Advances in Food Research

ADVANCES IN FOOD RESEARCH VOLUME 18

Contributors to This Volume

J . C . BAUERNFEIND STEPHEN S. CHANG MARIONL. FIELDS KARL 0. HERZ D. M . PINKERT PAUL J. SCHEUER HARRYE. SNYDER

ADVANCES IN FOOD RESEARCH VOLUME 18

Edited b y E. M . MRAK

C. 0. CHICHESTER

U ni ljerB i t y of’ Ca 1 i f i )r 1iu Duois, Culiforniu

University of Californiu Davis, Culifbriiia

G. F. STEWART U ti ioersi t !I oj‘ Cal ijo rii in Duois, Culifbriiiu

Edit o ria 1 B oa rd E. C. BATE-SMITH W. H. COOK M . A. JOSLYN

S . LEPKOVSKY

EDWARDSELTZER W. M . URHAIN J . F. VICKERY

1970

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CONTENTS CONTRIBUTORS TO VOLUME18

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vii

Meat Flavor

KARL 0. HEHZ AND STEPHENS . CHANG I. I1. 111. IV .

...... Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of Anteniortem and Postmortem Factors on Meat Flavor . . . . . . . . . . ...... Raw and Processed Meat Flavor ................................ ...... Some Proldems of Research on Meat Flavor ..................... V . Knowledge of Heated Meat Flavor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Newer Findings on and Related to Beef Flavor . . . . . . . . . . . . . . . . . . . . . . . . VII. On the Origin of Meat Flavor Compounds-Current Views . . . . . . . . . . . . . VIII . Interaction of' Compounds and Perception of Flavor . . . . . . . . . . . . . . . . . . . IX . Areas for Further Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 4 16 23 27 45 51 64 68 69

Microbial Sources of Protein

HARRY E . SNYDER I . Introduction ........................................................ I1. Review and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Additional Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

85 88 128 131

Toxins from Fish and Other Marine Organisms PAUL

I. 11. 111. IV . V.

J . SCIiEUEW

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Toxins from Fishes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Toxins from Shellfish-Saxitoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .............................. Other Marine Toxins . . . . . . . . . Research Needs . . . . . . . . . . ............................... References . . . ...... ..................................

141 143 155 157 158 159

V

CONTENTS

vi

The Flat Sour Bacteria

~ I A H I O NL. FIELDS I. 11. 111. IV. V. VI. VII. VIII.

Introduction ................................... Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Distribution of Spores of Flat Sour Bacteria . . . . . . . . . . . . . . . . Resistance of Spores to Lethal Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 Sources of Contamination in Canning P . . . . . . . . . . . . . . . . . . . . . . . . . 190 Control of Flat Sour Bacteria . . . . . . . . Biology of F l a t Sour Bacteria . . . . . . . . Research N e e d s . . . . . . . . . . . . . . . . . . .

...................................

Food Processing with Added Ascorbic Acid

1. c. f3AUEHNFEINI) 1. 11. 111. IV. V. VI. VI I ,

VIII. IX. X. XI. XII. XIII. XIV. XV. XVI.

ANI)

13. k1, PINKERT

. . . . . . . . . . . .,220 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .221 Methods of Addition to Food . . . . . . . . . . . . . . . . Ascorl)ic Acid a s iui Added Nutrient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,238 Ascorbic Acid Reactions . . . . . . . . . . . Synergist in Fat Protection . . . . . . . . . ................ Preventive o f Fruit Browning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preventive of Vegetable Discolorations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .256 Inhibitor of Oxidative Rancidity in Fish . . . . . . . . . . . . . . . . . . . . . . . . ,266 Stal)ilizer of Meat Color . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . “2 Flour or Bread I m p r o v e r . . . . . . . . . Oxygen Acceptor i n Beer P r o c e s s i n g , . . . . . . . . . . . . . . . . . . Reducing Agent in W i n e . . . . . . . . . . Oxidation Inhibition in Dairy Produ ............... ................ Miscellaneous Uses . . . . . . . . . . . . . . . . . . . . . Regulations on the Use of Ascorbic . . . . . . . . . . . . . . . . . . . . . . . . ,297 I.-Ascorl,ic Acid vs. Erythorbic Acid References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,304

sUt3JECX I N D E X

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317

CONTRIBUTORS TO VOLUME 18 Numbers in parentheses indicate the pages on which the authors' contributions begin.

J . C. BAUERNFEIND, Chemical Research Department, HoffmannLaRoche lnc., Nutley, New Jersey (219) STEPHEN S. CHANG,Department of Food Science, Rutgers, The State University,New Brunswick, New Jersey (1) ~ I A R I O NL. FIELDS,Department of Food Science and Nutrition, University of Missouri, Columbia, Missouri (1 63)

KARL 0. HERZ," Department of Food Science, Rutgers, The Stute Uniuersity, New Brunswick, New Jersey ( 1 ) D. M . PINKERT,Chemical Research Department, Hoffmann-LaRoche Inc., Nutley, New Jersey (219) PAUL J . SCHEUER,Department of Chemistry, University of Hawnii, Honolulu, Hawaii (141) HARRY E . SNYDER, Depurtment of Food Teclanologiy, lowu Stnte U n ive rsi t y ,A mes, 1ow CL (8.5)

'Present address: Wallerstein Laboratories, Staten Island, New York

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ADVANCES IN FOOD RESEARCH VOLUME 18

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MEAT FLAVOR* BY KARL 0. H E R Z ~AND STEPHEN S. CHANC Department of Food Science Autgers, The State Univer.yity, New Brunswick, New Jersey

I. Introduction

. . . . . . . . . . . . .. . .. . . . . . .. .

...............

2 2 3 3 4 4

B. The Meat Industry ............................. C. P u r v i e w . . . . . . . . . . . . . . . . . . . . . . . ....... . . . . . 11. Effects of Antemortem and Postmortem Fact A, Age, Breed, Sex, and Use. . . . . . . . . . . . . B. Feeding and Other Treatments . . . , . . . . . . . . . . . . . . . . . . . . . . . . . 6 C. Carcass Condition and Meat Characteristics . . . . . . . . . . .. . . 7 D. Slaughtering and Aging Conditions . . . , . . . . . . . . . . . . . . . . . . . . . 9 E. Freezing and Freezer Storage ...................... 14 111. Raw and Processed Meat Flavor. . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 A. Raw and Cooked Beef ............................ 16 ooked-Meat Flavor . . . . . . . . . . 18 B. Factors Causing C. Canned, Dried, ............................ 19 . . . . . . . . . . . . . . 21 D. Cured and Smo ............ E. Irradiated Beef .................................... 21

..................

23 23 . . . . . . . . . . 24 .......... 27 A. Flavor Precursor Studies . . . . ' .................... 28 B. Studies on Model Systems . . ........................... 32 C. Studies on Formed Meat Flavor. . . . . . . . . . . . . . . . . . . . . . . . . 33 D. On the Origin of Meat Flavor Compounds - Early Views . . . . . . . . . . . . . . 38 . . . . . . . . . . . . . . . . . . . 45 VI. Newer Findings on and Related to Beef Flavor A. Artificial Meat Flavors-A Clue? . . . . . . . . . . . . . . . . . 46 B. Beef Flavor Isolate Constituents - New Findings . . . . . . . . . . . . . . . . . . . . . 48 A. Precursor Studies . . . . . . . B. Formed Flavor Studies. . . . . . . . . . . . . . . . .

*Paper of the Journal Series, New Jersey Agricultural Experiment Station at Rutgers, The State University, New Brunswick, New Jersey. This investigation was supported by Agricultural Research Service, U.S. Department of Agriculture, Grant No. 12-14-1007669 (73) from the Eastern Utilization Research and Development Division, Philadelphia, Pennsylvania. +Present Address: Wallerstein Laboratories, 125 Lake Avenue, Staten Island, New York.

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KARL 0. HERZ AND STEPHEN S. CHANG

VII. On the Origin of Meat Flavor Compounds-Current Views . . . . . . . . . . . . . . . . 5 1 A. Nonmeaty Compounds in Beef Flavor Concentrates . . . . . . . . . .. . . . . . . . . 5 1 B. Classes of Compounds Likely to Influence Meat Flavor. . . . . . . . . . . . . . . 5 4 C. Compounds from Meaty Aroma GC Fractions.. . . . . . . . . . . . . . . . . . . . . .. 5 8 D. Other Nitrogen Ring Compounds Found in Foods . . . . . . . . . . . . . . . . . . . .60 E. Maillard Browning in Foods-A Digression . . . . . . . . . . . . . . . . . . . . . . . . .61 VIII. Interaction of Compoiu1ds and Perception of Flavor . . . . . . . . . . . .. . . . . . . . . 6 4 A. Chemical Compounds, Concentration, and Interaction . . . . . . . . . . . . . . . . . . 6 4 B. Flavor Perception . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,66 1X. Areas for Further Research. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , 6 8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 9

I. INTRODUCTION When used alone, the word “meat” is most frequently taken to mean beef in all cultures where beef is consumed. This review will focus on flavor of beef meat, though relevant information from studies of other meats will be included. Beef is consumed throughout the world as a nutritious and flavorful food. Its flavor is one of the few readily recalled and usually associated with a satisfying experience. Though this flavor should therefore have been studied widely, efforts have been in fact rather few. Up to 1960, the literature on beef flavor composition, formation, and impact on the human senses was minute. A few compounds had been reported present, but their relationships to flavor were unknown or obscure. During the last decade in the United States in particular, demands for knowledge of meat flavor have been stimulated by economic civilian and military needs.

A. POSSIBLE USES F O R W O W L E D G E OF MEAT FLAVOR

As the world food problem becomes more critical, specialists are veering toward the conclusion that meat will become a luxury item in the diet of most peoples. T h e conversion of plant protein and energy to meat is wasteful, and increasing pressure for maximal utilization of food sources will demand that forage, grains, and other animal feed be processed more efficiently into human food. If the compounds and reactions responsible for creating the satisfying aroma and mouth (taste and feel) sensations of beef can he harnessed to make possible production of closely analogous synthetic beef flavor, a tool will be available to the food industry for imparting this satisfying flavor to appropriate nonmeat nutritious substrates that can be made more efficiently and cheaply.

MEAT FLAVOR

3

In current-day meat technology, information on the compounds and the reactions involved in the formation of desirable flavor could well be used in several ways: (1)to provide conditions for storing and processing fresh meat that will prevent or minimize deleterious changes and optimize advantageous changes eventually affecting flavor; (2) to restore flavor quality and acceptable intensity to meat processed in ways that cause flavor loss; ( 3 )to impart desirable flavor to wholesome beef from animals whose meat is flavor-deficient; and (4) to aid geneticists in breeding cattle for quality of lean-meat flavor. B. THE MEAT INDUSTRY In the United States and in many other countries of the world, the meat industry holds a prominent place among the food processing industries. In some regions, religious or other taboos forbid consumption of meat or of beef. Wherever meat is part of the diet, it is recognized as a superior food from the standpoints of nutrition, flavor, and physiological eating satisfaction. Information on the meat industry in the United States has been published (American Meat Institute, 1967).T h e wholesale dollar value of beef produced in 1966 was $18 billion. The number of cattle slaughtered was 30 million. Employment in the industry stood at about 190,000, including 147,000 in production. In 1963,3000 slaughtering plants were operating. Worldwide in 1967, total production of meat and meat products reached 124.3 billion pounds, more than half of which was beef and veal (U.S.D.A., 1968a). Both U.S. exports and U.S. imports of beef and veal were above 5 billion pounds in 1967 (U.S.D.A., 196%). Annual consumption of beef in the United States is estimated at 20.1 billion pounds, or 104 lb per capita (American Meat Institute, 1967). Worldwide in 1965, four countries had higher per capita beef consumption than did the United States, namely, Argentina, Paraguay, Uruguay and New Zealand; Australia was a close sixth (U.S.D.A., 1966). All of these countries are meat producers. Most Western European countries show a per capita consumption of less than half that of the United States. Developing countries generally have a very low per capita consumption of beef. C. PURVIEW

This review will include the early work that represents attempts to relate various factors antemortem (age, breed, sex, use, feed) and post-

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KARL 0. HERZ A N D STEPHEN S. CHANG

mortem (slaughtering, carcass handling, aging, storing, processing) on general flavor acceptability of the cooked meat. Lack of adequate means for characterizing any but the grossest changes and differences makes the considerable information collected over many decades of questionable value in searching for the nature of cooked beef flavor, though of reasonable value in indicating directions for research once this flavor is bereft of all mystery. Known differences between raw and processed meat that have a bearing on flavor will be covered. Much work has been done on heated beef flavor itself, through precursor studies, model-system and analytical investigations. The available information will be examined to see where a decade of more intensive research has brought our knowledge of the biochemical reactions and the compounds that are responsible for meat flavor. Where does flavor perception fit into the picture to be developed? What are the remaining gaps in our knowledge deemed fruitful for further research? These are the topics that will conclude this review.

II. EFFECTS OF ANTEMORTEM AND POSTMORTEM FACTORS O N MEAT FLAVOR Since the compounds particularly concerned with cooked meat flavor are insufficiently known, the effects of various conditions and treatments on the flavor cannot be established unequivocally. Nitrogenous, carbohydrate, and lipid substances are possible flavor sources. Any treatment that alters the constitution of the animal, or the composition of meat after slaughter, might effect qualitative or quantitative changes in flavor precursors and compounds. Important differences - not all clearly related to flavor - caused by the particular nature or treatments, such as age, breed, sex, feed of the animal; and aging, freezing, cooking, and irradiation of beef will be reviewed. The discussion will chiefly be limited to beef although important points brought out in work on other meat aimals and foods will also be included.

A. AGE, BREED, SEX, AND USE Age of the animal at slaughter is related to flavor of the meat when cooked but the relationship is not rigid. Part of the reason for this is that individual animals differ in genetic and metabolic makeup (Blumer, 1963; Lowe and Kastelic, 1961). This difference is responsible

MEAT FLAVOR

5

for maturity being reached relatively earlier or later. Degree of maturity and force of metabolism have the greatest influence upon flavor. Young beef animals (calves) up to about 11 months have neither a typical beef flavor quality nor the expected flavor intensity (Barbella et al., 1939; Lowe and Kastelic, 1961).Maturing and changing metabolism are important factors in the development of normal beef flavor. Various studies have shown that age has no significant effect on flavor in steers between the ages of 18 and 30 months, where the bulk of cattle raised for meat fall (Barbella et al., 1939);Dunsing, 1959a; Simone et al., 1959). Barbella et al. (1939)reported an increase in desirable flavor intensity to 30 months with little change thereafter to 42 months. This trend was confirmed in a study in which steaks from 18month-old steers were rated higher in all palatability respects except flavor and juiciness when compared to steaks from 30-month-old steers (Simone et al., 1959).Scores for flavor of lean, aroma, and flavor of fat were not greatly different for commercial grade beef slaughtered at age 18 or 42 months (Lowe and Kastelic, 1961).A study comparing female beef animals of ages 18,42, and 90 months showed no significant difference in flavor evaluation by a taste panel (Tuma et al., 1962,1963). Jacobson and Fenton (1956)obtained similar results. If flavor is related to the content in the muscles of metabolic products, an increase in intensity and a change in profile up to an age of little metabolic change should be expected. Flavor normally is not tested in isolation, but is part of a profile that includes tenderness. Studies of the effects of breed and of sex have been few because of the difficulty in obtaining suitable groups of animals. Comparison of the work of different investigators is somewhat suspect because of variability in the nature of the samples used. One study made on a scale large enough to permit valid conclusions for a number of factors was reported by Barbella et al. in 1939. Herefords, Angus, Shorthorns, and Brahmans were included in the 728 heifers and steers from which data were obtained. Variation in intensity of lean-beef flavor due to sex was not significant at any age, but there was some significance between sex and quality of flavor of the fat. Breeding was the second greatest source of variability for flavor of lean beef and was highly significant statistically. A similar study with today’s major breeds of beef cattle is needed. Studies have been made to evaluate the effect of breed on tenderness of meat (Alsmeyer et al., 1959; Carpenter et al., 1955; Cartwright et al., 1957; Sharrah et al., 1965a); these confirm that breed may be expected to affect eating quality of meat at least with respect to tenderness.

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KARL 0. HERZ A N D STEPHEN S. CHANG

Still on a grand scale were the experiments reported for 335 beef carcasses by Kropf and Graf (1959); however, these workers chose homogeneous material according to grade, without regard to breed or age of animals at slaughter. Also, feeding history was not reported. Among the 161 steers, 94 heifers, and 80 cows, each type was represented by choice, good, and commercial carcasses. A range of carcass weight also was included in the sample, each grade having carcasses in the 4-5 cwt and 6-7 cwt range, but only choice having carcasses in the 8-9 cwt range. Panel flavor and juiciness scores gave no basis for ascribing a significant difference to either sex, grade, or weight. An interesting study was made on 10 pairs of bull and steer twins (Bryce-Jones et al., 1964b). Steer meat was definitely more tender and also more juicy and flavorful than that of bulls. Callow (1961)found that intramuscular fat is markedly lower in beef cattle than in dairy cattle. A comparison of beef-type (Hereford) and dairy-type (Holstein) cattle raised under similar conditions was made by Branaman et al. (1962). Although no significant differences were found in general desirability of lean flavor between beef and dairy cattle, intensity of desirable flavor was rated significantly greater for the beef-type cattle. Actually, whether differences observed are d u e to use or breed cannot be adequately answered from this study.

B. FEEDINGAND OTHERTREATMENTS Feeding programs and types of treatment vary widely among cattle producers. Feed may be predominantly pasture, sileage, or grain suitably supplemented, and treatments may involve hormone and antibiotics administration. Little is known about the effects of these variables on meat flavor (J. A. Bennett and Matthews, 1955). Hormone treatments are normally applied to hasten fattening of the cattle to market weight; they are more effective on steers than on heifers (U.S.D.A., 1964). T h e hormone most frequently used is diethylstilbestrol. This treatment results in lean beef (Lawrie, 1966; Tuma, 1963) with heavier carcasses (Everitt and Jury, 1964), and increased weight of the semitendinosus muscle fiber thickness, and increased nitrogen content (Paul, 1962b). The effect of hexoestrol implant treatment on eating quality of meat from steers differing in grade indicated that results depend on grade (Tuma, 1963). In better grade steers, treatment evoked no significant differences in eating quality, but in the best grade a lower content of intramuscular fat was noted. Roast rib muscle of low-grade steers improved in flavor and tenderness upon treatment, but the opposite effect was observed in the case of stewed neck muscles.

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7

Feeding of antibiotics or tranquilizers could affect meat flavor. The effect on rumen or intestinal flora may be important in the case of antibiotics. Whether the rations have a significant influence on beef flavor has not been established. Wanderstock and Miller (1948) have provided the best evidence that pasture feeding exclusively may have an adverse effect on most carcass characteristics, including flavor. In studies extending over 3 years, yearling Hereford steers brought in the spring from Texas were put on a known feeding management in the summer, brought to normal market weight, and compared for carcass and meat characteristics. At purchase, the animals were graded good to choice, and at slaughter, carcasses were graded commercial to choice. Of the five lots of steers used in each set, two lots were grazed on pasture and then full-fed in dry lot, one was grazed on pasture and supplemented with grain, one was solely full-fed in dry lot, and one was solely grazed on pasture. Major differences were found in grade on hoof, carcass grade, and aroma and flavor of rib roasts for pasture-only steers, which were consistently rated considerably lower than steers from the other lots that had received some grain. Based on carcass evaluation, the effect of a low milk ration on retardation of growth was substantially reduced in a 2-year pasture feeding period and practically negated during fattening in the feed lot (van Marle, 1963). Various eating qualities were examined in beef from steers fed in groups on high or low planes of nutrition in winter and long or short grass pasture in summer (Houston et uZ., 1965). Most effects reported were on tenderness and juiciness of the meat, but a loss of flavor was revealed in beef that had been on a low plane of nutrition during winter followed by grazing on short-grass pasture that became fully defoliated. Workers are generally agreed that substantial special fats supplied in the feed of pigs results in changes in the depot fat fatty acids composition (H. 0. Doty, 1958; Chung and Lin, 1965), but a similar effect is questioned for ruminants (Siedler, 1963). On the other hand, Privett et al. (1955) actually reported little effect of soybean oil meal and yellow corn feed in pigs, but greater differences ascribable to the oil content of the feed in steers. More information is needed. C. CARCASSCONDITIONAND MEAT CHARACTERISTICS Market demand in the United States has led to a fairly uniform supply of steer and heifer animals for slaughtering at ages between 14 and 30 months and at weights somewhere near 875 Ib for heifers and

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KARL 0. HE'RZ AND STEPHEN S . CHANG

near 975 Ib for steers (U.S.D.A., 1964). Nevertheless, producers cannot control the quality a s graded by appearance as well a s they can age and weight, although a very large proportion of cattle raised for slaughter does fall into the three acceptable consuiner beef animals grades of prime, choice, and good. An extensive investigation (D. M. Doty and Pierce, 1961) was made into the relationship of carcass grade and weight (and also certain aging conditions) to raw and cooked meat quality characteristics. Carcass grades included in this study were heavy prime (about 800 Ib), light prime (500 lb), heavy good (650 lb), light good (400 lb), and commercial cows (650 lb). A number of characteristics were compared, including aroma of broiled steaks, flavor of fat in rib steaks, and lean meat flavor. No indication was found in the 3-year study that the aroma of broiled steaks was influenced by carcass grade or weight. Similarly, the fat flavor in rib steaks (longissimus dorsi) was not affected initially, but poorer grades showed flavor quality deterioration in the holding period following the first 2 weeks. A significantly better lean meat flavor was observed for rib steaks from prime grade animals; aging for 2 weeks improved the lean meat flavor of steaks from good and commercial carcasses, but not to the level of the prime steaks, and holding past 2 weeks led to deterioration in the flavor of steaks from the lower grade carcasses. However, broiled semitendinosus (bottom round) did not show significant differences in lean meat flavor due to either carcass grade or weight. Information on age of the animals at slaughter was not published in this study. In a comparison of 18- and 30-month-old steers of choice and good carcass grades (Simone et ul., 1959),flavor and juiciness of roasts from rib and round were consistently rated higher for choice than for good, in both age groups. In addition to bearing out the reported (D. M. Doty and Pierce, 1961) difference in flavor between rib and round, Simone et ul. indicated that significant differences existed among portions of the same (semimembranosus- top round) muscle. Another comparison was made by Lowe and Kastelic (1961) for pairs of low choice and low commercial grade carcasses of 18-monthold beef animals of the same sex and breed. Although one heavy choice carcass was always superior in scores (aroma, flavor of lean, flavor of fat), suggesting that perhaps at this level weight does have a bearing, scores for the other choice carcass were generally near or below those for the commercial grade carcasses. In a previous study, Lowe et ul. (1952) had shown that roasts from choice and good carcasses were rated about the same, but roasts from commercial car-

MEAT FLAVOR

9

c a s e s were scored somewhat lower. I n a consumer panel study (Dunsing, 19591>),steaks from animals graded choice were preferred to steaks from animals graded good. In assigning grades to meat, fat finish and marbling were considered indicative of better eating quality. These factors affect juiciness, but their effect on desirable lean beef flavor has not been firmly established. Barbella et u1. (1939)’by covariance analysis showed that most of the variation in lean beef flavor was due to fat, determined as ether extract of the edible portion of rib (longissimus dorsi). Similarly, Simone et ul. (1959) concluded that ether extract could serve to indicate the quality of the rib, but not of other muscles or of the whole carcass. There is some question whether beef muscle fat - marbling 4iould be considered as part of the desirable flavor complex of cooked beef (Blumer, 1963; Gilpin et al., 1965).Alternatively, only particular fat fractions may play a role, since Ostrander and Dugan (1962) demonstrated differences in the fatty acid composition of subcutaneous, intermuscular, and intramuscular fat; these workers also showed that phospholipids are present in some beef muscles and not in others. N o significant effect of marbling on eating quality of beef meat has been reported. Dunsing (1959b) showed that marbling, a s determined by a household consumer panel, was not a sensitive indicator of desirable eating quality of steaks. Tunia et (11. (1962),in a study of 24 Hereford females of varying ages, demonstrated the absence of a significant relationship between marbling level and taste panel flavor and juiciness scores. Also, in the work reported b y D. M . Doty and Pierce (1961),lean flavor did not significantly differ for the well-marbled 12th rill and the little-marbIed 10th rib for steaks from either prime or good carcasses. The pH of meat may affect flavor, but since pH depends on antemortern treatments and postmortem holding conditions, such relationships will be discussed in the following section. L). SLAUGHTERING AND AGING: CONDITIONS

The effects on flavor of antemortem conditions have not been investigated. Certain indexes of quality have been adversely affected b y such antemortem conditions as crowding and hasty feeding during trnnsport to the abattoir; drastic temperature changes, crowding, arid hasty feeding while holding for slaughter; and administration of drugs, enzymes, or sugar. At slaughter, herding, method of stunning

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KARL 0. HERZ AND STEPHEN S . CHANG

and killing, and stimulation or degree of bleeding could have some influence on metabolism and the products accumulating in the tissues at time of kill. High temperatures may have an adverse influence both before and after slaughter, and the time held before carcass chilling is started and temperature to which carcass is chilled may also be important. Effects of poor antemortem conditions on certain characteristics, specifically carcass weight, are well known. Electric shock treatment at 20-min. intervals during the 24 hr preceding slaughter invariabl?. increased the pH of cooked steaks from Angus or Hereford steers, compared with control steaks from paired animals receiving no shock treatment (Lewis et nl., 1967). A few reports have dealt with such conditions as starving and adrenaline injection. The evolution of hydrogen sulfide during cooking of beef was greater in poor-grade starved cattle than in well-fed animals (Penny et ul., 1964);this was a pH effect, since the pH of meat from starved carcasses norinally is relatively high, and since raising the pH of meat artificially led to increased evolution of hydrogen sulfide. A relatively high pH may also result from injecting adrenaline just antemortem (Johnson and Vickery, 1964). This was tried in experiments designed to find the best conditions for processing meat b y the British AFD (accelerated freeze-dry) method. The treatment p r o x d generally worthwhile, particularly in regard to tenderness, but a definite loss of flavor was indicated in panel tests. Antemortem feeding of sucrose, on the other hand, lowered pH and increased lactate content of pork from pigs that had been transported 340 miles in 24 hr; the same effect was not noted in animals transported only 3.1 miles before slaughter (Gunther and Schweiger, 1966). Similar studies with beef have not been reported, although 110 doubt considerable practical knowledge is available at major abattoirs. Postmortem increase i n acidity from metabolism of glycogen to lactate in muscle (as contrasted to carbon dioxide and water in the live animal) has been known for a long time (Hoagland et u l . , 1917). Even after aging for considerable periods, however, some residue of glycogen remains in the muscles. N o clear effect of muscle pH 011 pidatability could b e demonstrated in several studies (Lowe and Kastelic, 1961; D. M. Doty and Pierce, 1961; Bodwell ct f i l . , 1965b),although it11 effect on color was noted. D. M. Doty arid Pierce (1961) found no relationship between carcass grade or weight and p H of the meat. Meat with high pH (about 7.0) was quite dark. Aging for 14 days at 34°F led to an increase of 0.2 pH unit, on the average, above the low reached post-rigor. Rib cores removed from five Hereford heifer car-

MEAT FLAVOR

11

casses at less than 10 minutes postmortem ranged from pH 6.90 to 7.07 (Bodwell et ( I / . , 1965b). Average pH at 48 hours was 5.46, and at 20 days, 5.57. Bodwell et al. (19651))also demonstrated that reducing sugars and lactic acid are formed in proportion approximately stoicliiometrically to the disappearance of glycogen. Thus the glycogen content of the L. dorsi muscle was 57.6 p M immediately postmortem and decreased to between 17.5%and 20% of this value i n 2 days of holding. Lactic acid values averaged 13.1 p M after slaughter, increased to 82.4 p M in 2 days, and remained on this general level until the end of the test period, 20 days postmortem. German workers (Gunther and Schweiger, 1966) did not find a simple correlation between pH and lactilte accumulation i n beef t i 1 u scl e ; con s id erab 1e variation i 11 I actate con te ti t and accu i n ti la t i on was noted while the pH drop pursued a generally similar course. A similar trend is also apparent from the data of Hodwell et ( I / . (196%). The pH dropped rather rapidly i n 48 hr, from near neutral to 5.57 (average for five animals), while lactate rose to near its niaxiniiini value, to 82.4 p M / g fresh tissue. Sulisequently, the low point of pH 5.36 was reached at 96 hours postmortem, followed by a rise to pH 5.59 at 14 clays postmortem, when lactate content was at practically the 48 h r level, namely, at 82.7 pM/g. Other fktors liesides lactic acid concentration play a significant role i n determining muscle pH, particularly later i n the aging period. To be perceived a s aroma in the cooked meat, compounds of relatively low molecular weight and consideralile volatility must lie present. Early work (Howe and Barbella, 1937) suggested that the 01)sttrved increase i n flavor from meat a s it is aged could he due to formation and retention i n the muscle ofsolu1)le protein products. However, if water solul)ility is a criterion of the contents of such smaller protein fragments, this has lieen shown to lie decreased by lmth aging and freezing of muscle (Awad et n l . , 1970; Rodwell c t d . ,1965a; Kronman and Winterl)ottom, 1960; Frijimaki and Deatherage, 1964). These water-soluble proteins a s a whole have enzymatic character, and enzyme activities decrease generally with aging of beef. Zeiider ot MI. (1958) a r i d others (Balls, 1960; D. M. Doty and Pierce, 1961) have cons i dere d that degradation may occii r i n part th ro ti gh hydrolysis Iirough t on liy the naturally occurring proteolytic enzymes (cathepsins) of 11ill scl e . Locker ( 1960) studied natural proteolysis during aging of beef throrigh changes in N-terminal groups and levels of free amino acids. H e concluded that proteolysis was not a significant factor i n the ten-

12

KARL 0. HERZ A N D STEPHEN S. CHANG

derizing effect of aging. A definite small increase in free amino acids during aging was noted. Niewiarowicz (1956)and also Leinati (1957) showed that the amino acids alanine, glutamic acid, leucine, and cystine are chiefly involved in this increase. In contrast to Locker’s results, Solovjev et uZ. (1962) showed that there is a considerable increase in N-terminal groups of pure myosin aged 6 days at 10°C. Locker’s finding of low N-terminal groups in muscle could b e explained by reactions of transpeptidation and peptide linkup mediated by enzymes active during aging, as suggested by Bodwell et ul. (1965b). More recently, Davey and Gilbert (1966) studied proteolysis during holding at 2°C for 30 days. T h e increase in nonprotein nitrogen during this period was calculated to correspond to a degradation of 2.3?h of the meat protein. However, the finding that proteolytic changes were confined to sarcoplasmic (not myofibril) components of muscle supports Locker’s conclusion that proteolysis does not influence tenderness. An effect on flavor cannot be ruled out. D. M. Doty and Pierce (1961)suggest that the increase in nonprotein nitrogen during aging of beef muscles by proteolysis could be an important factor in the flavor changes ascribed to aging processes. T h e findings of Davey and Gilbert (1966)are put somewhat in question by the report (Go11 et ul., 1964) that sarcoplasmic proteins are most soluble immediately after slaughter, and far more so if the muscle is excised than if left on the bone. The possibility exists that lower solubility may have resulted from a change in the protein by degradation to nonprotein fractions not determined by protein tests. Particular nonprotein nitrogen compounds have been followed to detect changes in concentration during aging of beef. Creatine and creatinine in muscle were studied by D. M. Doty and Pierce (1961). Creatine was present in raw L. dorsi muscle at concentrations ranging from 1.9 to 4.3 mg/g; individual values sometimes decreased slightly and sometimes increased slightly during aging i n an inconsistent manner. Cooking considerably lowered the creatine content of the steaks compared to the raw muscle. In contrast, creatinine content (range 0.08-0.44 mglg) in raw muscle definitely increased during aging, and normally doubled as a result of cooking. T h e implication is that creatine is changed to creatinine during cooking. Although a specific flavor effect was not assigned to either compound, a statistically significant correlation was found between increasing creatine content and lessening desirability of lean beef flavor, whereas desirability was positively correlated with creatinine content. Creatine phosphate levels postmortem were studied in five Hereford heifers b y Bodwell et u1. (19654. A decrease to one-fifth the ini-

MEAT FLAVOR

13

tial level was observed at 12 hr postmortem, and a zero level was present at 24 hr. The rapid depletion of creatine phosphate occurs before either the pH or the ATP concentration change greatly. Creatinine levels were not reported by these workers. The significance of these findings is not clear. Adenosine triphosphate and associated compounds and their degradation products have significance in areas other than flavor, and therefore have been examined for behavior during aging. Bodwell et al. (1965b) found little change in the level of about 10 pM/g fresh tissue at 6 hr postmortem, but a halving occurred by 12 hr and zero level was reached 24 hr postmortem. ATPase activity in splitting off phosphate is unabated while substrate remains ( D . M. Doty and Pierce, 1961). During aging for 20 days, total soluble phosphate content of beef muscle did not change appreciably, but orthophosphate content increased 50% (Bodwell et al., 1965b). The flavor-potentiating effect of certain nucleotides, and the use of inosinic acid in reproducing meat aroma from certain fractions isolated from water extracts of beef muscle (Batzer et nl., 1960) have raised the question whether such compounds might be very much involved in cooked beef flavor development. Phosphatases are present and presumably active in muscle during aging, so that nucleotides are rapidly decomposed in vivo (Terasaki, 1966; Kassemsarn et al., 1963).The flavor of inosine monophosphate has been described as meaty (Wood, 1961), whereas removal of the last phosphate gives bitter inosine which may be further degraded to bitter hypoxanthine. The impact of these reactions on cooked meat flavor is not clear, particularly in view of the observation of Davey and Gilbert (1966) that breakdown products of nucleosides actually decreased rather than increased during aging for 30 days, as shown by absorbance at 250 mp. If nucleic acids were substantially degraded during aging, absorbance would be expected to double. Hence the absence of an increase during a period known to cause improved cooked flavor can be taken a s inferring that nucleic acid breakdown is not directly responsihle for desirable flavor. Activities postmortem of other enzymes also have been investigated, without uncovering clues that could be helpful in pinpointing sources of flavor or flavor changes (D. M. Doty and Pierce, 1961; Bodwell et al., 1965a). Temperature is not normally a real variable, since this must be kept close to the freezing point to prevent microbial attack (Borgstrom, 1955). The time to cool a carcass may have a decided effect on a number of processes in the muscle, but such effects have not been

14

KARL 0. HERZ AND STEPHEN S. CHANG

clearly established; normal cooling is accomplished by 20 hr postmortem (Bodwell et al., 1965b). Time of aging was not a factor in palatability when differences of 2 days and 14 days were compared (Gauducheau. 1928), but meat aged 14 days was preferred slightly over meat aged 28 days. Certain treatments have been claimed to influence flavor properties of aged beef meat (Gauducheau, 1928);nearly 40 years ago, one patent claimed that injection of sauces into the bloodstream immediately postmortem would cause the muscles to take up the aroma of the sauces. Recently, injection of the mycelium from Aspergillus niger was claimed to result in flavor modification leading to speedier and more intense desirable flavor. A new process of aging meat (Anonynious, 1963) depends on growth of a particular group of microorganisms (Thmnnidium sp.) directly on the meat held at the rather high temperature of 72°F for 32 hr. Development of desirable flavor is speeded, diffusion through the muscles is claimed, and the entire aging process becomes more rapid and controllable. T h e significance and practical value of these treatments remain to be fully established, although the last has received commercial trial. Proteolytic enzymes have been successfully injected antemortem into the bloodstream, or stitch-pumped postmortem into muscles, in order to tenderize the meat. A qualitative effect of one such treatment with papain on beef steak flavor could not be demonstrated for the lower boiling aroma volatiles, although there were some differences in the quantities of individual compounds (K. 0. Herz, 1963).

E. FREEZING AND FREEZER STORAGE A vast industry has grown in the United States as home freezers have become more commonplace, and meat items are among the primary foods purveyed. The basis for this growth is the relatively slow rate of change in quality of properly prepared, packaged, and stored me at . Good quality raw meats keep for several months in freezer storage without loss of palatability (Weir, 1960). Actual storage life or extent of changes can be influenced by the feed on which the beef animal was raised; b y postmortem aging time, pH, metal contamination; and b y the temperature of holding before freezing. On the first of these factors, a study of effects of feeding regimen on various beef characteristics (Wanderstock and Miller, 1948) showed no significant differences in aroma or in flavor of lean in roasts from animals that had at some time received grain feed, but the already poor scores for meat from

MEAT FLAVOR

15

nninials fed pasture only were quite significantly further depressed a s a result of freezer storage for 6 months. A delay in freezing may lead to development of rancidity in the fat (Ritchey and Hostetler, 1964). This also occurs at higher freezer temperatures. Thus meat became rancid i n 3 mciiths when stored at 10"-15"F,while O"F storage protected its quality longer than 14 months. Industrial experience (Boyd, 1966) indicates that off-flavor development in good raw meat may begin after 3 months' storage; it becomes significant after approximately 6 months in the freezer, and is much more noticeable in the meat following cooking than in the defrosted raw meat. Temperature affects the rate of off-flavor development and is much greater at 5°F than at -10°F. With large pieces such as quarters of beef, size may also have an influence. If freezing of the interior portions is slow, off-flavor may develop there and work its way outward. One benefit of using a low temperature initially is that freezing of interior portions is more rapid. Pearson and Miller (1950) showed that increasing length of freezer storage does induce a gradual loss in acceptability ratings for aroma and flavor. Free fatty acid content rises from 1.6to 9.1%in frozen storage, ascribed primarily to phospholipid hydrolysis (Awad et al., 1970). Recently, Law et al. (1967) studied changes in L. dorsi and semimembranosus (top round) muscle from eight good grade carcasses kept in freezer storage at -18" to -23°C for 6 and 9 months. While loin steaks were not affected at 9 months, top round steaks actually were rated higher than fresh steaks after 6 months freezer storage, and lower at 9 months. This confirms an observation from industry (Boyd, 1966), that off-flavor development proceeds at different rates in different cuts or muscles. Rate of freezing fresh meat does not affect flavor (Pearson and Miller, 1950; Anonymous, 1964; Wanderstock and Miller, 1948; Dunker and Hankins, 1953). The rate should be sufficiently fast to ensure rapid freezing of the entire meat piece. Recommended is a rate of more than 0.2 cm/hr, attainable normally using freezer temperatures of-15"C or lower (Anonymous, 1964). One major drawback of freezer storage of beef is weight loss on thawing through increased drip. Maximum drip occurs when the muscle has been stored at just below its freezing point temperature (Moran and Hale, 1932). Most of the increased drip is thought to be water. The rate of thawing affected keeping quality of meat frozen for 3 to 6 months (Marginescu, 1935). Meat frozen 3 months and thawed slowly actually had better stability than comparable fresh meat, but after 6 months in freezer storage keeping quality was poor even when

16

KARL 0. HERZ A N D STEPHEN S. CHANG

thawing was slow. Rapid thawing invariably gave poor keeping quality. Inhibition of microbial spoilage is probably the single most important factor promoting freezing preservation of meat. A temperature of 21°F (-5°C) does not inhibit microbial development (Haines, 1931), but below -4°F the microbial count on meat is decreased and microbial development is inhibited. Commercial freezer storage normally is near-10°F (Boyd, 1966). The use of aluminum foil as wrapping for raw meat has been advocated on the grounds that it is responsible for formation of bacteriostatic complexes near the meat surface (Caserio et ul., 1963). Only a minute amount of Al"+ enters the meat, but this is apparently sufficient to cause considerable reduction in surface microbial populations as compared to other packaging materials. Although overall freezing rate is slower than for other packaging materials such as kraft paper and cellophane, the high heat conductivity of aluminum ensures that the meat surface contacted by aluminum will freeze extremely fast, leading to efficient growth inhibition or destruction of surface microorganisms (Bratzler and Tucker, 1963).

111. RAW AND PROCESSED MEAT FLAVOR Meat is not ordinarily consumed in the raw state. All processing starts with fresh meat, but the different forms lead to distinctly different products. Part of this individuality is derived from organoleptic properties, including aroma and flavor. The process used most often for direct consumption of fresh meat is dry or moist heat. Other processes are designed to extend the time of wholesomeness for the meat, and in doing so often produce new meat foods. Some- such as canning, vacuum-packaging in pouches, and most freeze-drying- include cooking as part of the preservation process. Others, such as curing and irradiation treatment, are carried out on the fresh meat. Raw meat flavor has not been adequately characterized, and it is therefore not surprising that differences in flavor between raw starting material and finished processed meat product have not been elucidated. A. RAW AND COOKED BEEF

The odor of fresh raw meat is usually very slight in intensity and reminiscent of commercial lactic acid (Weir, 1960) or bloodlike

MEAT FLAVOR

17

(Crocker, 1948). Crocker (1948) established that the flavor is in the juices of the meat and not in the fibers. He noted that human saliva apparently has no enzyme capable of releasing taste-producing substances when raw meat is taken into the mouth and chewed. Odor generally increases with the age of the animal (Crocker, 1948). Sex also may influence raw meat odor; mature males have a “staggy” odor (Weir, 1960). The sex odor of pork from boars was studied by Craig et (11. (1962) and has recently been traced to a steroid component. Patterson (1968) isolated 3a-hydroxy-5a-androst-16-ene and showed its relationship to the sex odor taint of boar pork meat. Off odors from fat oxidation are characteristic of the type animal, or in the case of a “fishy” odor, of the predominant feed material. Some off odors carry over during cooking or are even intensified by heat treatment. Amine volatiles in raw beef are 99.9% ammonia (Burks et al., 1959). Meat flavor after cooking has nothing reminiscent of raw meat odor. Both raw and cooked meat flavor substances can be extracted with water to the full extent (Kramlich and Pearson, 1958; Batzer et ul., 1960). Water solubility of flavors may cause some of the difference between boiled and roasted beef, in addition to the higher temperature and degree of dehydration in meats cooked in dry heat or with fat. Water solubility is the basis for studying meat flavor through relatively flavorless precursors. Fractionating and/or regenerating flavor by heat from lyophilized water extractable substances of lean meat produced essentially the same flavor profile (Batzer et ul., 1962; Hofstrand and Jacobson, 1960; Hornstein et ul., 1963)for beef, lamb, and pork, which suggested that the same substances and the same reactions may be responsible for the lean cooked flavor. Species differences in cooked meat flavor include volatile compounds produced during cooking in the presence of both muscle fiber and fat (Landmann and Batzer, 1966). Crocker (1948) suggested that meaty flavor is developed by cooking because of chemical changes in the fibers rather than the juices. Kramlich and Pearson (1958, 1960) studied changes in the flavor attributes of raw- and cooked-beef-water slurries, meat-press fluid and filter cake, and raw- and cooked-meat water leachings. Press fluids from raw beef had, after cooking, a highly concentrated flavor. However, flavor was stronger when the unpressed meat was cooked, suggesting that full flavor development requires heating the juice and the fibers together. Perhaps a clue to the nature of the flavor precursors in water extracts of lean meat is that they are dialyzable, hence must be of relatively small molecular size without being volatile.

18

KARL 0. HERZ A N D STEPHEN S. CHANG

B. FACTORS CAUSING DIFFERENCES FROM NORMAL COOKEDMEAT FLAVOR Such factors have been examined. After slaughter, the capability to produce nonnal meat flavor upon cooking is gained by the muscle during storage of a week or more. The desirable flavor is not present in cooked unaged beef, which is metallic and astringent (Weir, 1960). Antemortem stress appears to have some effect on cooking and evaporation loss as well as on the surface temperature of the meat during cooking (Lewis et al., 1967). Compared with meat from unstressed animals, loss was lower and surface temperature was higher after heating; internal temperature also was slightly higher. If flavor formation depends on temperature, as has been suggested (D. M. Doty, 1961; Hornstein et al., 1960; Hamm, 1966), such antemortem stress could influence flavor composition or intensity. However, over a limited range of oven temperatures, aroma and flavor of roasted meat were not influenced significantly by cooking at 135”, 150”, or 165°C (Tilgner, 1965). On the other hand, cooking to internal temperatures, of 70” and YWC, respectively, did show a significant preference in aroma and flavor of fat and lean for roasts cooked to the lower temperature. The effect of the cooking method on flavor has not been studied to a great extent. Ground L. dorsi cooked in fat produced three times as much carbonyl compounds as found when meat was cooked in water (Sanderson et al., 1966). Flavor precursor fractions gave different meaty flavors, reminiscent of roast beef when heated in fat, and of broth when heated in water (Batzer ei al., 1961; Hornstein et al.,

1960). Microwave cooking of beef has also been investigated. Generally, most characteristics were scored poorer for top rounds cooked in a microwave oven (Marshall, 1960). Shorter cooking time was obtained with a loss of juiciness and flavor, higher cooking and evaporation loss, and uneven drying out. Law et al. (1967)confirmed this with top round steaks. Loin steaks and top round steaks were preferred when cooked by the conventional methods (broiler for loin steaks, oven for round steaks), although both drip loss and total cooking loss were higher for broiled loin steaks. Ordinarily, cooked fresh beef is consumed within a reasonable time after cooking. If it is stored, refrigerated or frozen, a distinctive stale odor develops (Santoro e t al., 1960; Weir, 1960)that has been termed “old hamburger” or “warmed over” off-flavor (Boyd, 1966). A cause

MEAT FLAVOR

19

for this development has not been clearly established, although it has been suggested that myoglobin-catalyzed fat oxidation is responsible (Weir, 1960). Practically no information is available on the changes in volatile compounds emitted during cooking and normally lost to the atmosphere. At least in boiling, the aroma above the meat-and-water mixture before ebullition commences -and for a short time thereafter- is of a sharp somewhat unpleasant nature, not too close to typical boiled meat flavor (K. 0. Herz, 1966). D. M. Doty (1961) suggested that one function of cooking was the purging of undesirable flavor compounds from the meat. With chicken meat, Kazeniac (1961) showed that compounds of hydrogen sulfide and ammonia were important for flavor giving and flavor masking of the aroma components.

