker than Light Amber but not darker than Amber Color Standard. Honey that is darker than Amber Color Standard.

Color range Pfund scales (mm)

Optical densityb

8 or less

0.0945

Over 8 to and including 17 Over 17 to and including 34 Over 34 to and including 50

0.189 0.378 0.595

Over 50 to and including 85

1.389

Over 85 to and including 114 Over 114

3.008 -

Taken from USDA (1951). density (absorbance) = log ,o (100/percentage transmittance), at 560 nm for 3.15 cm thickness for caramel-glycerin solutions measured versus an equal cell containing glycerin. I’

* Optical

tarius Commission, 1969) into their national honey legislation, with a few minor exceptions. The United States participated in the development of the standards but does not accept them. In the standard, honey is defined as “the sweet substance produced by honey bees from the nectar of blossoms or from secretions of or on living parts of plants, which they collect, transform, combine with specific substances, and store in honey combs.” Honey is classified according to origin as blossom or nectar honey and honeydew honey, and by processing mode as comb, extracted, or pressed honey. The compositional criteria are shown in Table XXIV. Other rules relate to flavor, absence of fermentation, extent of heat treatment, addition of acid, cleanliness, and labeling. Honey not meeting the criteria for diastase and HMF content or flavor, fermentation, and extent of heating must be sold as “baking honey” or “industrial honey,” generally at lower prices. Methods of analyses are specified for the parameters listed in Table XXIV. Information on and a comparison of grading rules and regulations for many countries are given in an article by Fasler (1 975).

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JONATHAN W. WHITE, JR.

TABLE XXlV CODEX ALIMENTARIUS: ESSENTIAL COMPOSITION AND QUALITY FACTORS FOR HONEY"

Compositional Criteria Apparent reducing sugar content, calculated us invert sugar: Blossom honey, when labeled as such: Honeydew Honey and blends of Honeydew Honey and Blossom Honey: Moisture content: Heather Honey (Calluna) Apparent sucrose content: Honeydew Honey, blends of Honeydew Honey and Blossom Honey, Robinia, Lavender and Eanksia menziesii Honeys: Water-insoluble solids content: Pressed Honey: Mineral content (ash): Honeydew honey and blends of honeydew honey and blossom honey: Acidity: Diastase activiry and hydroxymethylfurfural content: Determined after processing, blending; diastase figure on Gothe scale: Provided the hydroxymethylfurfural content is: Honeys with low natural enzyme content, e.g. citrus. diastase content on Gothe scale: Provided the hydroxymethylfurfural content is:

not less than 65% not not not not

less than 60% more than 21% more than 23% more than 5%

not not not not

more than more than more than more than

10% 0.1% 0.5% 0.6%

not more than 1.O% not more than 40 meqllOOO gm

not less than 8 not more than 40 mg/kg not less than 3 not more than 15 mgkg

From Codex Alimentarius Commission (1969).

C. SPECIFICATIONS Honey is generally traded by sample, but difficulties are sometimes encountered when either the sampling is not done in a representative fashion, or improper storage of the lot between sampling and delivery brings about an increase of color or possible fermentation. Variations can exist from drum to drum, or even in layers of a single drum, depending upon producers practices (Smith, 1967). Purchase from processors is generally not subject to these problems. Honey purchased for food manufacturing use should be required to meet the appropriate U S . Grade (A or B), and should be specified by color (Pfund or USDA) and, if appropriate, by floral blend, in general terms. Further requirements, if established for specific manufacturing use, should avoid unnecessary detail as to specific composition limits (other than moisture), since a needless burden is thus placed on the supplier for analytical services not ordinarily done.

HONEY

36 1

An example of specification guidelines for purchase of honey for use in baking is given below (Smith and Johnson, 1951, 1952, 1953a,b; Johnson and Smith, 1953). General 1.

2. 3.

All honey containers should be clearly labeled, showing grade, floral source, moisture content, and color in mm Pfund, as well as U.S. Department of Agriculture color standards. Honey for bakers’ use should be “U.S. Grade A” or “B,” according to U.S. standards for grades of extracted honey, effective April 16, 1951. Honey should be heat-treated at 71°C for 30 minutes to retard granulation and enzyme activity. White or Whole Wheat Bread

The Pfund colorimeter reading should not exceed 70 rnm for honey to be used in white bread. 5. Predominant floral sources of Eastern buckwheat, fall flowers, heartsease, and tupelo honeys should not be used in white bread, except in blends as noted in item 7. 6. Eastern buckwheat, fall flowers, heartsease, and horsemint honeys should not be used in whole wheat bread, except in blends as noted in item 7. 7. Blends of acceptable honeys containing 10%of Eastern buckwheat, or 15% of heartsease, fall flowers, or tupelo honeys are acceptable.