C. CANNED,DRIED,AND FREEZE-DRIED BEEF

The change in organoleptic properties of meats that have undergone extensive heat treatment such as is required in canning is pronounced and well known. Frequently, the product resembles an overcooked product in regard to both texture and flavor. Differences in flavor have not been reported in the literature between normally cooked and canned meat. There seems to be some evidence that hightemperature-short-time (HTST) processed meat is less damaged from the flavor point of view than retorted meat (D. M. Doty, 1961). However, this was not observed by Luh et al. (1964), who compared HTST-treated strained meat with the retorted product. In this work, the HTST (150OC) product had a “raw” taste, and both hydrogen sulfide and methyl mercaptan were found among the volatile substances present. On the other hand, the retorted (122OC) product had a full, good canned-meat flavor, considerably more hydrogen sulfide and no detectable methyl mercaptan. Also under somewhat unusual conditions, Brennan and Bernhard (1964) found a number of compounds in “canned” meat in broth that they consider could be responsible for differences in canned- and cooked-meat flavors. Dehydration by hot air drying is not suitable for pieces of meat, but can be employed effectively with cooked ground meat (D. M. Doty, 1961). This process is not nomially employed to a great extent, since most meats suffer loss of flavor and tenderness. Removal of moisture by freeze-drying appears more feasible and involves less flavor loss but has some danger of off-flavor develop-

20

KARL 0. HERZ AND STEPHEN S. CHANG

ment. Raw meat is usually not freeze-dried, because off-flavor development is more rapid and intense (Boyd, 1966; El-Gharbawi and Dugan, 1965b), although a claim has been made that a satisfactory product is obtained if moisture is reduced below 1.7% (Thomson et al., 1962). Lipid oxidation of freeze-dried raw beef is a real problem, even if storage is under nitrogen (El-Gharbawi and Dugan, 196513).Phospholipids are oxidized first, the more saturated tissue triglycerides later. Water extracts of freeze-dried meat contained enzymes of the glycolytic pathway and the citric acid cycle, other sarcoplasmic proteins, and myoglobin. ATPase and lipase/lipoxidase activity remains after freeze-drying, although ATPase activity continuously decreases during storage of the freeze-dried meat. In addition to the probability of enzymatic action, deterioration may result from autoxidation of fats in the freeze-dried meat (Hamm, 1964). With the drop in moisture content oxygen absorption increases. In storage studies, free radicals were found, such as are formed during autoxidation of fats. Other workers (Thomson et al., 1962) consider that deterioration of raw freeze-dried beef in storage results chiefly from oxidation of reducing compounds and the depletion of carbohydrate compounds in browning reactions facilitated by the absence of water. Artificially increased pH of meat postmortem (by adrenaline injection before slaughter) was helpful in conjunction with the British accelerated freeze-drying process: it gave a product improved in many respects, but suffering from considerable loss in flavor (Penny et al., 1964). Examination of volatiles obtained from raw freeze-dried meat revealed no unusual compounds (El-Gharbawi and Dugan, 1965a). Cooked freeze-dried meat likewise must be dried below 2% moisture content. Lack of flavor or early development of off-flavor is considered the greatest weakness of cooked freeze-dried meat products. Comparisons with canned or frozen counter-parts of freeze-dried meat in various forms indicated that the freeze-dried products were at times rated as good, but almost always a comment was added concerning lack of flavor or presence of off-flavor. Off-flavor development in cooked beef that has been freeze-dried is well known (Bird, 1965). It is ordinarily present after 3 weeks of storage at room temperature; after 6 months if stored under nitrogen. Offflavor also develops if the freeze-dried meat is frozen, and it is detected and intensified when cooked. It has been variously described as autoxidized fat flavor or cardboard flavor. Nonenzymatic browning and lipid oxidation reactions during freeze-drying of food products have been discussed by Karel(1963).

MEAT FLAVOR

D. CURED

21

AND SMOKED hlEAT

There is as y e t no information as to how much of the distinctive flavor of cured or smoked meat is caused by the curing or smoke ingredients and how much is developed in the meat (Wilson, 1960; Landmann and Batzer, 1966). Nitrous oxide developed during curing and involved in cured pigment formation is a very reactive compound that could lead to formation of flavorous compounds. In smoking, certain compounds isolated both from smoke and from smoked ham contribute to the typical flavor, but there is no agreement on which compounds actually are responsible (Landmann and Batzer, 1966). W. W. Foster and Simpson (1961) showed that smoke consisted of a particulate and a vapor phase. The principal effect of smoking was produced by vapor phase substances dissolving in surface or interstitial moisture of the food; absorption led to combination with food constituents and then fixing. Support for this view comes from commercial preparation and sale of smoke flavoring made by absorbing smoke vapor in water (Hollenbeck and Marinelli, 1963). Phenolic compounds in smoke appear to be important to smoke-flavored meats (Draudt, 1963). T h e processes of curing and smoking alter flavor markedly from the base of cooked meat, but until this base flavor is known, the part played by flavor development in the meat cannot be assessed. A particular problem of cured meats is salt-catalyzed oxidation of lipids in frozen storage (Zipser et al., 1964; Cross and Ziegler, 1965). A satisfactory antioxidant has not been found, but the problem has been circumvented by coating salt with fat to avoid contact with meat lipids during freezer storage (Chang and Hoynak, 1966). Heme compounds, such as myoglobin and hemoglobin, act as the principal catalysts of lipid oxidation in raw and uncooked processed meat products (Zipser et al., 1964).

E. IRRADIATEDBEEF The promise of a meat product stable at room temperature is the reason behind the perseverance of workers studying the feasibility of irradiated meats. For many years, irradiation-induced off-flavors remained an insurmountable obstacle. Attempts to identify the compounds responsible for this off-flavor were only partially successful and were particularly difficult because the compounds responsible for desirable beef flavor were not known (Wick, 1963). Beef seems to be particularly sensitive to development of off-odors

22

KARL 0. HERZ AND STEPHEN S. CHANG

by irradiation even at relatively low doses (Evans and Niven, 1960; Wick, 1963; Hedin et al., 1961). The odors have at times been described as reminiscent of “wet dog” and “wet chicken feathers” (Hedin et al., 1961). Some workers consider that protein degradation or changes are responsible in some way for production of most of the off-odor (Wick, 1962; Hedin et al., 1961; Snyder, 1966; Ambe and Tappel, 1961),but others have concluded that changes in nitrogenous constituents are not related to changes in sensory characteristics of irradiated beef (Artar et al., 1961), or that the nonprotein nitrogen compounds are responsible for compounds contributing to irradiation off-odor (Bellamy, 1959). Wick et al. (1961a, 1965; Wick, 1963) noted that volatiles increased with increasing radiation. Sulfur and nitrogen containing compounds were responsible for much of the irradiation-induced odor, and methional was claimed to be a major contributor to the unpleasant type of irradiation odor. The bulk of the off-flavor originated from substances extractable from tissue b y water (Evans and Niven, 1960). Beef protein fractions differed in their ability to generate odor upon irradiation, but some did produce “wet dog” or “wet chicken feather” odors (Hedin et al., 1961). Degradation of sulfur-containing amino acids has been held responsible for irradiation off-odor production (Snyder, 1966). Solutions of amino acids undergoing oxidative reactions following irradiation were monitored for malonaldehyde formation (Ambe and Tappel, 1961). This was greatest for glutamic acid and arginine, ,onsiderable for methionine, and noted to some extent with methionine sulfoxide, phenylalanine, serine, and valine. Burks et al. (1959)considered that nonprotein nitrogen compounds were precursors of the volatile amines and ammonia produced during irradiation of beef. They held that this origin is more plausible in view of the 38-fold greater amino end groups present in the nonprotein compounds. Efforts at lessening or eliminating off-flavor in irradiated beef are bringing some results. Intensity of irradiation odor decreases continuously during storage at room temperature for 6 months (Snyder, 1966). Lowering the temperature of the beef during irradiation has a marked effect on off-flavor development (Anon., 1965; Snyder, 1966; Harlan et al., 1966). Steaks brought to a temperature of -196°C before irradiation had little off-flavor if warming to -80°C had been slow. Apparently, free radicals, stable at room temperature, can be formed at these very low temperatures maintained for several days, followed by careful slow warming until -80°C has been reached. Shorter ultralow-

MEAT FLAVOR

23

temperature holding, or allowing the frozen sample to warm u p quickly within the range of -196" to -8O"C, leads to loss of stable free radicals and development of off-flavor (Harlan et al., 1966). A patent (Alexander and Harriman, 1963) claims that irradiationproduced off-flavor can be removed from beef if the meat is immersed in minimum 0.85% saline during irradiation. When the salt solution is later discarded, undesirable off-flavors are removed with the solution.

IV. SOME PROBLEMS OF RESEARCH ON MEAT FLAVOR Two types of approaches can be used: (1) Analysis of the formed flavor; and (2) investigation of the precursors of the flavor. Both methods have been used on meat flavor.

A. PRECURSORSTUDIES Many workers have stated that the compounds in raw beef that produce flavor on heating are water soluble and can be leached into water. Others have contended that the compounds isolated from cooked meat, including the flavor, also are water soluble at the low concentrations encountered. Thus it seemed reasonable to try to at least complement knowledge of meat flavor through precursor studies on leachings from raw meat muscle. However, the heat treatment of meat continues to evolve flavor over quite a period of time. This observation suggests that substances are released from the meat fibers during heating. Whether these substances are the same as those leached into water in normal precursor sample solutions is not known. Although exhaustive leaching may leave a muscle residue that will not produce meat flavor on heating, this may quite possibly be because one or more essential reaction partners have been leached out. Certainly, if fat is involved in any way, aqueous leaching will have little influence on lipid material and preferentially lipid-soluble compounds. Thus, though fractions have been obtained in precursor studies that produce meaty aroma on heating, many questions must perforce remain concerning the role of the constituents of these fractions in meat flavor formed under normal conditions from heated muscle.

24

KARL 0. HERZ AND STEPHEN S. CHANC

B. FORMEDFLAVORSTUDIES In the development of desirable aroma and flavor of meat by heat treatment, gas is normally released from the meat. Hence, atmospheric components (in air or dissolved in a heating medium such as water or fat) probably are not involved in the formation of flavor, except perhaps at the surface of the meat. Flavor development inside the meat piece would therefore depend on (1)amount and location of substances involved in producing flavor compounds; (2) temperature at each location; and (3)water activity at each location. Meats and the way they are prepared as food pose some problems that are of little concern in flavor studies on such foods as fruits and nuts. These problems concern the uniformity and reproducibility of samples prepared as the material for flavor research, the unique nature of meat (kinds of fibers and of volatile compounds evolved), and the demands that these special meat characteristics make on sample form and flavor isolation equipment and procedures.

1 . Material for Research One desirable type of flavor frequently associated with beef is that of cooked steak; another is that of roast beef. Method of cooking steaks or roasts involve great variability in the degree of doneness, distribution of flavor b y type and quantity at different locations of a cut and in the leachings and drippings, and browning reactions at surfaces heated to high temperatures (K. 0. Herz, 1963). u. Uniform and Reproducible Sample. Cooking of steaks or roasts to the same degree of doneness is exceedingly difficult. In one cooking, the flavor potential is quantitatively not even approached, and once isolation of formed flavor has been attempted, the experience with leached raw meat suggests that perhaps the same flavor compounds will not again be produced when the deflavored residue is heated. Thus steaks or roasts cannot readily give the uniform and reproducible samples desirable for meat flavor research. The use of boiled beef as a material for flavor isolation avoids the problems of steaks and roasts. Although the flavor of boiled beef is different from that of roasted or broiled meat, some evidence shows that both arise from the same precursors (Batzer et al., 1960; Macy et ul., 196413; Wasserman and Gray, 1965). Boiled beef has a desirable type of meaty flavor; it is consumed extensively (for example, in “TV dinners”). Among the advantages of using boiled beef in flavor research are the

MEAT FLAVOR

25

possibilities of developing flavor uniformly and reproducibly, and of redeveloping the same flavor after one or more attempts at flavor isolation from a sample have been made (K. 0. Herz, 1968). b. Form of Sample. For nearly all the usual means of flavor isolation, the comminuted form is the most advantageous, because the need for diffusion of volatiles from interior portions of a food (e.g., meat) to the surface is substantially reduced. In one vacuum-isolation procedure used on boiled beef (K. 0. Herz and Chang, 1966), comminuting to a fine particle size was indispensable to permit metering of a slurry into the vacuum-stripping system. Meat fibers appear to cause greater problems of metering than are encountered with most other types of food. A fine slurry also facilitates rapid and uniform heating of the sample for redevelopment of flavor (K. 0. Herz, 1968).

2. Flavor Isolation Means Workers in flavor investigations have devised a number of different means for isolation. Aside froY+c!ontainer headspace gas examination, most systems have relied upon either reduced pressure or entrainment by inert gas to separate vaporizable material (presumably including flavor compounds) from the bulk of the food material. The method of gas entrainment requires fairly long intervals for removal of flavor compounds from solutions or slurries and demands considerable cooling capacity for trapping; it has not been employed with much success on meat. Numerous systems have been used for stripping volatiles from food substances with the assistance of low pressure-vacuum (Dimick and Makower, 1951; Eskew et al., 1959; Merritt et al., 1959; Weurman, 1961; Wick et al., 1961b; Chang, 1961; Smith et al., 1963; Roger and Turkot, 1965; Hobson-Frohock and Lea, 1965; Wick, 1965; Smouse, 1965; Mendelsohn et al., 1966; Krishnamurthy, 1966; Deck, 1965, 1967; K. 0. Herz and Chang, 1966). Some of these systems have met with a degree of success in isolating flavor from substances more tractable than meat, whereas others have shown indifferent effectiveness unless used with liquid foods or foods of relatively open structure, such as apple slices. The more effective systems used on meat involved some heating or preheating, movement of a slurry when this was the sample, trapping by effective means (solid carbon dioxide plus liquid nitrogen traps) to maintain low pressure in the system, and in some cases the aid of a stripping agent such as steam. Simple exposure of pieces of meat to an evacuated environmeiit is ineffective in the presence of the normal

26

KARL 0. HERZ A N D STEPHEN S. CHANG

moisture content of meat, because the temperature at the surface of the meat drops rapidly and impedes volatilization of flavor compounds (K. 0. Herz, 1963). The volume of water removed with flavor substances in gas sweeping or vacuum stripping is very large. T h e need to condense this water along with the flavor volatiles taxes the capacity of traps of most designs. Effective condensation is indispensable to prevent loss of volatiles during isolation as well as to maintain the lowest possible pressure at the point of volatilization of flavor compounds from the slurry of meat. Controlled zone freezing or zone melting may offer alternative isolation methods in the future (Huckle, 1966).

3. Approaches to Flavor Evaluation For flavor evaluation to be meaningful, the isolate must have the same flavor as the cooked meat from which it originated. If this same flavor persists following extraction (or other means of removing water) and concentration of the extract, the concentrate can be presumed to contain the compounds responsible for the flavor. a. Gas Chromatography. The most effective means developed to date for the separation of a mixture into components that are volatile or capable of being volatilized is the gas chromatograph. It has become the most widely used tool for the necessary separation step in the analysis of formed meat flavor concentrates. The possibility of changes or reactions at elevated temperatures within the instrument (Van Lunteren et al., 1968) poses some question concerning the degree of certainty with which compounds detected and subsequently identified can be said to exist in the concentrate and the food. b. Flavor Profile Differences. The large number of components shown to be present in flavor concentrates from meat, and the known lack of contribution to flavor by many of these, suggest that only a few substances giving peaks on gas chromatograms actually contribute to the major flavor notes. Therefore, if major compounds could be deleted from the whole spectrum of a given flavor in small groups, compounds actually involved in giving the flavor sensation could be 10cated and identified. This approach can be questioned from the aspect of the uncertainty of assigning a “no-effect” label to compounds. It has been shown that compounds may be present in a mixture below individual threshold for perception, but in combination they make a definite contribution

MEAT FLAVOR

27

to aroma. Also, some cornbinations of compounds exhibit synergistic or antagonist flavor effects. A similar difference approach relying on the nose rather than on GLC has been taken in work on chicken aroma (Klose et nl., 1966). Aroma as formed was carried by nitrogen gas through absorbent liquids or solids that would remove or hold certain portions of the aroma without in any way reacting with the unabsorbed fraction. The technique clearly demonstrated again (cf. Kazeniac, 1961) that hydrogen sulfide and ammonia make contributions to chicken aroma even though neither can readily b e shown to have an effect in the complete aroma. The variety of odors that can be produced from a single starting material depending on the conditions makes this work most difficult (Hedin et al., 1961). V. KNOWLEDGE OF HEATED MEAT FLAVOR Meat flavor is a complex stimulus to the human senses (Crocker, 1948; Kauffman et d.,1960; Lea, 1963; Lawrie, 1966) involving chiefly aroma and taste (D. M . Doty, 1961), but having also such characteristics as body and mouth satisfaction (Kazeniac, 1961).Although the overall flavor sensation may depend to some extent on compounds that are essentially nonvolatile (Kauffinan et al., 1960; Landmann and Batzer, 1966),volatile compounds comprise the more important part of the total ineat flavor profile (Crocker, 1948; Lawrie, 1966). Despite considerable work, great gaps remain. Vickery (1966) recently summed up this state of affairs, “. . , many volatile components probably contributing to meat flavor have been isolated and characterized, but we still do not know the major ones contributing to the characteristic flavor spectrum.” Flavor substances present as volatiles in cooked meat may not be pre-existent in the raw meat; evidence indicates that they are generated during cooking from nonvolatile precursors that are either watersoluble or fat-soluble. Apparently, however, the role of heat is not merely to convert precursors to volatile flavor. Heat may have such auxiliary and continuous functions as (1)releasing flavor or flavor precursors from fatty structures; (2) enabling mixing of fat-soluble and water-soluble compounds to take place as fat melts and becomes part of the meat juices; and (3) favoring browning reactions through evaporative and exudative dehydration and through protein degradation. Recent views on the components comprising the meat-flavor com-

28

KARL 0. HERZ AND STEPHEN S. CHANG

plex (Landmann and Batzer, 1966; Hornstein, 1967) consider that water-soluble materials in both raw and cooked meat contribute to a basic meaty flavor, whereas fat-soluble comFounds are responsible for flavors defining species characteristics. Among these compounds in raw meat are both volatiles and nonvolatiles, and crossing over during heating occurs from both categories. Nonvolatile flavor precursors give rise to both a nonvolatile- and a volatile-flavor fraction during heating. Many compounds in meat are capable of entering the flavor formation steps. Although proteins do not contribute to flavor, their degradation products can join free amino acids, sugars, minerals, lipids, nucleic acids, and their degradation products in producing meat flavor components. Three approaches have been taken in the past: (1) study of meat flavor formation from precursors and of the composition of the flavoryielding fractions; (2) experiments with model systems, built up chiefly on the basis of information obtained in precursor studies; and ( 3 )analysis of the composition of fully developed isolated meat flavor. A. FLAVORPRECURSORSTUDIES Kramlich and Pearson (1958) were among the first to record that leaching of raw and cooked beef in water at 4°C resulted in removal of the flavor from the muscle fibers. Ice-cold water extraction of flavor precursors from fresh lean meat has been used successfully b y a number of workers (Batzer et d., 1960; Hornstein and Crowe, 1960, 1963; Wasserman and Gray, 1965; Batzer et al., 1962). Dialysis applied to isolate flavor precursors from other water-soluble material led to the conclusion that the precursors must be of relatively low molecular weight, since they were present in the diffusate. Hornstein and Crowe (1960) described a method of obtaining a flavor powder concentrate by lyophilizing a cold distilled water extract of raw ground L. dorsi muscle. On heating the dry powder, an odor reminiscent of roast beef evolved. Heat applied to an aqueous solution of the powder produced the aroma of boiled beef. Extensive work on meat flavor precursors has been carried out at the American Meat Institute Foundation (summarized in Landmann and Batzer, 1966) and more recently at the U.S. Department of Agriculture’s Eastern Regional Laboratory (Wasserman and Gray, 1965; Zaika e t d.,1968).In the work at AMIF meat flavor precursors were isolated in fractions from lean beef water extract by diffusion first through dialysis tubing and subsequently through sausage casing. The stable d i h s a t e obtained could be separated further into two fractions by

29

MEAT FLAVOR

Sephadex gel filtration. One of these two fractions, designated Aa,, had an ultraviolet spectrum typical of a normal protein; it did not give rise to meaty flavor upon heating. Fraction Aaz, obtained as a white fluffy powder following gel filtration on Sephadex G-25, produced the aroma and flavor of cooked beef when heated. The composition of this fraction is qualitatively indicated in Table I, left column. Landmann and Batzer (1966) hold that the essential components of the flavor are inosinic acid and the “glycoprotein” or its complete hydrolysis products, among which glucose has been identified as the carbohydrate moiety. Among amino compounds, serine, glutamine, and asparagine seem to be necessary, and the last two seem to be the main sources of ammonia found in the volatiles of cooked meat. All compounds listed have little volatility. Wasserman and Gray (1965) attempted to check the work done at AMIF. In repeating the dialysis procedure, they found that their batch of Visking sausage casing did not effect the separation achieved by AMIF workers. Therefore, both dialyzate and d i h s a t e were further fractionated, the one on Dowex-50 resin, and the other on Sephadex

TABLE I CONSTITUTION OF REEF FLAVOR PREcunson FRACTIONS I N AQUEOUS EX1‘HACTS OF 1iAW LEANhlUSCLE L,andmann and Batzer, 1966 Precursor fraction Aa,

Hydrolyzate of glycoprotein

C;lycoprotein ------ - - - - I Inosinic acid Serine Taurine Glutamic acid Asparagine GIycine Clutamine Alanine Anserine Isoleiicine Carnosine Leucine Creatine p-Alanine Quiltennary Proline w i i n e s (3) Levulinic wid Glucose Inorgmic phosphate

Wasserman and Gray, 1965 Precursor fraction Aa,

Precursor fraction Al),

Hydrolyzate of fraction All2

H ypoxan th i ne In 0 s in e Alanine Arginine Glut;n~iicacid GI 11tam ine GI y c in e Hydroxy proline Leucine (iso) Methionine c;lncose Deoxyribose

H ypoxanth i 11e In osi ne Almine Arginine Glutamic acid C:lntamine G l yci n e Leucine (iso) M e th i on i n r

Ammonia Aspartic acid Threon i ne Serine C.lnt;uniic acid Proline Glycine Alanine Val i n c Meth ionine Isoleiicine Leuci n e Tyrosine Phenyla1;unine Lysine Histidine Arginine Tryptophan

30

KARL 0. HERZ AND STEPHEN S . CHANG

G-25. In the latter fractionation, a fraction corresponding to the Aa, fraction reported by the AMIF workers was obtained. Its qualitative composition is indicated in the second column of Table I. (Two additional fractions were obtained by Sephadex gel filtration, in contrast to only one reported from the AMIF; both fractions did not give rise to meaty flavor upon heating.) Ion-exchange chromatography of the dialyzate also gave rise to three fractions, one of which turned out to be all glycine. A second fraction contained chiefly carbohydrate (glucose, deoxyribose, succinic acid) and traces of glycine and isoleucine. Neither fraction gave rise to meaty flavor upon heating. The third fraction from ion-exchange separation, designated Ab2, did give rise to meaty flavor upon heating. Its composition is indicated in column 3 of Table I, based on paper chromatographic evaluation. The absence of carbohydrate is striking, dealing a blow to the reported indispensability of sugar for flavor formation by browning-type reactions. In all samples, hypoxanthine was the predominant purine compound, rather than the inosinic acid (or inosine P,) reported necessary for meat flavor development b y A M I F workers. Hypoxanthine is a degradation product of inosine; the breakdown could have occurred enzymatically during aging of the meat or chemically as a result of the procedures to which the extract was subjected in testing. Fractionation of fraction Ab, on Dowex-l yielded a fraction eluted with water and another eluted with dilute acid. The water eluate contained only amino acids (no hypoxanthine), whereas the dilute acid eluate was made up of hypoxanthine and three amino acids. Both fractions on pyrolysis evolved an aroma described as meat-like. The paper chromatography technique used for amino acids determination was questioned by Wasserman and Gray (1965) themselves. The use of an automatic amino acid analyzer demonstrated the presence in fraction Ab, of twenty components (compared to the seven amino acids determined by paper chromatography). These are listed in the last column of Table I. Histidine was not resolved from a component making up a major part of the sample. Present in relatively the largest amounts (pmoles per milligram) were, in order, ammonia, alanine, serine, glycine, and leucine. Another striking conclusion to be drawn from Table I is that the indispensability of a sulfur compound in the development of meat flavor must be seriously questioned. Although the AMIF workers suspected that one of two unidentified ninhydrin-positive spots could be due to a sulfur-containing compound, and two substantial compounds among the amino acids found by Wassennan et al. remain unidentified, there has been no indication that sulfur is actually present. The latter

+

MEAT FLAVOR

31

workers do report, however, that fraction Ab, appears to contain peptides or low-molecular-weight proteins in addition to the free amino acids. Macy et al. (1964a) prepared and studied the composition of a less highly fractionated lyophilized water extract of lean beef round. Paper chromatography using three different solvent systems was used, and additional confirmation was sought by ion-exchange chromatography. The numerous compounds demonstrated in the extract (which gave a meaty flavor when heated) were similar to those reported elsewhere (Batzer et ul., 1960; Wasserman and Gray, 1965). In addition, the sulfur-containing amino acids cystine, cysteine, and methionine and three phospho compounds (phosphoserine, phosphoethanolamine, and glycerophosphoethanolamine) were present. Analogous work b y Macy et al. (1964a) with lamb and pork meat revealed a generally similar pattern of compounds in water extracts, confirming a conclusion reached earlier by Hornstein and Crowe (e.g., 1964). These workers contend that species-specific flavors reside or are developed from substances in the lipid tissues of the animals. Volatiles were cold-trapped and identified from heat-treated lyophilized water extract of beef, among them ammonia, methylamine, hydrogen sulfide, methyl mercaptm, formaldehyde, acetone, and acetaldehyde, and also lactic acid and its ammonium salt. Wasserman and his colleagues have continued their work on precursors of beef flavor using milder techniques of separation, particularly column chromatography on the cross-linked polyacrylamide BioGel P-2 (Zaika et ul., 1968). The first of seven fractions obtained had meaty aroma. Though the aroma of the second fraction was described as meatlike, a grassy note was also present. Table I1 indicates the composition of the two fractions; compounds common to both are listed at the top. Though fraction 2 contained inosinic acid, fraction 1 did not include nucleotide derivatives. The most intense aromas were given by fractions that contained both amino acids and sugars. Flavor persisted when the phosphate was removed from either fraction, and ore.at'me or creatinine also could be shown not to be involved in the aroma-producing reactions. T h e hypothesis that species-specific flavor is in some way due to fat or fat-soluble compounds (Hornstein et al., 1960; Hornstein and Crowe, 1960, 1963, 1964) found perhaps its most obvious support in comparison of the lipid fractions extracted with chloroform-methanol from lean beef tissue and lean whale tissue (Hornstein et al., 1963). Beef triglycerides had fatty acids of 18 carbons or less, whereas 3770 of whale triglycerides had fatty acids in the 20- and 22-carbon

32

KARL 0. HERZ AND STEPHEN S. CHANG TABLE I1

1 AND 2 OBTAINED IN CHROMATOGRAPHY OF AQUEOUS LEAN MUSCLE EXTRACT ON BIO-GEL P-2. COMPOSITION OF FRACTIONS

Precursor fraction 1 Traces only: Asparagine Creatine Glutamine Phosphate Serine Taurine Arginine Cysteic acid More than traces: Alanine Camosine Glutamic acid Glycine Methionine Organic phosphates

Precursor fraction 2 More: Asparagine Creatine Glutamine Phosphate Serine Taurine

Alanine Carnosine Glutamic acid Glycine Methionine Organic phosphates

Glucose-6-PO, Unknown sugar phosphate Lactic acid

Glucose Fructose Ribose

Anserine Camitine Choline Histidine Isoleucine/leucine Lysine l-Methylhistidine Proline Valine

Aspartic acid Creatinine Inosinic Acid Phenylalanine Urea

range, many with a high degree of unsaturation. The data for cephalins and lecithins are somewhat difficult to interpret. The linoleic acid content of the cephalin fraction decreases greatly during cooking (Roberts and Campbell, 1970).

B. STUDIES ON MODEL SYSTEMS Few studies have been made to simulate conditions reflecting the situation existing during meat flavor development. Most of these have

MEAT FLAVOR

33

followed studies on flavor precursors. More work has been done with flavor-producing reactions, particularly browning. Hornstein and Crowe (1960) followed up the suggestion that reducing sugars present in muscle might directly react with proteins and in so doing produce meat flavor. Meat flavor did not result from vacuum-pyrolyzing mixtures of glucose with either gelatin or egg albumin. The large quantity of lactic acid present among meat extract components prompted these workers to investigate a function of this acid in meat flavor formation. Aqueous solution of egg albumin (10 g), glucose (2 g) and lactic acid (100mg) were carried through the same procedure that had been used to isolate lean meat flavor as a lyophilized powder. The preparation obtained did have an odor, but it did not resemble cooked meat flavor. Batzer et al. (1960) in their work on precursors isolated a fraction designated Aaz that gave meat flavor when heated. A major constituent of this fraction was a glycoprotein. This was separated and heated in fat together with inosinic acid and glucose. Provided the relative concentration ratios of the reacting substances were kept within closely confined limits, meat flavor reportedly was developed in many trials. However, when the amino acids recovered after hydrolysis of the glycoprotein were separately heated with inosinic acid and glucose, the same aroma was not produced. Hornstein and Crowe (1960, 1964) carried isolation of precursors from raw ground meat through steps of dialysis and ion-exchange separation to obtain two fractions, one reported to contain reducing sugars, the other amino acids. Heating each fraction separately did not yield meaty aromas, but these were produced following recombination of the two fractions prior to heating. Further support for the thought that sugar-amino acids reactions are involved in producing meat-type flavors came from Wood (1961). He prepared a synthetic mixture of the components of fresh muscle extract as determined in an exhaustive earlier study (Bender et al., 1958). Heating an aqueous solution of this mixture with glucose produced a meaty flavor and browning, but neither was observed when the glucose was omitted.

c. STUDIES ON

FORMEDMEAT FLAVOR

1, More Volatile Flavor Compounds An attempt has been made in Table I11 to collect information on volatile compounds present among beef flavor isolates obtained by

34

KARL 0. HERZ AND STEPHEN S. CHANG

Com 1,011 nd

Ref.('

Comporiiid

Ref."

Acid . v

Formic Acetic Propanoic H u tnnoic 2-Methylpropanoic Lactic (various Refs.) A1deli !Ides

(diphatic)

Compound

Ref."

R i n g Co m p o t i 11 (1.y

( 6 - m e m l ~rings) r 1 9 Methanol 9 Etlranol 1,8,9 Benzene 9 2-Propcinol 8 Benzaldehyde 2.6 Hutallol 6.8 Plienylacetnltleliytle 2-Butanol 8 8 N i t r o g c ti 1,9 Pen t;1nol Hexanol 8 c o 1 ) I po II i 1 d S 8 Ammonia H eptanol 8 Methylamine Octanol 8 3-hlethyll~ntant11 s uu;i I' c o 1IlI"' I1 tt da

Form;ildehyde Acetaldehyde Propanal Pentanal IIexanal Heptanal Octanal

Propanone Ace tone Methyl ethyl ketone 2-Hiitanone Acetoin Diacetyl

Nonnnal

Undec~uid Methional?

Hydrogen sulficlc 1,3,9 2,3,4 Methyl merc,iptan 1 1,6,8 Ethyl mercaptai: 8 5,9 Propyl mercaptan Buty l m e rcap t i i n Dimethyl sulfide

8 8 8

3,9 3

1,2,3,7,9 1,2,4,7

1,2,4,7

2 2

1,4,7,9

7 2-Metliylpropmal 3-Methyll1utanal Esters

Methyl formate

2

Butane Pentane Hexane He ptan e Octane

5 Dimethyl disulfide 5 5 5 5

3-Methylbutane

6

8

"References and material from which isolated are as follows: 1. Bender (1961, 1962); Bender and Ballance (1961). Ox meat during boiling for meat extract. 2. Hrenrian and Bernhard (1964).Canned beef in broth. 3. Hornstein et 01. ( 1 9 6 0 ~ Hornstein ; and Crowe, 1960). Lean muscle extract, lyopliilized. 4. Krainlich and Pearson (1960).Boiled beef in broth, nitrogen entrainment. 5. Merritt (1966). Enzyme-inactivated beef in cans (slightly boiled). 6. Sanderson et al. (1966).Ground beef heated in water or fat. 7. Self et u1. (1963b). Boiled beef. 8. Wick (1963, 1965);Wick et (11. (1967). Enzyme-inactivated beef. 9. Yueh and Strong (1960). Boiled beef in slurry.

MEAT FLAVOR

35

different techniques up to 1966. Improvements in instrumental measurement are reflected in the larger number of components observed arid identified in later work. Differences in materials, heating techniques, and analysis are briefly indicated in the footnotes to Table 111. No definite statement is possible on compounds or groups of compounds in this list to tie them to meat flavor. No group of compounds may be omitted from consideration in light of our current knowledge. A few general observations have been made, for example, on development of various flavors during cooking. D. M. Doty (1961) suggested that heat treatments may remove undesirable flavor in its early stages. During the early stages the prominent flavor impression is entirely different from that given by cooked meat. Wasserman and Gray (1965)describe a series of flavor impressions obtained from the water extract of lean beef upon boiling. The cold extract had a serum- or bloodlike odor. At boiling, the aroma was described as brothy and buttery or oleaginous. Constituents remaining when the extract was taken down to dryness and heated further underwent browning and pyrolysis accompanied by emanation of various odors and culminating in an aroma resembling that given off by broiling steak. Macy et al. (1964b) heated lean beef water extracts in a boiling water bath for 1 hr. Brothy flavor was continuously given off, and this flavor increased in strength with time of cooking (cf. Landmann and Batzer, 1966). After the l-hr treatment, analysis showed that there were left sufficient nonvolatile reserves to continue producing flavor compounds. Desirable flavor continues to be given off during heating of either finely divided lean beef slurries (K. 0. Herz, 1968)or flavor precursors extracts (Macy et ul., 1964b). Since diffusion of reactants cannot be a serious deterrent under these conditions, the rate-limiting step(s) causing continuous evolution of volatiles must be in the conversion of precursor to reactant and/or in the flavor-producing reaction itself. The similarity of lean meat flavor preparations obtained from different animal species has been mentioned. There is no evidence that intramuscular fat, without the water leachable substances, can produce meaty flavor in the extracted residue (Wasserman and Gray, 1965; Landmann and Batzer, 1966; Kramlich and Pearson, 1958).The same conclusion could be drawn for the meat-fiber residue (Homstein and Crowe, 1964; Wasserman and Gray, 1965).However, the absence of flavor development does not preclude participation of compounds evolved from water-insoluble meat material during heating. In other words, flavor development may take a different course when lean

36

KARL 0. HERZ A N D STEPHEN S. CHANG

meat is heated than when the extract is heated. This was the view held by Crocker (1948). An influence is possible even from the medium used to heat precursor fractions to produce meat aroma. This was shown by workers at AMIF (D. M. Doty, 1961). Cooking temperatures, dehydration effects, and atmosphere during cooking and eating are other factors that could influence development, type or quantity of volatile compounds. These problems have not been systematically investigated. The suggestion has been made that higher boiling components of meat-flavor isolates, coming off programmed GLC columns at temperatures above 70"C, collectively have more meat flavor than the lower boiling (earlier eluting) components (Hornstein et al., 1963).In recent work, the very highest boiling components, those eluted above 200"C, had a somewhat meaty odor and a mouthfeel that lingered much like cooked meat does after eating (K. 0. Herz, 1968).

2. Nonvolatile Flavor Compoonds Extracts of lean beef muscle contain a large variety of ordinarily nonvolatile compounds exhibiting little flavor. During preparation of commercial meat extract, concentration by evaporation in the presence of air could give rise to compounds different from those encountered in normal desired cooked meat flavor. However, good commercial beef extract has certain flavor notes reminiscent if not coincident with those of cooked meat flavor. Water extracts and commercial extracts of beef muscle have been studied (Bender et al., 1958; Wood and Bender, 1957; Macy et d., 1964b). Another ordinarily nonvolatile fraction of meat muscle, the lipids, also has been examined (Hornstein et al., 1963). Bender and his colleagues (Wood and Bender, 1957; Bender et al., 1958) exhaustively analyzed commercial ox muscle extract. A variety of nitrogenous compounds were isolated and identified accounting, according to these workers, for 82% of the materials in the extract. An unexpected finding was that only 2% (approximately) of the total extract was made up of amino acids, including methyl histidine, serine, methionine, alanine, leucine, isoleucine, histidine, taurine, and citrulline. Other types of compounds present were peptides, guanidines, purines, pyrimidines, camitine, choline, urea, ammonia, organic acids, protein material, and inorganic salts. During industrial preparation of the extract, most components decreased (in absolute quantity per batch) tipon heating, much of the loss presumably escaping as volatile compounds into the atmosphere. Some compounds,

37

MEAT FLAVOR

notably alanine, inosinic acid, and ammonia, were enriched in the finished extract. Reducing sugar was not detectable in the extract. Macy et (iZ. (1964b) analyzed the water extract from fresh beef muscle before and after heating. Calculated to 100 g of tissue, unheated extract contained only 161 mg of amino acids and related compounds, and this total dropped to 70.5 mg after heating. Anserine and carnosine made up 56% of the total in both unheated and heated extract, quantitatively next in order being alanine and taurine. Glucose was the predominant sugar (44 mg/100 g tissue) in the carbohydrate fraction from lyophilized diffusates of fresh beef extract, followed by fructose (3.56 mg) and ribose (1.09 mg). Heating least affected the fructose content, nearly halved the glucose, and reduced ribose to a trace. An increase in phosphoethanolamine content upon heating of beef extract suggested to Macy e t al. (1964b) that phospholipids were being hydrolyzed. Giam et al. (1965) extracted free and bound lipids from raw and cooked freeze-dried beef. Since chloroform-methanol was the extracting solvent for bound lipids, phospholipids would be among the extracted compounds. While differences were evident between free and bound lipids composition of fatty acids, little change was noted between raw beef lipids and cooked beef lipids. The fatty acids composition of meat tissue lipids has been examined by Hornstein et uZ. (1961). Trimmed muscle was extracted with chloroform-methanol, and the extract was fractionated by column chromatography into triglycerides, cephalins, and lecithins + sphingomyelins. Total lipids in 100 g of tissue weighed 4.57 g, the greatest portion of which (3.55 g) was triglyceride in nature. Phospholipids, present to the extent of 1.00 g, were associated with protein. More than 80% of the triglyceride fraction was made up of oleic, palmitic, and stearic acids. These compounds accounted for only 44% in the cephalins, and 54.5%in the lecithins sphingomyelins; in cephalins arachidonic acid predominated (33.30/0),and linoleic acid was high in both phospholipid fractions (16.2and 23.6%). An attempt was made by Hornstein et al. (1961) to assess the aroma of freshly isolated lipid fractions when heated in air. Beef triglycerides evolved an aroma of fried fat. Cephalin vapors smelled fishy, probably due to the high arachidonic acid content. Lecithins sphingomyelins also had the fishy odor present, but it seemed to be superimposed on an aroma suggestive of liver, The conclusion was reached that phospholipids probably do not contribute to desirable meat flavor; in excessively lean meat, they could contribute to poor flavor or off-flavors.

+

+

38

KARL 0. HERZ A N D STEPHEN S. CHANG

Inorganic salts in meat tissue contribute a salty taste which is more evident as a cooked beef slurry or a commercial extract is concentrated. Conceivably, the relative increase in salt concentration could, by a dehydrating mechanism or simply by an increased “salting-out” effect, raise a larger number of flavor molecules to a volatile state under the influence of heat, or induce otherwise less volatile compounds to join the train of aroma compounds in flavor isolation steps.

1). ON THE ORIGIN OF b‘lEAT

FLAVOR COMPOUNDS-EARLY VIEWS

Landmann and Batzer (1966) considered that cooked meat flavor is a complex mixture of volatile and nonvolatile compounds resulting from reactions induced by heat in raw meat flavor precursors, both nonvolatile and volatile. Water-soluble substances in raw meat give rise to the basic meaty flavor, and fat-soluble substances provide upon heating the characteristics associated with the flavors of different species. The entire meat muscle can be considered as possible precursing flavor material. In quantity, protein is highest in the nonaqueous portion of lean muscle, followed by fat, and small quantities of nonprotein nitrogen compounds, carbohydrates, and mineral ash.

1 . Mineruls und Carbohydrates Minerals are only auxiliary as far as odor is concerned; but they contribute a basic salty taste. Cations act as catalysts in many chemical reactions and may perform a catalytic function in enzymes with which they are associated. Their presence could be important in bringing about flavor-producing reactions, for example, by dehydration of protein water hulls to facilitate degradation, or by acting a s foci for reaction in liquids containing precursor material leached from the muscle. The negative and positive ions also may make possible the release of flavor compounds during splitting off from a larger molecule by replacement bonding to the larger fragment. A “salting-out of solution” type of enhancement of volatility of most volatile compounds would result from the large number of ions in the flavor-carrying solution. Very little polymerized carbohydrate is present in meat after aging; most of the glycogen in muscle has been converted to lactic acid, probably the major carbohydrate. Lineweaver (1961) suggested the plausibility of a contribution to cooked chicken meat flavor from carbohydrate. Kazeniac (1961)found lactic acid to be a major constituent of cooked chicken broth, but suggested a possible role in taste, not in

MEAT FLAVOR

39

aroma of cooked meat. Dryden et al. (1970)commented that lactic acid had, if anything, an undesirable effect on muscle palatability. Jones (1961)considered lactate in fish as essentially nonflavorous. He commented on the presence of pyruvic acid, a compound which is highly odoriferous. No references on the effect of pyruvic acid in muscle on flavor have been found, but perhaps this compound so important in metabolism has been reduced to lactate after slaughter. Lactic acid also made up the bulk, some 90%, of the less volatile of two fractions obtained by vacuum pyrolysis and fractionation from lyophilized water extracts of beef, pork, or lamb (Hornstein et ul., 1963). This fraction initially had a pleasant fniitlike aroma which changed on standing to a desirable meaty aroma. These workers did not suggest a role for lactic acid in the aroma of the fraction; they merely reported its predominant presence therein. Glucose, fructose, and ribose are the principal monosaccharides present in muscle; of these, ribose is the least stable to heat, and fructose the most stable (Macy et al., 1964b). Though naturally occurring quantities of pentoses in beef meat are less than 0.1% of wet-weight muscle, ribose in free form actually increases during storage (Fredholm, 1967). This would parallel the situation in fish muscles, where Tarr (1965) has shown that all pentose and pentose formed postmortem originates from nucleotide breakdown rather than through carbohydrate metabolism. Zaika et al. (1968)reported two sugar phosphates present in a fraction isolated from lean beef aqueous extract that gave meaty aroma on heating; lactic acid was also present in this fraction. Self (1967), in discussing potato flavor, suggested that the concentration of sugar may well be more important than that of amino acids for development and character of the cooked potato flavor. 2. Lipids und Cnrbonyls Hornstein et al. (1960; Hornstein and Crowe, 1961) showed that the aroma associated with the species of animal resides in the fat of beef, pork, or lamb. Lamb fat developed a “mutton” aroma when heated a t 100°C in vacuum or in air, but beef fat heated in vacuum had an applelike aroma, in air a deep-fat fried aroma. T h e odors from beef fat are similar to those given, respectively, by freshly prepared and slightly older 2,4-decadienal. Pork fat heated in vacuum smelled like cheese, in air like bacon. Reef had the least species-specific aroma from fat which therefore could come from the protein by degradation of the carbon skeleton, as suggested by Landmann and Batzer (1966).

40

KARL 0. HERZ AND STEPHEN S. CHANG

Studies by Hornstein et .al. (1960; Hornstein and Crowe, 1961) made on depot fat from different animals shed no light on a possible contribution to flavor from intramuscular fat and associated material. In work on cooked chicken volatiles (Minor et al., 1965b),aroma concentrates ceased to smell like chicken when carbonyls were removed. Few or none of these carbonyls may, however, have originated from lipid tissue, since Pippen et a2. (1969)observed that poultry aroma of fat is due to compounds derived from the lean and migrating into the fat during cooking. Generally, lipid degradation has been associated with the development of off-flavors rather than desirable flavor (Watts, 1954; Hornstein et al., 1961). Carbonyl compounds have been found among nearly all meat flavor isolates although their role in desirable meaty flavor is not yet clear. Sanderson et al. (1966) found the same carbonyls were evolved when ground beef was heated in water or in fat, but more carbonyls were evolved from fat-cooked beef. An inverse quantitative relation between butanal and 2-butanone observed by these workers suggested that such differences may be responsible for the characteristic flavors of wet and dry heated beef, respectively. Quantitative differences in aldehydes also may cause differences in flavor character of oxidized fat from different meat animals (Sulzbacher et al., 1963). Descriptions of flavor contributed by pure carbonyl compounds are not uniformly reported by different investigators, and do not include meaty flavors.

3. Protein-Derived and Other Nonnucleic Acid Nitrogen Compounds Since protein is the chief component of meat muscle that is denatured by heat treatment, the compounds formed probably are building units for meat flavor, if not the complete flavor. Dialyzable peptides and amino acids are components of flavor precursor fractions isolated from raw lean meats (Batzer et al., 1960; Macy et al., 1964b) as well as of commercial meat extract (Wood and Bender, 1957; Bender et al.,

1958). The content of amino compounds is rather low, however (160 mg/100 g tissue); some of these are not normally considered degradation products of proteins. The largest proportion of amino compounds in lyophilized difisate from lean beef water extract were anserine, carnosine, and taurine (Macy et al., 1964b). Upon heating and during development of a meaty flavor from a solution of d i h s a t e powder, nearly all amino compounds showed considerable loss, and with evo-

MEAT FLAVOR

41

lution of hydrogen sulfide during cooking the cystine content dropped to zero. This work has recently been extended (Macy et al., 1970). This observation indicates participation of meat fiber protein in flavor formation (cf. Pippen, 1967, on chicken flavor), since H,S evolution continues for long periods during cooking of beef, whereas the cystine in the leachable fraction probably was exhausted before an hour of cooking the difFusate powder solutions. Taurine (P-aminoethanesulfonic acid) in the diffusate was lowered only 55 YOby the heat treatment, and the small quantity of methionine present was lowered by 63%. Taurine may be formed by oxidation of the -SH group of cysteine followed by decarboxylation (Brewster, 1953). Cysteine is not ordinarily isolated from natural protein materials, and its absence in aqueous meat extracts may indicate that oxidative reactions are occurring either before extraction of water-solubles or upon lyophilizing the extract. Even the presence of taurine does, however, not offer a very convincing source for hydrogen sulfide evolution since reduction does not occur during heating of precursor solutions. More than 60% of the d i f i s a t e powder prepared by Macy et al. (1964b) consisted of the histidine dipeptides anserine and carnosine, and of a small quantity of free histidine and methyl histidine. These amino compounds also decreased greatly during heating (except histidine), but their contribution to meaty flavor is not known. Substantial amounts of creatine and creatinine as well as of guanine are present in commercial ox meat extract (Wood and Bender, 1957; Bender et al., 1958).Creatine generally is largely converted to the anhydride creatinine form under the influence of heat. These compounds may contribute to the taste spectrum of flavor, and to mouthfeel and satisfaction, but do not appreciably affect aroma (Zaika et al., 1968). Glutamic acid has enjoyed a reputation as enhancer of meaty flavors. Kazeniac (1961)compared several combinations of amino acids for their effect on mouth satisfaction. Glutamic acid was most effective with lysine, but also with carnosine and arginine. Histidine and glutamic acid had a sharp astringent taste; phenylalanine a sweet taste, and arginine a somewhat meaty taste. Singly, alanine was reported to impart a sweet taste to broth, and taurine a semmy, somewhat astringent taste. Kiely et al. (1960) observed odors formed when amino acids were dissolved in dilute HCI and heated in the presence of isatin-an aromatic imino-diketo compound- to enter into a Strecker degradation yielding an aldehyde having one carbon less than the parent amino acid. No odor was produced by glycine, tryptophan, arginine,

42

KARL 0. HERZ A N D STEPHEN S. CHANG

histidine, lysine, aspartic acid, serine, threonine, and tyrosine. With alanine the odor was malty; with valine, applelike; with leucine, malty; with isoleucine, apple-malty; with proline, reminiscent of mushroom; with phenylalanine, like violets; with cystine, like H,S; with methionine, cheesy-brothy; and with glutamic acid, like bacterial agar. These workers concluded that the Strecker degradation of amino acids may play an important role in flavor formation, particularly cheese flavor. The breakdown of methional, a Strecker degradation product of methionine, was investigated closely by Ballance (1961). Heating with ninhydrin, the chief product was methyl mercaptan, with traces of acrolein and dimethyl sulfide also present. Ballance suggested that methionine from foods such as meat can break down beyond methional [which itself can have a cheesy-brothy flavor (Patton, 1956; Patton and Barnes, 1958)] to the more strongly odorous methyl mercaptan, and so contribute to the volatile sulfur compounds formed on cooking. Destruction of methionine and evolution of H,S appear to go hand in hand in heat-processed meat products (Kagan, 1961).Dimethyl sulfide is produced in small amounts by heating a solution of methionine (Casey et d., 1965). The reaction-degradation products generated when either methionine or cysteine was heated with ribose have been claimed to possess a meatlike aroma (Zoltowska, 1967). Heating the peptide glutathione in water produced an aroma reminiscent of meat (Bouthilet, 1951b),and when the heated material was neutralized using NaOH the mouth taste also was that of meat. Paper chromatography showed that three compounds were present in the mixture arising from heating glutathione. Siedler (1961)reported that little or no loss of methionine, tryptophan, or lysine occurred during conventional cooking. Presumably this observation included the vast reserve of these amino acids present in the muscle fibers, against which the very small amounts leached out, e.g., in the experiments of Macy, et aZ. (1964b) are negligible. Pepper and Pearson (1968) determined sulfur compounds in adipose tissue, particularly kidney fat and trimming (intermuscular and external carcass) fat. Extraction with water, phosphate buffer, or ammonium sulfate solutions gave protein-containing fractions that, upon heating, evolved copious amounts of hydrogen sulfide. The suggestion was advanced that protein in the adipose tissues is a major source of H2S released upon heating, and that this gas comes chiefly from sulfhydryl groups during cooking of meat. Protein-derived compounds were shown to migrate into the fat

MEAT FLAVOR

43

during cooking of poultry meat (Pippen et al., 1969). Sulfur compounds in particular (but including only a very small quantity of H2S) were able to be taken up by the fat and were apparently retained therein to a greater extent than in the aqueous broth. After cooking, the fat had the characteristic aroma of the meat. These workers concluded that fat contributes to poultry aroma indirectly through its ability to dissolve and retain aroma components formed during cooking. Ammonia is one of the few compounds of a volatile nature that is formed in the raw muscle (Dvorak, 1961; Burks, 1960). Although the initial rapid accumulation derives from deamination of nucleic acids, following this there is a slow continuous increase in ammonia resulting from deamination of glutamine and of protein constituents. Ammonia is present in nearly all isolates of cooked meat flavor (see Table 11). Presumably at this point it does not chiefly derive from degradation of nucleic acids but rather from proteinaceous material.

4 . Nucleotides and Their Degradation Products Ever since Batzer et al. (1960)discovered inosinic acid (or inosine plus inorganic phosphate) among the compounds in flavor precursor fractions isolated from raw muscle, and demonstrated in a partly synthetic model system the need for this nucleic acid in flavor development, attention has been turned to the possibility that meat flavor may in part depend on such compounds. Shimazono (1964) claimed that inosine-5‘-monophosphate (IMP) is the most important constituent of meat flavor, adding that such compounds offer a means of control of the flavor. It is with this latter comment that other investigators agree (Caul and Raymond, 1964; Kurtzman and Sjostrom, 1964; Landmann and Batzer, 1966;Woscow, 1966). Caul and Raymond (1964)showed that inosinic acid blended flavor notes, enhancing the total flavor impression in soups. Others at Arthur D. Little (Kurtzman and Sjostrom, 1964) ascribed to sodium inosinate an effect as bodying agent, imparting an “impression of greater viscosity.” Modification of flavor was not always favorable. Spices and treatments giving corned beef hash its character were suppressed. Excessive astringency was noted in tuna and eggs. Slight alterations in the flavor profile were noted, for example, with cottage cheese and baked beans. Atypical brothiness was introduced in milk. But beef and chicken flavor were always enhanced, and sulfury notes in canned meats and bouillon were suppressed. Kurtzman and Sjostrom (1964) also reported that sodium inosinate

44

KARL, 0. HERZ A N D STEPHEN S. CHANG

apparently did not affect the basic tastes in a consistent manner. Woskow (1966), however, found that 1:1 mixtures of disodium inosinate and disodium guanylate consistently enhanced the saltiness of a sodium chloride solution and the sweetness of a sucrose solution. The strongest effects were in quenching the bitterness of a quinine sulfate solution, and also in suppressing sourness in a citric acid solution. Woskow suggested use of 5'-nucleotides to mask off-flavors. In water extracts of beef, 5'-nucleotides are present (Wismer-Pedersen, 1966; Takeda Chem. Industries, 1966)at levels roughly equivalent to those found for amino compounds by Macy et al. (1964b). Wismer-Pedersen (1966) found 210 mg 5'-nucleotides per 100 g raw beef muscle; this level decreased to 160 mg following roasting. Detailed composition of various 5'-nucleotides in beef has been reported (Takeda Chem. Industries, 1966) as shown in Table IV. Dannert and Pearson (1967) reported inosine 5'-monophosphate as the major 5'nucleotide in muscle of beef, pork, or lamb, with lesser amounts of adenosine 5'-monophosphate (AMP) also demonstrated to be present. Aging and storage conditions appeared to affect nucleotide concentration (Dannert and Pearson, 1967; Takeda Chem. Industries, 1966). Attachment of the phosphate moiety to carbon4 of the ribose sugar is imperative for flavoring action of nucleotides (Sakaguchi et d., 1963; Takeda Pharm. Industries, Ltd., 1963). Purine and pyrimidine bases of the nucleotides, the combination of such bases with ribose to give nucleosides, and even nucleotides having the phosphate moiety attached to ribose carbon-:! or c a r b o n 3 have little or no flavoring activity compared to the 5'-nucleotides. IMP is the predominant nucleotide of meat muscle in rigor mortis (Lee and Newbold, 1963),breaking down slowly, by loss of the phosphate, to inosine. In the presence of orthophosphate, inosine further degrades to form hypoxanthine, a phosphorylase mediating attach-

Com~'ollllrl

mg/100 g tissue

"From Takeclii Chcm. Intltistries, 1966.

MEAT FLAVOR

45

nient of inorganic phosphate to ribose at the carbon-1 position. The pathway by which IMP is formed from ATP has been described (Bendall and Davey, 1957), and inosine can also be fomied enzymatically from adenosine (Howard and Miles, 1964). In human metabolism, hypoxanthine is oxidized to uric acid and excreted. A breakdown of h>ypoxanthineby heat has not been reported. Though Wasserman and Gray (1965)isolated from lean beef muscle extract a fraction consisting of hypoxanthine and amino acids (no sugars detected) that produced a meaty aroma on heating, later work b y this group (Zaika et d., 1968) showed sugars to be present in two fractions isolated by mild techniques that produced meaty aroma on heating, and nucleic substances to be absent in the fraction that gave the best meat aroma. The view that nucleotides and their degradation products are to be considered as flavor enhancers rather than as flavorants is now more generally accepted by workers in the field (Macy et d.,1970). One important function of the 5’-nucleotides is believed to be the masking or suppressing of sulfury, fatty, or burnt flavors (Kuninaka, 1967).

VI. NEWER FINDINGS ON AND RELATED TO BEEF FLAVOR Knowledge of heated beef flavor was still very incomplete b y 1966, when the compounds listed in Table 111 (p. 34) were the only known constituents of flavor concentrates from beef cooked various ways. Most of these constituents had also been found in isolates from other foods. Not surprisingly, therefore, the common explanation of heef flavor at that time was that carbonyl compounds together with H,S and perhaps NH,, are the responsible agents, with quantitative relationships detenning the aroma that was indeed not present as a characteristic of any of these compounds individually and that had not been produced by mixing proposed components. The “carl,onyls-H2S-NH:, proposition” was not considered the final word even b y the U.S. Department of Agriculture whose research staff had originated the concept. The USDA supported further research into the nature of beef flavor. A program was initiated late in 1965 at Rutgers University to study the nature of beef flavor and the effect on it of certain processing conditions (freezer storage, freeze drying riiw and cooked beef). Results from this work are given in Section VI, B. The flitvor industry also did not appear to accept the “carbonylsH,S-NH,, proposition,” judging from continuing efforts evident in the patent literature.

46

KARL 0. HERZ AND STEPHEN S. CHANG

A. ARTIFICI4L MEAT FLAVORS -A CLUE? Patent claims for synthetic meat flavors were relatively few in the decade leading up to 1963. Jacobs (1956) claimed that 3-mercaptomethylpropionaldehyde imparts a brothlike flavor. May and Morton (1956) made a flavoring for use in meat products by reacting an aldehyde (mono, di, or P-hydroxy type) with cysteine. May and Akroyd, (1959)reacted liquid wood smoke with cystine and produced a flavoring said to simulate pork. Morton et al. (1960)showed that a meat flavor can be produced by heating pentose sugar with cysteine in excess water. If certain other amino acids are also present, a fuller flavor more like beef can be obtained, whereas ribose and cysteine alone produce a flavor resembling pork. Broderick and Linteris (1964) stated that basic meat-flavor-imparting preparations are obtained by reacting mercaptoacetaldehyde with certain compounds in such a way that at least one acetal-type linkage is formed. Reaction with xylose resulted in a particularly effective meat-flavor compound. Many early workers (e.g., Bouthilet, 1950, 1951a,b)felt strongly that meat flavor was produced either by a sulfiir-containing compound or by the breakdown products of such a compound present in the animal body. The two patents cited seem to lend some substance to such a hypothesis. Formation of hydrogen sulfide from meat during cooking is well known, and reaction products of this compound with the ammonia also released can give rise to odors reminiscent of neither of these two compounds (Kazeniac, 1961). Flavorings available commercially are of fairly good quality for chicken and ham imitations, but less so for beef flavor. Such beef flavor preparations apparently are composed chiefly of hydrolyzed vegetable and/or animal protein matter or yeast extract. They presumably do resemble somewhat the flavor profile deriving from the nonvolatile substances, but a lack of volatiles is immediately observed upon opening a sample bottle that has been closed for some time. A number of different flavors are produced when amino acids and sugars are reacted in a Maillard-type reaction. Anson (1962) claimed that b y correct choice of the browning partners, a meatlike flavor can be produced. He suggested that if meat flavor is not wholly d u e to such reaction products, it is so at least to a considerable extent. Harking back to Bouthilet’s finding (1951b) that glutathione can produce meaty aroma on heating, the patent by Kleffer (1968) claims the composition produced by heating cysteine and glutathione with pentose or hexose in concentrated solution at about 90°C as a meat flavoring agent. Addition of lactate or nucleotide enhances the flavoring quality.

MEAT FLAVOR

47

Fat is the important focal point in two recent patents. Hoersch (1967) claims a synthetic meat flavor produced by reacting a mixture of sodium nitrite and seven amino acids (asparagine, lysine, histidine, glutamic acid, valine, proline, and cysteine-HCI) at pH 6 and 120°C carried out under 2 layers of oil. The oil is presumed to hold volatiles produced in the reaction and to prevent or limit access of oxygen. Fat is said to be the material that is responsible for specifically meaty flavor in another patent (Kyowa Hakko Kogyo Co., Ltd., 1968).When amino acids, glucose, and phosphoric acid are heated, delicious food aroma is produced, but it is not meaty. If triglyceride fat or fatty acids are present, the flavor produced is that of good cooked meat. The same result can be obtained by heating a small quantity of thiamine with fat, and if to this one adds amino acids, hexose, and phosphoric acid, the flavor of good cooked meat is produced. Thiamine is rapidly emerging as one of the likely precursors of meat aroma. Three patents were issued to International Flavors and Fragrances Inc. (IFF)in 1968that involve the use of thiamine. To produce a beef flavor, Giacino ( 1968a)heated carbohydrate-free protein hydrolyzate and a 1 : l weight ratio of cysteine (or cystine, or sodium sulfide) and thiamine HCI. For a sweetish note, p-alanine is added to the reaction mixture. Pyruvic acid or pyruvic aldehyde is added if a roasted note is desired. For pork flavor, methionine would replace cysteine, and for chicken flavor, thiamine concentration would be one fifth of that used for beef and p-alanine would be present. The reaction proceeds during refluxing of aqueous mixtures of the necessary componen t s . The second IFF patent (Giacino, 1968b) calls for reacting thiamine first with an S-containing polypeptide (or the amino acid mix derived from it by hydrolysis). At some stage, ketones (preferably diacetyl, acetylmethylcarbinol, acetylpropionyl) and aldehydes (preferably pentanal, hexanal, heptanal) are added. Heating is done preferably in triglyceride fat. p-Alanine and nucleotides may be included in the reaction mixture, and when glutathione was used as the peptide with thiamine HCI, a good chicken flavor was produced. The third IFF patent (Bidmead et al., 1968) adds carboxylic acids (succinic, adipic, lactic, acetic, propionic, oxalic, tartaric, or malonic) again preferably to cysteine-HC1-thiamine-HCI and carbohydrate-free protein hydrolyzate. The result is said to be a roasted meat flavor. Interesting is the disclosure in some of the later claims that other sulfur compounds, especially 3-acetyl-3-mercaptopropanol-1, can be used to replace thiamine. Concerning the fate of thiamine in meat, Kiernat et (12. (1964) showed that loss under standard cooking procedures is in the range of

48

KARL 0. HERZ A N D STEPHEN S. CHANC

10 to 15% of an already low content. Heat degradation products of thiamine include several compounds (Arnold, 1968) similar to some reported in the following section among the constituents of boiled beef flavor concentrates. B.

BEEF FLAVORISOLATE CONSTITUENTS - NEW FINDINGS

Although the investigations on meat flavor begun in 1965at Rutgers University are continuing, certain information has been developed to date that can help to put our knowledge into a new light. Most of the work completed deals with fresh boiled beef and with freeze-dried boiled beef. No evidence has been brought to light (K.0. Herz, 1968) that freeze-drying causes a substantial quantitative or qualitative change in the gas chromatographic profile of flavor concentrates prepared identically from fresh boiled beef and from beef that has been freeze-dried either before or after boiling. The presence of over 100 compounds in a fresh boiled-beef flavor concentrate was established in gas chromatographic work, and fractionation on a suitable capillary column would probably show the presence of many additional components. Approximately 50 compounds in the flavor concentrate have been identified and reported (K. 0. Herz, 1968; Hirai et al., 1968; Chang et d.,1968). Those compounds identified in this work and not before reported in the literature (i.e., not in Table 111)are listed in Table V. The three sulfur-containing heterogeneous ring compounds had aromas that were consistently described as meaty, though not like the boiled-meat aroma in the total flavor concentrate; the aroma of the nitrogen-containing compound (an oxazoline) was closer to that of boiled beef. All originated from gas chromatographic fractions that had a decided meaty aroma. Work in The Netherlands (Tonsbeek et al., 1968)recently led to the identification of two additional heterogeneous five-member ring compounds and several fatty acids not previously reported: 4-Hydroxy-5-methyl-2-dihydrofuran-3-one 4-Hydroxy-2,5-dimethylfuran-3-one Pentanoic acid (valeric acid) 2-Methylpentanoic acid (isocaproic acid) These compounds were present in a flavor isolate from clear beef broth. Note should be taken here of another aspect of the work (K.0. Herz, 1968) done at Rutgers University: the nature of the aroma evolved from meat during cooking. Gas chromatography of concentrates pre-

TABLE V PREVIOUSLY ~INREPORTEI)~ : O h I P O UNl ) S IN FLA\’OR ~ S O l . A T E SFHOhI 1jO11.El) BEEF‘’

Conipountl

Identificationb

Aricls Hexanoic

Coin pound /h%(’il1’

P

r i /deh!/dcy

Identificationb

~~’~l/fl/i~Jl~ll~/.Y

Tolricne Propyll~ellzene

Paradiclilorobenzene 3-Xletl~yll~enz~~ldel~ycl~

(diphiitic) Ht.xadecand

P

2-Octenal

11

fi-Metliylhepten-l-;II

T

P P P T

S ti l f i i r C o r i i Iwrc tit1.s (diphatic) Slethylpropyl sulfide Methylallyl sdfide Diiillyl sulfide

T T T

Esters I A l C t O t1e,5

Ethyl acetate

P y-Vu1erol;ictone

P

E t I1 1, T.Y Hetcrogerieorts R i n g Cottt-

Pentyl ether

H

rilcol~0l.s

I)oiitids (0,S,N)

2-llethyl tetrahydrofitran-3-011e

P

Propanol

P

5-Thioirieth yl furfural

rr

2-Metliylprop~inoI

P

2-Pen ty 1furan

P

1-I’entene-3-01 2- H exenol 1-Octene-3-01

P T P

Thiol~lienci~rl~oxy-2-aldehytleliy~le

1’

2,S-Diinethyl-1,3,4-tritI~iol~iii

1’

2,4,S-Trimethylox~1zoline

P

Ketot1e.v

4-Octanone 3-Non;inone 3-l>odecanone

H €1

H

If ydroctirlx)11s Dodecane Pen tadecane Hexadecane Octadecane

P P P P

Uiidecene- 1 Pen tadecene- 1

P P

“Herz, 1968; Hirai et al., 1968; Chang et u l . , 1968. *Identification P = positive; T = tentative from IR or MS spectra; H = homologous series plot of compounds whose functional groups are known from IR spectra.

50

KARL 0. HERZ AND STEPHEN S. CHANG

pared from the material trapped with the vapors leaving the boiling vessel showed numerous.peaks including some that were coincident with peaks of the beef flavor concentrate. The large size of many of the peaks support the conclusion that a large quantity of volatile material is lost during cooking of meat, and, except at the very earliest stage of cooking, the volatiles lost include desirable beef aroma compounds. The smell of the concentrate was distinctly that of beef flavor, but with a harsh note superimposed thereon. Future work on identification of components of flavor concentrates from material collected during cooking of meat may yield information on the compounds formed when heating is first begun. If these largely undesirable compounds can be separated from those carrying the desirable meaty aroma- or can be discarded before collecting volatiles - the material ejected into the atmosphere could be an important industrial source of cooked meat flavor preparations. Large quantities could be obtained in commercial preparation of TV dinners and of institutional or military precooked meats. The origin of many of the compounds so far reported in beef flavor isolates will be discussed in the following section. Two compounds listed in Table V do not seem to be part of the beef flavor profile: 2,6di-tert-butyl-paracresol (commonly referred to as BHT) and paradichlorobenzene. It can only be surmised at this time that the antioxidant is an unmetabolized residue absorbed from the feed, and that the larvacide is a similar residue possibly arising from application to the carcass (Natl. Acad. Sci., 1959). Experiments with chickens fed BHTsupplemented fish fat (Liljemark, 1965) led to an accumulation of 0.04% BHT in the subcutaneous fat of the chickens. Paradichlorobenzene could possibly be a partially metabolized residue of such widely used herbicides as 2,4,5-trichlorophenoxyacetic acid, or of such insecticides as benzene hexachloride. Arnold et al. (1966) found a dichlorobenzene in the odor concentrate from sterilized concentrated milk; no explanation for the presence of the compound was offered. This finding of compounds in muscle that are present as a result of absorption again raises the question of the influence of feed components on the quality (composition) of meat flavor. Beef does not taste like pork, lamb, or chicken. Ruminant digestion and its main units (volatile fatty acids, chiefly propanoic) differs from that of nonruminants. Though skeletal muscle protein composition is rather invariant among meat animal species, certain branched-chain fatty acids are characteristic of ruminant fats alone (Hubbard and Pocklington, 1968). T h e conversion of phytol into fatty acids is held responsible for the

MEAT FLAVOR

51

presence of these acids in ruminant fat. Since most of the digestion of ruminants is carried on by the organisms in the rumen, number, kind, and activity of each participant will be a factor in digestion of what food and to what absorbable unit. A gross examination (Harris et al., 1968)of the role of bacteria in the digestive tract of chickens has made a similar point. Meat from conventionally reared chickens had a stronger and more characteristic flavor than that from germ-free chickens. The authors conclude that certain flavor compounds that are metabolites of bacteria in the intestine can be absorbed and carried to the muscle while the bird is still ‘11’ive.

VII. ON THE ORIGIN OF MEAT FLAVOR COMPOUNDSCURRENT VIEWS

The presumed origin of many of the compounds listed in Table I11 - those reported up to 1966- has been discussed in Section V. The evidence has been either too scant or too general and not sufficiently conclusive to lead to an understanding of the reactions responsible for the development of meat aroma compounds. The recent patent literature (Section VI,A) and the further findings of constituents of beef flavor concentrates (Table V) have given the first evidence that we may be coming closer to an understanding of the compounds and reactions leading to meat flavor, and that the previous concept of meat flavor is in all probability erroneous. Much still remains to be learned concerning the reactions of compounds in muscle that give rise to the compounds present in beef flavor concentrates from cooked meat. The finding of gas chromatographic fractions that have definite meaty aroma has shifted the emphasis in meat flavor research to looking for compounds that generate the aroma. This section will very briefly cover the classes of compounds that - while they may contribute to the general background against which the meaty notes stand out-are not believed to contribute directly to desirable meat aroma. Compounds believed to be involved in providing meaty flavor notes will be discussed in greater detail as to origin and reactions. These compounds are chiefly sulfur or nitrogen-containing ring compounds. A. NONMEATYCOMPOUNDS IN BEEF FLAVORCONCENTRATES The compounds in beef flavor concentrates that are presently thought not to contribute to desirable meaty aroma include hydrocar-

52

KARL 0. HERZ AND STEPHEN S. CHANG

bons, acids, alcohols, esters, an ether, aldehydes, and ketones. Since many of these compounds are volatile and have specific aroma, they may influence meat flavor perception without directly affecting the quality of the meat flavor.

1. Acids, Esters, Ether Straight-chain acids can be presumed to be present in muscle d u e to anabolic or catabolic metabolism chiefly of lipids. High molecular weight fatty acids are not particularly volatile and will not be found in meat flavor concentration that depends on volatilization. Methyl formate and ethyl acetate, as well as the fatty acids found, would not contribute to desirable meat aroma. The pentyl ether identified is virtually odorless. Whether the esters and the ether found in concentrates are present as such in the flavor profile of meat is not known. A number of compounds that contain methyl as a branch on the carbon skeleton have been found in meat flavor isolates (2-methylpropanoic acid; 2-methylpropanol; 2-methylpropanal; 3-methylbutanol, 3-methylbutanal; and 6-methylheptenal). Though the methyl group confers greater volatility on the compound compared to the straightchain compound with an equal number of carbons, none in the group has meaty aroma. These compounds may be fragments of protein, lipid, carbohydrate, or nucleic acid degradation, or they may represent undegraded (or perhaps deaminated) synthesis units accumulated in the cells for construction of tissue.

2. Hydrocurbons

The saturated compounds in this class have virtually no odors and the odors of unsaturated compounds are neither agreeable nor meaty (Merritt, 1966). Some hydrocarbons have been found in the raw meat, and this class of compounds increases greatly during irradiation of beef and especially of beef fatty tissue (Merritt e t al., 1967; Wick e t al., 1967). Lipids are thought to be the origin (or possibly the destination?) of the hydrocarbons in meat. 3. Alcohols Ethanol and propanol are present in cooked beef in considerable quantity. These may be adjuncts of in vivo metabolism, as they occur

MEAT FLAVOR

53

in many reactions known to take place in tissue. Other alcohols reported are present in small quantity with one exception; they include unsaturated 5- and 6-carbon alcohols. T h e origin of these compounds is not clear. The boiled-beef flavor concentrate of K. 0. Herz (1968)contained 1octene-3-01 in fair quantity. This compound has a decided mushroom odor when eluted from the GLC column. Stevens et al. (1967)thought that the rich beany aroma of canned Blue Lake beans was due chiefly to certain concentrations of cis-hex-3-ene-1-01 and l-octene-3-01, 1Octene-3-01 has been reported as a major constituent of volatiles in corn oil decomposition products during fat frying of foods (Krishnamurthy and Chang, 1967) and in reverted soybean oil volatiles (Smouse and Chang, 1967). Mechanisms of its formation have been discussed by Smouse and Chang (1967).

4 . Carbonyl Compounds The most numerous members of any class identified in meat flavor concentrates are the carbonyl compounds. Hexanal is quantitatively a major component (Wick, 1963; K. 0. Herz, 1968).Heptanal is present in volatiles from enzyme-inactivated beef (Wick et al., 1967)and both heptanal and hexadecanal were found in boiled beef flavor concentrate (K. 0. Herz, 1968). The unsaturated 2-octenal and the branched unsaturated 6-methyl-2-heptenal were present in boiled beef flavor (K. 0. Herz, 1968);a branched heptanal was tentatively reported in boiled chicken volatiles by Nonaka et al. (1967). Five 3- or 4-carbon ketones were reported in earlier work plus acetoin (Table 111), and three higher ketones were found subsequently (Cs, Cg,C,,,,Table V). Foul ketones of 8,9,or 10 carbon atoms were also identified in chicken volatiles by Nonaka e t al. (1967). Lipids suggest themselves as a likely origin of aldehydes and ketones, based on many studies of oxidative and thermal degradation. Possible reaction mechanisms in vegetable oil have been discussed b y Chang and colleagues (Chang et u l , , 1961; Mookherjee and Chang, 1963; Silveira e t al., 1965; Smouse, 1965; Kawada et d . ,1966, 1967; Krishnamurthy and Chang, 1967; Smouse and Chang, 1967).There is some question whether the same kinds of reactions can take place in the environment represented by the much lower lipid content of lean muscle. Carbonyl compounds containing up to four carbons may, however, have originated with sugars (Casey et d., 1965) or amino compounds

54

KARL 0. HERZ AND STEPHEN S. CHANG

via Maillard-type reactions (Hodge, 1953; Burton and McWeeny, 1964). The large pool of lactic acid known to be present is another possible source of 3-carbon carbonyl compounds.

5 . Benzene Ring Compounds Of the nine benzene compounds so far identified in beef flavor concentrates, one could be an insecticide residue and three are aromatic hydrocarbons not expected to contribute to desirable aroma. Phenylacetaldehyde has a floral scent (violets). Benzaldehyde, present in considerable quantity, represents the aroma of almonds. The presence of compounds of this type is not unexpected because several amino acids (phenylalanine, tyrosine, tryptophan) have the benzene ring as part of their structure. €3. CLASSESOF COMPOUNDS LIKELYTO INFLUENCEMEAT FLAVOR

Three types of compounds may offer a more direct contribution to the meat flavor profile, though none of the representatives found in flavor isolates has characteristic meaty aroma: lactones; furan ring compounds that do not contain sulfur; and aliphatic sulfur compounds.

1 . Lnctones Only y-valerolactone has been found in beef flavor isolates (K. 0. Herz, 1968).The compound does not have a meaty aroma; it is used by perfume makers to accent floral bouquet. But lactones have been shown to cover a wide spectrum of flavors (Fioriti et d., 1967) and ylactones have been found in milk fat and peach flavor isolates. Lactones may possibly originate from lipid metabolism or degradation, but they can also be formed from protein fragments such as a-ketoglutaric acid or in the degradation of sugar molecules. A compound identified as a-hydroxy-P-methyl-y-hexenolactone has been confirmed as the bouillon-like flavoring principle developed upon aging of a-ketobutyric acid (Sulser et al., 1967). a-Ketobutyric acid had previously been considered as the flavoring principle of vegetable protein hydrolyzates, but the finding of its spontaneous and acid-catalyzed reaction to form the flavoring compound by dimerization, lactonization, and decarboxylation r,roves the absence of the flavor note in the original &carbon acid. The base-catalyzed degradation of fructose by heat (Shaw et al.,

MEAT FLAVOR

55

1968)yielded among the reaction products a minor amount of y-butyrolactone and more substantial quantities of l-substituted cyclohexenolone and 4-substituted cyclopentenolones. The 2-hydroxy-3-methyl2-cyclopenten-l-one among these compounds has been found in flavor isolates from maple syrup (Filipic et al., 1965) and stored orange powder (Tatum et aZ., 1967). 2. F u m n Ring Compounds As research on the flavor of heated foods proceeds, furan compounds are being discovered in flavor isolates particularly from roasted or dry heated material such as coffee and cacao. Furan conipounds had not been reported in beef flavor isolates until the work of K. 0. Herz (1968). Different furan compounds that d o not contain sulfur have a wide variety of aromas, none of them meaty. Furan compounds that contain sulfur in the side chain will be discussed with 5member ring compounds. One of the two furan compounds listed in Table V, 2-pentylfuran, has a licorice flavor when eluted from the gas chromatographic column or when in concentrated solution; but in vegetable oil at concentrations in the range 1-10 ppm, it imparts a beany and grassy flavor (Smouse and Chang, 1967). This compound had heen found previously in heated corn oil (Krishnamurthy, 1966) and in reverted soybean oil (Smouse, 1965). Chung et (11. (1966) believed that this compound was responsible for the characteristic reversion flavor. Nonaka et (11. (1967) found 2-pentylfiiran in chicken flavor and suggested that it may be an oxidation product of 2,4-decadienal. 2-Pentylf11ran is present in moderate amount in the boiled beef flavor isolate (K. 0. Herz, 1968). Its flavor effect i n the aqueous environment of \,oiled beef may warrant further study. Another compound, having a saturated furan ring, was present in small amount. It had the odor of onion when eluted from the GLC column. The coinpound, 2-rnetliyltetrahydrof~ira1i-3-one,h a s previously been reported in roasted coffee aroma isolates (Cianturco c t ( i l . , 1966; Gautschi et ul., 1968). Recently it also was shown to be present in C O ~ O Rvolatiles (Van Praag et (il., 1968),together with the fi.:tpiien t 2-niethyltetrahydrofiiran. Of the two furanones isolated recently from beef broth (Tonsheek e t ul., 1968), the 4-liydroxy-2,5-cli1iietliyl-2-dihyclrofuran-2-one hud ii caramel odor, and the 4-hydroxy-5-metliyl-2-diliydrof~1ran-3-nne had a roasted chicory root odor. Other workers (Shaw et (/l., 1068) clescrihecl the dimethyl compound as having a sweet cotton-candy type of odor.

56

KARL 0. HERZ AND STEPHEN S. CHANC

Tonsbeek et (11. (1968)cite laboratory preparation of the monomethyl homolog by reaction of pentose and primary amine (Severin and Seilmeier, 1967). Shaw et a1. (1968)found the dimethyl homolog among the products of base-catalyzed fructose degradation. Gautschi et al. (1968) suggested that the furan compounds in coffee volatiles originate from sucrose. A 2-methyl 3-furanone was among the compounds isolated from roasted cocoa bean volatiles (Van Praag et al., 1968). A recent compilation of the volatiles known to be present in roasted coffee (Stoffelsma et al., 1968) lists 28 furan compounds that do not contain sulfur.

3. Hydrogen Sulfide and Straight-Chain Sulfur Compounds Aliphatic sulfides and disulfides are major flavor components of onion and garlic (Carson, 1967; Bernhard, 1968),and such compounds along with mercaptans have also been found in pineapple (Silverstein, 1967), coffee (Gautschi et al., 1968; Stoffelsma et al., 1968), tea (Kiribuchi and Yamanishi, 1963), cheeses (Day, 1967), potatoes (Self et al., 196313;Gumbmann and Burr, 1964), and cabbage (MacLeod and MacLeod, 1968). Identified in beef flavor isolates (Tables I11 and V) were hydrogen sulfide; the methyl, ethyl, propyl, and butyl mercaptans; the dimethyl, methylpropyl, methylallyl, and diallyl sulfides; and dimethyl disulfide. Chicken flavor isolates (Minor et al., 196513)were said to contain methyl, ethyl, propyl, and hexyl mercaptan; dimethyl, methyl ethyl, diethyl, methylisopropyl, and dipropyl sulfide; and methyl disulfide. All of these aliphatic sulfur compounds have strong odors and are generally perceived at very low concentration, but they do not carry meaty aroma. Pippen (1967) suggested that the presence in poultry volatiles of thiols and sulfides linked to alkyl groups up to hexyl is an indication that sulfur-containing amino acids break down by a mechanism more complex than the Strecker degradation. Earlier work by his group (Mecchi et al., 1964) had shown that cystine and cysteine residues in muscle protein are the chief precursors of hydrogen sulfide evolved when chicken is cooked. Glutathione at the amount found in muscle caused H,S production of less than 10% of that given by whole muscle, and isolated leg protein was also less efficient as H,S producer (80%)).A most interesting finding was that a nonprotein isolate from leg muscle began to produce H,S only after 2% hr of boiling. In very recent work (Pippen et al., 1969) the role of H,S in chicken flavor was examined more closely. The threshold value of 10 ppb in

MEAT FLAVOR

57

water is greatly exceeded by the 35 ppb found in fresh chicken broth and by the 180-730 ppb in fresh cooked chicken. Hence, hydrogen sulfide should contribute directly to the flavor profile. An indirect role was also discovered. H2S reacted with acetaldehyde in hot chicken fat to form one or more sulfur compounds that imparted a sauerkrautlike aroma to the fat (Pippen and Mecchi, 1969). The high reactivity of H,S with mesityl oxide -a compound formed from acetone in presence of a dehydrating agent-was demonstrated by Aylward et al. (1967). Mesityl oxide reacts with H,S even at room temperature and in dilute aqueous solution to form a vile aroma due to among other compounds, 4-methyl-4-mercaptopenta-2-one; the compound has the offensive and pervasive smell of the urine of a tomcat. The odor was produced when a number of vegetables were heated together and a very small quantity of mesityl oxide was added. Gumbmann and Burr (1964), in connection with their work on potato volatiles, postulated pathways for production of such compounds as dimethyl disulfide and branched sulfides. The degradation of the sulfur-containing body substances thiamine, biotin, CoA, and glutathione gives simple sulfur compounds. Methionine produces chiefly methyl mercaptan. Because of the very low flavor threshold of most simple sulfur compounds, these workers believed that such compounds are a secondary flavoring characteristic for many foods in which they are produced by cooking. They also pointed out again the well-known effect of concentration particularly of these compounds on the quality of the flavor perceived. Concerning the flavor compounds of cabbage, which include sulfur compounds as well as thiocyanates, MacLeod and MacLeod (1968)revived the earlier view that proportions of the various compounds determine the perceived flavor, but offered the addendum: together with small quantities of more specific flavoring compounds probably of higher boiling point. Much work has been done on the development of flavor in garlic and onion upon disruption of the tissue (cf. Carson, 1967). This has been shown to be an enzymatic reaction resulting in formation of alkyl di- and trisulfides that carry characteristic aroma: chiefly ally1 in garlic and methyl and propyl in onion. Perhaps the most interesting finding from this work that may apply to meat flavor is the observation that thiamine acts as a trapping agent for thiosulfonates; upon reaction with cysteine, thiamine is regenerated and alkylthiocysteines are formed. To our knowledge, enzymes such as have been found in garlic and onion have not been reported in beef or other meats, but then again, have they been looked for? Because slow heating occurs during cooking of piece meats, there is ample time and juice substrate for

58

KARL 0. HERZ AND STEPHEN S. CHANC

enzymatic reaction; but other than proteolysis of muscle, this area seems not to have been investigated. We d o know of a host of enzymes active in vivo, and there seems to be no particularly good reason why some of these might not be active for some time during heating of meat. Recently, Schwimmer and Guadagni (1967)reported odor intensification of onion and garlic products (dehydrated, juice, suspensions) brought on by addition of cysteine. These workers referred to the enzymatic reaction leading to thiosulfinates and thiosulfonates and presumed that the role of cysteine is to interact with these compounds to produce stable disulfides, including some that carry characteristic aroma.

c. COMPOUNDS

FROM

MEATY AROMA GC FRACTIONS

Only a few compounds that have meaty aroma have been found in the recent work of the group of Chang at Rutgers University; all of these originated from GC fractions that gave off a decidedly meaty odor, but contained other substances along with the one identified in each case. One thing the four compounds isolated from the meaty GC fractions have in common is the 5-member heterogeneous ring structure, the hetero atom being 0, S, or N. The structure of the four compounds is shown below. We venture to suggest that efficiently produced meat flavor isolates will be found to contain a number of compounds similar to those identified and discussed here. H CH,S

c=o

5- Thiomethylfurfural

H Thiophencarboxy2 -aldehyde

Me Me

Me 3,5-Dimethyl1,2,4-trithiolane

2,4,5-Trimethyl3-oxazoline

1 . 5-Thinniethylfurfural A small GC peak that had a meaty aroma (though not that of the boiled beef isolate) was identified as 5-thiomethylfurfural (K. 0. Herz,

MEAT FLAVOR

59

1968; Hirai et ul., 1968). The compound is the S-analog of 5-hydroxyniethylfurfural, a well-known terminal Maillard reaction product. 5Thiomethylfurfural may possibly be derived from methionine.

2. Thiopheiicurboxy-2-aldehyde One very small GC peak, with a spicy meat odor, is the sulfur conipound analogous to furfural, namely, thiophencarboxy-2-aldehyde. The compound could possibly originate solely from amino acids, but may also be formed in Maillard-type reactions between amino acids and sugars. A thiophene compound, 2-niethylthiophene, has been reported in chicken flavor (Nonaka et ul., 1967), and thiophene compounds have also been found in coffee volatiles (Gianturco et u l , , 1966; Gautschi et u l . , 1968; Stoffelsma et al., 1968).

3. 2,5-Diniethyl-l,3,4,-trithiolune In greatest abundance among the 5-membered ring compounds that contain sulfur was 2,5-dimethyI-1,3,4-trithiolane (also known as 3,5dimethyl-l,2,4-trithiolane). This compound has a sulfide odor in concentrated form, but when dilute it gives a meaty aroma. It may well be an important contributor to the aroma of boiled beef. A dithiolane compound, lipoic acid, is a metabolic compound of muscle, indicating that such compounds can form in muscle substrate. Structurally, the trithiolane compound found is reminiscent of decarboxylated and deaminated cystine, with only another S atom needed to close the ring. With the exception of thiolane-2-one reported in coffee volatiles (Stoffelsma et ul., 1968),thiolane compounds have not been found in studies of food flavors. Asinger et a l . (1959; Asinger and Thiel, 1958) have described the synthesis of 3,5-diniethyl-1,2,4-trithiolane from acetone, hydrogen sulfide, and elemental sulfur in diisobutylamine solvent. Conditions simulating these experimentally produced ones could exist locally during boiling of beef, where evolution of both hydrogen sulfide and ammonia occurs. The role of the elemental sulfur could be played b y the sulfur or the thiomethyl group of active methionine (adenosylmethionine), and acetone has been shown to he generated from a number of compounds in heated meat. The case of multisulhr ring cornpounds as food amnia bearers was made stronger recently by the identification of such a compound a s the major flavor i n Shiitake mushroom (Wada et ml., 1967). The authors gave to this compound the trivial name of lenthionine and repre-

60

KARL 0. HERZ AND STEPHEN S. CHANG

sented its structure as 2,4-dimethyl-1,2,5,6,7-pentathiolane. They offered no explanation for its natural formation, but synthesized the compound by reaction of sodium polysulfide with either formaldehyde or methylene chloride. 4 . 2,4,5-Trimethyloxazoline

This finding of an oxazoline is the first report of a nitrogen compound in meat flavor more complex than ammonia or methylamine, and it is the first of a 5-member ring that has two hetero atoms, N and 0. Its aroma is not very close to that of the boiled beef flavor isolate but the aroma of the broad GC fraction from which it originated was. The origin of this compound is not clear; it could be derived solely from amino acids, or it may be formed in Maillard-type reactions. T h e top ring of biotin, as conventionally represented, has the 0 and N atoms in positions similar to those found in the oxazoline, as do also some of the nucleic acid bases. Reports of oxazolines in the literature of food flavor have not been found. The compound 5-acetyl-2-methyloxazole was shown to be present in coffee volatiles (Stoffelsma et al., 1968). D. OTHER NITROGEN RING COMPOUNDSFOUNDIN FOODS The presence of the oxazoline suggests that other nitrogen ring compounds may be formed in heating of meat. The odor of pyridine was among those detected when sniffing the exhaust from the GC instrument during tests on boiled beef flavor isolates. This compound has not been identified. Pyridine has been said to give the Romano variety of canned beans its characteristic harsh flavor (Stevens et al., 1967). Pyrazines are another class of compounds that could be present in meat flavor, but have not so far been demonstrated therein. Deck et (11. (1965)isolated from potato chips a pyrazine compound that provided potato flavor. Pyrazines have been found in roasted coffee volatiles (Gianturco, 1967; Stoffelsma et al., 1968) and in cocoa volatiles (Van Praag et al., 1968). Methyl-substituted pyrazines are formed readily in heating neutral amino acids with fructose or with a variety of the small-molecule fractions obtained in Maillard-type reactions (Van Praag et nl., 1968).Amino acids are not considered essential to formation of pyrazines; ammonia is sufficient. A patent for pyrazine derivatives (Firmenich and Cie., 1964) to be used as flavoring to provide roasted hazelnut, peanut, or almond flavor to foods cites a method

MEAT FLAVOR

61

that leads to production of 2-methyl pyrazines having a mercaptomethyl substituent on carbon 3,5, or 6. A third class of compounds found in coffee (Stoffelsma et al., 1968) and cocoa (Van Praag et al., 1968)volatiles are the pyrrole compounds. Recent studies of the browning reaction between D-xylose and amino compounds (Kato and Fujimaki, 1968) have shown that pyrrole-2-aldehydes are formed with either amino acids or primary amines. The compounds formed were said to be rather stable and not to be intermediates in steps to malanoidin production.

E. MAILLARD BROWNINGI N FOODS - A DIGRESSION Several investigators believed that meaty flavor does not form from a single type of muscle substance, such as protein, or lipid, or nucleotides, but that molecules derived from different substances react to produce flavor compounds. Since flavor appears to be formed predominantly in the juices or broth (Landmann and Batzer, 1966), contact among different species of molecules presents no problem. Even browning reactions, known to occur best when little or no water is present, may be facilitated b y having the reactants first in solution or dispersion. Several workers have concluded that meat flavor development is chiefly a by-product (or product) of a Maillard-type browning reaction involving amino acids and sugars (Batzer et al., 1960; Wood, 1961; Jacobson and Koehler, 1963; Macy et ul., 1964b, 1970; Hornstein, 1967). Study of the reactions leading to browning in foods, with emphasis on reactions between aldose and amino compounds, has been considerable (Burton and McWeeny, 1963, 1964; Hodge, 1953,1967; Anet, 1964; Reynolds, 1963, 1965; Song and Chichester, 1966, 1967a,b; McWeeney et al., 1970; Rothe and Voigt, 1963). In the scheme of Hodge (1953), small carbonyl and amino compounds condense to give melanoidins. A scheme in which no fragments smaller than 6 carbons are produced (Reynolds, 1965) leads to furhral and was considered the main route to melanoidins. Burton and McWeeny (1964) concluded that melanoidins must be formed by several routes, that simple aldosyl-amine compounds decompose even on moderate heating forming carbonyl compounds that react readily with glycine to yield melanoidins. Montgomery and Day (1965), using model systems, considered that carbonyl condensation and eventual pigment formation was mediated by amino compounds such as glycine, but that the pigments actually did not contain ni-

62

KARL 0. HERZ AND STEPHEN S. CHANG

trogen (glycine is regenerated in the scheme they propose) and that alk-2-ends were present. Aromas formed when 1:1 amino acid:glucose mixtures are heated at 100”and 180°C have been studied by Barnes et al. (1947), Herz and Shallenberger (1960),and Kiely et al. (1960).A summary of the aromas have been provided by Hodge (1967).T h e cysteine-HC1:glucose mixture was the only one reported (Kiely et al., 1960) to give a “meaty” aroma. Aroma and browning in systems of pentose and amino acids have also been studied. Taurine-ribose solutions produced the greatest browning on heating various amino acids in phosphate buffer with either glucose or ribose (Saisithi and Dollar, 1966). Rothe and Voigt (1963) concluded from an in.tensive study that certain amino acids heated with xylose tend more toward producing browning rather than flavor compounds (measured as volatile aldehydes), whereas the reverse was the case with other amino acids. Browning with little or no aldehyde production was characteristic of serine, lysine, threonine, glycine, glutamic acid, proline, arginine, and histidine (listed in decreasing order of browning-product intensity). Alanine produced both intensive browning and considerable aldehyde. Considerable aldehyde production and relatively little browning was given by isoleucine, leucine, valine, methionine, and phenylalanine (in decreasing order of volatile aldehyde evolution). Some amino acids did not contribute significantly to either browning or aldehyde development, namely, cystine and cysteine, tyrosine, and tryptophan. Rothe and Voigt (1963) concluded that there is no direct relationship between development respectively of brown pigment and of flavor. Xylose is not an important sugar in meat, and the nature of the oxidant (sugar) may be significant in the quantitative aspects of the reaction (Wickremasinghe and Swain, 1964), as was demonstrated by measuring the aldehydes produced during heating of valine, leucine, and isoleucine with either catechin, autoxidized catechin, or glucose. Catechin produced no aldehydes, but its autoxidized product was more efficient than glucose. In other work (Casey et al., 1965),a larger quantity of volatile product was invariably given when fructose (rather than glucose) was the sugar reacting with individual amino acids. Methionine was degraded rapidly in the presence of fructose, whereas little reaction occurred with glucose. However, since reaction was judged by formation of a breakdown product of methional (acrolein) rather than the aldehyde, it is not clear whether the primary reaction to methional was affected.

63

MEAT FLAVOR

Carbonyl amino reactions have also been studied in relation to flavor from flour-based foods. Wiseblatt and Zoumut (1963) reacted dihydroxyacetone with proline and obtained a strong crackerlike aroma of the type given off by boiled preferment for bread. Rooney et (11. (1967) studied model systems related to bread and Salem et al. (1967) extended this work to bread systems. Strecker degradation aldehydes were obtained in heating alanine, valine, leucine, isoleucine, phenylalanine, or methionine with xylose, glucose, or maltose (the sugars decreased in reactivity of the system in the order listed). Glutamic acid and proline gave little browning and little aldehyde, and arginine and histidine gave strong browning and no aldehyde. Lysine also produced strong browning and no aldehyde, but gave a pleasing aroma. In the bread-system studies, all amino acids tested decreased production of hydroxymethylfurfural except methionine, which increased this known end product of some Maillard reactions. In studies on the flavor of canned meat, Zoltowska (1967) showed that methionine had greater sensory effect in Maillard-type reactions than any other amino acid. These findings would seem to support the conclurion inter alia of Burton and McWeeny (1964) that melanoidins are formed by more than one route and that the Maillard reaction is a conglomerate of concurrent reactions. Casey et al. (1965) showed that many volatiles evolved from foods during boiling or cooking can be produced in nearly the same amounts by boiling dilute mixtures of individual amino acids with either glucose or fructose. Table VI lists volatiles and precursors examined under such conditions. Browning in meat has been little studied. Pearson e t al. (1962,1966) followed brown color development during the heating of pork slurries TABLE V1 ALI)EHYDESEVOLVEI) HY BOILINGS O I . U T I ~ NOF S AMINO ACIDS A N D SU(;AHS I N hlODEL SYSTEhlS"

Rate of evolution Aldehyde measured

Precursor amino acid

Acetaldehyde Propionaldehyde 2-methylpropan;il 3-1iietliyll)utanaI Acrolein

Alanine a-Ainiiiobutyric acid Val i n e Leucine M e t h i on i tie

"Casey et ul., 1965.

(nmolesIhr) using Glucose Fructose

14.6 20.4 10.3 26.0 0.05

48.1 38.9 28.4 57.7 51.1

64

KARL 0. HERZ AND STEPHEN S . CHANG

to dryness at 100°C in approximately 25 hr. Degree of brownness was related to the level of reducing sugar present in the meat. Most browning was due to amino-sugar reaction, but a definite small amount was brought on by pyrolysis of the indigenous carbohydrate in meat. Development of odors concomitant with browning was not studied.