4.

Yeast-Leavened Sweet Goods

8. Predominant floral sources of Eastern buckwheat, fall flowers, heartsease, or horsemint honeys are not recommended for use in sweet goods, except in blends containing not more than 10% Eastern buckwheat, or 15% fall flowers, heartsease, or horsemint. Other honeys of acceptable flavor are satisfactory in yeast sweet goods. Cake Products Predominant floral sources of Eastern buckwheat, fall flowers, heartsease, and horsemint honeys are not desirable for use in white, yellow, or chocolate cakes. 10. Only honey classified as white by the U . S . grade and color standards is recommended for use in white cake. 9.

Fruit Cake 11.

Predominant floral sources of Eastern buckwheat and fall flowers honeys are not recommended for use in fruit cake.

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JONATHAN W . WHITE. JR.

Cookies 12. Predominant floral sources of heartsease, Eastern buckwheat, tupelo, or eucalyptus are not recommended. It is highly recommended, however, that only pressure-filtered honey be purchased for food manufacturing use; the USDA grade specification for defects could conceivably allow traces of undesirable material to be present. D.

QUALITY CONTROL

The parameters ordinarily monitored by food manufacturers will normally be moisture, flavor, color, and cleanliness. The refractometer is recommended for moisture, according to AOAC method 3 1.11 2 (Horwitz, 1975). Honey color is generally evaluated in the honey industry by the Pfund Honey Color Grader, since it provides a continuous scale of color, useful in blending and assigning prices to bulk honey. The instrument is available from beekeeping supply houses. The less expensive (and easier to use) USDA honey color classifier can be used to assign a sample to its color class. The use of a commercially available photometer for color classification was proposed by Townsend (1969); he used white light in an instrument accepting 19-mm test tubes and obtained acceptable correlation between absorbance and Pfund readings. He also demonstrated the use of the instrument for blending honeys. Flavor must ultimately be judged subjectively, though Merz (1963) has proposed that GLC examination of an ether extract provides a simple procedure for objective assessment of honey flavor. He found the HMF peak to dominate in assays of extracts of honeys of “satisfactory” flavor and to be but a minor constituent of those of organoleptically unsatisfactory flavor. Cleanliness is specified in the USDA grades as “shall be at least as free from defects as honey that has been strained through a No. 80 sieve (Grade A). , . No. 50 sieve (Grade B). . . No. 18 sieve (Grade C) at not over 130°F (54.4”C).” For food use, as noted above, pressure filtered (i.e., not simply strained) honey should be specified. The Codex requires a maximum of 0.1% in water-insoluble solids for honey, determined by filtering a 20-gm sample diluted with 80°C water through a tared sintered glass crucible (pore size 1540 microns) and washed sugar-free with 80°C water, drying and weighing. A filtered honey should have negligible residue by this test. A need for determining other compositional factors such as sugars, acids, and ash is not foreseen for quality control; methods are available in the AOAC book of methods (Horwitz, 1975). For defense against possible substitution of other syrups, the resorcinol test (AOAC Method 3 1.138, 3 1.139) or determination of HMF (Winkler, 1955) for acid inverted syrups and the AOAC test for corn syrup

HONEY

363

(Method 37.134-6) are recommended. Samples with HMF values over 20 mg HMFl100 gm must be suspected unless a history of high-temperature storage can be proven. A definitive test for the adulteration of honey with corn sirups, including the new high-fructose corn syrup has been developed (White and Doner, 1978). It is based on the difference in the ratio of l3C to 12Cin the sample. Corn syrups are slightly enriched in 13C in comparison with honey; variability in the ratio for honey is the lowest yet found for any honey constituent or property. Since diastase is the more heat-resistant honey enzyme (Fig. 14), assay for diastase may be used when intended use of honey requires that enzyme activity be eliminated. Honey intended for export may be assayed for diastase to provide assurance of meeting Codex standards. Edwards et al. (1975) have compared a procedure using a proprietary chromogenic substrate (Amylochrome) with the Codex method. Agreement was excellent over the entire range and major savings in operator time resulted also. Use of this or a similar product should be considered for routine diastase measurement in honey.

X.

RESEARCH NEEDS

Honey has been an article of commerce for many thousands of years and an object of research for perhaps one hundred. Much of the literature is still only descriptive, reporting values and variability in composition. A large proportion is devoted to detection of falsification or quality deterioration, but in recent years some understanding of the chemistry and biochemistry of the product is beginning to emerge. Because of the great complexity of honey, advances in understanding must often await improvements in analytical methods. “Understanding” based on inadequate analytical procedures must be reviewed and corrected when the opportunity arises. Generalizations are often based on inadequate numbers of samples tested because of limitations of time or funding; these must be placed upon a wider data base. Current and anticipated developments in manufacture of corn and other syrups offer opportunity for falsification of honey that are increasingly difficult to detect. While current studies in this field may provide definitive methods, each improvement in commercial syrups must be examined in this light in order to maintain the integrity of the honey market. A few specific research needs are outlined below. 1 . A broader base for prediction of enzyme stability and HMF accumulation in full-density honey upon storage and heating. 2. Assays of true glucose oxidase activities of honey, after removal by dialysis (or other means) of materials reactive to the hydrogen peroxide pro-

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JONATHAN W. WHITE, JR

duced, in relation to peroxide accumulation values which are determined with diluted whole honey. 3. Development of an accurate method for gluconolactone and gluconic acid in honey. 4. Determination of the heat capacity of honey in the temperature range of processing interest and verification of published data on heat transfer coefficients. 5 . Extension of the objective color classification of honey to photometers using a 1-cm cell to increase availability of equipment usable for this purpose. 6 . Development of uses for honey in the food industry where its attributes cannot be duplicated by other syrups so as to broaden industrial use of honey, seriously eroded by less expensive refined sweetening agents. Developments to maintain and strengthen markets for domestic honey can have an impact greatly exceeding the value of the honey itself. A strong honey market is of the greatest national importance because honey provides a large fraction of the beekeeper’s income; beekeeping is vital to pollination of billions of dollars worth of food, feed, and fiber crops in the United States.

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Biino. L. 1971. Ricerca di alcuni aminoacidi in due varieti di miele. Riv. Ital. Essenze Profurni 53(2). 80-84. Borus. L.. Kalinowski, J., and Zalewski, W. 1966. [Production and chemical composition of locust honey from the vicinity of Cigacice.] Pszczelnicze Zrsz. Nauk. 10(l/4), 113-122. Braunsdorf, K. 1932. Zuckerfiitterungshonig and Diastaseherkunft. Z . Unter. Lebensm. 64, 555558. Brice, B. A., Turner, A.. Jr.. and White, J. W., J r . 1956. Glass color standards for extracted honey. J . Assoc. Off. Agric. Chem. 39(4), 919-937. Brice, B. A., Turner, A.. Jr.. White, .I.W., Jr., Southerland. F. L.. Fenn, L. S., and Bostwick, E. P. 1965. Permanent glass color standards for extracted honey. U.S. Dep. Agric.. Agric.. Res. Sew. AIC 73-48, 1-6. Browne. C. A. 1908. Chemical analysis and composition of American Honeys. U . S . Dep. Agric., Bur. Chem. Bull. 110, 1-93 Bulman. M. W. 1955. Honey as a surgical dressing. Er. Bee J . 83, 664-665. Burgett, D. W. 1974. Glucose oxidase: A food protective mechanism in social hymenoptera. Ann. Entomol. Soc. Am. 67(4). 545-546. Cale, G . H., Banker. R . , and Powers, J. 1975. Management for honey production. In “The Hive and The Honeybee” (R. A. Grout. ed.), pp, 355412. Dadant and Sons. Hamilton, Illinois. Cavanagh. D.. Beazley. J . , and Ostapowicz. F. 1970. Radical operation for carcinoma of the vulva. J . Obstut. Gynecol. B r . Commrtnw. 77(1 I ) , 1037-1040. Chataway, H. D. 1932. Determination of moisture in honey. Can. J . Res. 6, 532-547. Chataway. H. D. 1933. The determination of moisture in honey by the hydrometer method. Can. J . Res. 8, 435439. Cocker. L. 1951. The enzymic production of acid in honey. J . Sci. Food Agric. 2(9). 41 1 4 1 4 . Codex Alimentarius Commission. 1969. “Recommended European Standard for Honey.” CAC/ RS-12-1969. Jt. FAO/WHO Food Stand. Program. Rome. Reprinted in Bee World 51(2), 79-91 (1970). Codounis, M. I. 1962. [“The Crystallization of Honey.”] Min. Agric.. Athens. Conrad. E. C.. and Palmer, J. K. 1976. Rapid analysis of carbohydrates by high-pressure liquid chromatography. Food Technol. 30(10). 84, 86, 88-92. Crane, E.. ed. 1975a. ”Honey: A Comprchensivc Survey.” 608 pp. Heinemann. London. Crane, E. 1975b. The World’s Honey Production. In “Honey: A Comprehensive Survey” (E. Crane, ed.), pp. 141-153. Heinemann. London. . of honey. In “Honey: A Comprehensive Survey” (E. Crane, ed.), pp. Crane, E. 1 9 7 5 ~ History 439488. Heinemann. London. Crerner. E. and Riedmann, M. 1964. Identifizicrung von gaschromatographisch getrennten Aromastoffen in Honigen. Z. Narurforsrh. B 19, 76-77. Curti, R., and Riganti, V. 1966. Ricerche sugli aminoacidi del miele. Rass. Chim. 18(6). 278-282. Davies. A. M. C. 1975. Amino acid analysis of honeys from eleven countries. J . Apic. Res. 14(1), 29-39. Davies. A. M. C. 1976. The application of amino acid analysis to the determination of the geographic origin of honey. J . Food Tec.hnol. 11, 515-523. Dean, G . R. 1974. An unstable crystalline phase in the D-glucose-water system. Carbohydr. Res. 34, 3 15-322. De Boer. H. W. 1934. De invloed van den ouderdoni opde samenstelling van honig. Chem. Weekbl. 31, 482487. Deodikar. G. B., Thakar, C. V.. Phadkc, R. P.. and Shah. N . P. 1957. Thixotropy in honey of Carvia callosa. Indian Bee J . 19, 71-72. Detroy, B. F. 1966. Determining film coefficient for a viscous liquid. Trans. ASAE 9(1). 91-2. 93, 97.