VIII. INTERACTION OF COMPOUNDS AND PERCEPTION OF FLAVOR Understanding of the perception of meat flavor by the human senses is complicated by reactions or other effects involving several or many flavor components, the nature of the flavor sensation, and the conditions under which flavor is perceived. A. CHEMICAL COMPOUNDS, CONCENTRATION, AND INTERACTION

The effect of concentration of some pure compounds on odor perceived is well known. For example, diacetyl gives the typical buttery aroma in dilute solution; concentrated diacetyl has a harsh acid smell (Eakle, 1963). Two other flavor compounds reported in meat flavor isolates (Table 111), hexanal and 2,4-decadienal, are relatively pleasant when diluted, but objectionable in high concentration (Minor et al., 1965a). The effect of dilution with miscible and immiscible solvents on gas chromatographic detection of volatiles in container headspace has been reported and discussed by Nawar (1966) and Van Lunteren et al. (1968). The human detector, the nose, exhibits vastly different sensitivity toward different compounds, and threshold values can vary among people depending on a number of complex characteristics and conditions, many of them incompletely known or understood. For any one nose, threshold perception can be affected by changes in structure of compounds (Guadagni et al., 1963), medium or solvent (G. Bennett et al., 1965; Patton, 1964; Nawar, 1966), or the presence of other compounds (Guadagni et al., 1963;Bennett et al., 1965). Threshold values are important to flavor perception because compounds perceived in extremely low concentration may contribute aroma out of proportion to their relative concentration. A well-known example is methyl mercaptan, reported present in meat flavor (Table

MEAT FLAVOR

65

111), which is perceived when present in water solution at 0.02 ppb. Guadagni et al. (1963) reported mean thresholds for n-alkanals from C, to C,, in water solution as being between 0.1 ppb for decanal and 12 ppb for pentanal. Threshold for perception was lowered considerably when a methyl group was added, such as is present in branched-chain aldehydes. Thus the threshold was 0.15 ppb for 3-methylbutanal as compared to 9 ppb for butanal or 12 ppb for pentanal. Lower 2-enals had relatively high thresholds; 2-nonenal was again in the very low threshold range, 0.008 ppb. Threshold for fatty acids is in the ppm (parts per million) range, between 3 and 7 ppm for C, to C,,,acids, and 54 ppm for acetic acid (Patton, 1964). Patton (1964) also examined the effect of the medium or solvent on flavor threshold of fatty acids and suggested that bonding to solvent molecules restrains volatilization of acetic acid in water and of higher fatty acids in oil. Hence flavor contribution may depend upon relative distribution of compounds between aqueous and lipid phases. There is significance here for the situation existing during cooking of meat, when originally fat-soluble or fat-entrained compounds may be expected to enter an essentially aqueous meat juice, or derivatives of water-soluble compounds to accumulate in the fat. A similar effect of the medium on detector response in gas chromatography of headspace gas has been reported by Nawar (1966). Threshold perception for diacetyl also differs with the medium, the pH, and the presence of added compounds (Bennett et al., 1965). In skimmilk, the threshold value was lowest (5 ppb) when the pH was 6.0, near that normal for cooked meat. At pH 5.5, 50 ppb was needed for perception, and 200 ppb at pH 5.0. In cream, at pH 4.4 the fat content suppressed acid flavor sufficiently to permit diacetyl to be tasted at a concentration of 50 ppb. In sour cream, addition at near threshold values of formic, acetic, propionic acid, or acetaldehyde lowered the threshold for diacetyl, but the 2-carbon compounds were much more effective (less than 1 ppb of diacetyl being detected) than either formic or propionic acid. In butter, a somewhat harsh acid flavor contributed by diacetyl and acids derived from cultures is smoothed out by dimethyl sulfide (Day et al., 1964). Kazeniac (1961) commented that hydrogen sulfide and ammonia, volatiles evolved in large amounts during heating of meats, probably would tend to combine in unstable ammonium sulfide compounds that could break down to give a number of products. He reported that the rotten-egg odor of hydrogen sulfide was reduced in the presence of ammonia, and with a large excess of ammonia a sweetish

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odor and taste effect was induced. Similarly, the typical odor of m i monia could not be detected i n the presence of sulfide. I n his work with chicken, hroth from light meat had a sharp aldehydic odor, but addition of ammonia brought the odor close to that from dark meat with its more sulfury note. A further complication may arise from cumulative effects of a number of compounds, each of which is present i n sub-threshold concentration (Lillard et o l . , 1962; Nawar and Fagerson, 1062; Guiidagni et u l . , 1963). A number of mixtures tested by Guadagni ct (iZ. (1963) suggest an additive effect in mixtures of different members of one class of compounds (e.g., aldehydes), b u t additivity also was ollcerved in most mixtures comprising different types of compounds. A synergistic effect reported (Nawar et d., 1962) for methyl ketones present i n solution in subthreshold concentration was not found for the various mixtures studied b y Guadagni et (11. (1963). €3. FLAVORPERCEPTION Workers in food flavor research have recognized the complex nature of the sensation, involving physiological, psychological, physicochemical, and perhaps also genetic and other factors not yet brought to light or fully understood (Wick, 1965). One recent discussion of perception (Kauhian, 1967) quotes the work of J. J. Gibson on vision. Sensations, if they exist, are held to be mere by-products of perception and not to be basic to its occurrence. The apprehension of a room would be accomplished by detection of invariant properties that are revealed over time. It is tempting to draw a parallel to flavor sensation and perception. The situation would be more complex with sensation and perception caused by a flavor source because intensity of and among flavor constituents present in the inspired air are not likely to b e “invariant properties” nor does the olfactory perceiving apparatus maintain its sensitivity “over time” to even an unvarying stimulus. In 1961 some 5 years after gas chromatography had entered a phase of extensive use in flavor research (Dimick and Corse, 1956), Dr. B. S. Schweigert told a group of newspaper food editors: “So the uniqueness of the individual and his evaluation is a key part of flavor research, and perhaps psychology and physiology of t h e test subjectman-has as much to do with flavor research as our new gadgets to characterize those subtle amounts of those chemicals in the foods.” The ultimate judgment of a flavor comes from the olfactory mechanism of the individual, and in trying to master flavor knowledge and

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manipulation we can never hope to reach 11eyond the democratic aim of pleasing the largest number of noses. Interaction of compounds does not nece wily end with the cessation of heat treatment of meat. Although aroma perceived before eating probably is not likely to change i n character other thun to become weaker b y self-adaptation of the receptors (Moncrieff, 1960; Koester, 1965), the act of chewing mid generally taking the meat into the mouth may introduce new conditions that could alter the total flavor impression. (Certainly mouthfeel, chewing resistance, viscosity, and temperature sensations are evoked.) Saliva pH and enzyme and other constituents, the grinding activity of teeth in the preseiice of oxygen and carbon dioxide, and the existence of a forced stream of expired gas, chiefly containing carbon dioxide, passing outward through the osnioreceptor region, are examples of conditions that might have an effect. To illustrate, certain green leaves and fruits such a s strawberries form 2-hexenal and hexanal when comminuted in air, but not when ground under nitrogen (Major et u l . , 1963; Winter and Willhalm, 1964; Drawert et ul., 1966). Linolenic acid was the precursor of 2-hexenal and linoleic acid gave rise to hexanal. Toiig~ietaste may have an effect superimposing on that of aroma and suppressing or accentuating certain aroma notes (Beidler, 1963; Dastoli and Price, 1966). Degradation and conversion of nonvolatile precursor material may produce flavor in the mouth, e.g., by enzymatic action. A thought-provoking circumstance concerning the role of flavor potentiating 5’-mononucleotides arises in connection with enzyme action in the mouth. Phosphatases that split off the phosphate at carbon-5 of ribose convert potentiating 5’-nucleotides into ineffective iiucleosides (Takeda Pharm. Industries, Ltd., 1963).Could the flavor in the mouth of say 5’-IMP depend on in s i t u enzymatic hydrolysis or transfer of phosphate? Theories of olfaction have been proposed on various bases, but none to date has received unanimous endorsement or proof (Fullman, 1963; Amoore, 1963; Dravnieks, 1967). Until we can describe flavor elements in concrete units such as we can now for color (wavelengths) and sound (frequencies),the only basis for judging the effect of flavor stimuli will be the individual’s response when exposed to them (Burr, 1964; Wick, 1965). Olfactory analogs devised so far do not provide the objective measurement or description. Certainly, the modern tool of flavor research, the gas chromatograph, is far from being correlated with the response of the human nose (Wick, 1965).

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IX. AREAS FOR FURTHER RESEARCH We need to know more than we know now about the origin and nature of the flavor compounds and the reactions that produce them; about in vivo and postmortem occurrences in the beef animal that bear on flavor; about the influences of feed and other conditions on cattle raising; about the physical and physiological events that lead to human perception of meat flavor quality and strength. Perhaps knowledge of the compounds and reactions involved are the more urgent areas for research, since many of the other areas can be studied more effectively when criteria for judgment (such as appearance or loss of flavor compounds or precursors) are available. Research on flavor-producing reactions may need to concentrate on the chemistry of such compounds as hydrogen ‘sulfide and ammonia under the conditions of their release from proteins by heat in the presence of other compounds such as carbonyls. The molecular environment needs to be studied. The possible role of fat-aqueous interfaces (droplet surfaces) in accumulating, catalyzing, or even participating in reactions leading to flavor compounds has not been considered. Investigation of the chemistry of browning in a mixed fat-aqueous system at internal temperatures in the range of 40”-80°C could provide needed insight. We also could profit from more information on what happens in fatty tissues during prolonged heating in this range. To what extent does flavor formation depend on transport of protein breakdown products to or into melted fat? What is the aqueous:fat distribution of the compounds involved in flavor development? What breakdown products from fat or stored in fat are significant? Then, from the flavor compounds we need to trace the steps back to the precursors (or forward to flavor compounds in the event that the more chance-dependent precursor studies proceed more quickly to results). Control of beef flavor is probably achieved in a more desirable way by directing live animal metabolism to yield the precursors (or feed them?) than by adding these or the formed flavor to the carcass or to the meat cut. With a knowledge of precursors and flavor compounds, we can study and assess the influence of slaughter, aging, and processing conditions on meat flavor quality and intensity. We can then, of course, also produce more effective and closely similar imitation flavor preparations for use with either beef-containing or meatless food products. And we may provide cattle breeders with a means to “select” for flavor quality, though much experimentation lies ahead of such a goal.

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Finally, the elusive areas of the physiology and physics of flavor perception - as well as the psychology of flavor satisfaction - remain to be more fully explored for meat flavor as for flavors generally. At the point of eating, what is the role of atmospheric oxygen? of saliva enzymes or other constituents? How much do background aroma compounds modify flavor perception of the meaty notes? Flavor in the case of meat may need to be resolved into time-dependent factors of initial stimulatory nose impact (high volatility of compounds/high sensitivity of receptors), in-mouth impact (overlapping aroma, taste, and tactile factors), and in-mouth aftertaste (low volatility of compounds/high adsorptive capacity). Such a separation does little more than isolate the first and last factors, leaving the in-mouth impact about as complex as before. But this approach may be a manageable beginning.

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Tuma, H. J., Hendrickson, R. L., Odell, G . V., and Stephens, D. F. 1963. Variation in the physical and chemical characteristics of the Longissimus dorsi muscle from animals differing in age. ]. Animal Sci. 22,354. USDA 1964. Finishing beef cattle. U . S .Dept. Agr., Farmers’ Bull. 2196. USDA 1966. “World Agricultural Production and Trade Statistical Report.” U.S. Dept. Agr., Foreign Ayr. Serv., Washington, D.C. USDA 196th “World Meat Production Higher in 1967,” Livestock and Meat Circ. FLM 7-68. U.S. Dept. Agr., Foreign Agr. Serv., Washington, D.C. USDA 1968b. “World Agricultural Production and Trade Statistical Report.” U.S. Dept. Agr., Foreign Agr. Serv., Washington, D.C. Van Lunteren, G., Van Straten, S., and Weurman, C. 1968. Irregularities in gas chromatographic direct vapour analysis. Proc. 3rd Intern. Colloq. Chem. C o f e e , 1967, Trieste. Assoc. Sci. Intern. Cafe, Paris. p. 191. van Marle, J. 1963. Effect of three levels of milk-feeding on the growth, development and carcass quality of Friesland steers. S. African]. Agr. Sci. 6,475. Van Praag, M., Stein, H. S., and Tibbetts, M. S. 1968. Steam volatile aroma constituents of roasted cocoa beans.]. Agr. Food Chrni.16, 1005. Vickery, J . R. 1966. Meat research-aspects of an Australian venture. Food T e c h ~ ~ o l . Austruliu 18,375. Wada, S., Nakatani, H., and Morita, K. 1967. A n e w aroma-bearing substance from Shiitake, and edible mushroom.]. Food Sci. 32,559. Wanderstock, J. J., and Miller, J. I. 1948. Quality and palatability of beef as affected by ~ 13,291. method of feeding and carcass grade. F O C JRes. Wasserman, A. E., and Gray, N. 1965. Meat flavor. 1. Fractionation of water-soluble flavor precursors of beef.]. Food Sci. 30,801. Weir, C. E . 1960. Palatability characteristics of meat. I n “The Science of Meat and Meat Products.” Chapter 6, p. 212. Freeman, San Francisco, California. Weir, C. E., Doty, D . M., and Auerbach, F. 1960. Meat preservation. In “The Science of Meat and Meat Products,” Chapter 9, p. 280. Freeman, San Francisco, California. Weurman, C. 1961. An all-glass laboratory apparatus for concentrating volatile compounds from dilute aqueous solutions.]. Food Sci. 26,239. Wick, E. L. 1963. Volatile components of irradiated beef. I n “Exploration in Future Food Processing Techniques” (S. A. Coldblith, ed.), Chapter 2, p. 23. M.I.T. Press, Cambridge, Massachusetts. Wick, E. L. 1965. Chemical and sensory aspects of the identification of odor constituents in foods -a review. Food Techno/.19,827. Wick, E. L., Yamanishi, T., Wertheimer, L. C., Hoff, J . E., Proctor, B. E., and Goldblith, S. A. 1961a. Radiation effects on beef, and investigation of some volatile components of irradiated beef.]. Agr. Food Chem. 9,289. Wick, E. L., Hoff, J. E., Goldblith, S. A,, and Proctor, 8.E. 1961b. T h e applicatioti of radiation-distillation to the production and isolation of components of beef irradiation flavor.]. Food Sci. 26,258. Wick, E. L., Koshika, M., and Mitzutani, J. 1965. Effect of storage at ambient teniperature on the volatile components of irradiated beef.]. Food Sci. 30,433. Wick, E. L., Murray, E., Mizutani, J., and Koshika, M. 1967. Irradiation flavor and the volatile components of beef. I n “Radiation Preservation of Foods, Am. Chem. Soc.” Ser. No. 65, p. 12. Wickremasinghe, R. L., and Swain, T. 1964. T h e flavour of Black Tea. Chem. b Ind. (London)p. 1574.

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Wilson, G . D. 1960. Smoke and smoking: Generation, constituents, deposition discussed at conference in Poland. Natl. Provisioner, p. 18. Winter, M., and Willhalm, B. 1964. Research on aromas. On the aroma of fresh strawberries-analysis of carbonyl compounds, volatile esters and alcohols. (In French.) H e l ~Chini. . Actu 47, 1215. Wiseblatt, L., and Zoumut, H. F. 1963. Isolation, origin and synthesis of a bread flavor constituent. Cereal Chem. 40, 162. Wismer-Pedersen, J. 1966. Use of carbazole to determine 5-ribonucleotides in meats. J . Food Sci. 31,980. Wood, T. 1961. The browning of ox-muscle extracts.]. Sci. Food Agr. 12,61. Wood, T., and Bender, A. E . 1957. Analysis of tissue constituents. Commercial oxmuscle extract. Biochem.J.67,366. Woskow, M. 1966. Flavor modifying properties of a mixture of 5-riboniicleotides. 26th Anti. Meetitig Ztist. Food Technologists, Portlmnd. Yueh, M. H., and Strong, F. M. 1960. Some volatile constituents of cooked beef. J . Agr. Food Chem. 8,491; Dissertation Ahstr. 22,730. Znika, L. L., Wasserman, A. E., Monk, C. A., Jr., and Salay, J. 1968. Meat flavor. 2. Procedures for the separation of water-soluble beef aroma preci1rsors.J. Food Sci. 33,53. Zender, R., Lataste-Dorolle. C., Collet, R. A., Rowinski, P., and Mouton, R. F. 1958. Aseptic autolysis of muscle: Biochemical and microscopic modifications occurring in rabbit and lamb muscle during aseptic and anaerobic stroage. Food Res. 23,305. Zipser, M. W., Kwon, T. W., and Watts, B. M. 1964. Oxidation changes in cured and uncured frozen p0rk.J. Agr. Food Chem. 12, 105. Zoltowska, A. 1967. T h e effect of thernial processes upon the flavour of canned meat with special reference (to) the changes of some amino acids. Roczniki Znst. Prsem!lslu Mlecz. 4(1),91.

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MICROBIAL SOURCES OF PROTEIN

BY HARRYE. SNYDER Depurtment of Food Technology lowu State Unicersity, Ames, lowu

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . A. World Food Situation . . . . €3. Microbial Protein Sources 11. Review and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

88

A . Yeasts and Bacteria.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Additional Research Needs ............................. A. Palatability and Digestil B. A Different Approach to Single-Cell Protein . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

116 128 128 ,130 131

I. INTRODUCTION Two circumstances have combined to lend urgency to the subject of microbial sources of protein. The first of these is the continued failure of our ability to provide sufficient food, particularly protein, for the world’s growing population. The second is the realization that microorganisms can transform petroleum hydrocarbons into microbial protein, protein that could and should be put to good use by the world’s hungry people.

A. WORLD FOODSITUATION There have been many recent symposia, books, articles, and reports on the world food situation. The large amount of published and oratorical material has even caused the subject “to be obscured by rhetorical overkill” (President’s Science Advisory Committee, 1967). There is unanimity of opinion on the seriousness of the situation but there is no consensus on our abilities to cope with the problem. The optimists say that the problem of hunger has always been in the world, but only 85

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recently have we developed the capability of eliminating hunger forever. Others say it is already too late, and disastrous famines are no more than 5 or 10 years away (Paddock and Paddock, 1967). An excellent study of the world food situation and its complexity has been made by the President’s Science Advisory Committee (1967). Volume I is a summary of the study and gives a very readable account of the immensity and intricacy of the problem. Also in 1967, a report was submitted to the Economic and Social Council of the United Nations that recommended an action program for alleviating world protein shortages. This report has now been published (United Nations, 1968)and is recommended a s a good though brief source of information. In studies of the ability of our planet to support and sustain people, the findings indicate that existing hunger is not due to inherent limitations in land area or photosynthetic capacity (Schmitt, 1965; dewit, 1967). Rather, the shortages of food are blamed on economic and sociological restraints. Coffey (1968)has stated that the food problem cannot be treated in isolation and is inseparable from the problem of general economic development. This view is shared by many and is undoubtedly true. Yet, the concept that food problems cannot or will not be alleviated until a country begins to pull itself up b y its bootstraps is frustrating to those who are trying to help. If the cycle of poverty-hunger-diseaseignorance-poverty is to be broken, its weakest link is hunger. By attacking at that point with cheap and nutritious food, there is certain to be better resistance to disease and there is growing evidence that mental development of young children will be spared a permanent impairment (Scrimshaw and Gordon, 1968).An attack at this weak link in the cycle could provide the key to better human development and the spark to economic development. Within the broad scope of world food shortages, the critical need is adequate protein for young children. Many proposals have been made for increasing protein supplies or for bettering the nutritional adequacy of protein. The President’s Science Advisory Committee (1967) reported, in order of priority, ten methods for improving o r increasing food protein supplies: (1)forti@ing cereal grains, (2) genetic improvement of cereal grains, ( 3 )increasing animal protein, (4)recovering oil seed protein, ( 5 )ocean fishing, (6) inland fishing, (7) fish protein concentrate, (8) single-cell protein, (9) leaf protein, (10) algae. This review will concentrate on what is known about microorganisms, including algae, as potential contributors of nutritious protein.

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B. MICROBIALPROTEIN SOURCES

The concept of eating substantial quantities of microorganisms as a foodstuff is perhaps 60 years old but has not become widespread. Because of the unpleasant connotations of the words “bacterial” or “microbial” in connection with food, the phrase “single-cell protein” was coined at the Massachusetts Institute of Technology in 1966 to depict the idea of microorganisms as food (Scrimshaw, 1968). The phrase has been widely accepted, and I will find it convenient to use despite the fact that marly compounds other than protein are found in single cells. In October 1967, ail International Conference on Single-Cell Protein was held at the Massachusetts Institute of Technology. The proceedings of that symposium were recently published, and I will make frequent references to that pul)lication (Mateles and Tannenbauni, 1968). Microorganisms do represent a potential food or food source because they contain the proteins, lipids, carbohydrates, vitamins, and minerals that are common to arid required by many forms of life. In addition, microorganisms contain unique compounds that may be a problem in nutrition, but there is 110 definite evidence of harmful compounds being present other than the well-known toxins produced by only certain species. Two compelling reasons for thinking seriously i t h i t single-cell protein are the very rapid growth rate and the degree of control one can exert over the growing conditions. The reproduction of microorganisms is such that bacteria and yeast can double in mass every hour or two, and algae, although slower, take less than 1 day to double. This extremely rapid growth 1)ecomes ttiore apparent when compared to growth of conventional agricultural species. Thaysen (1956) calculated that a beef animal weighing 1000 11) could produce 1 111 of new protein per day. The same weight of starting material a s soybeans would yield 80 l b of new protein per day if the total yield were prorated over the growing season. In contrast 1000 111 of yeast coiild yield 50 tons of new protein in one day. &din in comparison to conventional agl-icultiire, the degree ofcontrol over the growing conditions for single-cell protein is unique. With continuous or batch cultures, controls are available for the concentration of nutrients, the pH, the temperature, the oxygen concentration, and the cell concentration. Thus, the uncertainty of the environment that plagues agriculture with droughts, monsoon failures, floods, late frosts, etc. would not affect single-cell protein. Of course, there are problems unique to the cultivation of niicroor-

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ganisms such as maintaining pure cultures and minimizing the effects of metabolic products. One of the problems in mass cultivation of microorganisms is the availability of large quantities of a suitable carbon source that would allow taking advantage of the high potential growth rate. With the realization that hydrocarbons from petroleum or from natural gas can serve as carbon sources for many bacteria and yeasts, the chances of developing a feasible single-cell protein appear much better. At present there is no successful production of microorganisms to serve as a substantial part of our total food supply. I am not certain of the reasons for this failure (or even if it is a failure), but my purpose in this review will be to survey what is known about growing microorganisms on a large scale and about their nutritive properties. As more people become familiar with the problems and the potential of singlecell protein, we will be better able to take advantage of the unique opportunities and to make a significant contribution to world protein needs.

II. REVIEW AND DISCUSSION

The groups of microorganisms that have been considered as possible sources of proteins will be discussed in three sections: (A) yeasts and bacteria, ( B ) fungi, and (C) algae. It is convenient to group yeasts and bacteria together because the conditions for propagation, harvest, and drying of these microorganisms are similar. The major emphasis will be placed on yeasts and algae, and they will be discussed from the viewpoints of (1)useful species, (2)conditions of propagation, and ( 3 )nutritional value. A. YEASTS AND BACTERIA

Most of the literature, experience, and commercial production of single-cell protein has been with yeast. There are several reasons for the emphasis on yeast. For centuries men have made use of yeast for brewing beer, fermenting wine, and baking bread. This close association between established desirable foods and the smell and taste of yeast makes it relatively easy to consider yeast as a food. Some fungi are recognized as familiar foods but in most cultures bacteria and algae are not considered edible. A second important reason for the emphasis on yeast is a wealth of

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experience in large-scale production techniques gained through the bakers’ yeast industry and industrial alcohol production. Prescott and Dunn (1949) indicate that yeast was being produced commercially for bakers in the late 1800’s, and the intervening years have provided knowledge of carbon sources, nitrogen sources, salts, aeration requirements, pH control, fermentor design, and harvesting techniques that is directly applicable to production of yeast for food purposes. I n addition, yeast has been known and used in western countries as a vitamin and protein supplement for at least 40 years. Thus, people have ingested yeast, as such, for healthful purposes, and there have been no harmful side effects. This extended experience with the safety of food yeast is important for convincing those concerned with toxicology problems in new foods. And yet, with a good public image, technological competence and guaranteed safety, yeast has not been used as a food to any large extent. The reasons for this lack of utilization should be fully explored by those interested in promoting microbial sources of protein, because if yeast is not suitable for single-cell protein, bacteria and algae may be even less suitable. Some possible reasons for the lack of acceptance of yeast are failure to produce a palatable appealing food from yeast, no room in the very competitive market place for a new major food-particularly in those countries that have the technology for yeast production, and insufficient effort in selling the concept of yeast as food. Information on amounts of yeast currently being produced have been compiled b y Suomalainen (1966)and Peppler (1968).The total world output in 1963 was 180,000 tons (dry weight) of food and feed yeast with Russia, East Germany, Poland, and the United States as the major producers. The amount has not increased appreciably since 1963, but Bunker (1968) indicated Russia has plans to quadruple the present world production of food yeast. In relation to major food crops, yeast supplies negligible quantities of protein. Altschul(l965) indicated cereals furnish 1 X loxtons of protein annually and animals supply another 2 X lo7 tons. The protein from yeast would be approximately 1 x 10”tons or less than 0.1% of the total supply. Bacteria have been considered as possible food or feed for much less time than yeast. Roberts (1953)showed the feasibility of using E . coli as a feed supplement, and the current interest in using petroleum hydrocarbons as carbon sources for microbial protein has focused attention on bacteria. But at present no bacteria are being produced as food or feed except in laboratory scale experiments.

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1 . Useful Species Of the tens of genera and hundreds of species of yeast, few have been useful commercially and still fewer have been proposed as possible protein sources. Two species in particular have been used for food and feed yeast, Catidida utilis and Saccharoinyces curlsbergetisis. Candida utilis is the current name for the yeast that was first mentioned b y Hayduck and Haehn (1922)as mineral yeast. Cutididu utilis had been named Torulopsis utilis until Lodder and Kreger-van Rij (1952)confirmed earlier reports that a pseudomycelium is formed and consequently transferred it to the genus Candida. It is also known as torula yeast and is frequently grown as a primary yeast, i.e., a yeast grown for the purpose of producing cells rather than a fermentation product. The Germans grew C. utilis during World War I1 as a food supplement (Locke et d . , 1945). This primary yeast is frequently used because it grows well on substrates such as sulfite waste liquor and wood hydrolyzates. One of the advantages of C. utilis is that it will use pentoses as carbon sources. Other advantages are that it requires no accessory growth factors and it competes well with bacteria so that contamination is not a serious problem when producing C. utilis. Succliuroniyces curlsbergetisis is brewers’ yeast that can be recovered, processed, dried, and sold a s a nutritional supplement. Hence, S. carlslxrgeiisis is an example of yeast being used for a secondary purpose. After the beer fermentation is completed, brewers’ yeast is flavored with some of the bitter compounds from hops. To make it more palatable to humans, brewers’ yeast is debittered b y treating it with alkali, but the treatment has to be rapid arid the temperature kept low to prevent vitamin losses (Singruen and Ziemba,

1954). Locke et al. (1945) in discussing the German yeast production during World War I1 mention the use of Cutididu urboreu. According to Lodder and Kreger-van Rij (1952),C. arhorea has not been described, but of the two yeasts in their collection called C. urborea both came from German food yeast plants. On further study, one yeast was classified as C. utilis and the other as C . tropiculis. Catidida tropicalis is of considerable interest now because it is frequently used in studies of yeast production on hydrocarbons. Candiclu lipolyticu can also use hydrocarbons as a carbon source (J. B. Davis, 1967). Species of bacteria considered useful for protein production have been selected mainly on the basis of their ability to use hydrocarbons.

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If methane is the substrate, Pseudomonas metlzanicu (Leadbetter and Foster, 1958) might be the organism of choice, but for longer chain hydrocarbons such as n-hexadecane or n-octadecane Nocardiu, Mc~cobacterium, or Micrococcus species might by used (M. J. Johnson, 1967a). Mateles et al. (1967) used a different criterion for selection of a bacterium that may be useful for single-cell protein. By enrichment cultures, they were able to isolate from several sources a thermophilic Bacillus (probably B. stearothermopliilus) that can use n-alkanes at 70°C. Such an organism would be particularly useful if cooling of the fermentation vessel was a large part of the cost of single-cell protein production. Also, growing this Bacillus at high temperatures on hydrocarbons would minimize contamination by other organisms.

2 . Conditions for Propagation This section contains the kind of information that makes up the field of bioengineering or biotechnology. Since textbooks have been written on this subject (Aiba et al., 1965; Brakebrough, 1967), no attempt will be made to cover the original literature thoroughly. But some understanding of the process of growing microorganisms on a large scale is necessary to appreciate the potential and restrictions of the concept of single-cell protein. The majority of research work in this field has been done with submerged fermentations. The production technique makes use of a fermentation vessel with a capacity of thousands of gallons and with fittings for adding nutrients and sterile air, for sterilizing and cooling the medium, for agitation and for pH control. As Gaden (1964) pointed out, this highly mechanized technology may not fit the economic and technical capacities of countries most in need of protein supplements from microorganisms. Simpler techniques similar to those used in silage production might be more fitting for less developed countries. Still, it is possible by the techniques of submerged fermentations for a few highly skilled people to make food for many, and this may be one answer for the food production problems of any developing nation. u . Substrute. The main carbon and energy source used by microorganisms is termed the substrate, and for the food and feed yeast currently being produced in the world, the substrates are molasses and sulfite waste liquor. Molasses and sulfite waste liquor are both waste products (of the sugar refining and paper pulp industries, respectively) and consequently are of low cost. Since molasses can serve for bakers’ yeast production, it does have a market value, but sulfite waste

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liquor is a waste disposal problem because of the fermentable sugars it contains. Consequently, yeast growth on sulfite waste liquor is partly waste treatment and partly yeast production. Over the years many proposals have been made to use other wastes such as milk whey, citrus wastes, and cannery wastes for yeast production. These proposals usually have not worked because of the problem of collecting wastes when the volume is insufficient or because of the seasonal availability of the wastes. Peppler (1968) described the growth of yeast in cottage cheese whey at a plant in California. Saccharomyces fragilis was the yeast used because of its ability to grow on lactose. Wood hydrolyzate has been used as a substrate for yeast growth and for industrial alcohol production (Locke et al., 1945). This took place in Germany during World War 11, but it is doubtful if the process was justified economically. Most of the reviews on food yeast have considered conventional substrates and these may be consulted for further details (Thatcher, 1954; Thaysen, 1956; Wiley, 1954; Rose, 1961; Bunker, 1963). Considerable impetus was given to the single-cell protein idea when Champagnat et al. (1363) advocated growth of yeast on the paraffin existing in fuel oil. The original purpose was to upgrade the fuel oil by removing long chain paraffins that greatly increased viscosity. But the by-product of the reaction was vitamin- and protein-rich yeast that was quickly recognized as a valuable food or feed. Since the growth of microorganisms on hydrocarbons is a relatively new and exciting possibility, the emphasis in this section on substrates will be placed on hydrocarbons. The knowledge that microorganisms can use hydrocarbons for energy and carbon goes back to Sohngen (1906), but only recently have we understood how the carbon is assimilated and the energy obtained. Beerstecher (1954) and J. B. Davis (1967)have written reviews that relate hydrocarbon utilization to the broad field of petroleum microbiology. McKenna and Kallio (1965) and van der Linden and Thijsse (1965) have reviewed the mechanisms by which microorganisms degrade hydrocarbons. Fuhs (1961) and Foster (1962) have emphasized the microbiology of hydrocarbon utilization, and M. J. Johnson (1964, 1967a) has emphasized cell yields and bioengineering aspects of microbial growth on hydrocarbons. Recently Wang (1968a) and Humphrey (1968) have reviewed the bioengineering and economic aspects of producing protein from petroleum. (1) Mechanism of attack on hydrocarbons. ( a ) Bacteria. A discrepancy exists in the literature between the amount of interest in yeast

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as a source of single-cell protein from hydrocarbons and the amount of research done to determine how yeast attacks hydrocarbons. Most of the research on microbial breakdown of hydrocarbons was done with bacteria and was stimulated by a basic interest in hydrocarbon metabolism. Some of the earliest information about the initial attack on hydrocarbons came from Stewart and Kallio (1959).They showed that growth of a Gram negative coccus (later identified as Micrococcus ceri$icans) on n-alkanes of 14- to 18-carbon chain lengths led to the accumulation of waxes with the alcohol portion having the same number of carbon atoms as the n-alkane used. Stewart et al. (1959)showed accumulation of cetyl palmitate from growth of M. cerificans on n-hexadecane and were able to demonstrate by using I8O that 75% of the oxygen in the wax came from the atmosphere. They interpreted the results as shown in Fig. 1. The postulated hydroperoxide, which results from 0, attack 2 CH,(CH,),,CH,

c

+

2 0;'

2 C H,( C H,) ,,C H,O' '0"H

2 CH,(CH2)l,CH,018H

CH,(CH2),,CHOL8 i

H,OE

I

CH,(CH,),,CHZO8H

/

0 : ' Incorporation = 75%

FIG.1. Mechanism postulated by Stewart et al. (1959)for the initial attack on n-hexadecane by M . cerijcans. The experimental finding of 75% incorporation of "'0into cetylpalmitate is explained by this mechanism.

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at the terminal methyl group, has not been isolated but higher alkyl hydroperoxides can be used by microorganisms (Finnerty et ul., 1962). Further evidence that the initial attack is at a terminal methyl group was obtained by Finnerty and Kallio (1964). The pattern of labeling in cetyl palmitate and myristyl palmitate derived from hexadecane-l-l‘C and tetradecane-I-’% showed that the initial oxidation was at the carbon-1 atom. Palmitic acid could arise from tetradecane by the addition of a 2-carbon fragment to the 14-carbon chain. This work was also done with M . cerijicans. Leadbetter and Foster (1959)studied atmospheric ‘‘0incorporation by bacteria attacking short chain n-alkanes and found that Pseudomonus methanica, an “ethane” bacterium and a “propane” bacterium would incorporate atmospheric lXO when growing on methane, ethane, or propane. As a result of these kinds of studies on n-alkane oxidation b y microorganisms, the conclusion is that terminal methyl groups are oxidized by atmospheric oxygen. Subsequent oxidation products are the alcohol, the aldehyde, and the acid corresponding to the chain length of the original n-alkane. The acid can be oxidized further by @-oxidation thus providing the cells with energy and carbon through conventional pathways. A study of storage products accumulated by a Nocurdici species when grown on n-alkanes of varying chain length showed that Cl:% to C z ohydrocarbons resulted in aliphatic waxes and triglycerides containing acids and alcohols of the same chain lengths as the substrate (J. B. Davis, 1964a). But when the Nocardia species was grown on propane or butane no waxes were accumulated. Propane led to an accumulation of P-hydroxybutryate, and butane gave a similar, partially unsaturated, storage polymer (J. B. Davis, 1964b). Although there is agreement that n-alkanes can be oxidized by atmospheric oxygen, other mechanisms of oxidation have been proposed. Azoulay et al. (1963)postulated that the initial attack of Pseudomonas aeruginosa on heptane was a dehydrogenation with nicotinamide adenine dinucleotide (NAD) as the oxidizing agent. M. J. Johnson (1964) criticized this hypothesis on the basis of the free energy changes involved, and the inability of NAD to oxidize a saturated n-alkane. Van der Linden and Thijsse (1965) pointed out that there is nothing in the data of Azoulay et al. (1963)that would exclude an initial oxygenase attack on n-heptane. But Senez and Azoulay (1961) reported the anaerobic oxidation of heptane by resting cells of P . aeruginosa (previously grown on heptane) using pyocyanin or NAD as a hydrogen acceptor. So there is evidence for an initial attack on n-

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alkanes with the formation of a l-olefin but the nature of the oxidizing agent is in doubt. The enzymes catalyzing subsequent oxidation of heptanol and heptaldehyde were isolated, partially purified, and studied by Azoulay and Heydeman (1963) and Heydeman and Azoulay (1963). Earlier, Azoulay and Senez (1960) had demonstrated the adaptive nature of enzymes responsible for heptane oxidation by P . aeruginosa. Baptist et n1. (1963)and Gholson et al. (1963)studied octane oxidation by Pseudornonas oleovoruns. They demonstrated the oxidation of octane to octanol and octanoic acid by cell-free soluble enzyme preparations that require either NAD or NADH but did not work with NADP or NADPH. Although bacterial species are known that attack intermediate chain length n-alkanes (C, to C,,,),the majority of microorganisims oxidizing hydrocarbons attack long chain (C,2and longer) compounds or short chain gaseous alkanes. M. J. Johnson (1964) postulated that failure of many organisms to grow on concentrated n-alkanes of intermediate chain length is probably due to disturbance of the permeability properties of the cell membrane. The excellent lipid solvation of these alkanes would be sufficient to render them toxic in high concentrations. There is abundant evidence of microbial utilization of short chain gaseous alkanes. Methane is readily attacked and organisms oxidizing ethane, propane, and butane have been found. Methane oxidation is particularly interesting for those concerned with microbial protein production because of the cheapness of methane as a substrate. Humphrey (1967) listed the cost of natural gas at 0.254 per pound. Leadbetter and Foster (1958) showed that many pseudomonads could use methane. These organisms were characteristically pigmented and would not grow on long chain alkanes or conventional carbon sources. They did utilize methanol as a carbon and energy source and could incorporate glucose. Working with P . metlaanicn, Leadbetter and Foster (1960) found that cells grown on methane were capable of oxidizing ethane, propane, and butane but could not use ethane, propane, or butane to support growth. This oxidation of compounds that could not be used as substrates was named “co-oxidation” and was emphasized by Humphrey (1967) a s a potentially useful process in bioengineering. The incorporation of labeled methane into cell material was studied by Large et al. (1962) and by P. A. Johnson and Quayle (1965).Both labeled methane and methanol were found in phosphorylated hexoses as the first stable products of metabolism by P . methnnica (P. A. Johnson and Quayle, 1965).

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HARRY E. SNYDER

( b ) Yeasts. Although most of the work on mechanisms of hydrocarbon attack has been done with bacteria, some studies have been done with yeast. Stewart et aZ. (1960)found that Candida Zipolytica would attack the double bond of 1-olefins to yield a-glycols. This tended to confirm an earlier report by Bruyn [cited by van der Linden and Thijsse, (1965),p. 5011 that C. lipolytica produced hexadecanediol-l,2 from growth on n-hexadecane. Klug and Markovetz (1967)investigated the oxidation of C,, to C,, nalkanes and 1-alkenes by C. Zipolytica. T h e n-alkanes were oxidized to alcohols and fatty acids of the same chain length. From 1-alkenes, 12 diols and w-unsaturated acids were found. These products indicated that both an oxidation at the double bond and an oxidation at the terminal methyl group were possible. Later, Klug and Markovetz (1968) extended their results on 1-alkenes and concluded that three oxidative mechanisms are possible: (1) methyl group oxidation, (2)double bond oxidation, and ( 3 )subterminal oxidation. (c) Unique problems with hydrocarbon studies. Some characteristics of hydrocarbons, when used as microbial substrates, are quite different from conventional sugar substrates. Fuhs (1961)pointed out that the high vapor pressure of short chain n-alkanes (C, to C,J made it difficult to keep them in the side arm of a Warburg flask for respiratory experiments. The low solubility of hydrocarbons and problems resulting froin it were discussed by M. J. Johnson (1964) and by Humphrey (1967). Bamah et al. (1967) found that liquid hydrocarbons can best be incorporated in agar-containing media by adsorbing the hydrocarbon on finely divided silica. T. L. Miller et aZ. (1964) studied yeast growth on long chain alkanes by dissolving them in pristane (2,6,10,14-tetramethy1 pentadecane). The pristane was not attacked by the yeast and greatly increased the availability of the insoluble alkanes. Later, Wodzinski and Johnson (1968)reported large yields of bacteria growing on pristane. A problem associated with experiments on the ability of a microorganism to use various hydrocarbons is the purity of the substrate. Special purifications may b e necessary to insure that the main hydrocarbon and not an impurity is serving as the carbon and energy source. Many hydrocarbon substrates other than n-alkanes have been studied for their ability to support microbial growth. The results with branched chain alkanes and aromatic hydrocarbons have been reviewed by van der Linden and Thijsse (1965) and by McKenna and

MICROBIAL SOURCES OF PROTEIN

97

Kallio (1965).Coal tar fractions as well as petroleum fractions can be used by microorganisms (Silverman et al., 1966). Most of the results reported have been with aerobic growth on hydrocarbons. It is possible for microorganisms to use hydrocarbons under anaerobic conditions if oxidizing agents such as sulfate or nitrate are available. Novelli and ZoBell(l944) reported that Desulfouibrio could attack n-alkanes with chain length greater than C,,, in the absence of air. J. B. Davis (1967) mentioned thermodynamic limitations in the oxidation of n-alkanes of less than 10 carbon atoms under anaerobic conditions. (2) Cell yields on hydrocarbons. Relatively few studies have been reported on cell yields with purified hydrocarbon substrates as compared with the number of studies on mechanisms of microbial attack. And even fewer studies have been reported on optimum conditions for cell yield when cells are grown on crude substrates that would be used in any practical single-cell protein scheme. Raymond and Davis (1960) studied cultural conditions, cell yields, and cell composition of a Norcardiu sp. grown on n-alkanes. Cell yields (including the weight of an extracellular slime layer) of 95% based on weight of n-alkane were found. Omitting the extracellular material, cell yields of 70-80% of n-alkane were found. The yields varied with nitrogen source, amount of n-alkane and amount of oxygen. The chain length of n-alkane had a striking effect on cell composition. When a short chain alkane (n-hexane or n-tridecane) or glucose wa5 the substrate, no appreciable wax and approximately 28% lipid was found in the cells. With n-hexadecane or n-octadecane as the substrate, the cells accumulated approximately 50% lipid of which 60% was triglyceride and 40% was wax. T. L. Miller et u1. (1964) found that a Cnndida sp. did not accumulate large amounts of lipid when grown in liquid n-alkanes. n-Alkanes with chain lengths of C,2 to C,, were tested as substrates, and cell yields, growth rates, and cell composition were determined. A pH of 4.5 to 5 gave the fastest growth but total yields were not greatly affected by pH changes using hexadecane as a substrate. Cell yields on the basis of substrate utilized ranged from 60% for C,, to 8 0 4 3 % for C,, through C,8. Glucose in comparison gave a cell yield of 35%. Generation times were found to vary with chain length of n-alkane. The longest generation time of 7 hr occurred with C,2alkane, whereas octadecane gave a generation time of 4.5 hr. In comparison, the cells had a generation time of 2 hr with glucose as substrate. All generation times were determined at pH 5 and 30°C.

98

HARRY E. SNYDER

M. J. Johnson (1964)discussed an interesting point about the effect of chain length of n-alkanes on their solid or liquid state and hence availability of the hydrocarbon to microbial cells. An experiment was done with octadecane as a substrate for Candida at 25" and at 30°C. At 25"C, octadecane is a solid and at 30"C, it is a liquid. When the temperature of the growing culture was changed from 30" to 25"C, the cells continued to grow but the growth rate gradually decreased. By raising the temperature to 30" and rapidly decreasing to 2592, a growth rate increase was achieved which persisted after the temperature was lowered. The conclusion was that solid hydrocarbon can be used b y Cundidu but growth rates are affected by the surface area exposed. The surface area slowly decreases (and with it the growth rate) due to agglomeration when liquid hydrocarlmn is solidified b y a temperature decrease. T. L. Miller and Johnson (1966a) found that a stable mixed culture of Candictn intermedin and Cundidu lipalytica attacked n-alkanes more readily than C. intermedia alone. In the mineral salts plus n-alkane medium, C. lipolyticu would not grow alone. Cell yields based on substrate ranged from 74 to 87%. The C,, and C,, n-alkanes dissolved in pristane supported growth with the shortest generation times, 3.5 and 3.0 hr, respectively. The results of studies on cell composition showed cell nitrogen of 6.8 to 8.8% corresponding to protein contents of 34 to 48%. The oxidation of n-alkanes by resting cells of C. intermedia showed no lag regardless of the substrate on which the cells were grown. Hence, the enzymes for oxidizing n-alkanes were assumed to be constitutive. It is necessary to study the growth of microorganisms on highly purified n-alkanes to learn about the mechanism of attack and how well the cells grow on the various components of complex mixtures of hydrocarbons. But for production of single-cell protein, purified n-alkanes would be far too expensive as substrates, and crude petroleum fractions would be used. Some data exist on cell yields with a petroleum fraction known as gas oil (the fraction distilling between kerosene and lubricating oil). T. L. Miller and Johnson (196613) reported that the mixed culture of C. intermedia and C. Zipolytica did utilize the n-alkanes present in several different gas oil fractions. Cell yields were in the range of 70-90% based on n-alkanes oxidized, but were about 10% based on the weight of gas oil used. The cell nitrogen was high, about 10% but this resulted from using organic solvents for the harvest of cells. The solvents extracted part of the cell lipids. Another study of yeast growth on gas oil fractions was made by Dos-

MICROBIAL SOURCES OF PROTEIN

99

talek et ul. (1968a). They investigated the growth of C . lipolyticti on three different gas oils containing various amounts of n-alkanes. Cell yields were linearly related to n-alkanes utilized, but the freezing point change in the gas oil fractions depended on the proportion of short and long chain hydrocarbons. Although the yeast utilized short chain n-alkanes rapidly, these had less effect on the freezing point of the gas oil fraction than the long chain n-alkanes. In contrast with the studies on microbial yields done with batch cultures, Dostalek et a l . (196814 described the continuous cultivation of C. lipolyticn on gas oil. At the optimum dilution rate, a productivity of 1.7 g/liter-hr was obtained. Evans (1968) descrilled two continuous processes being used by the British Petroleum Company Ltd. One is Iiased on n-paraffins as the substrate and the other makes use of a gas oil substrate. Yields were not given but apparently both aseptic and nonaseptic continuous fermentation processes have been used with success. Since methane or natural gas is available in huge quantities and at low cost, some studies have been made of bacterial growth on methane for the purpose of producing useful protein. Wolnak e t al. (1967) found that a Bucillus species could be grown on a mixture of 40% methane, 40%) oxygen, 15% nitrogen, and 5% carbon dioxide plus a solution of mineral salts. The cell concentrations were low, about 1 g of cells per liter after a growth period of approximately 100 hr. Cell protein was approximately 35%,and analyses were made of the amino acid composition and vitamin content of the cells. Hamer et al. (1967) investigated the growth on methane of an original bacterial isolate with the characteristics of Methanomonas (motile, Gram negative, rod-shaped). Taking into account the potential explosiveness of oxygen-methane mixtures, a safe mixture which would support growth was found. However, cell yields under these conditions were quite low. (3) Other substrates. For comparative purposes, some studies of yeast propagation on substrates other than hydrocarbons will be reviewed. The only large-scale, economically feasible yeast propagation process for vitamin and protein production is the sulfite waste liquor treatment. This process was well described by Inskeep e t al. (1951)and the essential features of the process are outlined in the flow diagram of Fig. 2. Detailed yield data were not given by Inskeep e t al. (1951); however they mentioned that 1% dry yeast can be produced with a concurrent decrease in fermentable sugar from the original level of 1.5% to a concentration of 0.2%. This would amount to a cell yield of

HARRY E. SNYDER

100 SPENT LIQUOR FROM PULP MILL 1.5% ?JGAR

BLEND -TANKS

f

WASHING STAGES

RIVER

HEAT EXCHANGER

EFRIGER 50 OR 100 POUND BAGS

FLAKER DRUM DRYERS

FIG.2. A diagram showing flow of materials for production of yeast from sulfite waste liquor. Adapted from Inskeep et al. (1951).

7770 based on fermentable sugars. The yeast used for treating sulfite waste liquor is C . utilis, which is particularly useful in this process because of its ability to use pentoses. Veldhuis (1952) studying growth of C . utilis on citrus press liquor found that cell yields varied from 70 to 26% depending on the initial concentration of sugar. The high cell yield of 70% was obtained with 0.6% initial sugar concentration, and the low yield of 25% occurred with 3.8% of sugar present initially. The amount of sugar used was above 94%for all concentrations tried.