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Dittmar, J. H. 1935. Hygroscopicity of sugars and sugar mixtures. Ind. Eng. Chem. 27(3), 333-335. Dold, H., Du. D. H., andDziao, S. T. 1937. Nachweis antibakterieller, hitze- und lichtempfindlicher Hemmungstoffe (Inhibine) im Naturhonig (Bliitenhonig). Z. Hyg. Infektionskr. 120, 155167. Doner, L. W . 1977. The sugars of honey-a review. J . Sci. Food Agr. 28,443-456. Dorrscheidt, W., and Friedrich, K. 1962. Trennung von aromastoffen des Honigs mit Hilfe der Gas-Chromatographie. J . Chromatogr. 7(1), 13-18. Duisberg, H . , and Gebelein, H. 1958. Uber die Kontrolle von Erhitzungsschaden bei Honigen. Z . Lebensm.-(Inters. -Forsch. 107(6), 489-501. Duisberg, H., and Hadom, H. 1966. Welche Anforderungen sind an Handelshonige zu stellen? Vorschlage auf Grund der Statistischen Auswertung von ca 1600 Honig-Analysen. M i f f .Geb. Lebensmirrelunters. H y g . 57(5), 386-407. Duisberg, H., and Wamecke, B. 1959. Erhitzungs- und Lichteinfluss auf Fermente und lnhibine des Honigs. Z . Lebensm.-Unters. -Forsch. 111, 1 11-1 19. Dustmann, J. H. I97 la. Messung von Wasserstoffperoxid und Enzymaktivitat in mitteleuropaischen Honigen. Z. Bienenforsch. 9(2), 66-73. Dustmann, J. H. 1971b. Uber du Katalaseactivitat in Bienenhonig auf der Tracht der Heidekrautgewbchse (Ericaceae). Z . Lebensm.-Unters. -Forsch. 145, 292-295. Dustmann, J. H. 1972. Einfluss der Dialyse bei der Bestimmung der Saccharase-Aktivitat in Honig. Lebensm.-Wiss. Technol. 5(2), 70-71. Dyce, E. J. 1935. Honey process and product. U.S.Patent No. 1,987,893. Dzialoszynski, L., and Kuik. K. 1963. Aktywnosc kwasnej fosfatazy a-amylazy i katalazy w miodach z okzolic torunia. Pszczelnicze Zesz. Nauk. 7(1), 33-39. Ekhigo, T., and Takenaka, T. 1973. Changes in erlose contents by honeybee invertase. Nippon Nogei Kaguku Kaishi 47(3), 177-183. Edwards, R. A., Faraji-Haremi, R., and Wootton, M. 1975. A rapid method for determining the diastase activity in honey. J. Apic. Res. 14(1), 47-50. Elser, E. 1924. Beitrage zur quantitativen Honiguntersuchung. Arch. Bienenkd. 6, 1 18. Erlenmeyer and Planta. 1874. Uber die Fermente in den Bienen, im Bienenbrot, u. im Pollen und iiber einige Bestandteile des Honigs. Chem. Zentrulbl. p . 790. Fabian, F. W., and Quinet, R. I. 1928. A study of the cause of honey fermentation. Mich., Agric. Exp. Stn. Tech. Bull. 62, 1 - 4 1 . Farnsteiner, K. 1908. Der ameisengesauregehalt des Honigs. Z. (Inters. Nahr.- Genussm. Gebrauchsgegensraende 15, 598604. Fasler, A. 1975. Honey Standards Legislation. In “Honey a Comprehensive Survey” (E. Crane, ed.), pp. 329-354. Heinemann, London. Feinberg, B. 1951. Ash in honey. Am. Bee J . 91, 471. Fiehe, J. 1932. Uber die Herkunft der Honigdiastase. Z. (Infers.Lebensm. 63, 329-331. Gauhe, A. 1941, Uber ein glukoseoxydierendes Enzym in der Pharynxdriise der Honigbiene. Z. Vgl. Physiol. 28(3), 21 1-253. Gautier, J.-A,, Renault, J., and Julia-Alvarez, M. 1961. Recherche du sucre interverti dans le miel. Premiere partie: critique des reactions de Fiehe et de Feder. Annis. Falsf. Fraua‘es 54, 177-193. Geddes, J. P. 1964. Packs honey in quantity. Food Eng. 36(1 I ) , 100-101. Gennaro, A. R., Sideri, C. N.. Rubin, N., and Osol, A. 1959. Use of honey in medicinal preparations. Am. Bee J. 99, 492-493. Gillette, C. C. 1931. Honey catalase. J. Econ. Entomol. 24, 605-606. Giri, K. V. 1938. The chemical composition and enzyme content of Indian honey. Madras Agric. J. 26, 68-72. Glaubau, C. A. 1944. Honey-its use in cakes and cookies for the holiday season. Bakers Wkly. Oct. 16, pp. 40-42; Oct. 23, pp. 44-46; Oct. 30, pp. 56-58, 66; NOV.6, pp. 48-50; NOV.13, pp. 56-58, 69.

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Glabau. C. A. 1945. How the various kinds of honey breads are made. Parts 1-5. Bakers Wkly. July 2, pp. 52-53; July 9, pp. 50-51; July 16, pp. 4 4 4 5 ;July 23, pp. 50-51; July 30, pp. 34-35. Goldschmidt, S . , and Burkert, H. 1955. Die Hydrolyse des cholinergischen Honigwirkstoffes und anderer Cholinester mittels Cholinesterasen und deren Hemmung im Honig. Hoppe-Seyler’s Z. Physiol. Chem. 301, 78-89. Gonnet, M., and Lavie, P. 1963. Study of the adhesiveness of crystallized honey to the walls of glass vessels. Abstr., Int. Beekeep. Congr., 19th, Prague Pap. No. 38. Gontarski, H. 1948. Ein Vitamin C oxydierendes Ferment der Honigbiene. Z. Naturforsch. B 3, 245-249. Gontarski, H. 1954. Fermentbiologische Studien an Bienen. I. Das physikochemische Verhalten der kolenhydratspaltenden Fermente. (A) invertierende Enzyme. Verh. Dtsch. Ges. Angew. Entnmol. 12, 186-197. Gothe. F. 1914. Die Fermente des Honigs. Z. Unters. Nahr.- Genussm. Gebrauchsgegenstaende 28, 273-286. Green, G. W. 1951. The granulation of honey and its relation to the laws of crystallization. Int. Beekeep. Congr., 14th, Leamington, Engl. Pap. No. 18. Greenleaf, C. A., and Browne. C. A. 1929. Some observations on the Fiehe test. J. Assoc. Of. Agric. Chem. 12, 319-323. Griebel, C. 1938. Vitamin C enthaltenden Honig. Z. Unters. Lebensm. 75, 417420. Griffith, J. H. 1934a. Honey can be used in practically all baked goods. Bakers Helper, July 28, pp. 112-1 14, Aug. 1 I , 189-191. Griffith, J. H. 1934b. Rolls made with honey. Bakers Helper, pp. 470, 474. Griffith, J. H. 1934~.Varieties of bread made with honey. BakersHelper, Dec. I , pp. 876-877.910. Gubin, A. F. 1945. [The Beekeeping Institute during the war: Honey in medicine.] Pschelovodstvo 1, 25-29. Giinther, F., and Burckhan, 0 . 1967. Bestimmung der sauren Gesamtphosphatase in Honig. Dtsch. Lebensm.-Rundsch. 63(2), 41 4 4 . Guilbault, J. 1965. Crystallization of honey. Thesis. Ontario Agric. Coll., Guelph. Gundel, M.. and Blattner, V. 1934. Uber die Wirkung des Honigs auf Bakterien und infizierte Wunden. Arch. Hyg. Bakteriol. 112, 319-322. Hadorn. H. 1964. Enthalten Orangenbliiten und Lavendelbliiten honige enzymhernmende Stoffe? Ann. Abeille 7(4), 31 1-320. Hadorn, H., and Kovacs, A. S. 1960. Zur Untersuchung und Beurteilung von auslandischem Bienenhonig auf Grund des Hydroxymethyl furfurol und Diastase gehaltes. Mitt. Geb. Lebensmittelunters. Hyg. 51, 373-390. Hadorn, H., and Ziircher, K. 1962. Zur Bestimmung der Saccharase-Aktivitkit in Honig. Mitt. Geb. Lebensmittelunters. Hyg. 53, 6-28. Hadorn, H., and Ziircher, K . 1963. Uber Zuckerfutterungshonig. Mitt. Geb. Lebensmittelunters. Hyg. 54, 322-330. Hadorn, H., and Ziircher, K. 1974. Zuckerspektrum und Kristallisationstendenz von Honigen. M i f t . Geb. Lebensmittelunters. Hyg. 65, 407420. Hadorn, H., Ziircher, K., and Doevelaar, F. H. 1962. Uber W h e - und Lagerschadigunsen von Bienenhonig. Mitt. Geb. Lebensmittelunters. Hyg. 53(3), 191-229. Hahn, H. 1970. Zum Gehalt und zur Herkunft der freien Aminosauren in Honig. Dissertation, Univ. Stuttgart, Stuttgart. Haydak, M. H. 1936. A prolonged test of milk and honey diet. Minn. Med. J . 19, 774-776. Haydak, M. H. 1955. The nutritional value of honey. Am. Bee J . 95(5), 185-191. Haydak, M. H . , Palixr, L. S., Tanquary, M. C., and Vivino, A. E. 1942. Vitamin content of honeys. J . Nutr. 23, 581-588. Haydak, M.H., Palmer, L. S., Tanquary, M. C., and Vivino, A. E. 1943. The effect of commercial clarification on the vitamin content of honey. J. Nutr. 26,(3), 319-321.