MICROBIAL SOURCES OF PROTEIN

101

Another agricultural waste product, protein waste water from a potato starch plant, was investigated by Reiser (1954) for suitability to support C . utilis growth. T h e net yeast yield was found to be about 45% and cost estimates indicated that yeast could be produced for 5c per pound. Marr (1968) has reviewed the basic principles of microbial growth including the effect of nutrition on growth rate and composition, the effect of nutrient concentration on growth rate, and the yield coefficient. Microbial yields based on batch experiments are not particularly useful in determining optimum conditions for yield during continuous microbial cultivation. If yield is calculated on a weight basis, the use of n-alkanes as substrates will give higher yields than carbohydrates. This is because of the large incorporation of oxygen needed to convert alkanes to the lipid, protein, and carbohydrate that make u p microbial cells. b. Other Nutrients. Besides a source of carbon and energy, microorganisms require nitrogen, phosphorus, and a wide variety of other elements. Usually the species of yeast or bacteria is chosen for its ability to utilize an inorganic source of nitrogen such as ammonium sulfate or liquid ammonia. Since the purpose is to produce protein, it would be futile to work with an organism that required certain amino acids. Phosphate is supplied in a form that will not greatly change the pH of the medium like diammonium or disodium phosphate. Other salts such as potassium chloride and magnesium sulfate may be added for potassium, magnesium, and sulfur requirements. The remaining trace elements - iron, cobalt, manganese, and copper-are usually present as contaminants of the water or of the carbon source in high enough concentration to satisfy microbial growth. If such is not the case, one or more of these trace elements may have to be added to the medium. The sulfite waste liquor fermentation requires the addition of liquid ammonia, diammonium phosphate, and potassium chloride for the growth of C. utilis (Inskeep et ol., 1951). For growth of the mixed culture of C. intermedia and C . lipolyticu on gas oil fractions, T. L. Miller and Johnson (1966b) added 5.0 g NH4H,P04, 0.7 g KH2P04,0.4 g MgS04.7H20,0.1 g NaCI, 0.1 g CaC12.2Hz0,710 p g ZnS04.7H20,670 p g MnS04.H20, 200 p g Fe(NH4),(S04),-6H,0, and 40 p g CUS04-5H,0per liter of medium. c. p H . The hydrogen ion concentration is an important variable since cell yields vary with pH, and in some fermentations the pH can be adjusted to minimize contamination by other microorganisms. T. L. Miller et al. (1964) studied the optimum p H for growth of C. intermedia on n-alkanes and found minimum generation times at pH 4.5

102

HARRY E. S N Y D E R

and 5.0. Yeasts generally are able to grow well at acid pH values, h i t bacteria frequently are inhibited b y a pH of 4.5 to 5.5. Hence, pH can be controlled to minimize bacterial contamination of yeast cultures but the converse is not true. A pH of 6.0 to 8.0 is favorable to both bacteria and yeasts. During the growth of yeast on both carbohydrate and hydrocar1,on sulistrates, the pH decreases. The pH decrease can be attributed to ( 1 ) the production of small amounts of organic acids like succinic acid during growth on carbohydrate, (2) the absorption by the medium of carbon dioxide produced by the cells, or ( 3 )use of basic compounds such a s ammonia hy the cells. Studies with hydrocarbon substrates have not conclusively demonstrated the production of extracellular acid products; however, T. L. Miller et t r l . (1964)and T. L. Miller mrd Johnson (196621) were unable to account for all the carbon utilized as cells and carbon dioxide. Some of the missing carbon may be present a s acidic metabolic products. Since the pH does change during cell growth, most fermentation processes have provision for controlling t h e pH. The sensor for pH is a specially constructed glass electrode that can be sterilized with steam tinder pressure. The response to the sensor is monitored contiirriously on a recorder and is used to maintain the pH automatically at the optimum (Aiba et ( i l . , 1965). d. O x ~ / f i e i iOne . of the most important variables in the production of yeast o r 1)acterial cells is the availability of oxygen. Because of the difference i n metabolism that leads to a great increase in available energy when cells are grown aerol)ically, the supply of oxygen is most important for efficient use of the carlion source. Under anaerobic conditions, some microorganisms are able to continue growing but a portion of the carbohydrate is not oxidized completely. The incompletely oxidized substrate accumulates as ethanol, acetic acid, lactic acid, etc., and is lost a s a potential source of energy for the cells. Resting cells under aerobic conditions completely oxidize the carbon source to carbon dioxide and water, and the energy available is approximately 19 times more than the enerky available anaerobically. Aerobically 38 moles of high energy phosphate is produced per mole o f glucose oxidized, and anaerobically only 2 moles of high energy phosphate is ol)tained per mole of glucose. Aiba et (11. (1965, p. 62) point out that with growing cells, a s compared to resting cells, approximately 5 times as much yeast is obtained aerobically a s anaerobically. T h e decrease in advantage of growing cells coinpared with resting cells is due to incorporation of carbon for biosynthesis in growing cells, carbon that is not oxidized to carbon dioxide and does not provide energy.

MICROBIAL SOURCES OF PROTEIN

103

Several good reviews exist on the theory and practice of supplying oxygen to microorganisms growing in submerged culture (Finn, 1954, 1967; Arnold and Steel, 1958; Lockhart and Squires, 1963; Aiha et ul., 1965). Since the supply of oxygen is particularly important for growth of microorganisms on hydrocarbons, the subjects of aeration and agitation will lie considered for the benefit of those food technologists and microbiologists who are not usually concerned with the mass transfer of oxygen. ( 1 ) Acrntion. As shown in Fig. 3 oxygen passes through a series of resistances in going from air to the sites of consumption in the cells. The greatest resistance on the supply side is the stagnant liquid film acljjacent to an air bubble, I/&. Oxygen must diffuse through this film, the thickness of which can lie miniinizecl h i t not eliminated by agitation. On the demand side the greatest resistance is apparently within the microbial cell. Finn ( 1954) calculated an oxygen concentration d r o p of only 2 x 1 0 - ~rnmole across the liquid film immediately adjacent to the cell, and Borkowski and Johnson (1967) experimentally determined the oxygen concentration change ;icross the film to he 1.3 x ni m 01 e . If the oxygen supply to respiring microorganisms is continually increased, a critical concentration, Crri,,will be reached, and no further increase in respiration will take place above the Crrit. Resistance that limits oxygen demand above the Ccritis thought to lie the availability of reduced metabolic intermediates, particularly, NADH, and of ADP (adenosine diphosphate).

--

-+\

n

--. \

AIR BUBBLE

, MEDIUM

FIG.3 . A diagram showing the different resistances eiicountercd b y oxygen ;IS it is transferred from an air bul)l)Ie on the left through the mediuni t o the inicrol)ial cell o n the right.

104

HARRY E. SNYDER

In the region of low oxygen supply where respiration rate depends on the quantity of oxygen, the limiting factor in S . cererjisiae was thought by Winzler (1941) to be saturation of respiratory enzymes. M. J. Johnson (196713) studied C . uti2is with acetate as a substrate and concluded that diffusion of oxygen within the cell was probably the main limiting factor. Regardless of which of the specific resistances is most significant in limiting supply and demand of oxygen, if the cultivation of cells is such that a steady state prevails, the rate of mass transfer of oxygen can be shown to be proportional to the difference in oxygen concentration between the liquid in equilibrium with the gas phase and the bulk liquid. Mathematically,

Na = K , a (C"-Cl)

(1) where Nu = rate of mass transfer of oxygen in mmoles/liter-hr, KI,= an overall conductance of oxygen from the gas phase to the bulk liquid, a = area of contact between gas and liquid, C" = the concentration in mmoles/liter of oxygen in liquid in equilibrium with the gas phase, and CL= the concentration in mmoles/liter of oxygen in bulk liquid. Since K , and a cannot be determined independently, they are treated as a single constant with the units of l/hr. The constant KLahas the properties of a conductance, in that higher values mean more rapid transfer of oxygen. By appropriate techniques (see reviews by Finn, 1954, 1967),KLa can be evaluated for a specific fermentor and is assumed to be an inherent characteristic of the fermentor, although changes in the characteristics of the medium during growth of microorganisms may affect KLu. If a steady state exists, then the rate of supply of oxygen as defined above will also be the rate at which oxygen is consumed by the growing cells. The rate of oxygen consumption can be expressed, as the product of C,, cell concentration in milligrams per milliliter, and Q f O z (the respiration rate in millimoles rather than microliters of oxygen uptake). For steady-state conditions CcQoz = K,a (C*-C,,) (2) The respiration rate can be measured and multiplied by the cell concentration. C" can be calculated from the oxygen concentration in

the supplied gas from

C" =- 1 HmM

Po'

(3)

MICROBIAL SOURCES OF PROTEIN

105

For air measured in atmospheres, pOz is 0.21 and C* is 0.2 mmolesl liter at 3WC, so the proportionality constant, 1/H' is approximately 1 for these specific conditions. C,, can b e measured directly by various polarographic means, and thus all the values in Eq. (2)except K L u can be measured. This method of calculation of KLu depends upon a QO2 value which may be for resting cells rather than for growing cells. A more direct measurement of K L u can be achieved by measuring in the fermentor the rate of change of O2 (and consequently the demand for O2 by a growing culture) at CIAlevels above Ccrit.The direct measurement of CIAcan be made easily since the introduction of membrane oxygen probes. Clark et al. (1953) reported on a unique type of polarographic measurement of oxygen that made use of a membrane covering the cathode. The membrane has characteristics of allowing oxygen to penetrate but not water. When a potential of 0.8 volts is applied to the electrodes, oxygen is reduced, and the current generated is proportional to the concentration of oxygen. The small amount of oxygen soluble in the membrane prevents the accumulation of reduction products at the cathode; the amount of oxygen passing the membrane is proportional to the concentration in the bulk fluid. The membrane probes can be sterilized by steam, and a moderate flow of fluid by the membrane is sufficient for accurate results. Finn (1967) discussed this technique for measuring oxygen in solution. It is possible to measure the variables necessary to calculate K L u without measuring dissolved oxygen, C L . If one measures the total amount of oxygen being used in a fermentation by means of a material balance on the incoming and outgoing gas, then the rate of oxygen utilization can be determined, and if the cell concentration and availability of oxygen is such that very little oxygen is present (C, < Ccrit,CL can be assumed to be zero. T h e rate of oxygen utilization under these circumstances is equal to K L u C', and K,a can be readily calculated. A technique that has been widely used for measuring K L u is the sulfite method (Ecker and Lockhart, 1959). The fermentor is charged with a neutral or slightly alkaline solution of sulfite plus a copper catalyst. Oxygen is rapidly reduced under these conditions so that CL is essentially zero. The rate of oxygen supply to the fluid can be determined by periodically sampling and titrating the remaining sulfite iodometrically. As before, the rate of oxygen supply equals K L u C * , and C" can be calculated based cn the partial pressure of oxygen and its solubility in the sulfite solution. The question of oxygen availability is particularly applicable for

106

HARRY E. SNYDER

cells growing on hydrocarbon. Darlington (1964) and M. J . Johnson (1967a) have pointed out that approximately three times a s much oxygen is required for hydrocarbon a s for carbohydrate oxidation. For a femientor that is supplying oxygen at near maximum capacity, about one-third fewer cells can be produced in a given time. Since oxygen is more soluble in hydrocarbons than in aqueous solution, the medium with hydrocarbon may hold more oxygen, thereby increasing Cu and increasing the availability of oxygen from the supply side. It is not clear if cells growing on hydrocarbon would respond to an increased oxygen supply or if they are limited, as are other cells, by the availability of intermediates such as NADH and ADP. Toxicity of oxygen was reported, particularly for cells growing directly in gas (0.R. Brown and Hugett, 1968),and increasing oxygen availability by using enriched air or pure oxygen does not seem feasible at present. (2) Agitution. Agitation is an important factor in aeration of cells in submerged fermentation for several reasons. It minimizes the stagnant liquid film surrounding air bubliles; it breaks up hul)bles giving an increased air to liquid surface area; and by swirling bubbles it i n creases the path length and time necessary to reach the surface. The act of aeration will provide some agitation to any submerged fermentation but a better arrangement is to introduce the air just tielow a rotating blade or impeller. Also, vertical baffles in the fennentor are useful in providing more oxygen for the cells (increasing K,,cr). An empirical rule relating aeration with agitation is

K , a a(power/volume)".""

(4)

e . Temperature. Any microorganism will grow more rapidly as the temperature is increased, but the limits of temperature tolerance vary. Since heat is generated b y inefficient oxidation of carbohydrate, one of the main considerations in industrial fermentations is to cool the fermentor. When hydrocarbon is the substrate, more oxidation is needed and more heat is produced than with carbohydrate. T o minimize cooling requirements, Mateles et u1. (1967)looked for and found a Bacillus species that will grow well on hexadecane at 65°C. This thermophilic bacterium, if used as a source of single-cell protein, could be cultivated in tropical countries with no extra costs for refrigerated cooling of the fermentor. There are no yeasts that are able to grow at temperatures of 60" to 7WC, but Phaff et ul. (1966) mentioned that Sacchuromyces frugilis grows well at 45°C. Loginova et (11. (1966)isolated and studied strains of S. frugilis that were thermotolerant.

MICROBIAL SOURCES OF PROTEIN

107

f. Cell Iznrcesting. An important aspect of the total process of producing single-cell protein is the cell recovery or harvesting procedure. For an introduction to this subject there is a general review by Freeman (1964).The main techniques, which are discussed by Freeman, are centrifugation, filtration, flocculation, and spray-drying. Other techniques that are being investigated or used on a small scale are flotation, freeze-drying, ion exchange, and electrophoresis. Aiba et al. (1965) discussed the properties of the cells and suspended fluids that affected the sepwration process and gave quantitative relationships relating these properties. Their chapter should be consulted for engineering inform.'1t'ion. In comparing various attributes of yeasts, molds, and bacteria for single-cell protein, M. J. Johnson (19674 pointed out that centrifugation is the technique of choice i n harvesting yeast, and other considerations being equal, yeast would sediment approxiniately 25 times faster than bacteria in a centrifugal field. The velocity of sedimentation of a particle is proportional to the particle diameter squared, so the size of yeast cells is a distinct advantage over bacteria i n economy of harvesting. Wang (19681,) discussed some of the unique problems of recovering cells from hydrocarbon fermentations. If cells are grown in purified nalkanes, there will be very little hydrocarllon residue and a purification fennentor may be sufficient to decrease the residues. For cells that are cultivated on crude gas-oil mixtures a solvent extraction procedure may be needed. 3 . N u t r i t i o n d Value Single-cell protein is a worthwhile concept only if humans benefit from it nutritionally. The emphasis in this review is on protein, and there are several good general references to protein nutrition in addition to the appropriate chapters of nutrition textbooks. The report of the Joint FAO/WIiO Expert Group on Protein Requirements (World IIealth Organization, 1965) is concise and authoritative. An exhaustive study is the two-volume work edited by Munro and Allison (1964). W. D. Brown (1967) wrote a very readable review of some of the current problems and concepts in protein nutrition. An extensive bibliography of the nutritional work done with yeast during the period 1917 and 1949 has been collected by de Diaz (1951). The vitamin content of yeast is an appreciable nutritional asset, and many uses of yeast as a food supplement are \,:wed on the vitamin content rather than protein. Because of the emphasis in this review on protein, only incidental reference to vitamin content will be made,

108

HARRY E. SNYDER

but yeast traditionally has been more important as a vitamin source than as a protein source. a. Composition. To evaluate the nutritional properties of yeast we need to know its composition. Table I gives data for C. utilis grown on sufite liquor. A similar analysis for bacterial cells would yield similar results with perhaps a higher percentage of crude protein but a lower content of vitam ins. The protein content of microorganisms is consistently calculated from the Kjeldahl nitrogen times 6.25. Undoubtedly, this results in an overestimate of the protein since nitrogen from nucleic acids and hexoseamines is included. Stokes (1958) estimated that 8-13% of the yeast nitrogen was from purines and about 4% from pyrimidines. Nevertheless, for comparative purposes the Kjeldahl nitrogen times 6.25 gives a useful estimate of protein content. The gross composition gives a rough idea of the food value of yeast, but to learn about the nutritive value of protein, information on the amounts of essential amino acids is necessary. The essential amino acids for human nutrition are lysine, leucine, isoleucine, valine, phenylalanine, tryptophan, tyrosine, and methionine with cysteine and tyrosine being able to contribute some of the methionine and phenylalanine requirement. For good protein nutrition a person must ingest sufficient essential amino acids in the correct proportion and sufficient nitrogen to allow biosynthesis of the nonessential amino acids. Table I1 compares the essential amino acid content of the protein TABLE I PROXIMATE AND VITAhlIN ANALYSESOF c. # t i / i . S CROWN ON SULFITE WASTE LIQUOR" Cross composition (%) Moisture Ash Phosphorus (as P) Calcium (as Ca) Crude protein ( N X 6.25) Crude fat (with prehydrolysis) Carbohydrate (by difference) Vitamins ( p g l g ) Biotin Folic acid Niacin Pantothenic acid Pyridoxine hydrochloride Riboflavin Thiamine

"Adapted from Inskeep et d.(1951).

6 9 2 1 47 5 27

2 21 417 37 33 45 5

109

MICROBIAL SOURCES OF PROTEIN

A

TABLE I1 AMNO A(:lDS IN ALCAE, A N D WHOLEEM:

COh.1PARISON OF ESSENTIAL

YEAST,

BACTERIA, Grams per 100 g amino acids

Grams per 100 g protein Amino acid

C. utilis"

Chlorella 7-11-05''

Whole egg"

Biicillus"

(Cystine) Isoleucine Leucine Ly s i n e Methionine Phenylalanine Threonine Tryptophane Val i n e (Tyrosine)

1.4 7.9 7.5 8.7 1.8 5.1

-

2.4 6.6 8.8 6.4 3.1 5.8 5.1 1.6 7.3 4.2

0.5 6.1 8.9 6.9 2.7 5.6 4.3

5.5 1.4 6.3 -

3.6 4.0 7.8 2.0 4.8 3.4 1.5 5.8 2.9

-

6.7 4.2

"Recalculated from Inskeep et ul. (1951). bLubitz (1962). 'World Health Organization (1965). dMateles et a / . (1967).

from yeast bacteria, algae, and whole egg. The egg protein is the reference protein recommended by the joint FAO/WHO Expert Group (World Health Organization, 1965). A wide variety of microbial proteins were analyzed for essential amino acids by Anderson and Jackson (1958). The main deficiency of the microbial proteins is sulfurcontaining amino acids. Chiao and Peterson (1953) studied the methionine and cysteine contents of 20 yeasts and found all of them deficient in these two amino acids. They attempted to increase the sulfurcontaining amino acids by increasing the nitrogen content of the medium and by adding cysteine, choline, and threonine but were unsuccessful. Nelson et (11. (1960) studied the methionine content of 271 strains of yeast and found a range of 0.4 to 1.7 g methionine per 16 g nitrogen. 6. Protein Nutritional Measurement. Knowledge about the amino acid composition is useful but has to be combined with knowledge of the biological availability of the protein. For example, microbial cells have protein a s structural components of their cell walls, and this protein is included in amino acid analyses, but frequently microbial cell walls are not digested by animals or humans. Therefore, information is needed on the amount of nitrogen ingested ( I ) , the nitrogen absorbed (A), and the nitrogen retained (B)by an animal or human during a feeding experiment.

110

HARRY E. SNYDER

The nitrogen absorbed (A) can be calculated from the nitrogen ingested (I) as follows: A

= Z - fecal

nitrogen

and the nitrogen retained (B) is given by

B

= A - urinary

nitrogen

The amount of nitrogen lost in the feces or urine should be corrected for the endogenous loss, i.e., the nitrogen being excreted at the end of a test period on a nonprotein diet. The retained nitrogen per unit of absorbed nitrogen B / A is called the biological value of the protein, and the absorbed nitrogen per unit of ingested nitrogen A/Z is called the digestibility of the protein. The net protein utilization (NPU) is the protein retained per unit of protein ingested or B/I. NPU is equivalent to biological value times digestibility:

B A

A Z

B Z

Another measure of biological response to protein is the protein efficiency ratio (PER), the weight gained per unit weight of protein ingested. Usually a set of standard diet conditions are prescribed, such as feeding the protein at a 10% level and comparing PER on the test diet with a standard protein source such as casein. c. Nutritional Studies. Sure and House (1949) reported a digestibility of 91 941,biological value of 66%, and NPU of 60%)for a debittered brewers’ yeast fed at a 5%’ level to rats. Evans (1968) studied yeast grown on hydrocarbons and indicated that the biological value and NPU were quite low. Siipplementation with methionine, however, gave 90%digestibility, a biological value of 89%,and NPU of 80%. Sure (1946) studied PER of brewers’ yeast fed to rats. When 1, 3, and 5%, yeast was substituted for the same quantity of wheat flour, PER of 1.19, 1.36, and 1.37 were found. Much of the earlier nutritional work with yeast was of this type, and it was reviewed by von Loesecke

(1946). More recently, Bressani (1968) wrote an excellent review on nutritional aspects of yeast. Bressani emphasized the nutritional benefits gained from using yeast as a protein supplement. When yeast is mixed in optimal amounts with wheat flour, corn, cottonseed, or sesame, PER increases dramatically. Yeast does not complement soybeans in the same way because both proteins lack sulfur-containing amino acids.

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Yeast must be killed prior to feeding because vial)le yeast is not digested (Hochberg et (il., 1945; Parsons et (if., 1945) and fails to yield the vitamins one would expect. In fact, viahle yeast may absorb vitamins as it passes through the gastrointestinal tract (Kingsley and Parsons, 1947). Although it has not been reported, one would expect that viable yeast could absorb amino acids i n the same way, so the ingestion of live bakers’ yeast is not a recommended practice. Even after killing, the nutritive value of microbial cells can be improved by processing to disrupt the cell wall. Tannenbauin e t ul. (1966)and Tannenbaum (1968) showed that digestibility of microbial protein is greater for cell contents than for whole cells, but the irnprovement is due to unavailable protein in the cell walls. When biological value or NPU was compared for whole and broken cells, there was not a dramatic improvement due to cell disruption. One way to avoid the digestibility problem is to prepare yeast extracts or yeast autolyzates. These products have been used as flavoring adjuncts and nutritional supplements. The Australians, for example, make use of a product that is spread on bread and consists mainly of yeast extract and yeast autolyzate. Farrer (1954) described the nutritive value of yeast extract, and Vosti and Joslyn (1954a,b) described the changes during the process of yeast autolysis. Extracts, hydrolyzates and autolyzates represent successful techniques for making maximum use of the nutritive value of yeast. d . Modi$ccition of Microbial Protein. One of the very interesting aspects of microbial protein is the possibility that protein content of the cells can be changed either by selection of suitable mutants or b y modifying the medium and other growth conditions. Related to changes in total protein is the possibility that the amino acid composition of the protein might be modified to IIetter satisfy human nutritional needs. Recently, the amino acid content of corn was modified genetically to provide ii higher content of lysine than previously (Mertz et d . ,1966),and if such a modification is possible with corn, it would seem certain that micro1)ial protein could be changed. Unfortunately, there is no good evidence that such changes are easy or even possible. Many authors have pointed out that the protein-to-fat ratio of microbial cells can be changed by changing the carbon to nitrogen content of the medium. If the amount of nitrogen is quite low in relation to the carbon source, for example, 0.46 g nitrogen/100 g glucose, the fat content of the yeast, Rhodotorula grcicilis, can amount to a s much a s 50-60% with 1 2 - 1 3 s protein on a diy weight basis (Enebo et (il., 1946). As the amount of nitrogen in the medium increases, the percentage of protein increases and percentage of fat decreases.

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These changes in percent do not reflect absolute changes in both fat and protein. Very probably the percentage changes are due to an accumulation of lipid in mature cells since fat droplets become visible and since the generation times are greatly increased with a low nitrogento-carbon ratio (Enebo et al., 1946). Protein percentages of microbial cells have been found to range widely (Anderson and Jackson, 1958), but there is no known way of increasing the amount of protein above that present in healthy, rapidly dividing cells. For C. utilis or S . cerevisiae the protein content is about 50%. Consequently, changing the constituents of the medium might be useful for increasing yields of lipid but cannot be used for increased protein production. A modification in amino acid composition of microbial cells might appear relatively easy, particularly with our increased understanding of protein synthesis and microbial genetics. But the majority of microbiological protein that is available to human beings is probably enzymatic (Stokes, 1958).The structural protein and amino acids present in the cell wall are thought to be not available to humans. If the usable microbiological protein is mainly enzymatic, it means that large numbers of different protein molecules are supplying the amino acids. This makes it extremely difficult to modify the amino acid composition. A genetic change that eliminates an amino acid or a protein molecule would only affect a very small percentage of the total protein. In contrast, changing enough enzymes to make a substantial change in the amino acid composition would probably mean loss of viability for the cell. For these reasons, it may be easier to modify the storage proteins of seeds or cereals (proteins that do not play a major role in the metabolism and viability of the plant) than to make major changes in the protein composition of microorganisms. e. Toxicity. It is probably as a nutritional supplement rather than as a major source of new food protein that single-cell protein will be successful in the immediate future. Studies on yeast as the sole source of protein, however, led to some interesting nutritional findings that are not completely resolved yet. Nickerson and Brown (1965) reviewed this situation. A discussion of these findings is pertinent to the role of microbiological protein in world food problems. (1) Dietary liver necrosis. Yeast protein is deficient in sulfur-containing amino acids. This fact was known to Hock and Fink (1943a) when they attempted to substitute yeast protein for milk or fish protein in experimental diets for rats. The rats grew less well on the yeast protein than on milk or fish protein, but the deficiency could be overcome by adding cystine to the yeast diet. Rather than being content with the

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growth data, Hock and Fink examined the organs of the animals on the yeast diet. They found that a diet of primarily yeast protein caused liver necrosis in addition to poor growth. And when the yeast diet was supplemented with cystine not only did the weight gains improve but there was no liver necrosis. These preliminary results with small numbers of animals were confirmed later the same year (Hock and Fink, 1943b). The discovery of Hock and Fink that diets containing yeast protein could result in liver necrosis caused concern and confusion for the next 15years. Dietary liver necrosis could be produced by diets low in protein (casein) as shown by GyGrgy and Goldblat (1949). They found that the liver damage took as long as 120 days to develop, and yeast diets (American brewers’ yeast) did not produce liver necrosis. T h e liver damage from low-casein diets could be alleviated by dietary supplements of sulfur-containing amino acids or of a-tocopherol. Schwarz (1944) had found that a-tocopherol would protect against dietary liver necrosis. The role of sulfur-containing amino acids was not clear because Glynn et a2. (1945) found that cystine or methionine from natural products had a protective effect but synthesized D, L-methionine could not protect against dietary liver necrosis. Another point of confusion was the failure of American brewers’ yeast to produce the disease as compared with British bakers’ yeast which caused liver necrosis. Lindan and Work (1951a,b) examined the difference between these two yeasts and attributed it to a lower content of sulfurcontaining amino acids and protein in the British yeast. This conclusion was based, however, on a sulfur analysis that was not very reliable. Some order was brought into this confusion by Schwarz (1951a) who found that primary grown Torula yeast could be used to reproducibly cause a rapid (20 days) appearance of dietary liver necrosis in rats. Using this technique, Schwarz (1951b) found that the difference between the brewers’ yeast and primary yeast could be attributed to a protective factor in the brewers’ yeast. T h e factor was called factor 3 (the other two factors were sulfur-containing amino acids and a-tocopherol) and it was also found in casein (Schwarz, 1952). The next significant development was the discovery that factor 3 was an organic selenium compound (Schwarz and Foltz, 1957). With this information, it was evident that the sulfur-containing amino acids alleviated dietary liver necrosis because they were contaminated with the selenium-containing factor 3, although Schwarz (1962) pointed out that sulfur-containing amino acids may still decrease the need for a-tocopherol in preventing liver damage. Trace

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amounts of selenium compounds may also explain the difference between cystine and methionine in preventing liver necrosis (Glynn et al., 1945). At present there are still questions about the role played by factor 3 and a-tocopherol in preventing dietary liver necrosis, but there should be no question concerning sulfur-containing amino acids or yeast protein in causing the disease. There is no danger in feeding yeast protein if it is properly supplemented with a-tocopherol or selenium compounds or both. The deficiency in sulfur amino acids will result in decreased growth if yeast protein alone is used. But this deficiency can be overcome by supplementing the diet with cystine and methionine. The deficiency in sulfur amino acids does not cause dietary liver necrosis. (2) Uric acid. Another question about toxicity of yeast was raised b y Carter and Phillips (1944) who thought that the high purine and pyrimidine content of yeast might increase the amount of uric acid in the blood. Bunker (1961)indicated that a limit of 15 to 20 g dry yeast as a safe daily intake was probably in error. This amount was calculated on the basis that a given increase of yeast in the diet would cause a proportional increase in blood uric acid. But the increases that have been noted are not proportional to the yeast eaten, particularly with large amounts of yiest. Humphrey (1967)mentioned that 100 g of yeast had been recommended as the maximum safe daily intake based on results of the Germans during World War 11. This amount of yeast would be sufficient to supply all the protein required by a 100-kg man (based on 0.5 g per kilogram daily requirement and 50% protein in the yeast). Hence, what is stated as an upper safe limit probably would never be reached owing to palatability problems. S. A. Miller (1968)though that large nucleic acid to protein ratios in yeast might cause increased uric acid levels in the blood, and that this constituted one of the most important subjects to be investigated. Recently, Waslien et al. (1968)did investigate the influence of pure ribonucleic acid added to human diets. They concluded that 10-20 g of yeast, used a s a supplement in a low-protein diet, would not cause a problem of increased uric acid in blood. ( 3 ) Human intolerance, Most experiments for evaluating toxic problems or nutritional advantages of single-cell proteiii have been done with laboratory animals. In contrast, Waslien et al. (1969)fed Hydrogeizonaonas eutropha and Aerobacter aerogenes to human volunteers and reported adverse effects. Dosages ranging from 6 to 17 g per person per day caused symptoms of vertigo, nausea, vomiting, and diarrhea. Similar or larger doses of the same organisms fed to chim-

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panzees, monkeys, and dogs did not result in these symptoms. The human intolerance to ingested bacteria is important to the concept of single-cell protein and should be confirmed. Oser (1968) raised the interesting point of how one tests for toxicity of a foodstuff when it comprises a major part of the diet. From experience with food additives, it is customary to feed a substance at a wide range of concentrations to determine the level at which toxicity symptoms begin to appear. Then a safety factor of about 100-fold is used to determine the level at which the food additive will be allowed. With a protein it is impossible to feed quantities which become toxic and then put in a 100-fold safety factor. It should be kept in mind also that some of our best protein sources. such as whole egg, contain antinutritional factors like avidin. Hence, absolute guarantees of safety and freedom from toxic factors are difficult if not impossible to achieve. f. Palatability. Some experience has been gained with levels at which human foods may be supplemented in large-scale feeding experiments. An addition of yeast as a vitamin supplement to conventional foods was tolerated well at a level of 6 g per 8-oz serving (Sunderman et al., 1945). More recently, Klapka et al. (1958)reported on the addition of yeast to food served i n a Wisconsin hospital and found that 10 g per person per day was not objectionable. The problem of introduing a new food to people, particularly a new food with a strange flavor and texture, is probably the biggest difficulty in making use of single-cell protein. We have the technology to produce single-cell protein and with sufficient markets the production could be economical. There is no nutritional barrier to utilizing single-cell protein; on the contrary, this food could be of considerable nutritional benefit. But the fact that its flavor, appearance, and texture are new and different could be sufficient to prevent the use of singlecell protein. This shortcoming may be more of a problem in the developing countries where the food is needed than in those countries where much of the money in the food business is made by successfully introducing new food products. Unfortunately, most of the attempts to use single-cell protein have consisted of harvesting cells, killing them b; drying at high temperatures, and feeding. When we consider the technology that is necessary to make a palatable food from wheat, we should not be too surprised that harvesting and drying yeast cells does not transform them into a highly palatable food. This represents a challenge for the food scientist and technologist- to make palatable foods from single-cell protein.

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The most promising immediate answer to the palatability problem is to use single-cell protein as a supplement to conventional foods at levels that are undetected or not objectionable. B. FUNGI It is possible to grow fungal mycelium in submerged culture under conditions similar to those used for the propagation of yeast. Fungi require a carbon source, nitrogen source, oxygen, and certain salts. The mycelium usually forms pellets or clumps in agitated, submerged culture, and it is difficult to supply oxygen to the inner cells of the mycelial clumps (M. J. Johnson, 1967a). Litchfield (1968) recently reviewed the progress in cultivating fungal mycelium and pointed out that yeast is just as good nutritionally but easier to grow. The highly desirable flavors, which make gourmet foods of the fruiting bodies of some fungi, can be produced in submerged culture, but actual fruiting bodies with their characteristic texture are not produced. The mycelium from species of Morchella is being grown as a flavoring material that sells for $3.60 per pound in 100-lb lots (Litchfield, 1968). But this would be of little use as a source of protein. Gray (1966) enthusiastically espoused the growth of fungi on agricultural wastes as a partial solution to world food problems. The familiarity of people with fungal fruiting bodies as a desirable food is a strong point in favor of fungi, but unfortunately fruiting bodies are not produced in culture. At present it appears that yeast grows faster and is better nutritionally than fungal mycelium, and therefore yeast is more likely to be useful as single-cell protein.

C. ALGAE Interest in large-scale cultivation of unicellular algae has gone through several phases during the last 20 years. In the late 1940’s and early 1950’s there was considerable interest in algae as a possible food source, and this interest culminated in the publication of a book that contains much of what is known about algal cultivation (Burlew, 1953). During the same time period, William Oswald and Clarence Golueke of the University of California initiated studies on the growth of algae in sewage oxidation ponds. The aim of this work was to produce oxygen that would speed up the oxidation of sewage. The usefulness

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of the algal cells as a food supplement was also recognized, and research is continuing on this increasingly important subject (Oswald and Golueke, 1968). When the United States became interested in space exploration, algae gained prominence as the most likely candidate for providing gas exchange on long-term manned space flights. Algae could use waste carbon dioxide, provide oxygen, and possibly provide organic nutrients as well. The considerable knowledge gained during this period about algal cultivation is contained mainly in symposia proceedings, e.g., Gafford and Fulton (1962)and McDowell and Leveille ( 1964).

Recently, the idea of using unicellular algae as food has received impetus from the discovery that Africans in the Chad Republic regularly use algae as food (Clement et al., 1967). It is fitting that a lessdeveloped African nation should provide a possible solution to one of the world’s food problems.

1 . Useful Species By far the majority of work on large-scale cultivation of unicellular algae, whether for food or for gas exchange has been done with Chlorella species, particularly Chlorella pyrenoidosa. The reason for this is the relatively rapid growth rate compared with other unicellular algae. Myers (1953)compared growth rate constants for typical bacteria, yeasts, protozoa, and algae. The algae are the slowest growers with doubling times in terms of days rather than hours or minutes. For a special high-temperature strain of Chlorella 7-11-05, doubling times of the order of 2 to 3 hr at 38°C were reported (Sorokin and Myers, 1953; Sorokin, 1967).The high temperature strain of ChloreZZa 7-11-05 has been extensively used for gas exchange and production studies because of its rapid growth rate at 38°C. Of the many other species of unicellular algae, Scenedesmus species are most frequently studied. Oswald and Golueke (1968)reported that S. quadricauda has persisted in a large-scale sewage oxidation pond for 7 yr. This occurred in spite of an initial seeding with Chlorella. According to Bunker (1968), the large-scale algal cultivation being explored b y the Czechoslovakians makes use of S . quadricauda, mainly because of its large and heavy cells. The interest in use of Spirulina (a blue-green alga) as a food source stems from two very practical considerations. First, the large spiral cells are relatively easy to harvest from a pond and can be sun dried. This practice obviates the need for expensive centrifugation for har-

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vesting and for special drying techniques. Second, the people of the Chad Republic have harvested and eaten Spirulinu for centuries hence there is less need for worry about a new food source with possible toxicological complications. Of course, many species of macroscopic algae continue to be used by man. Some of these are used as food or feed directly and others furnish food ingredients. Chase (1941) has compiled an interesting review of the use of algae b y man, particularly in the Far East. More recently, Zaneveld (1959) described the macroscopic algae being used in Southeast Asia. The most extensive algal collection in the United States is at Indiana University; cultures are readily available for a small fee. Starr (1964) has listed the species available and a supplemental list has been issued by the Indiana University Collection (Department of Botany, Bloomington, Indiana 47401).

2. Conditions for Propagation Much less experience has been gained with the mass culture of algae than with bacteria and yeast. Furthermore, the cultural conditions for photosynthetic autotrophic organisms are quite different than for heterotrophic organisms. For example, the use of light as an energy source has sonie unique economic aspects. Oswald and Golueke (1968)pointed out that the use of sunlight as an energy source is the only practicable means of mass culture of algae at present. With electricity costing at least l d k W h r and the yield of algae being 1-3 g/kW, the cost of algae would be prohibitive when grown under artificial illumination. Still there are possibilities for increasing the efficiency of the electrical conversion to light and for more efficient use of light by the algae. Because of its uniqueness in algae production, light will be discussed first. u . Light. ( 1 ) Artificiul illuminution. Strictly speaking the term light refers to that part of the electromagnetic spectrum to which our eyes are sensitive. This region of the spectrum is also responsible for photosynthesis (because the same kinds of electronic transitions are involved in vision and photosynthesis); hence energy measurements have often been made in psychophysical units of lumens and candles rather than in strictly physical units of jouleslsec (watts) or ergs/sec. The great number of different units involved can be confusing but usually either illuminance units [foot-candles or lumens per square meter (lux)], or irradiance units (watts per square centimeter or calories per minute per square centimeter) are used. Neither system takes

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into account the wavelength distribution of the radiation. In a series of papers on gas exchange by algae (Ammann and Lynch, 1965, 1966, 1967; Ammann and Fraser-Smith, 1968),use was made of the unique units of quanta per 10 nmeters per second per centimeter of lamp length in the 400- to 700-nmeter range. The efficiency of utilization of light by algae is a question of interest in deciding whether algae can be more useful to us as food providers than higher plants. Myers (1953)described the light intensity, I,, that saturates the photosynthetic machinery of the cells. T h e growth rate constant for algae continued to increase with increases in light intensity until the value of I , was reached. Further increases in intensity did not affect the growth rate. The actual value for I , was approximately 400 foot-candles. This light intensity is very much less than the 10,000 foot-candles that roughly corresponds to sunlight. The relatively low values of I , account for the fact that algae grown in dim light in the laboratory can be shown to be more efficient than algae grown in sunlight (Myers and Graham, 1959;Wassink et al., 1953). A possible technique for increasing efficiency of utilization of sunlight is based on the discovery of Kok (1953) that algal cells utilize light more efficiently when exposed to flashing light rather than continuous illumination (with light to dark time periods of the order of 1:10).Theoretically, it may be possible by creating strong turbulence to expose algal gels in a dense culture intermittently to continuous illumination and thereby create a flashing light effect. C. K. Powell et al. (1965)after a careful study of the possibility of moving algal cells in this way concluded that excessive power would be required for any efficiency gain that might be achieved. Another technique that has been explored for making use of the high light intensities available from sunlight, is the use of light diffiisers. By exposing the bases of cones to direct sunlight and exposing algae to the sides of the cones a large increase in area irradiated accompanied by a decrease in irradiance can be achieved. Some experimental work has been done with this idea, and Myers and Graham (1961)found a %fold increase in cell yield of C . ellipsoidea by using diffusers and irradiances comparable to sunlight but provided by artificial illumination for better control. Algae are not unique in being more efficient in dim light than full sunlight. Higher plants respond in the same way, hence the chloroplasts of algae and higher plants are essentially similar in their response to light. With algae, however, it is possible to achieve a density of cells that cannot be achieved with higher plants. Since the yield of algae depends upon the concentration of cells and the growth rate

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constant, increasing the concentration should increase the yield. This works only to a point, then further increase in concentration of cells greatly decreases the available light; if the light intensity falls below I,, the growth rate decreases. Also, dense cultures affect the chlorophyll content of the cells. As the cell concentration increases, the chlorophyll content increases, which still further decreases the availability of light (Myers and Graham, 1959).This secondary effect may be related to the bleaching of algae that occurs when cells are exposed to extremely intense light (Tamiya et aZ., 1953). It appears that algal cells respond to very low light intensities by manufacturing more chlorophyll, and they respond to very high light intensities by inhibiting chlorophyll synthesis. The increase in yield with increasing cell concentration followed by decreased light availability as cell concentration increases results in an optimum cell concentration for maximum yield. The optimum concentration for cell yield will be something less than the maximum cell concentration that can be achieved in algal culture. Working with high intensity light, Matthern and Koch (1964) and Matthern (1966) achieved cell concentrations of 24 g/liter on a dry weight basis. Fluorescent lights with intensities of 52,000 lumens were used to cultivate ChZoreZZa 7-11-05in a continuous system. But at high cell densities the yields of cells in grams per day were decreased (Matthern and Koch, 1964). Very high intensity lights, 200,000 lumens, were also used (Matthern, 1966) but on a yield basis there seems to be no reason to attempt to achieve very high cell densities. Prokop and Ricica (1968a,b) recently made an extensive study of yields of ChZoreZZa 7-11-05 in batch and continuous cultivators under artificial illumination. They achieved yields of 65-163 g per square meter per day, which is quite high, but the culture vessels were only 2-3 liters in volume and vigorous agitation was used. In contrast to the results of Myers and Graham (1959), Prokop and Ricica (1968b) found that chlorophyll content peaks at a cell concentration of 0.5 to 1g/liter and slightly decreases as cell concentration increases. (2) Sunlight. As already mentioned, the intensity of sunlight is far above the saturation intensity for algae. Consequently, it would be useful if the sunlight could be distributed throughout a deep culture either by vigorous stirring or by light difhsors. Mayer et al. (1964) experimented with both techniques and found stirring to be helpful in increasing yields in a deep (one meter) 2000-liter culture vessel. They also obtained increased yields with light diffusers but only under subdued daylight and not under bright sun. The 2000-liter culture apparently operated to select for particular species of algae under

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specific conditions of operation. For example, the appearance and continuity of C . pyrenoidosa as a predominant species was attributed to a large value of [somewhere above 2000 foot-candles according to Mayer et a1. (1964)l.This points out a property of light intensity, selection for specific algal species, that is not related directly to yield, but which may be extremely important in large scale algal cultivation using open cultures in sunlight. During experiments with growth of algae in sewage oxidation ponds, Oswald and Golueke (1968) reported a selection for S. quadricauda although the conditions responsible for selection were given as decreased susceptibility to rotifers rather than an effect of light intensity. Most experiments on the utilization of sunlight by algae have attempted to make best use of the sunlight by using thin layers of algal suspension. Tamiya et al. (1953) used glass tubing 3 cm in diameter for large-scale (40-liter) cultivation of algae under sunlight. They also established an inverse relationship between culture depth and growth rate of algae under artificial illumination. This relationship emphasizes the importance to cell yields of surface area exposed to light rather than volume of the culture. The report on pilot plant studies by Arthur D. Little, Inc. (Anonymous, 1953) cited yields in the range of 2-11 g per square meter per day for growth in sunlight with cultures that were 2-3 inches deep. These yields are less than for the deep culture studied b y Mayer et al. (1964)who obtained yields of 13 g per square meter per day. I n attempting to evaluate the growth of algae in sunlight for the purpose of gas exchange, Gafford and Fulton (1962) found that cell yields were independent of cell volume but did depend on the area exposed for 1-, 2-, and 4-cm depths of cultures. Their culture vessel was a hemispherical dome with an aluminum reflector on the inside. b. Nutrients. ( 1 ) Carbon dioxide. The carbon source for photosynthetic algae is carbon dioxide. Apparently, some differences exist between genera of algae in utilization of bicarbonate. Osterlund (1950) reported that Scenedesmus quadricauda would utilize bicarbonate but Chlorella pyrenoidosa would not. The amounts of carbon dioxide supplied are not extremely critical for the algae but may be critical economically. Myers (1953) indicated that 0.1 volume per cent was sufficient to saturate algal cells with carbon dioxide and toxicity problems are not encountered until the concentration is above 5%. Consequently, most laboratory experiments use 5% to obtain maximum transfer from the gas phase if there is a rapid uptake of carbon dioxide. With large-scale mass culture of algae the concentration of

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carbon dioxide would be an economic factor, and further information would be needed to determine the optimum carbon dioxide concentration from a cost-yield consideration. Carbonic acid has pK’s at pH 6.4 and pH 10.3 so normally very little carbonate ion would be present. As free carbon dioxide is bubbled through an algal culture, it will react with free base to form bicarbonate and will also dissolve. The total available carbon dioxide will depend therefore upon the pH of the medium as well as the rate at which carbon dioxide is bubbled through the culture medium. If any buffer exists to maintain the pH, then larger amounts of carbon dioxide will be retained and presumably be available to algae as a carbon source (at least to those species that can use bicarbonate). This result of increased carbon dioxide availability would be comparable to the trapping of carbon dioxide by buffers in the supernatant fluid from respiring algal cells (Sorokin and Mitrofanov, 1966). Mayer et aZ. (1964) found that carbon dioxide added intermittently to a 2000-liter culture vessel served just as well as continuous carbon dioxide addition, so the medium apparently has considerable carbon dioxide storage capacity. In the algae-production units advocated by Oswald and Golueke (1968) there is no need for carbon dioxide addition because sewage digestion is going on continuously at the bottom of the oxidation ponds. Bacterial decomposition of the sewage leads to slow but continuous production of carbon dioxide for utilization by the algae. Sorokin (1962) investigated the influence of carbon dioxide concentration on synchronously dividing ChZoreZZa 7-11-05. Measuring further cell division in the dark, after original cultivation in light, Sorokin found inhibition of cell division with carbon dioxide concentrations of 5 % and above. Some inhibition was found with 1% carbon dioxide. Pipes (1962) found that continuous cultures of algae could be regulated by the carbon dioxide being supplied. There is nothing to be gained, however, in making carbon dioxide limiting as compared with cultures limited by light. About 0.03%carbon dioxide has been found sufficient to support maximum growth. (2) Other nutrients. The most important major nutrient other than carbon is nitrogen (oxygen and hydrogen are major constituents of the algal cell but readily available from water through photosynthesis). Nitrogen sources most commonly used are nitrates, urea, or ammonium salts. Krauss (1953) showed that nitrate is rapidly taken up by Scenedesmus in relation to other major nutrients such as potassium, magnesium, phosphorus, and sulfur. As nitrate is used, the pH of the culture medium increases. If ammonium salts are used for nitrogen,

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the pH drops as the algae grow, and if urea is used, the pH increases slightly (E. A. Davis et al., 1953). There is no clear-cut advantage for any one of the several possible inorganic nitrogen sources except price. The other major mineral nutrients are supplied by readily available salts. Iron can be easily precipitated and thereby not be available to the growing algae. The incorporation of a chelating agent such as ethylene diaminetetraacetic acid (EDTA) has been recommended to keep iron in solution, but Eyster (1962) pointed out that EDTA may increase the need for zinc, manganese, and calcium in nutrient media for Chlorella. The need for iron by photosynthetic plants has been known for a long time and has been generally ascribed to the biosynthesis of cytochromes. The direct role of ferredoxin in photosynthesis is now better understood, and undoubtedly one of the main purposes of iron in algal nutrition is to aid in ferredoxin production. The trace elements required by algae for photosynthesis have been reviewed by Eyster (1962).For physiological experiments a complete trace element mixture such as used by Arnon (1938) is frequently added to the medium. For large-scale culture, the algae would probably have to depend upon the chance contamination of added nutrients and of water for trace elements. The ability of alae to accumulate inorganic ions was discussed by Myers (1953).Cells grown on Knop’s solution would have a typical ash content of 5% of their dry weight or 1.25%of wet weight. Hence, one liter of cells would contain 12.5 g of salts in comparison to 3.75 g of salts in the original medium. Some media for algal growth (Starr, 1964) include a soil extract. This medium adjunct was advocated by Pringsheim (1936), and Krauss (1953)pointed out that the most likely function of the soil extract is to provide iron chelation. Also, there is the possibility that some hormonelike compounds might be provided by the extract. c. Temperature. Tamiya et al. (1953) found that at high temperatures more growth response was obtained from an increase in the light intensity than at low temperatures. A related effect of temperature was a depression of growth due to combined low temperature (7°C) and high light intensity. The depression was related to fading or bleaching of the algal cells under these conditions. As the temperature was raised, the light intensity necessary to bleach the cells also increased. Another effect of temperature is on the enhancement of photosynthesis by flashing lights. Kok (1953) reported that as temperature decreased the relative efficiency gain from a flashing light source was less. Both of these temperature effects favor growth at high temperatures.