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peroxide accumulation system by light. J. Food Sci. 29(6), 819-828. White, J. W., Jr., and Walton, G. P. 1950. Flavor modification of low-grade honeys. U . S . Dep. Agric., Bur. Agric. Ind. Chem. AIC-272, 1-13. White, J. W., Jr., Petty. J.. and Hager, R. B. 1958. The composition of honey. 11. Lactone content. J. Assoc. Off. Agric. Chem. 41(If, 194-197. White, J. W., Jr., Riethof. M. L., and Kushnir, I. 1961. Composition of honey. V1. The effect of storage on carbohydrates, acidity, and diastase content. J. Food Sci. 26(1), 63-71. White, J . W., Jr.. Riethof, M. L.. Subers, M. H.. and Kushnir, 1. 1962. Composition of American honeys. US.,Dep. Agric., Tech. Bull. 1261, 1-124. White, J. W., Jr., Subers, M. H., and Kushnir, I. 1963a. How processing and storage affect honey quality. Gleun. Bee Culr. 91, 4 2 2 4 2 5 . White, .I.W., Jr., Subers, M. H., and Schepartz, A. I . 1963b. The identification of inhibine, the antibacterial factor in honey, as hydrogen peroxide and its origin in a honey glucose oxidase system. Biochim. Biophys. ACIU 73, 57-70. White, J. W., Jr., Kushnir, I., and Subers, M. H. 1964. Effect of storage and processing temperatures on honey quality. Food Technol. 18(4), 153-156. Willson, R. B. 1975. World trading in honey. In “Honey: A Comprehensive Survey” (E. Crane, ed.), pp. 355-377. Heinemann, London. Willson, R. B., and Crane, E. 1975. Uses and products of honey. In “Honey: A Comprehensive Survey” (E. Crane. ed.), pp. 378-391, Heinemann, London. Wilson, H. F., and Marvin, G. E. 1929. On the occurrence of yeasts which may cause the spoilage of honey. J. Econ. Entomol. 22, 513-517. Wilson, H. F., and Marvin, G. E. 1931. The effect of temperature on honey in storage. J. Econ. Entomol. 24, 589-597. Wilson, H. F., and Marvin, G . E. 1932. Relation of temperature to the deterioration of honey in storage. A progress report. J . Econ. Entomol. 25, 525-528. Winkler, 0. 1955. Beitrag zum Nachweis und zur Bestimmung von Oxymethylfurfural in Honig und Kunsthonig. Z. Lebensm.-Unrersuch. -Forsch. 102(3), 161 -167. Wootton. M., Edwards, R. A,, Faraji-Haremi, R., and Johnson, A. T. 1976a. Effect of accelerated storage conditions on the chemical composition and properties of Australian honeys. I . Colour, acidity, and total nitrogen content. J. Apic. Res. 15(1), 23-28. Wootton, M., Edwards, R. A,, and Faraji-Haremi, R. 1976b. Effect of accelerated storage conditions on the chemical composition and properties of Australian honeys. 2. Changes in sugar and free amino acid contents. J. Apic. Res. 15(1), 29-34. Zaiss. 1934. Der Honig in ausserlich Anwendung. Muench. Med. Wochenschr. 11, 1891. Zalewski, W. 1965. Fosfatazy w miodach. Pszczelnicze Zesz. Nuuk. 9(1-2), 1-34.

SUBJECT INDEX A Acid phosphatases, in tea, 235 Acids. in honey, 304-305 Actin SH groups in, 35-36 function, 38-39 Actinins, SH group role in, 40 Actomyosin, SH group role i n , 39-40 Aging, of meat, SH groups and, 55-58 Alcohol dehydrogenase, of tea, 235-236 Amino acids in honey, 308-312 of tea, 240-241 Amylase, in honey, 321-324 Anserine, role in histamine toxicity, 141-142

B Bacterial endotoxins, role in histamine toxicity, 145-146 Bacterial toxins, irradiation effects on, 186 Black tea aroma development in, 256-257 carotenes, in, 257 fatty acids in, 259-260 fermentation of, 253-260 firing of, 260-26 I grading of, 261 organoleptic properties of. 263-265 preconditioning of, 252 rolling of, 252-253 storage of, 267-268 volatile constituents of, 250-25 I withering of, 252

C Carbohydrates in honey, 299-304 radiation effects on, 174 Carnosine, role in histamine toxicity, 141 -142 Carotenoids, in tea, 246 Catalase, in honey, 329-330

Cereal grains, radurization of, 194-196 Chlorophyll, in tea, 245-246 Chlorophyllase, in tea, 235 Curing. of meats, effects on SH groups, 77-80

D 5-Dehydroshikimate reductase, of tea, 234 Denaturation, of meat, effect on SH groups, 58-63 Diabetes, honey use in, 353-354 Diets for laboratory animals, irradiation of, 185 special, irradiation of, 183. 185 Disulfide groups, determination of, 28-30

E Enzymes in honey, 312-330 of tea. 233-236 N-Ethylmaleimide. as SH group reagent, 22-24

F Fats, see Lipids Fatty acids, in meats, effects on SH groups, 57-58 Fermentation of black tea, 253-260 of honey, 351-352 Fish and fish products histamine in allowable levels, 146 detection. 130-135 enzymic formation, 121-122 spoilage role, 135-1 39 histamine toxicity from, 1 13- 154 histidine decarboxylases in, 122-128 Fluoronietry assay, of histamine. I3 I - I33 Food irradiation, 155-227 combined with other processes, 187-188 disinfestation, 198-200 economics of, 205-208 equipment for, 166 375

376

SUBJECT INDEX

Food irradiation, (cont’d) future of, 213-216 general effects on carbohydrates, 173 foods, 168-169 lipids, 170-173 proteins, 169-170 high-dose applications of, 174-1 88 historical aspects, 15.5- I63 laboratory plan for, 165 low-dose applications of, 188-205 radiation sources for, 163-168 radicidation, 197-198 radurization, 188- I97 in various countries, 158-162 wholesomeness of foods in, 209-2 I3 Freezing, of meats, SH groups in, 73-76 Fruits irradiation of, 183 radurization of, 194-196

G Gas-liquid chromatography, of histamine. 133 Glucose oxidase, in honey, 325-329 Green tea organoleptic properties of, 265-266 production changes in, 261-262 storage of, 267 Guinea pig ileum contraction assay, for histamine, 130- I3 I

H Histaminase, role in histamine toxicity, 144-145 Histamine bacteria responsible for formation of, 122I24 bacterial destruction of, 128 colorimetric assay of, 133-134 enzyniatic isotopic assay of, 134 in fish, detection of, 130-135 fluoroinetric assay of, 130- I3 1 gas liquid chromarography of, 133 guinea pig ileum contraction assay of, 130131 histidine as precursor of, 124-125 occurrence of precursors, of, 124

scombroid toxicity and, 139-140 thin-layer chromatography of, 134-135 toxicity from, in fish, 113-154 cases of, 115-120 early reviews, 120-121 symptomatology, 114-1 15 synergists or potentiators of, 142-146 Histidine, as histamine precursor, 124-125 Histidine decarboxylase(s) carbohydrate effects on, 127 in fish, 122 oxygen tension effects on, 127-128 pH effects on, 126-127 temperature effects on, 125-126 vitamin effects on, 127 Honey, 287-374 acids in, 304-305 analysis and composition of, 297-333 aroma of, 33 1-332 carbohydrates in, 299-304 color of, 332 crystallization of, 339-344 diabetic use of, 353-354 enzymes in, 312-330 fermentation in, 351-352 flavor of, 331 folklore of, 354 harvesting in, 293 hygroscopicity of, 338-339 market forms of, 295-297 melezitose crystallization from, 343-344 minerals in, 305, 354 moisture content of, 298-299 nutritive value of, 352-354 physical characteristics of, 333-344 pollination in, 293 processing of, 293-295 plant Iayout, 295 production and processing of, 289-292 production methods for, 292-293 proteins and amino acids in, 305-312 research needs on, 363-364 rheology of, 333-335 standards and quality control of, 358-363 storage changes i n , 344-352 thermal properties of, 335-338 toxic constituents of, 333 uses of, 354-358 as food, 354-357 as nonfood, 357-358 vitamins in, 332, 354