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Still another factor that favors high temperature cultivation of algae is the decreased need for cooling. Illuminated bodies of water heat up due to infrared radiation, and cooling is needed for temperature control. If the algae can grow at higher temperatures, the need for cooling is diminished. Most algae give optimum growth and yields at about 25°C so the discovery of a strain of Chlorella by Sorokin and Myers (1953) with a temperature optimum at 38°C gave rise to hopes that some of the problems of algal cultivation would be solved to the extent that mass cultivation of algae would be economically feasible. The Sorokin and Myers strain of Chlorella was given the number 7-1105 and has been used extensively for algal physiology experiments, for gas-exchange data, and for mass cultivation of algae. Other high temperature strains have also been found (Tamiya, 1957; Sorokin,

1967). Unfortunately, the earlier expectations for high yields from temperature-tolerant strains have not been fulfilled. Since yields depend more upon efficiency of light utilization than upon specific growth rate, the high growth rate constants of the temperature-tolerant strains are not a particular advantage (Myers and Graham, 1961).Also, an increased saturation intensity (I,) was anticipated for the high temperature strains but Myers and Graham (1959)pointed out that the high I, depends upon growth conditions and is achieved when dense cell cultures are exposed to the irradiance of sunlight. In any large-scale, outdoor algal cultivation such as in sewage oxidation ponds, light intensity and temperature would not be controllable directly. Some control may be exerted, however, by the frequency of harvest thereby controlling the amount of light penetrating into the depths of the oxidation pond.

3. Nutritional Value Fewer nutritional experiments have been done with algae than with yeast. But information exists from the long-term usage of macroscopic algae, and now from the Africans’ experience with Spirulina, that certain algae may be consumed with no ill effects. There is some information in the literature, however, about toxic algae. Gorham (1964) investigated two blue-green algae, species of Microcystis and Anabaena, that can cause sickness and death. The toxin of Microcystis was found to be a cylic peptide, but the toxin produced by Anabaena was not identified. Bernstein and Safferman (1966) showed that hypersensitive people will display an allergic reaction to extracts from unicellular algae, but

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there is no indication that allergies to algae are any worse than to other biological materials. Table I11 shows the gross composition of Chlorella 7-11-05 and the vitamin content. The gross composition can be changed by changing the culture conditions (Milner, 1948), but the data of Table I11 are typical and are surprisingly close to the composition of yeast in Table I. Vitamin contents of the two microorganisms show more pronounced differences. The protein content, as measured by Kjeldahl nitrogen times 6.25, contains nonprotein nitrogen and only about 80% of the amount shown would be true protein (Leveille et ul., 1962a). Protein is the component of most interest nutritionally, and the amino acid composition is shown in Table 11. Algal protein contains all the essential amino acids, but as with yeast and bacteria, it is low in methionine. Animal feeding experiments give better information on the nutritional adequacy of algal protein than do chemical analyses. Unfortunately, in the data that follow, the source of the algae and the cultural conditions are so ambiguous that comparisons between experiments are not valid. Several investigators have used algae from sewage treatment ponds for nutritive evaluation. Hintz et al. (1966)fed sheep, cattle, and pigs with rations containing CIilorella species, Scenedesmus obliquus, and Scenedesmus quadricauda. The mixture of several species resulted from harvests at different seasons from the sewage oxidation ponds described by Oswald et al. (1959).The ruminants digested the algal protein more readily than did pigs. Sheep showed a calculated digestTABLE 111 AND VITAMIN ANALYSISOF DRIED PROXIMATE AND POWDERED C ~ d O r e h 7-1 1-05" Crosv compositioti (%) Protein ( N X 6.25) Crude fat Carbohydrate Ash Moisture Crude fiber Vitamins ( p g l g ) Ascorbic acid p-Carotene Pantothenic acid Pyridoxine Thiamine "From Lubitz (1962).

55.5 7.5 17.8 8.25 7.0 3.1 14.6 50.2 11.2 3.0 7.7

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ibility of algal protein of 73%,and the digestibility was 74%for cattle. Pigs, in contrast, had a calculated digestibility of 5345% when algal protein was fed at 6 and 10%levels. Hence, the ruminants have some advantage over nonruminants in obtaining protein from algae. The algal rations were pelleted with hay or barley to insure consumption. If simple mixtures were used, the animals sorted the feed and rejected the algae. The authors concluded that sewage grown algae could be a valuable animal feed supplement due to the protein, p-carotene, and min era1s . A mixture of algal species from the same source was used by Cook et al. (1963)to evaluate PER in rats. In this study, thick gruels prepared from cereals plus nonfat dry milk were mixed with algae in various amounts, and the PER were determined. Also, various baked goods were prepared with algae as an ingredient and were evaluated for PER. Gruel-like mixtures containing oatmeal, cracked wheat, nonfat dry milk, and 55, 44, or 27% algae had PER that did not significantly differ from 2.5, the PER for 100%nonfat dry milk. The algae alone had a PER of 1.69 and a digestibility of 65%. Boiling the algae for 30 min improved the PER and digestibility but autoclaving for 30 min at 120°C significantly decreased PER and digestibility (Cook, 1962). Recently Erchul and Isenberg (1968) evaluated seven different samples of sewage grown algae. The species composition of each sample varied, but Chlorella and Scenedesmus were the predominant genera. Rats were the test animals, and diets provided algae at a level of 10% (based on 65% digestibility). The PER ranged from 0.68 to 1.98 for the seven different samples, and digestibility ranged from 51 to 75%. The variation in PER was due mainly to the differences in digestibility, but reasons for the variation in digestibility were not known. One factor that has been cited as complicating the evaluation of algae grown in open oxidation ponds is the presence of contaminating bacteria. Vanderveen et al. (1962)compared sterile Chlorella 7-11-05 grown heterotrophically and autotrophically with a contaminated culture of the same species. The sterile algae provided higher average weight gains and greater digestibility of energy and protein than contaminated algae, but the differences between sterile autotrophically grown Chlorella and the industrially grown Chlorella were small. This study does clearly demonstrate that the nutritional properties of the same species grown heterotrophically and autotrophically are not the same. The data of Cook et al. (1963)and Hintz et al. (1966) were obtained with diets in which algae were used as protein supplements. Leveille

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et al. (1962b) studied the PER and feed efficiencies of three algal sources of protein as the only protein in the diet. The test animals were chicks and rats. The three algal proteins were from (1) a mixture of Clilorella ellipsoideu and Scetiedesmus obliquus produced in outdoor ponds according to the techniques described by Kanazawa et (11. (1958) in Japan; (2) an industrially produced Chlorella p yrenoidosa; and (3) an industrially produced Spoiigiococcum excantricurn. The PER of all three sources were considerably lower than casein or soybean controls. But there was wide variation also between the algal sources. Amino acid supplementation of the diets indicated that methionine was a limiting amino acid in all three algal preparations. Lubitz (1962) studied the suitability of Chlorella 7-11-05 as a protein source for rats and found that rats could grow on Chlorella 7-11-05 as the sole source of protein, fat, and carbohydrate. Histological examination of the tissues of rats fed only Chlorella 7-11-05 and a salt and vitamin mixture indicated no gross histological changes at 21 o/o Chlorellu 7-11-05 but at 92% Chlorella 7-11-05 histological abnormalities were found in the pancreas and salivary glands. Since a large part of the nutritional inadequacy of algae appears to be related to low digestibility, Shefner et al. (1962) expeTimented with enzyme preparations that might aid in digestion. They found that purified single enzymes were of little use in attacking algal cell walls, but crude mixtures of enzymes such as those derived from the snail Helix pomatia could yield soluble, utilizable protein and carbohydrate from digestion of Chlorella 7-11-05. There have been some nutritional experiments done with humans on diets containing algal protein. Krauss (1962) summarized the data obtained by the Japanese using algae produced by the open air ponds described by Kanazawa et al. (1958). The same source of algae was used by R. C. Powell et al. (1961) to evaluate reactions of humans to algal food. The algae were given a drastic heat treatment of autoclaving at 160°C for 2 hr to insure freedom from pathogens. Healthy young men were the subjects, and levels of 10 to 500 g per day per person were fed. Even at the 10- to 20-g level, bitter spinachlike flavors were detected, and the algae were found most acceptable when incorporated in ginger bread, chocolate cookies, or chocolate cake. Amounts up to 100 g per day were tolerated by the volunteers, but gastrointestinal symptoms of eructation and flatulence were noted. Amounts larger than 100 g increased gastrointestinal distress, however, two men were able to ingest 500 g of algae per day as the only food source for 2 days. The gastrointestinal symptoms with the 100 g to 500 g per day amounts were severe, and Cook et al. (1963) questioned whether

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the symptoms could have resulted from the drastic heat treatment given to the algae. A prolonged nitrogen balance study was done by Dam et al. (1965) to evaluate algal protein in human diets. The species fed were Scenedesmus obliquus, grown specifically for this study, and Chlorella 7-1105, grown similarly to the Chlorella 7-11-05 of Lubitz (1962). With algae providing 90-95% of the protein in the diet, it was possible to achieve a positive nitrogen balance over a 10-day period. The Scenedesmus obliquus was tested both as the lyophilized green alga and as ethanol-extracted alga. Although the ethanol extraction removed almost all of the green color and bitter taste, the acceptance of ethanolextracted algae was not much better than for the unextracted material. However, ethanol extraction of Chlorella 7-11-05 had better effects, and no adverse comments of nausea, bloated feeling, or bitter taste were made with extracted Chlorella 7-11-05. A high fecal nitrogen and a low digestibility of 58-6870 were found in these experiments. Total amounts of algae consumed per day per person were in the range of 50 to 90 g. In a subsequent study from the same group, Lee et al. (1967) found that use of Chlorella 7-11-05 as a supplementary nitrogen source was far more satisfactory for humans than using algae as the sole nitrogen source. The digestibility increased to 71-75% when only part of the protein in the diet came from algae. With algae as with yeast the best nutritional responses are obtained when single-cell protein is used as supplemental protein. 111. ADDITIONAL RESEARCH NEEDS Single-cell protein is still far from being able to contribute substantially to the world’s food needs. And the potential of rapid and easily controlled protein synthesis remains to be exploited. Some of the problems and the challenges of bringing the concept of single-cell protein to fruition are examined in this section. A.

PALATABILITY AND

DIGESTIBILITY

To serve as a useful food, single-cell protein has to be palatable. The initial attempts to use single-cell protein as food have been to feed the harvested and dried cells directly to men or animals or to incorporate single-cell protein in a conventional food hoping to mask

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the strange and undesirable flavor and texture. If any pretreatment was used, it consisted of steaming or cooking the microbial cells. This approach has not resulted in a palatable product. Single-cell protein at its present stage of development should be considered a raw material from which palatable foods may be made. The harvested and dried cells are analogous to harvested and dried grain, not ready to eat foods but raw materials that will yield palatable foods after appropriate processing. A problem closely related to palatability is digestibility. If a palatable food can be made but is only incompletely digested, so that the carbon and nitrogen compounds that have been produced are again lost, little will have been accomplished. One of the big challenges to the food technologist is to work with single-cell protein as a raw material that requires processing to be fully useful as a palatable and digestible food. The most frequently suggested processing route is to feed single-cell protein to animals, so that conventional animal protein foods can be produced. This may be a feasible and acceptable solution, but inherent in animal feeding is the loss of efficiency that accompanies the introduction of another biological system into the food chain. If the economy of a nation can afford it, animal feeding may be a processing solution. A related possibility would be to introduce an animal system that might be more efficient in converting single-cell protein to animal protein than pigs, cattle, or chickens. For example, the enzyme systems in the gut of the snail H e l i x pomatia are able to digest algal cell walls. Undoubtedly, the snail has this ability because algal cells represent a natural food for the snail. Perhaps algal protein could be converted to snail proteins more efficiently than to pork, beef, or chicken. Furthermore, the large muscular foot of the snail is already a conventional food in some countries, and thus palatability and toxicological problems are greatly decreased. The degree of sophistication that is used for processing single-cell protein will have to depend upon the country or region involved. Because of wide differences in taste preferences and in ability to pay, there will be few, if any, worldwide solutions. It may be feasible in some countries to isolate a protein fraction from microorganisms, and to spin this into a fiber that is eventually incorporated into a meat analog. In other countries, fermentation of a carbohydrate food may serve directly to increase the protein and vitamin content and to result in a new food. Hesseltine (1965) has written a fascinating account of littleknown food fermentations.

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B. A DIFFERENT APPROACHTO SINCLE-CELLPROTEIN Mankind has been successful in utilizing microorganisms in many ways. Some of the obvious uses are alcoholic fermentations, antibiotic production, acid fermentations such as for cheese, sauerkraut, pickle, olive, and sausage production, organic acid production, and amino acid production. In each instance, some specific organic product has been produced by the microorganism and that product has been useful rather than whole cells. Perhaps we should take a clue from this historical precedent and attempt to produce protein as a product of cells rather than as a useful fraction of whole cells. It is well known that bacteria and yeast produce extracellular enzymes although the differentiation between true extracellular enzymes and lysis products needs to be done carefully (Pollock, 1962). Such an extracellular enzyme might conceivably be produced on a large enough scale to serve as a useful protein nutrient for humans. To those who are familiar with extracellular enzymes this proposal will seem fantastic for its lack of appreciation of the small amounts of enzyme produced and of the large amounts of protein required. But the idea is worth serious consideration. Demain (1966) has written an interesting review of the role of regulatory mechanisms in governing the kinds and amounts of metabolic products produced by microorganisms. He pointed out that under some circumstances (Clayton, 1962) it is possible for a bacterium to accumulate 25% of its protein as a single enzyme. At present, protein synthesis in microorganisms is invariably linked to cell division. But as we learn more about metabolic control mechanisms, it may become possible to hold large quantities of cells that are either not dividing or dividing very slowly to maintain the population. If the cells were held in a dense layer with medium flowing by, then the nitrogen and carbon sources might be channeled directly into synthesis and secretion of an extracellular enzyme. The enzyme could be harvested from the culture fluid without disturbing the dense layer of enzyme-producing cells. There are many advantages to this kind of protein production that do not exist with whole cell production. For example, the single enzyme that is produced could be selected on the basis of its amino acid composition, thereby ensuring that the essential amino acid requirements of man would be met. If the proper enzyme cannot be found, it should be possible to modify the amino acid composition of a single extracellular protein to provide the proper balance of amino acids. The modification of amino acid composition of whole cells does not

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seem feasible because of the large number of different proteins involved. There remains an awesome challenge to people of this generation. A challenge to put together in many different possible ways the factors needed to produce protein and to make that protein available to the people who need it. The challenge will and should be met by multiple solutions, and the people who will be best equipped to find the solutions are those who know some engineering, some agriculture, some nutrition, some biochemistry, and some microbiology. In other words -a food technologist.

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TOXINS FROM FISH AND OTHER MARINE ORGANISMS BY PAUL J. SCHEUER Department of Chemistry University of Hawaii, Honolulu, Hawoii I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Toxins from Fishes ........... A. Toxins That Cau B. Tetrodotoxin . . . . . . . . . . . . . C. Pahutoxin.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Other Fish Toxins .......................... 111. Toxins from Shellfish IV. Other Marine Toxins . .......................................... V. Research N e e d s . . . . . . . . . . . . .......................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. INTRODUCTION Sea creatures from virtually all marine phyla have been a source of food for humans from the earliest days of history-as soon as man began to explore his aquatic environment. Although fish and shellfish have always been the principal objects of man’s harvest from the sea, consumption of such diverse organisms as algae, echinoderms, or mammals has not exactly been negligible. Along with the exploration of marine food sources came the recognition that some species were tastier than others and that ingestion of certain other species caused discomfort, illness, or even death. A number of factors in recent years have caused us to focus more sharply on two interrelated problems: (1) making efficient use of the world’s oceans for food production and (2) recognizing those organisms that are toxic to man. Perhaps the most compelling reason has been the rapid growth of the world’s population without a parallel increase in food production. Many economists, sociologists, and political leaders claim this to be the world’s most pressing problem. The divergence of the population and food curves is aggravated by the fact that the highly developed countries, which are capable of efficient ter14 1

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restrial food production, continue to remove large acreages from agricultural utilization to make room for subdivisions, highways, airports, and industrial complexes. Another major factor that is playing a role in calling our attention to marine food resources has been our growing awareness that the oceans-inner space, as some call it-are a poorly explored and poorly understood part of the earth. Still another driving force for our new interest in these problems is the recognition that substances that are toxic, i.e., physiologically active, not only provide tools for an insight into the mechanism of this activity, but also represent potential sources of new drugs. Recent attempts in this country and elsewhere to manufacture a fish protein concentrate in order to help feed the world’s expanding population have directed attention to the question of marine toxins. Clearly it could be a dangerous undertaking of far-reaching consequences if one were to catch fish indiscriminately, process the fish into a meal, and distribute the concentrate without knowing whether any of the processed fish are toxic or whether the toxins survive the processing. Indeed of equal importance with the problem of producing a palatable and inexpensive fish protein concentrate is the problem of producing one that is safe to eat. Existing data on fish and other marine toxins are deplorably incomplete and a great deal of work needs to be done before our knowledge of toxic marine organisms approaches that of terrestrial species - plant and animal. Halstead’s monumental effort to gather in one place all current knowledge on the biology, pharmacology, and chemistry of poisonous and venomous marine animals (1965, 1967) has been an important contribution to this field, but it has also dramatized the many gaps, large and small, which exist in this area. Other general references and recent reviews, which provide background material on one or the other aspects covered in this chapter, include those by Banner (1967), Courville et al. (1958), Kaiser and Michl (1958), Keegan and MacFarlarie (1963),Nigrelli (1960), Nigrelli et al. (1967), Russell (1965, 1967), Russell and Saunders (1967), Scheuer (1964, 1969), and Shilo (1967). This review will attempt to present a picture of the current state of our chemical knowledge of marine toxins. Emphasis will be placed on toxins isolated from fish and shellfish, but toxins from other marine organisms that have been characterized will also be included. Many of these species are used as food and many are implicated in the food chain of the marine organisms themselves, a chain which leads from plankton feeders and other herbivores to the large carnivorous fishes.

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II. TOXINS FROM FISHES

A. TOXINS THATCAUSECICUATERA

Perhaps the greatest potential danger to the production of a safe fish protein concentrate comes from fishes which cause a disease known a s ciguatera. Among the reasons which make ciguatera so important to the seafood processor are the widespread occurrence of ciguateric fishes in tropical and subtropical waters, the implication of many species of fish; sudden outbreaks in areas with no prior history of ciguatera; and the heat stability of the toxins. The term ciguatera is of Spanish origin and was apparently first used by the Spanish conquistadores in Cuba to describe a disease which was caused by ingesting a toxic marine mollusk, Turbo pica, or cigua.” The name later expanded by usage to include intoxications by other marine organisms and is now the accepted term for a specific type of toxicity. One of the first outbreaks of ciguatera was described as occurring in the tropical Pacific in 1606 when the crew sailing with the Spanish explorer Pedro Fernandez Quiros off the coast of the New Hebrides ate infected fish -probably snapper of the family Lutjanidae - and came down with fish poisoning. Perhaps the best-known historical account describes the outbreak of ciguatera intoxication in 1774 when Captain Cook’s crew of the H.M.S. Resolution became ill in the New Hebrides after eating red snapper. The clinical symptoms of ciguatera, which have been described repeatedly in the literature, often present a bizarre combination of neurological and gastrointestinal phenomena. Vomiting and diarrhea, loss of motor ability, tingling of the extremities, and reversal of temperature sensation are mentioned most frequently. A typical ciguatera case may begin some 6 hours after ingestion of toxic fish and may last several days. Few authentic fatalities have been ascribed to ciguatera, but recurring symptoms -even several months after an acute intoxication and even as a result of eating fish that is patently nontoxic-are reported frequently. There exists at the present time no known antidote for ciguatera nor is there a simple rapid field test capable of distinguishing ciguateric from nonciguateric fish. Over the years a great many species of fish have been implicated or at least suspected in connection with ciguatera poisoning. Recently Bagnis (1967) in a careful clinical and epidemiological study made in French Polynesia found that 96% of some 2800 investigated cases could be traced to members of ten families of fishes: Acanthuridae, “

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Serranidae, Scaridae, Lethrinidae, Lutjanidae, Carangidae, Mugilidae, Labridae, Balistidae, and Murenidae. Poisoning b y acanthurids accounted for 65% of all cases cited by Bagnis. It must be borne in mind that in many island groups, where lutjanids, for example, are plentiful, the relative importance of a given family as a bearer of ciguatera will vary. This complex etiology of ciguatera is matched if not surpassed by the intricate and poorly understood ecological aspects of ciguatera. A number of papers have been published that deal with the ecology in various archipelagoes. Recent articles by Helfrich et ul. (1968)and b y Helfrich and Banner (1968), which are concerned largely with ciguatera in the Line Islands, constitute an excellent appraisal of the current state of our knowledge. Thus it may be stated unequivocally that no pelagic fishes have ever been known to cause ciguatera in man or, conversely, that only those species that live in shallow water within 30"latitude north or south of the equatorial reef areas have been implicated. Beyond this important basic observation, which links ciguatera to environmental factors, we find few substantiated facts. It is a reasonable hypothesis that ciguateric fishes acquire the toxin whether intact or as a precursor we do not know- from their food and that it is passed on from fungi or algae or other benthonic organisms via herbivorous fishes to the carnivores. Analysis of fish stomach contents and the fact that in many toxic areas both carnivorous and herbivorous fishes carry ciguatera provide evidence for such a hypothesis, but no toxin has ever been isolated from any ciguateric organism other than carnivorous fishes. The well-documented observations that some areas in a given archipelago are safe while others are not safe, that ciguatera in a given area may suddenly erupt and then gradually decline, and that no seasonal variations have ever been found to occur, lend an aura of credibility to the additional hypothesis that a newly created surface on the reef is the primary factor that disturbs a given ecosystem and leads to ciguatera. A number of outbreaks in the Pacific following World War I1 have variously been ascribed to war-connected dredging, shipwrecks, dumping of war materiel; circumstantial evidence doubtless exists for every outbreak. But these man-made disturbances in the reef can neither be the sole reason nor a sufficient reason for an outbreak of ciguatera: How could they possibly explain the occurrence of the disease in the New Hebrides in the 17th and 18th centuries? Furthermore, many man-made alterations of reef areas in the past 30 years have not resulted in ciguatera. Chemical research directed toward molecular structure determination of ciguatera toxin has been carried out for a number of years in our

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laboratories in collaboration with Professor A. H. Banner who is directing biological and pharmacological studies at the Hawaii Institute of Marine Biology. Except for some earlier work by Hessel (1963)we are apparently the only group working on ciguatera at the present time. Because of the variable biological and geographical parameters described above we have attempted to concentrate on one species of fish from one area. Depending on such logistic reasons as the accessibility of a toxic area and the abundance of fish, we have worked at one time or another with the muscle of the red snapper (Lutjatzus holzur) from Palmyra and Christmas Islands and with the liver of the shark (Cnrchurhinus menisorrah) from Johnston Island. However, the subject of our longest and most concentrated effort has been the muscle and liver of the moray eel (Cymnothoruxjnvanicus) from Johnston Island. Johnston Island is about 1100 km southwest of Hawaii and has been accessible because the U.S. Atomic Energy Commission maintains a base there. Eels are trapped arid returned frozen to the laboratory. Since only very few laboratory animals respond to ciguateric fish quantitatively and reproducibly, routine toxicity screening is carried out by feeding suspected fish flesh to a mongoose (Herpestes mutigo) at a level of 10?h of its body weight (Banner et ul., 1960). The fish receives a toxicity rating according to the reaction of the mongoose, which varies from no effect to death viu intermediate stages which are judged by mild, moderate, or severe loss of motor ability. Eel muscle is extracted in l-kg batches. Each batch is cooked and homogenized with acetone. The homogenate is filtered and the acetone removed. The resulting aqueous residue is extracted with diethyl ether. Further purification of the diethyl ether residue is achieved by multiple column chromatography, twice on silicic acid and once on alumina. The toxic fractions are eluted in chloroform containing some methanol and final purification is achieved by preparative thin-layer chromatography on silica gel G. In contrast to the initial screening of ciguateric fish, to which few animals respond, bioassay of the toxic extracts is possible with a variety of test animals. T h e entire routine extraction and purification procedure in our laboratory is monitored by intraperitoneal (ip) injection in mice as described by Banner et (11. (1961). T h e resulting toxin, which is isolated from the flesh of the moray eel in cu. 10 ppm yield, has a minimum lethal dose (ip injection, mice) of 0.5 mg/kg. It is a transparent, light yellow,viscous, relatively unstable oil that we have named ciguatoxin (Scheuer et d., 1967).Combustion data point to an empirical composition of C,,,H,,NO, for ciguatoxin.

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That the molecule is however at least threefold the size suggested by the empirical formula is indicated by high resolution mass measurement of some degradation products which contain three nitrogen atoms. Hydrolysis of ciguatoxin has led to the identification of glycerol and a series of long chain saturated and unsaturated fatty acids. The nitrogenous moiety of the molecule is even less stable to air and light than intact ciguatoxin. It is positive to Dragendorff and Jaffi. reagents and fails to yield a molecular ion peak in the mass spectrometer. The linkage of the nitrogenous portion to the rest of the molecule is as yet unknown. Ciguatoxin then has some of the features of a typical lipid in addition to an uncommon nitrogenous moiety. Case histories of ciguatera intoxication have long suggested that fish viscera, particularly the liver, are more toxic than the muscle of a ciguateric fish. Helfrich et al. (1968) demonstrated that this is indeed the case. Recent work in our laboratory has further shown that the livers of all Johnston Island eels are toxic regardless of their rating in the mongoose screening test. It is interesting to note that this liver toxin, when purified in a manner essentially parallel to the purification of ciguatoxin, produces the same symptoms in mice as does ciguatoxin, but yields no glycerol and essentially no fatty acids on hydrolysis. Mass spectral data indicate that this toxin from the liver of the moray eel is closely related or identical to the nitrogenous portion of the ciguatoxin molecule. Because of its large size ciguatoxin does not give rise to well-defined infrared (IR) and nuclear magnetic resonance (NMR) spectra. Therefore such diffuse spectra cannot be used as criteria of identity nor can they reveal much unequivocal structural information. These limitations must be kept in mind if one wants to evaluate the following observations. We have in our laboratory isolated the toxins from the flesh of the red snapper (Lutjanus bohar, Line Islands) and from shark livers (Carcharhinus menisorrah, Johnston Island). The toxins from both sources produce typical ciguatera symptoms in mice and the spectra of the toxins - diffiise as they are-exhibit striking similarity. Although this has not been proved, we believe that the molecular structures of the various toxins that cause ciguatera possess at least many common features and may well possess an identical portion responsible for the observed physiological activity. Ciguatera poisoning has been reported from the island of Okinawa in the Ryukyus. Hashimoto and Yasumoto (1965) extracted a toxin from the flesh of a grouper, Epinephelus fuscoguttcztus. The cat, which is the test animal used by the Japanese workers, developed typical ciguatera symptoms -vomiting, diarrhea, loss of motor ability -

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but the toxin showed no evidence of a lipid moiety and could not be extracted with or partitioned into organic solvents. The Japanese workers have named this water-soluble toxin ciguaterin. No additional work has been reported, and what if any structural relationship exists between ciguatoxin and ciguaterin cannot be determined at present. If one discounts the poorly authenticated “origin” of ciguatera, which traces at least the name of the disease to a marine mollusk (vide supra), no organisms other than fishes have until recently been known to cause ciguatera intoxication in humans. A recent report by McFarren et al. (1965) suggests strongly that oysters and clams may also cause ciguatera. An outbreak in 1962 in a few areas on the Florida coast was linked to a bloom of Gymnodinium breve (“red tide”). Although no conclusive evidence was found to establish this connection beyond doubt and although no toxin was isolated from the toxic shellfish, human and laboratory animal symptoms as well as the general characteristics of the toxic extracts paralleled those described for ciguatoxin. No further chemical studies have been reported, and the work that has been described is inconclusive. Nevertheless, the apparent occurrence of ciguatera in shellfish and the apparent link to a seasonal “red tide” phenomenon, has introduced two new parameters into a problem already richly endowed with poorly defined variables. It should be noted that this is not the only case of ciguatera poisoning that has been reported from the Florida coast. Larson and Rivas (1965) described a laboratory study of a toxic barracuda ( S p h y raena barracuda), which had poisoned five persons and given them typical ciguatera symptoms. The remaining portions of the fish were subjected to various bioassays that showed that the toxin had indeed typical properties of ciguatera. Hashimoto (1956) described a similar outbreak caused by toxic barracuda (Sphyraena picuda) which had brought about ciguatera symptoms in some thirty persons in Tokyo. A laboratory investigation was carried out on the uneaten portion of the fish and here, too, the general properties of the toxin and the results of bioassays were those we have since recognized as typical of ciguatera.

B. TETRODOTOXIN Virtually every aspect of puffer fish intoxication stands in sharp contrast to the corresponding results of ciguatera poisoning. The toxin, with a curious exception that will be mentioned below, is produced by members of a single family of fishes, the Tetraodontidae, and only

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the viscera, particularly the ovaries and the liver, contain the toxin. Production of the toxin shows marked seasonal variation. Fatalities from eating puffer are common in spite of elaborate precautions. The molecular structure of tetrodotoxin is known. Following the complete structural elucidation of tetrodotoxin, which was disclosed in the spring of 1964 at the International Symposium on the Chemistry of Natural Products in Kyoto, Japan, detailed papers were published by the several groups who were active in the field: Tsuda (1966), Goto et al. (1965), Woodward (1964), and Mosher et al. (1964). Chinese medical books published 2000 years ago tell of poisoning by puffer (fugu, globe, swell) fish, and puffer fish bones have been found in Japanese burial places that are 1500 years old. The Japanese literature of some 400 years ago describes puffer intoxication. Puffer has been considered a culinary delicacy in Japan, where some restaurants specialize in fugu dishes. In Japan fugu i s so well recognized as a public health hazard that only licensed personnel may handle the fish in the market and remove the viscera. In spite of these precautions 500 intoxications were recorded in Japan in 1958-1959, of which 294 proved fatal (Tsuda, 1966). A pure toxin, first named spheroidine, was isolated by Yokoo in 1948 from the puffer Sphaeroides rubripes. Much of the pioneering work in the structural elucidation of this toxin was carried out by Professor Tsuda at the University of Tokyo. Professor Hirata at Nagoya University and Professor Woodward at Harvard carried on independent investigations in the late 1950’s. Professor Mosher at Stanford also arrived at the structure of tetrodotoxin, but by a route that did not involve the use of puffer fish. Tetrodotoxin is a colorless crystalline substance, which is virtually insoluble in all but acidic media. It is weakly basic and has the composition C,,H,,N30,. The molecule is small (mol. wt. 319), but possesses the remarkable feature that the number of oxygen plus nitrogen atoms equals the number of carbons. An outline of the structural elucidation follows. Woodward (1964) was able to demonstrate that the three nitrogen atoms of tetrodotoxin are present in the molecule as a guanidine moiety by isolating guanidine as the picrate following vigorous oxidation of the toxin with aqueous sodium permanganate at 75°C.A variety of drastic degradations - warm aqueous sodium hydroxide, pyridine acetic anhydride followed by vacuum pyrolysis, phosphorus hydrogen iodide followed by potassium ferricyanide, and concentrated sulfuric acid - yielded closely related quinazoline derivatives of structute (I), where the nature of R and R’ depends on the exact mode of degrada-

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tion. These key compounds indicated strongly that six of the eleven carbon atoms of the toxin are contained in a carbocyclic ring.

OR (1)

In spite of its guanidine function tetrodotoxin is only weakly basic (pK,, 8.5) and many attempts to prepare crystalline salts failed. However, treatment of the toxin with 0.2 N hydrogen chloride in methanol-acetone did yield a crystalline O-methyl-Of, 0”-isopropylidenetetrodotoxin hydrochloride monohydrate, to which structure (11) was assigned on the basis of a complete X-ray crystallographic analysis (Woodward, 1964).If one subtracts from the molecular formula of (11) (ClsH,,,N,O,.HCI) the elements of methanol, acetone, and hydrogen chloride and adds the two molecules of water, which are eliminated in the methyl ether and acetonide formation, one arrives at CllH,,N30H,the precise formula of tetrodotoxin. Comparison of the NMR spectra of the two compounds further confirms their close structural relationship. The two compounds, however, differ in one important aspect. The hydrochloride is a lactone with appropriate IR absorption at 1751 cm-’, while the toxin itself lacks a lactonic infrared band. On the other hand, the IR bands assigned to the guanidine portion (1658, 1605 cm-l) remain unchanged in the two compounds, thus demonstrating that the hydrochloride cannot be a guanidinium salt. The basicity of tetrodotoxin (pK,, 8.5) is far too weak to be originating from the guanidine moiety. This fact, coupled with the observation that the pK,, of the hydrochloride increased to 9.2 in aqueous dioxane, led to the conclusion that the basicity of tetrodotoxin must be due to its zwitterionic nature and that one of the hydtoxyl groups is being titrated when the pK,, is measured. Increased pK,, is characteristic of hydroxyl ionization when one proceeds from a medium of high to one of low dielectric constant.

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The ensuing question, which of the hydroxyl groups in tetrodotoxin might be sufficiently acidic to furnish a proton to nitrogen, was solved by analysis of the NMR spectrum of heptaacetylanhydrotetrodotoxin. If the unmethylated precursor of (11)were to undergo acetylation, the product would exhibit three characteristic resonances in the NMR arising from protons on carbons which also bear acyloxy groups, viz., carbons 5,7, and 8. In fact, only one such resonance is present in the NMR spectrum of the heptaacetyl compound. This forces the conclusion that two of these three groups cannot be present as free hydroxyl groups in tetrodotoxin, but must be combined in a new entity. If one of the hydroxyl groups combines with the lactone to form a hemilactal,

Alcohol

Lactone

Hemilactal

only one characteristic proton resonance remains. Double resonance experiments proved that it is the carbon-5 hydroxyl group in tetrodotoxin that is part of the hemilactal (or a two-thirds orthoester) function. This last consideration then establishes structure (111) as the structure of tetrodotoxin. Once again this natural product revealed a complex function, a hemilactal, which had not previously been encountered in nature or in synthesis.

In the spring of 1964 when this complex structure had been elucidated, there remained a question whether tetrodotoxin is monomeric of composition C,,H,,N,O, or whether it is a 22-carbon dimer. This question has been decided unambiguously in favor of the monomeric structure by two approaches. Woodward and Gougoutas (1964) SUCceeded in preparing crystals of tetrodotoxin which were suitable for single crystal X-ray diffraction studies. Measurement of the unit cell

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dimensions and the density of the crystals, and consideration of symmetry requirements led to the unambiguous conclusion that crystalline tetrodotoxin is monomeric and contains two molecules per unit cell. The monomeric nature of tetrodotoxin in solution was ascertained by Goto e t ul. (1965) through a careful analysis of its titration curve. No synthesis of tetrodotoxin has been reported. The natural product is available for pharmacological research from puffer ovaries by a procedure described by Goto et d.(1965),which yields 1-2 g of crystalline precipitate from 100 kg of raw material. By a remarkable coincidence - hinted at above - Mosher et ul. (1964)isolated a neurotoxin, tarichatoxin, from egg clusters of the California newt Turichu torosu, an organism that is biologically totally unrelated to puffers. As Mosher’s structural studies proceeded, considerable similarities between tarichatoxin and tetrodotoxin became apparent and complete identity of the two toxins was demonstrated. This unusual organic molecule is thus derived from two widely divergent biological sources and the question of its biosynthesis is an intriguing one indeed. C. PAHUTOXIN Pahutoxin represents another entirely different picture from either tetrodotoxin or ciguatera. Ciguatera poisoning constitutes, among fish poisons, the greatest public health problem and the gravest potential danger to a safe fish protein concentrate despite its rarely fatal character because it is biologically and geographically so diffuse and because our knowledge is fragmentary. Tetrodotoxin, on the other hand, is a powerful poison, but is a threat only within narrow biological and geographical boundaries. We also know its structure and are beginning to understand its pharmacological mechanism of action. In contrast, pahutoxin is only mildly toxic to warm-blooded animals and there is no evidence of any human intoxication from eating boxfishes. A discussion of pahutoxin is included here because it is an interesting metabolite produced by a fish and because it adds to our understanding of fish toxins. Members of the trunkfish family (Ostraciontidae) are small, colorful, slow-moving reef fishes, resembling a miniature puffer fish, which is not surprising since puffers and trunkfishes belong to the order Plectognathi. There are reports in the literature that trunkfishes are eaten in some parts of the Pacific and there are also some references to their toxic nature. Herald (1961) reports that the trunkfishes are con-

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sidered a delicate food in some parts of the Pacific and are roasted in their carapaces much the way chestnuts are roasted. Malo (1898) in his treatise on ancient Hawaiian culture states that the pahu (boxfish) was kapu to Hawaiian women and eaten only by men. The emphasis here should be on the fact that the boxfishes were eaten and not that they were a sexually restricted food as, for example, the banana. According to Buddle (1930) boxfishes and cowfishes are eaten and even esteemed by the inhabitants of Singapore. He further states, however, that human consumption should be prohibited since many of these fishes are poisonous. Brock (1956) first reported the observation that the boxfish Ostracion lentiginosus when placed in an aquarium secreted a substance that rapidly killed other fish in the vicinity. Boylan and Scheuer (1967) isolated the crystalline toxin, named pahutoxin after the Hawaiian name of the fish, determined its molecular structure by degradation and synthesis, and studied some of its physiological activities. This unique metabolite is the choline chloride ester of P-acetoxypalmitic acid (IV). CH:, - (CHJI,

- CH(0Ac) - CH, -

+

CO? - (CH?), - N(CH:j):,CI-

(IV)

The boxfish were caught with a small net and “milked” by being placed in a container of distilled water. The shock of finding themselves in fresh water caused them to release copious quantities of a mucous fluid. The boxfish were then quickly returned to the sea. In order to prevent hydrolysis of the toxin the aqueous solution was immediately extracted with 1-butanol. The toxic butanol solution was purified by column chromatography on silicic acid, which yielded a white amorphous solid. Among many attempts to crystallize the toxin only one, for which no rational explanation occurs to us, was fruitful: passage of the toxin through an anion exchange column that had been treated with picric acid, followed by conventional crystallization from acetone. This procedure yielded long colorless hygroscopic needles, m.p. 74”-75”C, [a],) MeoH 3.05”. Pahutoxin is soluble in water, ethanol, chloroform, hot acetone, and hot ethyl acetate. One adult boxfish yielded up to 60 mg of toxin. Infrared and NMR spectra revealed the presence of quaternary nitrogen, an ester function, and a choline moiety. Weakly basic hydrolysis (1 N sodium bicarbonate at 50°C) led to the isolation and identification of choline chloride, 2-hexadecenoic acid, the choline chloride ester of 2-hexadecenoic acid, and 3-acetoxyhexadecanoic acid. Acid hydrolysis in 2 N methanolic sulfuric acid at 60” furnished only one

+

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product, methyl 3-hydroxyhexadecanoate. In a third hydrolytic procedure, designed to detect volatile fragments and employing trifluoroacetic acid in deuterium oxide at 60°, acetic acid was the only reaction product. As a result of these hydrolytic reactions, structure (IV) emerged as the most likely structure of pahutoxin. However, since combustion data of pahutoxin were variable and confusing, racemic pahutoxin was synthesized as follows. Commercial tetradecanol was oxidized with lead tetraacetate. The resulting tetradecanal was condensed with ethyl bromoacetate in the presence of zinc, leading after alkaline hydrolysis to 3-hydroxyhexadecanoic acid, m.p. 183.5"C. Treatment of this acid with acetic anhydride furnished 3-acetoxyhexadecanoyl chloride and from it esterification with choline chloride yielded racemic (IV) identical with the natural toxin in all respects except melting point and optical rotation. For comparison of biological activity (toxicity and hemolysis) the two lower homologs-the choline chloride esters of 3-acetoxytetx-adecanoic and 3-acetoxydodecanoic acids were also synthesized by the same procedure. The C,, compound proved slightly less and the C,, homolog significantly less active than the C,, pahutoxin. Biological assay did not differentiate between optically active (natural) and racemic (synthetic) pahutoxin. Two structurally related compounds have been isolated from marine organisms, but it has not been determined whether these compounds share with pahutoxin hemolytic activity or toxicity toward fish. One compound is the glycolipid (V) isolated by Nakazawa (1959)from a Japanese oyster. The other (CH:J,CH - (CH,),

- CH

= CH - (CH,),

- CO,

- CH,

+

- CH, - N(CH:J:,X-

(V)

is murexine (VI) isolated by Erspamer and Benati (1953)from the mollusk Murex trunculus and related species.

ty

0

CH=CH-CO,-CH,CH,-N(CH,),XO

H

(VI)

The fact that pahutoxin is essentially nontoxic to warm-blooded animals and shows specific toxicity toward fish suggests interesting possibilities for structure-activity research in the field of fish repellents. Whether the mechanism of the toxicity of pahutoxin is related to, or perhaps caused by, the hemolytic activity is an open question.

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T h e analogous shapes of the dose-activity curves of these two parameters would indicate that this is indeed the case (Boylan and Scheuer,

1967). Another ichthyotoxic skin secretion extracted from a fish has been described by Maretzki and Del Castillo (1967). T h e toxin was extracted from the soapfish Rypticus saponaceus and bears superficial resemblance to pahutoxin because of its ichthyotoxic and hemolytic properties. Its structure is not known, but the authors suggest that it may be a polypeptide or closely associated with a polypeptide. From two species of the same family of fishes, the Serranidae, Hashimoto (1968) has isolated identical or closely related toxins. The skin of the sea basses Pogonoperca punctata and Grammistes sexlineatus sexlineatus yields a bitter substance which is ichthyotoxic, hemolytic, and toxic to mice. It was shown not to be identical with pahutoxin, but may well be identical or structurally related to the toxic skin secretion of the soapfish.

D. OTHER FISH TOXINS From time to time there have appeared in the literature reports of fish poisonings which seem to differ from those that were described above. However, most of these reports are poorly documented and our knowledge is fragmentary at best. The following remarks will briefly indicate the nature of the available information. Clupeoid poisoning is so named since it is believed to arise from eating certain tropical sardines or herrings of the genus Clupeu and related genera. Outbreaks have been reported from Tahiti, New Caledonia, Fiji, and Indonesia. In Fiji the toxicity is said to be connected with the seasonal swarming of a marine worm (palolo),but aside from reported illnesses and fatalities the toxin has not been defined either pharmacologically or chemically. Scombroid poisoning, on the other hand, is much better understood than clupeoid poisoning, but it is not present in fresh or properly refrigerated fish. It is an allergic intoxication apparently caused by bacterial action on improperly stored tuna and other mackerellike fishes of the family Scombridae. This bacterial action, distinct from putrefaction, is believed to cause increased production of histamine and related substances that have been designated “saurine.” Victims of scombroid poisoning claim that the affected fish has a sharp or peppery taste. The action of the toxin is very rapid and occurs within minutes after the fish is eaten. Among the characteristic symptoms are intense headache, dizziness, skin eruption, and a swelling of the face.

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However, the symptoms are transient, generally lasting from 8 to 12 hours, and no fatalities have been reported in this century. The intense purgative action of certain fishes of the family Gempylidae is referred to as gempylid poisoning by Halstead (1967). T h e fishes that cause this action are few in number and the physiological action is caused by normal constituents of these fishes. Mori et al. (1966)have analyzed the oil of two such species from Okinawa, Lepidocybium jlavobrunneuni and Ruvettus pretiosus. These authors found that these fishes have a high oil content (20%) and that the oil consists predominantly of waxes. The major constituents of the wax after hydrolysis are hexadecanol (cetyl alcohol), 9-octadecen- 1-01 (oley1 alcohol), 9-octadecenoic (oleic) acid, and eicosenoic acid (site of unsaturation not determined). The same workers demonstrated that the oil caused diarrhea and seborrhea in rats. Perhaps the most elusive of all fish intoxications is hallucinogenic mullet poisoning. Occasional reports have appeared over the years that ingestion of members of the families Mugilidae and Mullidae (occasionally others) from restricted locations in the Hawaiian Islands during the summer months and from Norfolk Island in the Southwest Pacific causes nightmares and hallucinations. None of these reports have been substantiated by laboratory investigation to date. Fish poisoning of an entirely different character is represented by Minamata disease so called after Minamata Bay in Japan where it was first observed in 1953. It is not caused by a naturally occurring fish toxin, but stems from an environmental pollution problem. Minamata disease is worth noting here since it constitutes a potential danger to the marine food supply in proximity to industrial areas. The disease was caused by the effluents of a vinyl chloride plant in the city of Minamata and was traced to accumulation of mercury compounds in fish and shellfish. It was a serious outbreak with a high proportion of deaths among the affected population.

Ill. TOXINS FROM SHELLFISH -SAXITOXIN Sporadic outbreaks of poisonings from eating mussels and clams along the Pacific coast of North America have long been known. During the 1920’s it was shown that shellfish became toxic only during times of the “red tide,” which was caused by the bloom of the dynoflagellate Goizyuulax cutendla. California mussels, M ytilus californianus; Alaska butter clams, Suxidonius giganteus; and Bay of

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Fundy scallops, Pecten grandis have all been used for the isolation of the toxin. The early work has been ably reviewed by Schantz (1960). Most of the recent chemical work has been carried out by Schuett and Rapoport (1962) with toxin isolated from the clam, which retains the toxin in its siphon. The toxin is called saxitoxin. Neither saxitoxin, nor its dihydrochloride have been crystallized, but its purity has been established by several criteria. Saxitoxin is an optically active ( [aIu 130") diacidic base (pK, 8.1, 11.5) which shows no absorption in the ultraviolet. The toxin is liabile to base in the presence of air. Drastic oxidation with permanganate or periodic acid yields guanidinopropionic acid (VII).

+

H2N~(=NH)-NH~H,~H,-C0,H (VIU

Treatment of saxitoxin with phosphorus and hydriodic acid in acetic acid gave a 57Y0 yield of a weakly basic compound, C,H,,N,O, m.p. 100-102°C (IX). This degradation product was catalytically hydrogenated to a tetrahydro derivative, CXH,4N20, m.p. 129-131" (VIII). The structures of these two compounds were established by the following synthetic sequence. Synthetic (VIII) and (IX) were identical in all respects with the respective degradation products and thereby estab1,2lished structure (IX) as 8-methyl-2-oxo-2,4,5,6-tetrahydropyrrolo[ clpyrimidine.