SUBJECT INDEX Hydrogen sulfide. release of, during meat heating, 6 4 4 9 Hydroxymethyl furfural, in honey. 345-349

I Insects, radiation disinfestation of, 199-2OO Invertase. in honey, 313-321 Irradiation, of food, 155-227

L Laboratory animals, diets for, irradiation of, 185 Leucine a-ketoglutarate transaminase, in tea, 235 Linolenic acid, oxidation by tea leaf, 235 Lipids, radiation effects on, 170-173

M Malate dehydrogenase. of tea, 236 Meats cysteine in, 50-52 heat effects on nutritive value of, 63-64 hydrogen sulfide release from, during heating. 64-69 irradiation of, 174-182 radurization of, 189-192 sulfhydryl and disulfide groups in, 1-1 I I curing effects, 77-80 factors affecting, 55-58 freezing effects on, 73-76 irradiation effects on. 81-83 muscles, 45-50 organs, 52-55 processing effects on, 58-83 shelf-life effects, 84 toxicological aspects, 84-85 texture of, disulfide group effects on, 71-73 Melezitose, from honey, 343-344 Mercaptides. formation of, from sulthydryl groups, 13-22 Mercury compounds, as SH reagents, 19-22 Minerals, i n honey, 305 Mitochondria, SH groups in, 42 Monoaniine oxidase inhibitors, role in histamine toxicity, 145 Muscle fibers, SH group role in, 40 Muscle proteins, SH groups i n , 3 1 4 0

377

Mycotoxins, irradiation effects on, 186 Myofibrillar proteins SH groups in, 31-40 functional role. 36 Myosin SH groups in, 35 function, 36-38

0 Organs SH content of, 52-55 Oxidizing agents for SH groups, 8

P Parasites, radiation disinfestation of, 198- 199 Pectin methylesterase, of tea, 236 Peptidase, in tea, 234-235 Peroxidase, in tea, 234 Phenylalanine ammonia lyase, of tea, 234 Phosphatase, in honey, 330 Polyphenol oxidase, of tea, 233-234 Polyphenols, in tea, 236-240 Poultry. radurization of, 189-192 Proteins in honey, 305-308 radiation effects on, 169-170

R Radurization, of food, 188-197 Ribonuclease, in tea, 236

S "Saurine," histamine toxicity and, 129 Sausages, ripening of effects on SH groups, 80 Sarcolemma, SH groups in, 4 1 4 2 Sarcoplasmic matrix, SH groups in, 4 2 4 3 Sarcoplasmic reticulum, SH groups in, 41 "Scombroid poisoning," see Histamine, toxicity from Seafood irradiation of, 174-182 radurization of, 192-1 94 Senescence, radiation inhibition of, 201-204 Shelf life, of meats, SH group effects on, 84

SUBJECT INDEX

378

Skeletal muscle, myofibrillar proteins of, 31 Smoking, of meats, effects on SH groups, 80-8 I Spices, irradiation of, 183 Spoilage, of fish, histamine role in, 135-139 Sprouting, radiation inhibition of sprouting of. 20 1-204 Sugars. in honey, 302-303 Sulfhydryl and disulfide groups amperometric titration of, 13-22 color reagents for, 27 determination of, 3-6 methods for meats, 6-28 inmeats, 1 - 1 1 1

T Tea, 229-286 amino acids of, 240-241 aroma development i n , 256-257 black tea, 25 1-26 I carbohydrates in, 243 chlorophyll and carotenoids in, 245-246 clinical effects of, 269-27 I composition of, 232-25 I chemical and biochemical components, 233-249 factors affecting, 248-249 enzymes of, 233-236 fermentation of, 253-260 green tea, 26 1-263

history of, 229-230 host plant-pest relationships of, 271 instant type, 272-273 lipids in, 243-244 minerals in, 246-248 organoleptic propenies of, 263-267 phosphate esters, nucleofides,and caffeine in, 24 1-243 poiyphenols in, 236-240 potential by-products of, 268-269 processing changes in, 248-263 research needs on, 273 storage of, 266-268 triterpenoids in, 244-245 volatile compounds in, 248 Thin-layer chromatography of histamine, 134135 Tropomyosin SH groups in, 36 function. 3 9 4 0 Troponin SH groups in, 36 function. 39-40

V Vegetables irradiation of, 183 radurization of, 194-196 Viruses, food-borne, radiation effects on, 186

A 8

8

c

9

E

l

D O F 2 G 3 H 4 1 5 J 6