1

LiAlH,

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This work by Schuett and Rapoport (1962) still constitutes the entire published structural knowledge of saxitoxin. Although compound (IX) contains eight of the ten carbon atoms of saxitoxin and can accommodate the atomic chain of guanidinopropionic acid, most of the hetero atoms in saxitoxin are not accounted for. Russell (1967)in his review paper published a total structure of saxitoxin credited to Rapoport. However, this published structure must be erroneous since it does not encompass the molecular formula of the toxin. The toxicity of saxitoxin is roughly that of tetrodotoxin and it is therefore one of the most highly toxic substances of low molecular weight known. However, its genesis from the dynoflagellate Gonyaulax catendla is well understood and its occurrence is rather narrowly confined. IV. OTHER MARINE TOXINS Even a cursory examination of volume 1 of Halstead’s trea t’ise (1965) dealing with marine toxins derived from invertebrates, serves to show that an impressive number of marine animals elaborate substances that are toxic to man. However, there is no evidence at the present time that any of these - the holothurins and asterosaponins from echinoderms, nereistoxin from a marine annelid, aplysin from a mollusk, and many others which have been studied too little even to be named - are used as food and may therefore be a public health hazard, or play a role in the food web of marine vertebrates or edible shellfish. Algae, on the other hand, definitely are part - even if a poorly delineated part-of the food chain of marine animals. Toxic algae are known, but our knowledge of them is even scantier than that of toxic animals. The most notable exception is the involvement of the dynoflagellate Gonyaulux catenella in paralytic shellfish poisoning, which has been described above. Algae are suspected as originators or precursors of ciguatera toxins, but no single piece of evidence has been reported, which lends credence to this suspicion. An ichthyotoxic and hemolytic substance, prymnesin, has been isolated from the phytoflagellate Prymneisum purvum (Shilo, 1967). It is believed to be a saponin, but this has not been proved. Bishop et al. (1959)have isolated a toxic cyclic peptide from the blue-green alga Microcystis aeruginosn. Perhaps the most interesting algal toxin, chemically and biogenetically, was recently described by Hashimoto et al. (1968).A massive fish kill in 1962 in Lake Sagami near Tokyo was traced to extensive

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blooms of a dynoflagellate believed to be Glenodinium sp. and later identified as Peridinium polonicum. The substance responsible for the ichthyotoxic activity, glenodinine, was isolated as an exceedingly unstable solid, which however formed a crystalline reineckate, 111.p. 125-126°C. Glenodinine is active only at basic pH values and its isolation and purification procedure further shows its alkaloidal nature. Spot tests confirmed the presence of secondary and tertiary nitrogen and suggested that one of the functions in the molecule is a sulfhydryl group. The mass spectrum of glenodinine hydrochloride exhibits in the lower inass range considerable resemblance to that of ibogaine = 12-methoxyibogamine (X). This intriguing possibility that

a sulfhydryl alkaloid may be produced by this dynoflagellate will, if confirmed, shed new light on alkaloid distribution and pose new questions on the role of alkaloids in plant metabolism. Some caution with respect to the identity of this toxin with an alkaloid of the Apocynaceae seems to be in order since glenodinine hydrochloride is transparent in the ultraviolet and since no mass peak corresponding to the indole moiety appears in the published mass spectrum of glenodinine. The prominent peaks in the lower mass range are all assignable to an alkyl substituted piperidine derivative. Absence of further blooms in 1965 and 1967 has so fir prevented further research on this fascinating substance.

V. RESEARCH NEEDS Our knowledge of toxic factors from the marine environment lags far behind our knowledge of toxins elaborated by terrestrial organisms. The necessity of finding improved and new food sources from the world’s oceans makes it mandatory that we expand our research effort and simply increase our basic knowledge of these substances. More specifically, we need to know the biological origin of ciguatera; we need to develop a simple field test for its recognition; we need to know the molecular structure of the toxins; and we need to know whether these toxins survive the various processes that are used and contemplated for the production of fish protein concentrates.

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Although much valuable work has been done on algal photosynthesis and algal productivity, we know far too little about food chains on coral reefs or in the open ocean. Our knowledge of marine steroids, marine terpenoids, and even marine carbohydrates is likewise only fragmentary. Major research programs are therefore urgently needed to fill in the gaps in our knowledge of marine toxins.

ACKNOWLEDGMENT Much of the iinpul~lishedresearch on ciguatefii i n the writer’s lalmratory and at the Hawaii Institute of hlarine Biology has heen generously supported b y U.S. Pul)lic He;tlth Service Grant UI-00216.

REFERENCES h g n i s , R. 1967. Les eml”)issonnenieiits par le poissoii eii Polyntsir friinpiiise: Etude clinique et i.pidi.miologi[]iie. R r c . /i!/g, ,\led, Soc. 15, 619-646. Baiiiier, A. H . 1967. Poisonous innrine miini:tls, ;I synopsis./. Foreiisic Sci. 12, 180- 192. Baiiiier, A. H., Scheuer, P. J., S:is;tki, S.,tlelfrich, P., ; i d Alender,C . H. 1R60. Ol)servations on ciguiitera-type toxin in fish. Atiti. &‘.)’. i k t r d . Sci. 90, 770-787. B;inner, A. H., Sasaki, S.. Helfrich, P., Alender, C. 13., ;nid Scheuer, P. J. 1961. 13ioassay ofcigwter;i toxin. h‘trtrtrc, 189, 929-230. Hishop, C. T., Anet, E. F. L. J., ; i d Gorh;inr, P. R . 1H59. Isohtion and iclentification o f the fast-deuth hictor i i i Alicloc~!~.sfis trc,rrrgitio,scl N R C - I , C : t r t i . / . R i o c / i r i t r . P / i ! / s i o /37, . 453-47 1. 13oyl;iii, D. 13., ;nit1 Sc*heuer,P. J. 1867. l’ahutoxin: A fish poison. Scioic.c 155, 52-56. Brock, V. E. 1956. Possihle production of sul,stances poisonoris to fishes b y the box fish (htrficioii /etiligitio.strs Schneicter. C:o)ic,iu 111). IHFj- 15J6. Bntttlle, R. 1930. Soiiie coniinoii lioisonoiis fishes f o i i n t l i n Singapore waters. /. R o ! / . N ( I L N All>(/. / S0.t;. 16, 102-1 11. Corirvillc~,D. A,, I l d s t e a d , H. W., i i n d Ilessel, D. W. 18.58. hlarine hiotoxins: Isohtioil and properties. C,‘/ictti. Rct;. 58, 2.35-248. Erslxtnier, V., i i i i d I3riiati, 0. 1953. Itleiitificatiol, o f iiiurexine iis ~-[imitl;izolyl-(4)~xryl-choline. Sciutrce 117, 161- 162. Goto, T., Kislii, Y., Tiik;ihashi, 3 . , and I i i n i t a , Y. 1965. Tetiodotoxin, ‘I‘elrtr/ic~t/,ori21,

2059-2088. Hnlsteutl, 13. W. 1965. “Poisonous ;uid Veilornous hlin-ine A n i t i d s o f t h e World” Vol. 1. U.S. Chvt. Printing Office, Washiiigtoii, I1.C. H;ilste;itl, 13. W. 1967. “Poisonous and Vcnomoris Xl;iriiic, Aninids o f t h e World,” \’ol. 2, U.S. Covt. Printing Office, Wiishington, D.C. Hashimoto, Y. 1956. A irote oil the poisoil o f i i l)arriicudii, S ) ) / t ! / r u e i i u /iicitdcr 131och Q Sclineidei-.H I ~ / / . , / ~ I /Soc. J ~ I ~S uI .i . I:is/icric,.c 21, 1 1Y3- I 157. H;isliinioto. Y. 1968. 1’ersoii;il coniiiiiiiiic;itioii. Hashinioto, Y., ; i i d Yasninoto, T. 1965. A n o t e o i i cigu;iter;i poisoning i i r Okinawii and the toxiir 0 1 ii groiiper, Eliitrc~liliulits~ i c s c . o g ~ r t / n t rForsk3. ts H u l l . , / o p r i i . S o c . k’i,s/ic,ric,s 31, 452-458. Hashimoto, Y., Okaichi, T., Dang, L. I)., m d Noguchi, T. 1968. Glenodinine. Ichthyotoxic substance produced h y ii dinoflagellate, Pcriditiirrni / i o / o t I i c r c ~ t i Hull. . /u/)(I~I. Soc. Sci. l:i,s/ic,rivs 04, 528-534.

160

PAUL J. SCHEUER

Helfrich, P., and Banner, A. H. 1968. Ciguatera fish poisoning. 11. General patterns of Bishop Miiscurti 23,371-382. development in the Pacific. 0ccci.siotid P a p x ~ Helfrich, P., Piyakarnchana, T., and Miles, P. S . 1968. Ciguatera fish poisoning. 1. The ecology of ciguateric reef fishes in the line islands. Occusiotiml Pupers Bishop Museum 23,305-369. Herald, E. S . 1961. “Living Fishes of the World,” p. 278. Doubleday, New York. Hessel, D. W. 1963. Marine biotoxins. 111. The extraction and partial purification of ciguatera toxin from Lutjutitts bohrrr (Forskil);use of silicic acid chroniatography. I n “Venomous and Poisonous Animals and Noxious Plants of the Pacific Region” ( H . L. Keegan and W. V. MacFarlane, eds.), pp. 203-209. Pergamon Press, Oxford. Kaiser, E., and Michl, H. 1958. “Die Biocheniie der tierischeti Cifte.” F. Deuticke, Vienna. Keegan, H. L., and MacFarlane, W. V., eds. 1963. “Venomous and Poisonous Animals and Noxious Plants ofthe Pacific Region.” Pergamon Press, Oxford. Larson, E., and Rivas, L. R. 1965. Ciguatera poisoning from barracuda. Quurt. J. 1:lorida Acud. Sci. 28, 173-184. McFarren, E. F., Tanabe, H., Silva, F. J.. Wilson, W. B., Campbell, J. E., and Lewis, K. H. 1965. The occurrence of a ciguatera-like poison in oysters, clams, and Cynitioditiiuni bret;e cultures. T C J X ~3,C 1O1 1~ 123. Malo, D. 1898. “Hawaiian Antiquities.” [The 1898 translation was published as Bishop Museiini Spec. Pub/. No. 2 (1951).] Maretzki, A,, and Del Castillo, J. 1967. A toxin secreted by the soapfish Rypticus s c ~ ~ ~ o n U C B O I I S . TCJXiCO?l4, 245-250. Mori, M., Saito, T., Nakanishi, Y., Miyazawa, K., and Hashimoto, Y. 1966. The composit i . Sci. Fishtion and toxicity of wax in the flesh of castor oil fishes. Bull. J ~ p ~ Soc. eries 32, 137-145. Mosher, H. S., Fuhrman, F. A., Buchwald, H. D., and Fischer, H. G. 1964. Tarichatoxintetrodotoxin: a potent neurotoxin. Science 144, 1100- 1 1 10. Nakazawa, Y. 1959. New glycolipide in the oyster. V. The nitrogenous components and structures of the glycolipide. J. Biochem. (Tokyo)46,1579-1585. Nigrelli, R. F. 1960. Biochemistry and phannacology of compounds derived from marine organisms. A n n . N.Y. Acud. Sci.90,617-949. Nigrelli, R. F., Stempien, M. F., Jr., Ruggieri, G. D., Liguori, V. R., and Cecil, J. T. 1967. Substances of potential biomedical importance from marine organisms. Federation Proc. 26,1197-1205. Russell, F. E. 1965. Marine toxins and venomous and poisonous marine animals. Adwin. Murine R i d . 3,256-384. Russell, F. E. 1967. Comparative pharmacology of some animal toxins. Federutiori Proc. 26,1206-1224. Russell, F. E., and Saunders, P. R. 1967. “Animal Toxins.” Pergamon Press, Oxford. Schantz, E. J . 1960. Biochemical studies on paralytic shellfish poison. Ann. N.Y. Acud. Sci.90,843-855. Scheuer, P. J. 1964. The chemistry of toxins isolated from some marine organisms. Fortschr. Chem. Org. Nuturstoffe 22,265-278. Scheuer, P. J. 1969. The chemistry of some toxins isolated from marine organisms. Fortschr. Chem. Org. Nuturstoffe 27, 322-339. Scheuer, P. J., Takahashi, W., Tsutsumi, J., and Yoshida, T. 1967. Ciguatoxin: Isolation and chemical nature. Science 155, 1267- 1268. Schuett, W., and Rapoport, H. 1962. Saxitoxin, the paralytic shellfish poison. Degradation to a pyrrolopyrimidine. J . Am. Chem. Soc. 84,2266-2267.

TOXINS FROM FISH AND OTHER MARINE ORGANISMS

161

Shilo, M . 1967. Formation and mode of action of algal toxins. Hacteriol. Reo. 31, 180- 193. Tsrida, K. 1966. Uber Tetrodotoxin, Ciftstoff der Bowlfische. Noturwissenschu~ten53, 171- 176. Woodward, R. B. 1964. The structure of tetrodotoxin. Pure A p p l . Chem. 9,49-74. Woodward, R. B., and Cougoutas, J . Z . 1964. The structure of tetrodotoxin.]. A m . C h e n ~ . Soc. 86,5030. Yokoo, A. 1948, A toxin of the globefish. Bull. Tokyo Inst. Techno/. 13, 8-12; Chem. A/i,str.44,3622 (1950).

This Page Intentionally Left Blank

THE FLAT SOUR BACTERIA

BY MARION L. FIELDS Department of’ Food Science and Nutrition, Unioersity of Missouri, Columbia, Missouri

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164 . 11. Classification

B. M i l k . . .......................... C. Sugar and Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Wheat and Wheat Products IV. Resistance of Spores to Lethal

...................... ...................................

179

F. Morphology and Chemical Composition of Spores C. Enzymes of Vegetative Cells .................... H. Metabolism of Nitrogen Compounds. . . . . . . . . . . . . . References

.......... 163

164

MARION L. FIELDS

I. INTRODUCTION

In 1897 Prescott and Underwood published a paper which described the souring of canned sweet corn. These authors distinguished between two types of souring, one which resulted in swelling of the can and the other which did not. It is possible that some of the souring without swelling of the can may have been the flat sour spoilage, as we know it today. They stated that one part of a particular canner's single-day production was shipped to the northern part of the United States, whereas another portion was shipped to the southern part of the United States. The canned corn sent to the northern part was perfect at the end of a year, while the corn shipped to the South became sour. The original batch of canned corn could have had flat sour spores present which caused spoilage when the corn was stored at a warm temperature. Prescott and Underwood described several bacilli that were isolated from soured canned corn. The descriptions were short and did not seem typical of the flat sour bacteria. The bacilli isolated by the authors grew at 20°, and 3TC, with growth particularly at 37°C. However, Bacillus stearothermophilus, the bacterium which causes flat sour spoilage in corn, does not grow rapidly at 37°C and does not grow at all at 20°C. It is therefore probable that Prescott and Underwood did not isolate B . stearothermophilus, although they may have observed the growth of this organism in some of the commercial packs. It is possible that some of the corn may have been spoiled by Bacillus coagulans due to the fact that it received a mild heat treatment usually given corn at that time. The importance of thermophilic spore-forming bacteria in the spoilage of canned corn was discussed by Barlow in

1913. During a study of the coagulation of evaporated milk in 1915, Hammer, of the Iowa Agricultural Experiment Station, isolated an organism which he named Bacillus coagulans because it caused coagulation of the canned milk but did not produce a swell. The organism produced about 1% acid in cans of spoiled evaporated milk. Hammer described the flavor of the milk as faintly sour and slightly cheesy, but not at all disagreeable. The relationship of B . coagulans to flat sour spoilage in canned foods was not shown for several years after Hammer's original work. In 1917 Donk, while working at the research laboratory of the National Canners Association in Washington, D.C., isolated a thermophilic spore-forming rod from Maine-style corn (cream-style corn). He named this organism Bacillus stearothermophilus. This bacterium is the typical flat sour-producing microorganism of low-acid vegetables,

165

THE FLAT SOUR BACTERIA

whereas B. coagulans is the typical flat sour-producing microorganism in milk and tomato juice. Also in 1920, Bigelow and Esty reported on the thermal death point of several typical thermophilic spore-forming microorganisms, which were subsequently assigned numbers by the National Canners Association. Several of the isolates (cultures 1503, 1820, 1373, 1518, 1356, 1549, and 4109) used by Bigelow and Esty were classified by Smith et al. (1952) as B . stearothermophilus. Because of the heat resistance of the other cultures studied b y Bigelow and Esty in 1920, they also were probably B . stearothermophilus. Bigelow, in 1921, demonstrated the logarithmic nature of the thermal death-time curve with thermophilic spore-forming bacteria. The fact that the thermal death-time curves were logarithmic was the basis for part of the process calculations that were to be developed b y Ball (1923). Research on spoiled canned foods continued at the National Canners Association (NCA) in Washington, D.C. from which Esty and Stevenson issued a report in 1925 on the various types of spoiled canned foods. Their description of “flat sours” is given in Table I. TABLE I DESCRIPTION OF FLAT SOUR SPOILAGE IN NONACIDPRODUCTS UNDERSTERILIZATION“

BY

Significant features

Description

Condition of container Microscopic examination of direct smears from can

Flat Rods, with or without apparent spore formation; usually apparently pure cultures Usually sloppy in appearance; normal or sour odor Aerobic and anaerobic growth at 37°C and 55°C or at 55°C alone; no gas formation; usually evidence of spore formation on agar; microscopic examination should check direct smears No leaks on pressure testing; seams and general container good

Physical examination of contents Culture

Seam examination.

“Esty and Stevenson ((1925).Reprinted from the Journal of Znfectious Diseases 36, 486-500 by permission of the Editor.

166

MARION L. FIELDS

Cameron and Esty in 1926 classified the flat sour bacteria from nonacid foods. After studying 214 cultures of nongas-forming, sporeforming bacteria, they grouped the bacteria- according to their growth temperatures - into facultative and obligate thermophiles. Cameron and Esty’s cultures 1503 and 1356 (according to Smith et al., 1952) were B . stearothermophilus. Both cultures represented Cameron and Esty’s group 100 (the obligate thermophiles). In 1928 Cordes reported on bacterial action in the coagulation of evaporated milk (flat sours). H e attributed spoilage of the evaporated milk occurred after heat treatment to B.coagulans. In 1933, Berry of the Research Department of the American Can Company isolated an organism from off-flavored commercially canned tomato juice. H e considered it to be an unnamed organism and gave it the name Bacillus thermoacidurans. In studying this organism, he found that 37°C was the optimum temperature for producing the typical flat sour, off-flavor reaction which resulted when the bacteria was inoculated into nonacid products. The current economic importance of both the flar sour bacteria is as hard to evaluate in 1969 as it was in 1926 when Cameron and Esty made the following statement: Commercial experience does not afford reliable data concerning the relative frequency of loss from under-sterilization and leakage and nothing more than a rough estimate may be given on this basis. It should be borne in mind that figures on flat sour spoilage may be low due to the lack of external signs and consequent difficulty of detection.

Cameron and Esty (1926) in their laboratory examined 439 cans of food and found an average of 32.8%spoiled by flat sour bacteria (see Table 11). The NCA has made extensive field studies since 1926 pointing up contamination sources. Since that time, preventive measures as recommended by the NCA, the can companies, and the glass manufacturers have practically eliminated flat sour spoilage. Some of the reasons for this improved situation are (1) the elimination of wooden equipment, (2)better sanitation procedures, and (3)the use of “thermophile-free” or canners-grade sugar and starch. Better cooling procedures have also played a big part in the elimination of flat sour spoilage in commercially canned foods. However, it should be pointed out that laxity in any of these areas could again make flat sour spoilage a problem in low-acid canned food. According to Troy and Schenck (1960), flat sour spoilage of canned tomato juice was first encountered in 1931 and was described by them as follows:

167

T H E FLAT SOUR BACTERIA

TABLE I1 OCCURRENCE OF SWELLSAND FLAT SOURS UNDERSTERILIZED NONACIDC A N N E D FOODS^

THE RELATIVE FREQUENCY IN

OF THE

Spoiled Data, in Percentages

Product Corn Peas String beans Beets Spinach Milk Squash and pumpkin Swr et potatoes Pork and beans Hominy Miscellaneous (lima beans, succotash, asparagus) Total

Numberb samples examined

Number samples spoiled

Understerilization Flat sour

Swell

Spoiled through leakage

177 69 39 26 25 20

119 41 14 6 15 10

26.8 19.5 35.7 0.0 20.0 80.0

26.8 34.2 14.3 0.0 26.7 20.0

46.4 46.3 50.0 100.0 53.3 0.0

19 16 12 10

13 3 3 6

84.6 66.6 33.4 66.6

0.0 0.0 0.0 33.4

15.4 33.4 66.6 0.0

26

11

45.4

9.2

45.4

439

24 1

32.8

23.6

43.8

“Cameron and Esty (1926). Reprinted from the Journul of Infectious Diseuses 39, 89-105 by permission of the Editor. bA variable number of cans comprised each “sample.” Since that time, outbreaks of this type of spoilage have occurred almost yearly, many times reaching serious proportions and causing severe economic losses to the canner. Its occurrence has been erratic, occurring in one season’s pack and not in the next, even though the same processing methods are used.

Occasional flat sour spoilage caused by B . steurothermophilus still occurs. For example, the author isolated a strain of B . steurothermophilus from canned cream-style corn from a cannery in 1959.

II. CLASSIFICATION

The canning industry has described spoilage of low-acid foods by B . stearothermophilus as flat sour spoilage and spoilage of tomato juice by B . cougulans as aciduric flat sour spoilage. These bacteria produce acid but no gas from carbohydrates and produce the typical flat sour spoilage in canned foods. Although such a classification as flat sour

168

MARION L. FIELDS

bacteria is useful from an industrial point of view, a more detaile. examination is necessary in determinative bacteriology where morphological, cultural, and biochemical characteristics are used to designate the names of bacteria. These characteristics separate the flat sour bacteria into two species. A. Bacillus coagulans

Bacillus coagulans was isolated from spoiled evaporated milk in 1915 by Hammer. Although Cameron and Esty in 1926 did not give generic names to their groups of flat sour bacteria, they did give descriptions which fit the description of Smith et al. (1952)for B . coagulans. Berry isolated a spore-forming rod from spoiled tomato juice in 1933 and named it B . thermoacidurans. The interrelationship of these descriptions is listed in Tables I11 and IV. Comparison of each test made by the four authors is not possible since each worker did not make the same determinations. It is the belief of this author, like Allen (1953), that Cameron and Esty’s Group 80 is B . coagulans. Further proof that Group 80 is B . coagulans may be found by consulting U.S. Agriculture Monograph No. 16. Cultures 4578,1460, 1734, and 1215 were submitted to Smith et al. (1952)by Wallace Bohrer of National Canners Association in Washington, D.C. All of these were identified as B . coagulans and were listed in Cameron and Esty’s Group 80. B . coagulans is less heat resistant than B . stearothermophilus (Murrell, 1955). This was also illustrated by Cameron and Esty (1926).Organism 1608, which is in Group 80, is less heat resistant in corn juice pH 6.1 than cultures 1476, 1492, 1503, and 1592, which are representative of Group 100. Of these, culture 1503 is listed in U.S. Agriculture Monograph No. 16 as B . stearothermophilus which is more resistant than microorganism 1608. It is interesting to note that B . coagulans can cause spoilage in lowacid foods since it is usually associated with flat sour spoilage in acid products like tomato juice. Probably the reason that B . coagulans is found less frequently in low-acid foods is because of the low heat resistance of its spores and because of the high heat process given lowacid foods. B . coagulans can grow in media with as low a pH as 4.02 (Pederson and Becker, 1949),whereas B . stearothermophilus does not grow at or below pH 5.0 (Smith et al., 1952). In the past, many people have consumed flat sour tomato juice without ill effects. The author could find no references indicating that B. coagulans is in any way pathogenic or toxic. A personal communi-

THE FLAT SOUR BACTERIA

169

cation (1969) from Cleve B. Denny of the National Canners Association indicates that he is familiar with human feeding studies, conducted in California, which showed that neither B . coagulans nor B . stearothermophilus caused any adverse symptoms in humans. B. Bacillus Steurothermophilus

B . steurothermophilus was described by Donk in 1920. An extension of this description of this microorganism was made b y Smith et al. (1952). For comparative purposes the characteristics of the two flat sour bacteria have been listed in Table V. In the key of Smith et al. (1952), emphasis is placed on the fact that B . coagulans is Gram positive and the sporangia are only slightly swollen. These characteristics are not sufficient evidence for a sharp distinction, however, since 50% of the strains of B . coagulans had swollen sporangia. As shown in Table V, there are some variations where the vegetative rods may be Gram variable. Smith et al. (1952) made the statement that B . coagulans was placed in Group 1 (a group of similar species) because it resembled Group 1 more than Group 2.

c. ROUGH

AND SMOOTH VARIANTS

Both rough and smooth variants are found in B . coagulans and B . stearothermophilus (Table V). According to Bisset (1955),the smooth variant divides by constriction of the cell wall, whereas the rough variant forms complete cross walls which subsequently split. Bisset stated that these two methods of cell division correlate with the smooth and rough filamentous morphological forms. These morphological forms (rough and smooth) are also linked to other properties such as biochemical reactions (Henshelwood, 1947). It is because of these variant forms that there is considerable variation in the descriptions of B . coagulans and B . steurothermophilus.

111. DISTRIBUTION OF SPORES OF FLAT SOUR BACTERIA

A. SOIL .4ND WATER

Miquel (1888) isolated from the river Seine a thermophile which was able to grow at 73°C. Thermophilic bacteria have also been found in tropical soils (deKruyff, 1910), in desert sands (Nkgre, 1918), and in

TABLE I11

COMPARISON OF Author

~ ~ O R P H O L O C OF Y

B . couguluns Colonies on nutrient agar

Vegetative cells

Hammer, 1915

Rods, 1.6-1.7 p by 0.5-0.7

One-third width of veg. cell

Cameron and Esty, 1926 Group 80

Rods, 1.0-4.0 Gram-positive

Terminal to subterminal; elliptical 0.8 by 1.5 p (some bulging of rod)

Surface colonies 1-2 mm diam; shiny, white, nonviscid, round, entire (2-3 days)

Growth in nutrient broth Turbidity and sediment

Turbidity slight or absent; no sediment

5

8z r

Berry, 1933

Rods, 1.5-6.6 p by 0.3-0.9 p ; Gram-positive

Terminal to subterminal 1.2 by 2.0 p

Colonies 1-5 mm diam; circular, raised, glistening, smooth, opaque

Smith et al., 1952

Rods, 0.6-1.0 p by 2.5-5.0 p (variations: 0.51.2 p by 2.06.0 p filaments); Gram-variable

0.9-1.0 p by 1.2-1.5 p ; one thin-walled subterniinal to terminal (variations: 0.81.1 p by 1.22.0 P)

Usually small, round, opaque not distinctive

Not given by these authors

TABLE IV COMPARISON OF BIOCHEMICAL AND PHYSIOLOGICAL REACTIONS

OF

B. congulans

Author

Gas formation

Acid production from

Nitrates reduced

Growth temperature

Hammer, 1915

None

Dextrose, galactose, levulose, lactose, maltose, raffinose

Berry, 1933

None

Arabinose, dextrose, wlevulose, D-galactoSe, mannose, saccharose, maltose

No

37°C and 55°C

Cameron and Esty, 1926

None

Xylose, arabinose, glucose, galactose mannose, fructose

No

Max. 65°C; min. 30"-35"C; Opt. 45"-5OoC

Smith et nl., 1952

None

Glucose, variable from arabinose, xylose, lactose, sucrose, glycerol, mannitol

Usually negative, 18 strains positive

Good growth 33"-45"C;max. for most strains 55"-60"C; no growth at 65°C

37°C and 55°C

172

MARION L. FIELDS TABLE V COMPARISON OF SELECTEDCHARACTERISTICS OF B. coagulans stearothermophilus"

AND

B.

Character

B. coagulans

Vegetative rods

0 . 6 / ~ - 1 . 0by ~ 2.5~5.0; Gram-positive; Gram-variable; 0 . 5 ~ 1.2 by 2 . 0 - 6 . 0 ~

0 . 9 ~ - 1 . 0by ~ 2.5~3 . 5 ~Gram-variable; ; 0.6~ by 2 . 0 ~ - 5 . 0 ~

B. stearothermophilus

Sporangia

Definitely swollen in some cases; not swollen in others

Definitely swollen; racket-shaped

Spores

0 . 9 / ~ - 1 . 0by ~ 1.2~1 . 5 ~oval; ; thin-walled; subterminal to terminal (variations: 0.8-1.111 by 1.2p-2.0j1 bent or cylindrical)

1 . 0 / ~ - 1 . 2by~ 1 . 5 ~ 2 . 2 ~characteristically ; variable in size; oval; terminal to subterminal

Colonies

Usually small, round, opaque, not distinctive, smooth to rough

Not distinctive; penpoint to small; round to irregular; translucent to opaque; smooth to rough

Growth on proteosepeptone acid agar

Good

None

Production of acetylmethyl carbinol

Usually positive

Negative

Hydrogen sulfideb

Not given

Positive in tryptone broth

Temperature of growth

Good growth 33"-45"C; maximum temperature for majority of strains 55"-60°C; no growth at 65°C; poor growth, if any, at 28°C.

Good growth 50"-65"C; variable at 70°C and 37°C; no growth at 28°C.

"Smith et al., 1952. %tark and Tetrault. 1952b.

sea water (MacFadgen and Blaxall, 1894,1899).Bartholomew and Hittenburg (1949) even found thennophilic bacteria in deep ocean bottom cores. Marsh and Larsen (1953) studied isolates of bacteria from the hot springs of Yellowstone National Park. Out of 24 cultures, 21 were

T H E FLAT SOUR BACTERIA

173

found to be B . stearothernaophilus. Some strains had characteristics of both B . steurothermopkilzis and B . coagulnns. In addition to such obvious environments as hot springs, McBee and McBee (1956) found thermophilic aerobic bacteria in arctic soils and waters. Thermophilic bacteria were identified in 38 of 59 soils and waters collected near Point Barrow, Alaska. The presence of the thermophiles seems to be dependent on accumulations of organic matter other than peat. There was no association of the presence of these organisms with fecal contamination. Mercer et al. (1960), on the other hand, found that peat soil of one asparagus field in California contained 102,000,000 thermophilic flat sour spores per gram of soil. Other soils with less organic content had fewer spores, indicating that peat might have some influence on these organisms. The question arises: How do thermophiles grow in soils where the temperature is lower than their laboratory growth temperature? McBee and McBee (1956) suggested that thermophilic bacteria may be able to grow at a reduced temperature in the soil because of the presence of vitamins or other growth factors produced by other microflora. Thus, Campbell (1954) showed that unidentified growth factors in yeast extract allowed an “obligate” thermophile to grow at 36°C. In a study on 41 soil samples from the Hawaiian Islands, Fields (196813)found that all of the samples contained thermophilic, aerobic, spore-forming bacteria, although the number of spores was low (usually less than 100 per gram).

B. MILK Thermophiles are of interest to the dairy industry because of their growth in pasteurizing equipment, their presence in dried milk, and their implication in the spoilage of canned milk. Hussong and Hammer (1928),in a study of spoiled skim milk intended for drying in a milk-processing plant, found that the milk had been pasteurized at 71”-77”Cand held hot in a wooden tank for periods of 24 hours. They described the organism that caused coagulation in the milk and named it Bacillus culidoluctis. According to Hussong and Hainnier (1928), this organism grew at 75°C. Smith et al. (1952) renamed this organism B. cougulans, however. They stated that, of the five cultures of B . calidolactis that orginally came from Iowa State College, three were identified as B. coagulans and two as B . stearothermophilus. The growth temperature as given by Hussong and Hammer was actually that of B. stearothermophilus. Breed et al. (1929) suggested that even the cleanest milk may occa-

174

MARION L. FIELDS

sionally contain small numbers of thermophilic bacteria. They listed the following as places where these microorganisms might grow: (a) foam which may remain in t h e pasteurizing vat for hours; (b) in milk cooked onto the metal pipes or other equipment where the water or steam used in heating the milk is at a temperature above 150”-152”F;(c) dead ends which cause eddies in the current of hot milk; ( d ) gabkets, pitted metal surfaces and other rough places which offer conditions favorable for the development of these bacteria.

The thermophilic organisms are also of interest to the dairy industry because they showed up as “pinpoint” colonies when cultured at 37°C in an agar medium and consequently were of concern as to their significance. Prickett (1928) made a special study involving 480 cultures of both spore-forming and non-spore-forming microorganisms secured from a variety of sources. He concluded, as did Breed et al. (1929), that the growth of thermophilic bacteria in pasteurized milk produced undesirable changes that affected the commercial life and salability of the product. He found no evidence that these organisms were important from the public health standpoint. In 1932 Hansen studied eleven cultures of thermophilic bacteria that had grown in sterile skim milk. When this contaminated milk was fed to guinea pigs, the animals continued to grow and otherwise showed no ill effects, indicating that these bacteria were not of public health significance. B. coagulans, Strain 1460 of Cameron and Esty’s Group 80 ( B . coagulans according to Smith et al., 1952), and Strain 1356 ( B . stearothermophilus according to Smith et al., 1952) were included in the cultures isolated. Although the feeding studies he carried out were negative, Hansen felt that pasteurized milk should nevertheless be as free of harmless bacteria as possible. In studying the use of milk powders in the canning industry, Sorensen (1938) recommended methods by which flat sour spores could be detected. H e employed N/60 sodium or lithium hydroxides instead of distilled water in dilution flakes, which facilitated the dissolving of atmospheric roller process powder so that undissolved flakes and specks of undissolved material were eliminated. C . SUGARAND STARCH In the 1930’s considerable discussion centered around the role of sugar in the spoilage of canned foods. For example, Ingersoll (1930) stated that sugar alone was not responsible for the introduction of thermophilic bacteria in the canning process and cited one large canner who had never had any problem with sugar. This author then suggested that the probable answer to this problem was one of reason-

175

T H E FLAT SOUR BACTERIA

able care and sanitation in the processing of canned goods coupled with an intelligent understanding of what constitutes the relative importance of various sources of contamination. Dr. Cameron of the National Canners Association, in a letter to the editor of Food Industries, disputed the statements of Dr. Ingersoll that referred to spoilage caused by spores in sugar. Dr. Cameron asserted that these microorganisms could grow and develop in the processing plant. He was wrong, however, when h e said that it would be most unusual if flat sour bacteria were found in the soil. 0. B. Williams (1930) tested 55 samples of soil and found only 2 which contained flat sour spores, and these soils had low spore counts. It appears that he, too, did not recognize that soil-borne thermophilic spore-forming bacteria could seed equipment just as effectively as sugar and that, after this seeding, spores could be produced that were just as resistant as spores in sugar. Cameron and Bigelow (1931) studied thermophilic bacteria in sugar. They found that the "flat sour" group was the most frequently encountered and that flat sour spores were in essentially all of the cane sugar samples examined. A wide variation in number of spores in individual samples was observed. Table VI shows that the removal of

REMOVAL O F

Sample No. 1

2 3 4

5 6

7 8 9

10 11

TABLE VI FLAT S O U R SPORES B Y FIL1'HATION Description Original inoculated First syrup through press About 50 gal. (189.5liters) through press Average after all through first time During second time through press Average after all through second time Average after all through third time Average after all through fourth time Composite 50 gal sweet water Mother liquor after centrifuge Finished sugar (sporeslg)

A N D C21YS1'ALLIZA'IION"

Number o f flat sour spores per cc of syrup 25,000 9000 3500 2500 1000

I000

1700 1000 600 2200 238

"Cameron and Bigelow, 1931. Copyright 1931 by the American Chemical Society. Reprinted by permission of the copyright owner.

176

MARION L. FIELDS

flat sour spores by filtration and crystallization resulted in a decrease in spores up to a point, after which no reduction in the number of spores was observed. Cameron and Yesair (1931) showed that when sugar containing 2500 spores per 10 g was added to corn, 54.2% of the cans of corn spoiled after being heated 90 min at 250°F. The flat sour bacteria, although they are nonpathogenic, are nevertheless of economic importance to the canning industry. Therefore, a method for detecting flat sour spores in sugar was needed. Cameron of the National Canners Association published a procedure for the detection of thermophilic bacteria in sugar (1936). Cameron and Yesair (1931) suggested that sugar should contain no more than 75 spores per 10 g. Today’s standard states that for each 5 samples examined there should be a maximum of 75 spores and an average of not more than 50 per gram. The question of thermophilic contamination within the sugar factory was discussed by Calton (1936) with emphasis on beet sugar. In the introduction of his paper Calton summarized some of the information known on the subject at that time: 1. Filtration plays one of the most important roles in removal of thermophilic spores. 2. Accumulation may occur at points before or after filtration. 3. Crystallization, although less effective than filtration, excludes a large degree of contamination from the sugar. 4.Contamination is confined to the crystal surface and is greatest in clumps of crystals. 5. Contamination of simps bears no relation to purity. 6. Flat sour thermophiles can grow in concentrations as high as 40% sugar sirup; growth and acid production are rapid in concentrations up to 25%.

Calton (1936) also listed the most likely points of contamination within the sugar beet factory. These are given in Table VII. Calton also showed that growth of flat sour bacteria may occur in the pan storage tanks, in the liquor, and on the wall surface. Starch, as well as sugar, can be a source of flat sour spores in canned foods. When contaminated starch is used to can cream-style corn, flat sour spoilage and other types of spoilage may occur (Cameron, 1937). Spoilage of canned foods may also result from ingredients other than sugar and starch. For example, flour, spaghetti, beans and chowder, and dried milk and mushrooms were suspected of causing spoilage in creamed soups (Cameron, 1937). The number of flat sour spores in beet sugar decreased after it had been stored for several months. T h e influence of containers, the number of spores present, and sugar impurities were given by Hall (1938) as the reasons for this decrease. The holding of lots with high

T H E FLAT SOUR BACTERIA

POINTS Material House supply water Battery supply water Cosseltes (sugar beets) Diffusion juice First press juice Second press juice Thin juice

Thick juice

Wet sugar Dry sugar

OF

177

TABLE VII GENERAL CONTAMINATION" Location

Various locations Line to battery Belt Measuring tank Press spouts, receiver Press spouts, receiver Blow-up receiver, press spouts Evaporator Supply tank, heater Fifth-body evaporator Blow-up press, spouts Pan storage Centrifugals

Number of flat sours

2 8 5 6 265 1605 140 0 99 121 122 26,144 15 13

"Calton, 1936. Copyright 1936 by the American Chemical Society. Reprinted by permission of the copyright owner.

spore counts could be used, therefore, as a means of controlling spoilage. Clark and Tanner (1937) found that flat sour spores were the most prevalent type in sugar and starch. In commercially available sugar, 72 samples contained flat sour spore counts of 0-75, 13 samples contained 75-150 spores, and 6 samples contained greater than 150 spores per 10 g in sugar. In 20 samples of starch, the range of flat sour spores was 0-325 spores per 10 g of starch with an average of 139 spores per 10 g. In 1939 Hall made a study of the survival of thermophilic foodspoilage organisms in stored white beet sugar. H e found no relation with the type of container in which the sugar was stored and the decrease in spore counts. He suggested that the reason for the decrease in spore count was due to their dehydration caused either by the drying of sugar during manufacture or by the presence of hygroscopic impurities in the sugar. He further suggested that storage of sugar was a means of improving its bacteriological quality. According to C. B. Denny (1969), however, it takes several years of storage to reduce large numbers of spores to an acceptable level. The distribution of rough and smooth variants of aerobic, sporeforming bacteria in a sugar refinery was made by Fields (1968a) who

178

MARION L. FIELDS

found that the raw sugar cane from the mill contained 36 times more spores than the finished product. The spore population comprised 92% smooth variant which dropped to 40% after filtration of the raw liquor (Table VIII). This is in agreement with the data of Cameron and Bigelow (1931) (Table VI). The number of spores in the sugar cane (5.0 per g) was similar to the number of spores in beet sugar as reported by Calton (1936) (Table VII). The composition of a spore population (in terms of the number of rough and smooth variants) can be determined only after considerable study of the heat resistance of the spores of these variants produced under different environmental conditions. The bacteriological counts of sugar during the refining process as studied by Fields in 1968 (1968a) (Table VIII) when compared to those of Cameron and Bigelow in 1931 (Table VI) show an improvement in the quality of sugar. Undoubtedly, the pioneering and continuing educational work of the National Canners Association has done much to help improve bacteriological quality of sugar. An example is the talk by Bohrer (1955), presented before the Bone Char Research Workers, where he reviewed some of the properties of thermophilic bacteria encountered in sugar.

D. VARIOUSINGREDIENTS Richmond and Fields (1966) made a study of various food ingredients to determine the distribution of thermophilic, aerobic spore-forming bacteria. In their study the following ingredients were tested: sugar (brown, 8 samples; granular, 5 samples; raw sugar, 6 samples); corn starch (13 samples); cake flour ( 1 sample); TABLE VIII

EFFECTOF

S U G A R HEFININ(: OPERATIONS ON NUhZBEH A N D

OF

AEROBIC SI'OIWFORhlING

Sample 1 2 3

4 5 6

7 8

Raw sugar from mill Diluted raw sugar Filtered raw l i q i i o r After bone char Filter after 20 hr Filtcr after 80 hr After vacuuiii pan Finished sugar

"Fields. 1968a.

TYPESOF

VARIANTS

THERMOPHILIC SPORES"

Spore count sporeslg

% Smooth

180.00 128.00 2.20 0.05 5.60 1.20 2.30 5.00

92 69 40 0 90 91 90 80

variant

THE FLAT SOUR BACTERIA

179

oat flour (1 sample); potato starch (1 sample); spice (11 samples and 9 kinds); green split peas (1 sample); strain G yeast (1 sample); cocoa (1 sample); gelatin (1 sample); and dried soup (1 sample of tomato and 1 of vegetable). More B. couguluns was found than any of the other species, and it was found in 14 of the 18 food ingredients tested. As to the distribution of rough and smooth variants found in these ingredients, cornstarch contained more spores of the rough variant (82%),whereas black pepper had only 21% of the spores of the rough variant. Yesair and Williams (1942),who studied spice contamination and its control, found that different spices had aerobic spore counts of 0 to 3000. However, out of 38 samples tested, 21 had counts of 0. Black pepper had counts of 1200 and 1800 per gram, while a sample of ground curry had a count of 3000 per gram. Spices, if used in canned foods, should therefore be checked to see if they contain spores which may cause flat sour spoilage.

E . WHEATAND WHEATPRODUCTS Jones et uZ. (1953) made a study of 38 samples of durum and common wheats and their products and found that the majority of the spores were found on the surface of the grain. The flat sour counts made on three different dry wheat samples were 33, 5, and 6 per gram. Spore counts were also made on the wheat during various stages of the milling process and these counts ranged from 0 to 39 per gram. Thatcher et ul. (1953)also studied the sanitation of Canadian flour mills and its relationship to the microbial content of flour. In finished flour from 50 separate mills, the flat sour spore count ranged from 0 to 32 spores per gram. Samples from 27 of the mills had spore counts less than 10 per gram. Spore counts of “dead stock” (flour) in low-grade “boots” (part of the equipment) had spore counts similar to the finished flour. The data in both of these studies indicate that wheat and wheat products may be a source of flat sour spores.

IV. RESISTANCE OF SPORES TO LETHAL AGENTS

A. EARLYMEASUREMENTSOF THERMORESISTANCE Bigelow and Esty (1920) developed a method which is still used by some researchers today in spore-resistance studies. A constant tem-

180

MARION L. FIELDS

perature bath is filled with oil. (Although this author uses mineral oil, Bigelow and Esty (1920) recommended hydrogenated oil or paraffin for temperatures above 100OC.) The temperature is regulated by a thermostat and an electric stirrer. Sealed glass tubes (7 mm inner diam., 9 mm outer diam.) contain the bacterial spore suspension. These sealed glass tubes (hard glass) are placed in wire baskets so that multiple tubes may be removed at definite intervals during the heating process. The spores are usually suspended in phosphate buffer, food extracts, or water during heating. Sterility may be determined by plating or streaking on a suitable nutrient medium, such as nutrient agar, nutrient broth, or dextrose tryptone agar. If the spores are heated in a food extract, the contents of the tube may be grown in the same food extract (sterile). B. NEWER METHODS OF h'lEASURING SPORE RESISTANCE

C . C. Williams et al. (1937)described an apparatus for determining the heat resistance of bacterial spores. The original article should be consulted for further details on construction. C. C . Williams et al. (1937)used this apparatus to study the rate of death of B . stearothermophilus rather than absolute death times. A micromethod and apparatus for the multiple determination of rates of destruction of bacteria and bacterial spores by heat were described by J. A. Stern and Proctor (1954). These authors described a low-cost apparatus of temperature range 212"-284°F. in which the spores of B. stearothermophilus are subjected to heat in phosphate buffer (pH 7.0).A straight line was obtained by plotting the mean percent surviving organisms against time in seconds. Since multiple determinations can be made at one time, data can be acquired easily; thus statistical analysis can be applied which establishes the confidence limits of the slopes or D values. A micromethod and apparatus for the multiple determination of rates of destruction of bacterial agents were also developed by Wilder and Nordan (1957).This apparatus consisted of a device for filling capillary tubes, for evacuating the tubes, and for introducing culture media into the tubes after heating. The developers of this method stated that a large number of samples can be handled with a minimum of time and materials. The come-up and cooling time are minimized by using capillary tubes. Although other methods could be cited, the procedures described above seem to be generally representative of the group as a whole.

THE FLAT SOUR BACTERIA

c. ORDER

OF

181

DEATH

The logarithmic nature of thermal death-time curves was shown by Bigelow in 1921 in his work with B . stearothermophilus 1518 as well as other flat sour bacteria. He also presented a graph showing the average of 15 typical thermophiles. This, too, was a straight line when time was plotted on a logarithmic scale and temperature on an arithmetic scale. Youland and Stumbo (1953)studied the order of death of spores of B . coagulans and found it to b e logarithmic. This judgment was based on statistical analyses of D values for different spore concentrations and different temperatures. On the other hand, Frank and Campbell (1957)found the rate of thermal destruction of spores of B . coagulans to b e nonlogarithmic. D values from three initial spore levels of B . coagulans were heated at 101.2"C using the thermoresistometer tube method. The three spore suspensions had survivor curves which were parallel and nonlinear.

D. RESISTANCEOF SPORES

OF THERMOPHILIC

BACTERIATO

HEAT The spores of B . stearotliernio),hilus have a high degree of heat resistance. According to Murrell (1955),it takes 28 min at 250°F. to destroy the spores of this organism. At this temperature the spores of Bncillus cercus have a resistance of0.013 min, Bacillus megutherium 0.08 min, and Bucillus subtilis 0.16 min. J. A. Stern and Proctor (1954) found the thermoresistance of R. steurotlzermophilus to be as follows: at 268.7"F a D value of 0.153 min (9.18 sec) with 99.7%) confidence limits of 0.147 min (8.82 sec) and 0.16 min (9.60 sec). Reed et u1. (1951) studied the heat resistance of B. stearothermophilus - 1518 spores in phosphate buffer, asparagus, green beans, corn (frozen), corn puree, peas (fresh), peas (frozen), pea puree, pumpkin puree, spinach, shrimp, and sweet potatoes. The highest F,,,,c,, value in these products was 44 min; also, the rate of destruction curves for culture 1518 in phosphate buffer and green beans were not logarithmic. However, Collier and Townsend (1956) found that B . stearothermophilus 1518 had a z value (slope of thermal death time of 0.708 min in superheated steam and curve) of 26°F and an FssOoF that the survivor curves were logarithmic. In a later study, Murrell and Warth (1965) showed the relationship between the D value and the dipicolinic acid (DPA)content of spores.

182

MARION L. FIELDS

Bacillus coagulans had a DlOoOc value of 270 min and DPA content of 10.42%dry weight, whereas one suspension of B . stearothermophilus had a DIoVcof 459 min with DPA 9.77% dry weight. The second suspension of B . stearothermophilus had a DlooOcof 714 min with a DPA content of 13.55% dry weight. The DPA content does not determine the degree of heat resistance. El-Bisi et al. (1962) studied the death and release of DPA from spores of B . coagulans. Death progressed at a higher rate than the release of DPA and calcium from the heated spores. The calcium content of spores has also been shown to be associated with heat resistance of B . coagulans. In addition to calcium, manganese caused an increase in spore resistance, whereas magnesium had no effect. Increased concentrations of phosphate caused a decrease in spore resistance (Amaha and Ordal, 1957). El-Bisi and Ordal (1956) showed that neither the type of nutrient growth medium (tomato juice agar, corn concoction agar, and thennoacidurans agar) nor its p H (5,6,or 7) had a significant effect on the death rate of the spores of B . coagulans produced. They, too, found that a phosphate concentration of 1.0%H,PO, had a markedly depressing effect on the resistance of the spores. In 1954 0. B. Williams and Robertson studied the effect of temperature of incubation at which the B . stearothermophilus spores were formed on the heat resistance of the spores. Both the spores of facultative strains and obligate strains increased with increased incubation temperature. El-Bisi and Ordal (1956) and Lechowich and Ordal (1962) also found that the higher the temperature causing spore production in B . coagulans the greater the heat resistance of these spores. The suspending medium in which spores are heated also has an influence upon the heat resistance of the spores. When spores of B . stearothermophilus were heated in disodium phosphate buffer, an apparent increase in resistance with decreasing molal concentration of phosphate buffer over the range of M/15 to M/120 was observed b y 0. B. Williams and Hennessee (1956). Cationic environments also influence the thermal resistance of B . coagulans. Frank (1955) studied the effect of added sodium, potassium, calcium, magnesium, and iron in tomato juice (pH 4.4) and found a statistically significant decrease in the thermal resistance of B . coagulans spores. The deletion of calcium, magnesium, or iron did not change the thermal resistance. A. M. Cook and Gilbert (1965) determined that a 10% dry weight suspension of yeast in distilled water protected the spores of B . stearothermophilus during heating. El-Bisi et al. (1955) found that 100 ppm of fixed copper, maneb, captan, and dichlone (fungicides) in tomato

T H E FLAT SOUR BACTERIA

183

juice (pH 4.2) caused a significant decrease in the D value of the thermal death rate of the spores of B . coagulans. T h e effect of storage time on thermal destruction of spores of B . stearothermophilus was studied by Fields and Finley (1962). When the spores were young in age and used in inoculated pack studies with green beans, there were survivors after a process time equivalent to 250°F for 18 min, indicating the F value was greater than 18. However, after a storage period of 16 months at 4"C, the F value was 7. A. M. Cook and Gilbert (1968a) found that a storage period of 52 weeks at 4°C or 65 weeks at -16°C had little effect on the heat resistance of spore suspensions of B . stearothermophilus. The reason for the variance between the results of Cook and Gilbert, and Fields and Finley is not apparent. In a study on rough and smooth variants of B . stearothermophilus NCA 1518 b y Fields (1963), the smooth variant was more heat resistant than the rough. A study of the structure of spores of rough and smooth variants of B . stearothermophilus revealed that smooth variant spores (DzSwF=2.32min) had a thicker spore wall than the rough variant (D2s,0F=1.42min). Also, the layers of the spore wall seemed to adhere more closely in the smooth than rough variants. The difference in the spore coat may be associated with the heat resistance of the spores (Rotman and Fields, 1966). A. M. Cook and Brown (1965)described a procedure whereby the spores of B . stearothermophilus were placed on paper and used as indicators of a given heat process. Replicate experiments indicated that reproducible results can be achieved with these spore papers. If the spore suspension used to treat the papers was too low in spore counts, the spores lost viability. Counts of lo6 spores were needed if long storage periods were used. The spores of B . stearothermophilus were more resistant to ultraviolet light than those of Bacillus globigi, but were influenced less by the air temperature during irradiation. The thennophile was less mutable than the niesophile when compared as to streptomycin resistance, fermentation, and biochemical mutants (Mefferd and Campbell, 1952). Lategan (1966) found, however, that spores of B . stearothermophilus NCA 1518 were more sensitive to heat after pretreatnients with ultraviolet irradiation. This sensitivity increased with exposure to visible light. Lategan suggested that these results have practical importance in canning plants whereby irradiation of contaminated sugar solutions is followed by heat sterilization of'the canned product. Linear (semilogarithmic) and parabolic survival curves were derived

184

MARION L. FIELDS

after spores of B. stearothermophilus were exposed to heat and ultraviolet light, respectively. Increased salt concentration (1-8%)caused a progressive decrease in the thermal destruction of the spores of B. coagulans. Sucrose and dextrose (50%) enhanced the heat resistance; citric, acetic, and lactic acids (0.5, 1.0, and 2.070) increased the death rate of the spores, whereas 0.1 % sodium benzoate and 0.2% ascorbic acid had no effect on the death rate (Anderson et al., 1949). Allylisothiocyanate (at 10 ppm) in buffer (pH 4.0) in apple juice and in grape juice had a more distinctive effect than did oil of onion and oil of garlic, which had an influence upon spores of B. coagulans (Kosker et al., 1951). The effect of recovery conditions (incubation temperature, incubation time, recovery media, and diluent) were studied by A. M. Cook and Gilbert (1968b) using the spores of B. stearothermophilus. Maximum counts for heated spores were obtained at 45" and 50"C, whereas the maximum counts for unheated spores were at 50", 5 9 , and 65°C. Tests with incubation times of 1 , 2 , and 3 days showed no apparent differences in recovery. Recovery media had a significant effect on the numbers of spores recovered. The highest recoveries were made with antibiotic assay medium A with 0.1% starch added (AAMS) (pH 7.3).The AAMS medium had a significant influence on heat activation responses. Four- to fivefold increases in viable counts were obtained when AAMS media were used as compared to 1- to 2fold increases with dextrose tryptone agar. Sodium chloride in the recovery medium suppressed significantly the number of heated spores recovered. A. M. Cook and Gilbert ( 1 9 6 8 ~studied ) the effect of sporulating conditions and the nature of the heating medium upon heat resistance of spores of B. stearothermophilus. Spores produced with increasing temperatures were more heat resistant. For example, spores produced at 50°C had a D value of 12.2 min; spores produced at 55°C had a D value of 16.2 min; and spores produced at 60°C had a D value of 24.4 min. Only with 1000 ppm manganese sulfate in the sporulation medium were the spores less heat resistant. These workers also showed that in Sorenson's buffer (M/40)pH 7.0 and (M/15)pH 7.0, and in McIlvaine's buffer pH 7.0, the heat resistance of B. stearothermophilus differed.

E. INFLUENCE

OF

ANTIBIOTICSAND PLANT EXTRACTSON HEAT RESISTANCE OF SPORES

Spores heated in water extracts of carrots, tomato, and bean (leaves, stems, and edible portions which were sterilized by filtering through a

THE FLAT SOUR BACTERIA

185

Seitz filter) were less resistant than spores of FS 787 heated at 201°F in phosphate buffer. The spores of B . coagulans were more easily killed in 200 ppm of the following solutions than in buffer: cy-naphthaleneacetic acid, indol-3-acetic acid, P-naphthoxyacetic acid, and y-(indol-3) N-butyric acid (LaBaw and Desrosier, 1954). These authors also investigated the influences of such other compounds as 2,4-dichlorophenoxyacetic acid, 2,3,5-triiodobenzoic acid, 2,4,5-trichlorobenzoic acid, and O-chlorobenzoic acid. All of these were more toxic to the spores of FS 787 at 201°F than when the spores were suspended in phosphate buffer. The effect of subtilin on thermophilic flat sour bacteria was studied by 0. B. Williams and Campbell (1951)who found that a combination of subtilin and mild heat was ineffective for complete destruction of the spores of a number of obligate and facultative flat sour bacteria. Bacillus stearothermophilus NCA 1518 was included in the group studied. Michener (1953) also showed that subtilin had a sporicidal effect on spores, but some spores survived the mild heat treatment (5 min boiling). At 50 ppm subtilin (without heat) spores survived after 8 days, although their numbers had decreased. Neomycin, cleiomycin, and M-2517-6 were used in a study of the effects of antibiotics on the spores of B . coagulans (43-P) in tomato juice. Neomycin (250 pglml) prevented the growth of spores in unheated juice, but this antibiotic was ineffective when the juice was heated for 5 min in flowing steam (Kaufman et ul., 1954). These workers concluded that the use of these antibiotics seemed to have no practical value in the control of flat sour spoilage in tomato juice. Michener (1955)studied the action of subtilin on the heated spores of several species of Bacillus. He found that fewer spores of B . stearothermophilus and B . coagulans formed colonies after contact with 5 ppm subtilin and a heat treatment of 5 min at 100°C. Michener concluded that although subtilin is absorbed in the ungerminated spores, the spores are not affected by it until they are placed in an environment which causes them to germinate. Subtilin, methylol gramicidin, rhatany root extract, and tyrothricin were used at 20 ppm in pea brine to determine the influence on the spores of B . stearothermophilus NCA 1518. Five percent tyrothricin was also used in pea brine. Tyrothricin and subtilin at 20 ppm were also tested in corn brine against the spores of B . stearothermophilus. All of the antibiotic treatments were more effective than the control when heated at 240°F except subtilin (at 20 ppm) which approached the death rate of the control spores (C. B. Denny and Rohrer, 1959). T h e action of nisin on the heat resistance of B . stearothermophilus spores was studied by Thorpe (1960) who found that when nisin was

186

MARION L. FIELDS

removed from the spores by trypsin, the heat resistance of the spores was restored almost to normal. C. B. Denny et al. (1961) found that tylosin was more active than nisin against spores of B . stearothermophilus. Tylosin at 1.0 ppm prevented the spoilage of canned mushrooms when the product was processed for 10 min at 240°F. C. B. Denny et al. (1961) packed cream-style corn containing the spores of B. stearotherrnophilus and 1 ppm tylosin. Tylosin prevented the growth of this flat sour bacterium after 30 days at 130°F. These workers showed successful experimental use of tylosin to prevent early thermophilic spoilage of canned foods given a minimum health cook. Nisin does not influence the heat resistance of spores of B . stearothermophilus NCA 1518, but the carry-over of nisin in the recovery media reduces the count. Nisin in sufficient concentration can also prevent the spores from germinating (Tramer, 1964).

F. EFFECTOF CHLORINE ON SPORES LaBree et al. (1960) investigated the influence of chlorine on the spores of B. coaguluns. T h e sporicidal action increased with decreased pH values, increased temperatures, and increased concentration of chlorine. By increasing the temperature to 60"C,the sporicidal effect is in a practical range for use in a cannery during the clean-up period. The spores of B. stecirotlzermophi1U.E NCA 1518 were killed with 10 ppm chlorine at 26°C with a pH of 7.0 in 10 min. However, at 26°C and at p H 7.0 with 2 ppin chlorine, practically no spores were killed in 10 min (Kuhn, 1963). Mercer and Olson (1969) found that spraying tomatoes with 50 ppm chlorine moderately reduced the spore counts on the tomatoes, whereas this level drastically reduced spore counts in flume water. They also recommended that 5 to 10 ppm chlorine b e used to maintain the sanitary conditions of equipment. The lower levels of chlorine also drastically lowered the counts in flume water.

G. EFFECT

OF

IONIZING

AND

ULTHAVIOLET

RADIATION

ON

SPOHES

Photoreactivation of spores of €3. stearothe~~nophilus occurred after ultraviolet light irradiation. Ten percent sucrose in the recovery medium significantly effected the recovery of irradiated spores incubated at 50°C. Nonirradiated spores were more resistant to the salt at 65°C.

THE FLAT SOUR BACTERIA

187

than at 50°C. The reason for this phenomenon was not given by Lategan (1965). McArdle (1955) used a suspension of B . coagulans to calibrate a linear accelerator while Postweiler and Caldwell (1961) used the spores of B . stearothermophilus as a means of measuring electronbeam dosage. The survivor curve was linear over dosages of 0.2,0.4, 0.6, and 0.8 megarads. Germination of spores of B . cougziluns NURLD #770 after irradiation increased significantly after addition of biotin, thiamine, and nicotinic acid when compared to nonirradiated spores. Apparently, injured spores become more demanding in their germination requirements after gamma irradiation (Johnson, 1959). Billerbeck (1959) determined the death rate curve of B . coagulans NURLD #770 and found that the rate was nonlogarithmic when the spores were subjected to gamma rays and to heat (225OF). The death rate of spores of B. steurotlzermophilus NCA 1518 was measured by Kuhn (1963). H e found that spores in a gel (agar) had a and lower death rate than in buffer control or in glucose (lo-', lo-" M). Sucrose, on the other hand, had a protective effect at lo-', and lo-" M when compared to the buffer control but protected less than the agar gel. Glucose was superior to sucrose in agar gel systems in protecting the spores. The D value (90%reduction in the spore population) for spores in agar ( a s a sol) was 4.0 min, whereas the D value for spores in an agar gel was 9.4 min, thus showing the protective effect of the agar. Kuhn's data show that by blocking or reducing free radical diffusion in the intercellular solvent system with a colloidal gel structure the inactivation coefficients of the irradiated spores were greatly reduced. Since the death rate was less in a gel, the majority of the inactivation was initiated outside of the spore.

V. SOURCES OF CONTAMINATION IN CANNING PLANT A.

TOMATO JUICE

PROCESSING

The soil is the source of L3. conguluns in the processing plant. Doyle et n2. (1958),in a study on sanitation of canning equipment, found that soil from tomato fields contained as many as 358,000 themiophilic spores - spore counts being determined with tomato agar. These workers showed that there was a definite relation between the concentration of soil particles (in ppm) and the number of flat sour spores

188

MARION L. FIELDS

in tomato soak-tank waters. It is important, therefore, that an adequate source of potable water be available for washing of the tomatoes and that soak-tank and flume waters have a clean supply being added at all times to keep the spore counts as low as possible. Spore counts of tomatoes as received at the cannery were as high as 1060 spores/lb when detected on tomato juice agar (Doyle et al., 1958). In more recent study of tomato washing made by Mercer and Olson (1969), bacterial spore counts were found to be closely related to residual soil on tomatoes at every sampling point in a pilot plant study. The increasing use of mechanical harvesting of tomatoes has increased the soil load and therefore the bacterial spore load. If proper sanitation is not maintained in the processing plant, B . coagulans can establish itself in equipment and actually grow in the processing plant; for example, B . coagulans can grow at 28°F (Smith et al., 1952). Fields (1962) showed that B. coagulans can grow and produce spores in tomato juice in which Oidium lactis has already grown. Normal tomato juice is too acid for sporulation to occur, but if this fungus uses the acid, the pH is raised and sporulation of B . coagulans can occur. Oidium lactis may be a contaminant in tomato juice plants with poor sanitation. Because it grows on equipment, this fungus is called “machinery mold.” There are many points of possible contamination in the processing plant- some of the common ones are given in Table IX. According to Bohrer and Reed (1949)fillers, then extractors, were the source of most spores. B. LOW-ACIDVEGETABLES The low-acid vegetables such as peas, corn, potatoes, etc. may be spoiled by B . stearothermophilus. Sources of B . stearothermophilus TABLE IX SOURCESOF CONTAMINATION WITH B. coagulans I N TOMATO JUICE PROCESSING PLANTS Source Pockets of filler heads, extractors, preheaters, pumps, pipe lines, canvas flaps on entrance end of washer, brewer hose in juice line, dead ends. Poorly washed raw tomatoes, dead ends, wooden and porous materials, filling machines, conveyor belts. Field soil, conveying belt.

Reference Bohrer and Reed, 1949

Troy and Schenck, 1960

Doyle et al., 1958

189

THE FLAT SOUR BACTERIA

spores are similar to those of B . coagulans except the pH of the raw product is different. B . coagulans, which is less heat resistant than B. stearothermophilus, grows at a lower pH than B . stearothermophilus, a difference which is used in separating these bacteria taxonomically. B. stearothermophilus does not grow at pH 5.0, whereas B . coagulans does. These differences in heat resistance and pH of product are the reasons why only B . coagulans spoils tomato juice and B . stearothermophilus spoils the lower acid vegetables, which also are heated at the higher temperatures that easily destroy B . coagulans. Sources of contamination within the canning plant are given in Table X. As soil is also a common source of flat sour bacteria, high spore counts of flat sour bacteria were found in asparagus fields. Counts ranging from 1,400,000 spores per gram to 5,800,000 per gram (surface counts) were found by Mercer et al. (1960). Soil may be trapped beneath the brackets and inside the folded tip as the spear passes through the soil. Since the brackets and tips are included in the canned product, the spears must be washed thoroughly (Mercer et al., 1960). Blanchers have been the focal point of contamination of spores within the cannery because of the temperature and available nutrients necessary for spore formation. Fields (1966) showed that the rough variant of B . stearothermophilus formed spores in pea extract and on pea agar more readily than the smooth variant. Plant extracts, such as pea, support vegetative growth and sporulation. Food ingredients like sugar and starch may also contain flat sour spores which may be added to the product or be “seed” for contaminating the equipment. Although B . stearothermophilus does not grow well at low temperatures (some at 33°C and more at 37°C Smith et al., 1952) as well as B. coagulans does, growth of these organisms can occur in the canning plant in plant residues if moisture and proper temperature exist.

TABLE X SOURCESOF CONTAMINATION WITH E . steurothermophilus

IN

PROCESSING PLANT

Source

Reference

Soil Sugar, starch, wooden and porous containers, pea blanchers, pumpkin wilters, wooden paddles and rollers, pumps, pipes, extractors, raw product Raw product and pea blancher

Townsend, 1932 Townsend and Esty, 1939

Knock, 1954

190

MARION L. FIELDS

VI. CONTROL OF FLAT SOUR BACTERIA SANITIZINGAND CLEANINGPROGRAMS

A well-organized and implemented program of sanitation is a “must” if the manufacturer is to produce a high quality product. The National Canners Association (NCA) over the years has contributed much to many aspects of canning technology. In 1952 the NCA in cooperation with the Association of Food Industry Sanitarians, Inc. (AFIS) prepared a book, Sanitation for the Food-Preservation Zndustrim. For specific details on the various aspects of sanitation, the reader is referred to this book. In addition to a thorough cleaning at the end of each day, in-plant chlorination can do much to reduce the buildup of bacterial spores in the processing plant. Low concentration (2-5 ppm residual chlorine) may be sprayed on equipment and belts during operations. Not only will this treatment kill microorganisms, but this practice will also reduce the cleanup required at the end of the day. At these levels, no offflavors will occur (if no phenolic compounds are present and if no chlorinated water is used in syrups and brines). The inspection of ingredients is also important. The Oficiul and Tentative Methods of Analysis of the Associution of Oficiul Agricultural Chemists (A.O.A.C., 1945) gives detailed methods for determining flat sour spores in starch and sugar. In both starch and sugar (for 5 samples) there should be a maximum of 75 spores and an average of not more than 50 spores per gram. Bacteriological control of processing conditions should be maintained. Bacteriological surveys, which should be a part of the quality control program, should complement the sanitation program but at the same time not be the only control practice. When the results of bacteriological surveys are available, the product is already in the warehouse; therefore, emphasis must be placed upon a preventive program. The focal point of this program is an adequate sanitation program in the plant. As a check on the sanitation program, bacteriological surveys of the canning plant should be done. Some of the points under consideration are given in Table XI. If canned foods are stored at temperatures less than those required for germination of the flat sour spores, the spores (if any survived the process) will die after a holding time. Pearce and Wheaton (1952)have called this “autosterilization.” By proper quality control of the canned foods, spoilage can be prevented. Emphasis should be placed on pre-

191

THE FLAT SOUR BACTERIA TABLE XI POINTS IN BACTI.:RIOI.OGI(:AI~ SURVEYS OF TOMATO AND PEA CANNERIES

SAMPLING

Type of cannery

Sampling point

( 1 ) Extractor (2) Storage tank ( 3 ) Filler bowl (4) Closing machine ( 1 ) Raw peas before washing and

Tomato juice

Pea

Reference Hutchings, 1949

Frazier and Foster, 1959

blanching

(2) Blancher water ( 3 ) Peas after I,lanching (4) Brine from tank (5) Peas and brine after sealing and hefore sterilization

venting the entrance of flat sour spores rather than depending upon warehouse conditions to prevent spoilage.

VII. BIOLOGY OF FLAT SOUR BACTERIA A. DEFINITIONS

The reviews of Gaughran (1947) and Allen (1950) should be consulted for a general discussion of thermophiles. The growth of bacteria is influenced b y temperature. Some strains of B. stearotherinophilus are obligates (stenothermophiles),whereas other strains are facultative thermophiles (grow at 55" and 37"C, Cameron and Esty, 1926).Other designations for groups of bacteria according to temperature are discussed in Gaughran (1947)and Allen (1950).The temperature range for growth of B . coaguluns is 28"-65°Cand 33"-70°Cfor B . stearothermophilus (Smith et ul., 1952).

u.

W H Y IS

TI-IEI-IMOPHILY POSSIBLE?

In Allen's discussion of the aerobic, thennophilic spore-forming hacteria, she listed three factors that make thermophily possible. These are (1) the external environment, (2)the structural modification of the cell, and ( 3 )dynamic factors (Allen, 1953). The first of these was

192

MARION L. FIELDS

discussed under resistance of spores to lethal agents. Lamanna (1940) showed that as the growth temperature is raised in the genus Bacillus the size of the cell decreases. More attention has been given to the chemical structure of the thermophilic cells than morphological properties. Militzer et al. (1949, 1951) and Militzer and Burns (1952)demonstrated that B . stearothermophilus has thermostable enzymes. Koffler (1957) has reported on protoplasmic differences between mesophiles and thermophiles. The flagella of thermophiles (including B . stearothermophilus) were more thermostable than flagella from mesophiles. Allen (1953) pointed out that some thermophilic bacteria can grow at high temperatures without notable thermostable respiratory enzymes and that a high level of metabolism keeps repairing the cell so that growth and life can continue.

C. GROWTH OF FLAT SOUR BACTERIA

0. B. Williams (1936)reported on a tryptone medium for the detection of flat sour spores in sugar. H e found that a casein digest (tryptone) was superior to beef extract, nutrient agar, yeast extract, and yeast infusion media. The concentrations of bromcresol-purple indicator for the medium was 0.004%. A growth medium for B . coagulans which consisted of proteose peptone 5 g, yeast extract 5 g, glucose 5 g, K,HPO, 4 g, and distilled water 500 ml, with pH adjusted to 5.0 has been developed. In a separate flask, 20 g of agar are dissolved in 500 ml distilled water. The two portions are combined after they have been autoclaved. This medium is used for isolating B . coagulans from spoiled tomato juice and for producing spore crops and determining survivors in heat-resistance studies (R. M. Stern et al., 1942). In a study of the effectiveness of proteose-peptone acid agar as a medium for isolating B . coagulans from tomato juice and tomato juice equipment, Taylor (1953) found that of 41 original isolations of filamentous types only 27 were recovered on retransfer to fresh proteosepeptone acid agar. The organisms resembled an Actinomycetes. Only 12 of the 41 cultures formed spores. Because of the presence of these organisms, Taylor stated that proteose-peptone acid agar is not specific for B . coagulans when used to conduct bacteriological surveys of commercial tomato juice processing lines. To be certain that organisms growing on this agar are B . coagulans, slides should be made and spore stains carried out to determine if the organism is a sporeforming bacterium. Rice and Pederson (1954) studied the inhibitory influence of acidic

T H E FLAT SOUR BACTERIA

193

constituents of tomato juice and found that pH exerted a greater activity than total and titratable acidity, buffer capacity, and organic acids on the growth of B. coagulans. With a high concentration of spores, growth occurred in 0.8% citrate at pH 4.4, whereas with a lowspore inoculum growth occurred (2 out of 3 cultures) at 0.8% citrate (pH4.5). The influence of heat shock on the germination of spores and growth of B. coagulans in tomato juice was studied by Desrosier and Heiligman (1956a). Either a heat shock or a decrease in pH (less acid) permitted the growth of this organism. If the spores were heated and stored at 2"-4°C for a short time, no growth occurred in tomato juice. If, however, phosphate ions were added, germination occurred and growth resulted. These authors thought that phosphate ions played a role in spore germination. Neilson et al. (1959) studied the growth of B. stearothermophilus in the following medium: tryptic digest of casein 10% by volume of a 9% solution; Bacto yeast extract, 0.5%, K2HP04,0.25%; glucose, 0.1% (pH 7.2). Growth curves were established between 45" and 70°C and generation times as low as 10 min were found. Two periods of growth were observed at 65"C, whereas growth curves below 65°C were similar to Escherichia coli between 22" and 42°C. Growth of "obligate" thermophiles at 37°C was observed by Long and Williams (1959) to be a function of the culture conditions employed. At 37°C only tryptose basamin glucose permitted growth at this temperature. This medium apparently supplied essential nutrients which other media did not. These workers also found that there was a relationship between the surface to volume ratio of the growth media. The larger the surface to volume ratio the better the growth, which indicates the need for oxygen. Campbell (1954) was not able to grow one strain of B. stearothermophilus at 36°C in nutrient broth but was successful when a basamin medium was used. An adequate oxygen supply is needed for both vegetative cell growth and spore formation. Long and Williams (1960) found that aeration of broth culture of B . stearothermophilus at 37°C promoted growth and spore formation, whereas aeration at 55°C inhibited germination of spore inocula and growth. At temperatures higher than 55"C, spore formation was inhibited but vegetative growth was vigorous. These workers concluded from their study that there was no evidence indicating that increased aeration was required for growth and sporulation at elevated temperatures. Instead, the converse of this was indicated. Growth of rough and smooth variants of B . stearothermophilus was

194

MARION L. FIELDS

studied in pea extract by Fields (1966). H e found that the smooth variant grew faster from spore inoculum than the rough variant did and produced more acid than the rough when grown separately. However, when both were grown together the growth was similar to the smooth variant. With vegetative cells as inoculum, growth of the rough and smooth variants were similar to the rough, both in growth and in change in pH of the culture. The growth and interaction of rough and smooth variants of B.stearo thermophilus as influenced by oxygen tension and temperature were studied by Hill and Fields (1967a). Growth was not prevented by the exclusion of oxygen, but the generation time of the two variants was influenced. The “pure” rough (homogeneous population) had a shorter generation time when grown under anaerobic conditions at 55”C, whereas the generation time of the smooth variant was increased b y low oxygen tension. The amount of acid produced was also influenced by the oxygen tension. Temperature affected the generation time of the variants. T h e smooth variant was more sensitive to 45°C than the rough. At 55°C the generation time of the smooth variant decreased more than threefold, whereas at 65°C a twofold decrease in generation time of the smooth was noticed. A twofold decrease in generation time was observed for each increase of 10°C with the rough variant (Hill and Fields, 1967a). The type of growth media and the pH of the media have an influence on growth and interaction of rough and smooth variants of B. stearothermophilus. A slightly shorter generation time in a minimal broth occurred as compared to trypticase soy broth (TSA) devoid of phosphate, whereas the generation time of the smooth variant grown in the minimal medium was longer than that in TSA. The pH of the growth medium had an influence on the growth of the two variants. The lag time of the rough variant was only 3 hr more at p H 6 and 8 when compared to pH 7, whereas there was a lag time of 37 hr at pH 6.0 for the smooth variant and 47 hr at pH 8.0 as compared to pH 7.0. As the smooth variant grew, considerable acid was produced, whereas the rough variant produced little acid and considerable basic substances in the growth media. The nutrient requirements of B. cougulans and B. stearotfiermophilus have been studied by Cleverdon et al. (1949), Campbell and Williams (1953), O’Brien and Campbell (1957), and Marshall and Beers (1967). The nutrients required by these two species are shown in Table XII. T h e requirements varied by strain and incubation temperature. One group studied by Campbell and Williams (1953)had no

195

T H E FLAT SOUR BACTERIA TABLE XI1 NUTRIENT REQUIREMENTSOF B. cougtclans Organism

B . stecirotherttrophilrts

B . steurotlaeritiophi1 us B. cougu lu 11s

H . steurothentro-

philrrs

B. steurotherinop h ilrr s

AND

B. steorothermophilus

Requirements at 55°C

Reference

Aspartic and glutainic acids, cysteine, methionine, arginine, histidine, riboflavin, folic acid, niacin, serine, proline, leucine, adenine, (specific requirements varied with strain). Thiamine arid biotin required by dl Niacin , thi;im ine , and biotin Isoleucine, leucine. val ine, m e th ion ine, histidine, arginine, thiamine. nicotinic acid, biotin (spores). Vegetative cells the same except leucine and nicotinic acid not required Methionine, thiamine, biotin, folic acid (vegetative cell growth); spores require glutamic acid, histidine, isoleucine, leucine, valine Biotin, niacin, th iain i n e Histidine, thiamine, biotin, folic acid, leucine, methionine, tryptophan. nicotinic acid Leucine, thiamine, nicotinic acid, biotin, glutamic acid, histidine, methionine, pyridoxal, valine, folic acid Smooth variant required: arginine, histidine, isoleucine, methionine, valine, biotin, thiamine. Rough variant required: methionine, biotin.

Marshall and Beers, 1967

Cleverdon et ul., 1949 O’Brien and C a m phe I I, 1957

Cleverdon et d., 1949 Campbell and Williams, 1953

Hill and Fields, 1967b.

196

MARION L. FIELDS

differences in growth requirements regardless of incubation temperature, whereas a second group had additional requirements as the temperature increased. A third group required additional nutrients as the temperature was lowered. In a study of the nutritional requirements of rough and smooth variants of NCA 1518 of B. stearothermophilus, Hill and Fields (1967b) found that the rough variant was less demanding than the smooth, requiring only methionine and biotin for growth. In a glucose-mineral salt medium, growth of the rough variant was stimulated by arginine, histidine, isoleucine, valine, and thiamine.

D. SPORULATION OF FLATSOUR BACTERIA In the first volume of Spores, Ordal (1957)listed and discussed the following factors which are necessary for sporulation to occur: temperature, pH, oxygen requirements, carbon sources, nitrogen sources, nutrilites, and other factors. According to Ordal, the optimum temperature for sporulation is close to that for growth but in a narrower range. He reported that the optimum pH for B. coagulans NCA strain 43P was pH 7.0. Although Ordal did not give any data on oxygen requirements and carbon sources of the flat sour bacteria, he did give examples for other species of Bacillus. L-Alanine and p-aminobenzoic acid (PABA)stimulated the sporulation of B. coagulans. Fields (1962) showed that B. coagulans could grow in tomato juice with Oidium lactis and produce spores after 0.lactis utilized the acid in the juice. Bacillus coagulans grew and sporulated in tomato juice which had contained living or dead 0. lactis. Bacillus coagulans growing with 0. lactis produced spores, but when no living 0. lactis was present, sporulation did not occur although heavy vegetative growth was observed. Without 0. lactis, B. coagulans lowers the pH (made more acid) to a level where sporulation cannot occur. Sporulation of B. coagulans was stimulated on a peptone-containing agar medium when manganous sulfate, nickel sulfate, or cobalt sulfate were added (maximum stimulation being at 1 ppm) (Amaha et al., 1956). The presence of manganese broadened the pH range over which sporulation occurred. At 37"C, aeration increased the amount of sporulation of B. stearothermophilus; at 55°C it decreased the sporulation (Long and Williams, 1960).Rotman (1967) found that aeration was necessary for the smooth variant of B. stearothermophilus to sporulate in nutrient broth enriched with 0.4% yeast extract.

THE FLAT SOUR BACTERIA

197

Nutrient agar with manganous sulfate (1-30 ppm) was used to increase sporulation of B . stearothermophilus (Titus, 1957).Thermoacidurans agar (proteose-peptone agar) enriched with 10 ppm manganese (pH 7.0) was used by Fields and Jenne (1962a) for spore production of B . coagulans. Kim and Naylor (1966)suggested the following sporulation medium for B . stearothermophilus: nutrient broth, 0.8%; yeast extract, 0.4?h; MnCl2*4H20,10 ppm; Difco agar, pH 7.2. These authors also gave a preliminary procedure for use before inoculating the sporulation media. P. J. Thompson and Thames (1967)used a medium to study the sporulation of B . stearothermophilus composed of the following ingredients: tryptone, 30 g; 0.5 M K2HP04,61 ml; 0.5 M KH2P04,3.9 ml; 5.4 x lo2mM H2S04.H20,10 ml; and 1480 ml of water (pH 6.8).Maximal spore yields were obtained when the inocula were grown aerobically. The sporulation medium was aerated by bubbling air through the growth medium. Oxygen was required for rapid vegetative growth and sporulation. The addition of 15 to 30 ppm manganese stimulated sporulation. A study of sporulation of variants of B . stearothermophilus in pea extract and on pea agar was made by Fields (1966). When grown by itself in pea extract, the smooth variant reduced the p H of the medium and thus sporulation was inhibited, whereas the rough variant produced less acid and consequently more spores. The same general observations were made on pea agar. When the smooth variant was grown with the rough, the p H decreased less and the spores that were formed were those of the rough variant.

E. SPORE GERMINATION AND OUTGROWTH Germination can occur without the organism producing a colony on agar. When germination occurs and the vegetative cell divides and produces a colony, the process is called germination and outgrowth. Curran and Evans (1944, 1945) demonstrated systematically that heat at a sublethal level could induce dormant spores to germinate. They studied both mesophilic as well as thermophilic strains, including strain NCA 1518 B . stearothermophilus. Both Brachfeld (1955) and Titus (1957) showed that spores of B . stearothermophilus could be activated at temperatures above 100°C. In 1962 Finley and Fields studied heat activation and heat-induced dormancy of B . stearothermophilus spores. They noted heat-induced dormancy when spores of B . stearothermophilus were heated in dis-

108

MARION L. FIELDS

tilled water at 80", Yo", and 100°C. Only with temperatures greater than 100°C did activation occur. Maximal activation occurred at temperatures 100" to 115"C, the temperature depending upon the strain and suspension. Phosphate buffer (M/120), regardless of strain and suspension source, influenced the germination of B. stearothermophilus spores. Finley and Fields (1962) thought that the phosphate lowered the heat resistance of the spores to the extent that the heat treatments were lethal to a portion of the spore population. Further studies were made on the effect of carbohydrates in phosphate buffer on germination of B . stearothermophilus spores (Fields and Finley, 1963).In these studies it was found that different spore-germination responses occurred when spores were heated in monosaccharides, disaccharides, and polysaccharides in 0.0083 M phosphate buffer (pH 7.1).These differences were observed among various strains and among spore suspensions of the same strain. The differences were attributed to rough and smooth variants in the spore population and to osmosensitivity of spores of variants within the population when subjected to a heat shock of 110°C. Many times highly concentrated spore suspensions are produced for various studies and tests. Curran and Pallansch (1963)found that incipient germination in heavy suspensions of B. stearothermophilus NCA 1518 spores occurred at subminimal growth temperatures. This incipient germination was rapid down to 20°C but was slower and incomplete at 14°C. Dilution of the suspension prevented the rate of germination. Washing with deionized water had no appreciable influence upon early germination of spores. Calcium dipicolinate incorporated into tryptone glucose extract agar was used b y Busta and Ordal (1964) to enumerate the endospore population (total viable count) without heat activation. Their method, based on 50 mM CaCl, and 40 mM sodium dipicolinate as the active components, was used to germinate spores of B. subtilis, B. stearothermophilus, B . megatherium, and B . coagulans. Dipicolinate-induced germination of B. stearothermophilus spores was studied by Fields and Frank (1969). T h e optimum pH for spore germination of rough variant of strain M was found to be 5.5. Since at pH 5.5 the dipicolinate molecule is in the doubly ionized form, these researchers regarded dipicolinate anion as the effective inducer of germination. The addition of Mg2+,Ca2+,Mn2+,and Co2+ions to the germination system brought about an increasing inhibition in spore germination with 100% inhibition occurring with Co2+.Germination occurred over a temperature range of 30" to 60"C, with the greatest

THE FLAT SOUR BACTERIA

199

germination occurring between 40" to 50°C. Selected carbohydrates (glucose, fructose, sucrose, maltose, dextrins, and starch) and plant extracts (green beans, peas, spinach, and corn) were used to study the influence of these materials on heat activation of B . stearothermophilus spores (Fields and Finley, 1964). The concentration of the carbohydrates in which the spores were heated at 110°Chad an influence on the number of spores that germinated. With increased carbohydrate concentration (0.001,0.01, and 0 . M ) the dominant trend was for reduced counts. The spores were activated in spinach, pea, and corn extracts, whereas they were suppressed in bean extract. The decline in spore counts was attributed to an increase in osmotic pressure in both plant extracts and carbohydrate solutions. The increase in counts in the plant extract was not explained. The influence of osmotic pressure on the germination of B . stearothermophilus was continued by Fields (1964) who found that heat shocking of spores (1l0OC) in 20% sucrose solutions caused the rough variant to decrease in a mixed population (rough and smooth variants) of strain M. The rough variant of strain NCA 1518 of B . stearothermophilus also decreased in counts when heated (110°C) in 20% sucrose. The smooth variant, however, increased in counts in the presence of 20% sucrose. These data also suggest that spores respond to different osmotic pressures. In a study of dormancy and activation of bacterial spores, Lewis et al. (1965) showed that by subjecting the spores of B . stearotherniophilus to pH 1.5 for 80 min at 25°C the rate of germination increased from 18 to 80%. If the spores were exposed to 0.02M calcium ions (pH 9.7) both dormancy and heat resistance were restored to the spores. In a study of the relation between heat activation and colony formation of the spores of B . stearother)no),lzilus by A. M. Cook and Brown (1%4), it was found that only about 5% of the unheated spores developed into colonies, whereas this increased to about 50% with optimum heat activation. In a letter to the editor, Brown et aZ. (1968) stated that subjecting the spores to 0.5 N HCl at 25°C increased the colony count to the value of total count with a release of dipicolinic acid from the spores. D. W. Cook et ul. (1964) found that carbon dioxide was necessary for the initiation of growth of germinated spores of B . steurotliermop}iilus but that carbon dioxide was not necessary for spore germination. Heat activation of B . coagulans spores was accomplished by Desrosier and Heiligman (1956b).The maximum activation at four temperatures was as follows: 65"C, 40 min; 75"C, 20 min; 85"C, 5 min; and

200

MARION L. FIELDS

95"C, 2 min. In addition to studying activation, these workers also studied inhibitors of spore germination of B. coagulans. Sodium arsenate, 2,4-dinitrophenol, sodium azide, sodium arsenite, sodium fluoride, and iodoacetic acid inhibited spore germination. These respiratory inhibitors prevented the use of glucose or sodium pyruvate in the germination solution and hence oxidation of these compounds. Heiligman et al. (1956)also studied chemical compounds that stimulated germination of B. coagulans spores. L-Alanine was the only amino acid which stimulated germination. (L-Glutamic acid, L-aspartic acid, and tyrosine were also tested.) The highest rate of germination occurred when glucose, L-alanine, and adenosine were combined in the substrate. The influence of lysozyme treatment and age of spores of B. coaguZans strain 27 when heated (80°C for 15 min) in tomato juice at different pH's was studied by Fields and Jenne (1962b). Two-year-old, non-lysozyme-treated spores, when heated in samples of tomato juice at pH 4.1,4.5, 5.5, and 6.5 gave increasing recoveries with decreasing pH (highest recoveries at p H 6.5).There was no true activation since, at pH 6.5, the recovery, as computed as percent of the control, was only 64%. New, non-lysozyme-treated spores, when heated in the same menstruum, gave higher recoveries than the two-year-old spores, but still there was no true activation response when heated at the same temperature. New, lysozyme-treated spores were activated at pH 4.5,5.5, and 6.5, whereas only recoveries of 58%occurred at p H 4.1.

F. h'lORPHOLOGY

AND

CHEMICAL COMPOSITION OF SPORES

Berlin et al. (1963)used gas-displacement and gas-adsorption techniques to determine the chemical density and physical surface properties of B. stearothermophilus NCA 1518 held in the dry state. T h e lyophilized spores had a density of 1273 g/cm2. These researchers calculated the specific surface area and average pore radius for spores of this thermophile. The spores of B . stearothermophilus had a surface area (m'lg) of 5.20 as contrasted to a surface area of 2.97 for B . cereus and 5.04 for B. subtilis. The average pore radius for B. stearothermophilus was 95 A as contrasted to 236 A for B. cereus. Neither the density nor the surface area could be correlated with heat resistance; however, these workers concluded that the surface of dry spores was relatively smooth and impervious, having only a few pores which accounts for the permeability of the spores.

201

THE FLAT SOUR BACTERIA

The structure of spores of rough and smooth variants of B. stearothermophilus with special reference to their heat resistance was studied by Rotman and Fields (1966)who found that there were differences in morphology of the spores which could be attributed to differences in values of the smooth and rough heat resistance of the spore. T h e DZsrF variants were 2.32 and 1.42 min, respectively. A comparison between smooth and rough variants is shown in Table XIII. Inspection of thin sections of the spores of both variants revealed that there were layers in the spore wall of both variants. The adherence of these layers was greater in the smooth variant spores than in the rough variant with spore walls being thicker in the smooth variant. Also, in the smooth variant there were two layers of low electron density between the spore wall and cortex. Together with the spore wall, this structure may act as a barrier to heat and thus may help to explain the heat resistance of the smooth variant, although the chemical composition may also influence the heat resistance of the spores. Calcium dipicolinic acid has been associated with heat resistance but did not seem to be related to resistance in this case since the rough variant had more cortex than the smooth but less heat resistance. Using absorption spectroscopy, Rotman (1967)analyzed the spores of rough and smooth variants of B . stearothermophilus for calcium, manganese, magnesium, and zinc. The smooth variant spores were richer in calcium, manganese, and zinc; those of the rough variant contained more magnesium. At pH 14, the dipicolinic acid (DPA) content of lo8 spores was 41.4 pg, and for the rough variant it was 39.5 pg/lOs spores. No direct relationship was found between DPA, mineral concentrations, and heat resistance even though the rough variant had a D2srFvalue of 3.5 min, and the D,,,, value for the smooth was 2.33 min (Rotman, 1967). The amino acid content of the above spores was also determined TABLE XI11 COMPARISON BETWEEN ROUGH AND SMOOTH VARIANTS" (AVERAGE OF 1 0 SECTIONS) Variant Rough Smooth

Spore wall

Cortex

Spore core wall

(A)

(A)

(A)

1163.0 1338.3

1437.5 1137.8

856.8 b

"Rotman and Fields, 1966. Copyright @ 1966 by the Institute of Food Technologists.

"Couldnot be observed.

202

MARION L. FIELDS

(Fields and Rotman, 1968). The values are shown in Table XIV. T h e smooth variant spores were richer in amino acids except for ornithine and half-cystine. Since spores of the variants differed in heat resistance, the type of amino acids may play an important role in heat resistance, a fact which needs further investigation. Curran et ul. (1943) made spectrochemical analyses of vegetative cells and spores of bacteria and found that spores of B. stearotlierniopliilus were richer in calcium and lower in potassium than the vegetative cells from which they were derived. Vegetative cells, however, were richer in phosphorus but lower in copper and manganese than the spores. In general, these workers found that higher concentrations of calcium were associated with enhanced heat tolerance and heat resistance. Windle and Sacks (1963) used electron paramagnetic resonance to study manganese and copper in dry, lyophilized spores of €3. couguluns that had been produced on manganese-rich media. These TABLE XIV AMINO ACIIX OF THE SPORES OF Roucij AND SMOOTHVARIANTSOF Bacillus stecirotherino~Jliilu.se Amino acid

S m ootli

Rough

Smooth/rough

( / q j l O H spores) Aspartic acid Threonine Serine GI utam ic acid Proline Glycine Alanine Valine Half-cystine Methionine Isoleucine Leucine Tyrosine Phen ylalariine Omithine Lysine Histidine Arginine

73.6 39.9 28.1 132.3 43.0 63.9 94.1 86.5 13.5 20.1 44.1 90.6 61.1 44.1 3.8 54.2 23.3 54.5

55.0 27.4 19.2 108.0 38.1 43.4 76.9 58.1 6.2 16.1 26.8 64.0 44.3 28.6 8.1 36.0 16.1 38.4

Total

970.7

710.7

1.3 1.4 1.5 1.2 1.1 1.5 1.2 1.5 2.2 1.2 1.6 1.4 1.4 1.5 0.47 1.5 1.4 1.4 -~

1.4

“Fields and Rotman, 1968. Reprinted by permission of the American Society for Microbiology.

THE FLAT SOUR BACTERIA

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workers hypothesized that manganese is bound in at least two different ways in spores: to DPA and to the spore-coat protein by an ionic bond.

G. ENZYMESOF

VEGETATIVE

CELLS

The starch-saccharifying enzymes of B . stearothermophilus have been given considerable attention during the past few years - more than the other enzymes of this organism. Stark and Tetrault (1951) studied a saccharifying enzyme which was produced at 70”C, the upper limit of growth for this particular strain. Studies were made with a crude enzyme preparation on a 2% starch substrate (pH 7.0) at 90°C. The studies were carried out over a period of 12 hr. Maltose concentrations increased during the whole incubation time. Further research of Stark and Tetrault (1952) was reported. In this research, 35 cultures of B. stenrothermophilus were studied to determine if they could hydrolyze five types of starches under different cultural conditions. The breakdown of starch was independent of the type of starch and the initial pH of the medium studied. The hydrolysis of starch was better in liquid than solid media. Starch concentration and incubation time were important factors. Dextranization was a better index of hydrolysis of starch than production of acid or sugar. Soluble starch was better than insoluble starch in demonstrating hydrolysis. The type of protein in the growth media had an influence on the formation of amylase. Trypticase was the best protein source. Studies on thermal and p H stability of the amylase of B . stearotherniophilus were conducted by Hartman et al. (1955).The amylase was reasonably stable within a pH range of 5.25 to about 7.5 at 60°C. Campbell and his associates made a comprehensive study of a-amylase of B. stearothermoplziltrs. Manning and Campbell (1961), using crystallized a-amylase, studied some of the enzyme’s properties. aAmylase had a pH optimum of 4.6 to 5.1 with the optimal rate of hydrolysis occurring from 55” to 70°C. Calcium was necessary for enzyme activity. The enzyme had a large number of free acidic groups. The molecular weight was estimated to be 15,404 (based on osmotic pressure) or 15,600 (based on sedimentation and diffiision data). Manning and Campbell (1961) suggested that a-amylase “exists as a semirandom or random-coiled, well-hydrated molecule, with any secondary structure due to the presence of disulfide bonds.” Campbell and Manning (1961) gave the following chemical composition of aamylase: glutamic acid, 22 (amino acid residues); proline, 22; aspartic acid, 11; valine, 11; glycine, 9; leucine, 9; alanine, 8; threonine, 8; iso-

204

MARION L. FIELDS

leucine, 7 ; lysine, 6; phenylalanine, 6; serine, 6; half-cystine, 4; histidine, 4; arginine, 3; methionine, 3; tyrosine, 3; and amide, 3. Campbell and Cleveland (1961) determined the terminal amino acid residues of crystalline a-amylase of B. stearothermophilus. Using the fluorodinitrobenzene and phenylthiohydantoin methods, two moles of NH,-terminal phenylalanine per mole of enzyme were determined. Their data suggested that the enzyme is composed of two polypeptide chains having identical NH,-terminal and COOH-terminal residues. Apparently these residues are held together by disulfide bonds. a-Amylase synthesis can be induced in growing cultures of B . stearothermophilus. In a growing culture, the enzyme was produced during the logarithmic phase of growth with the amount of enzyme produced being proportional to the rate of growth. The poorer the carbon source for growth (glycerol, 109 units of enzyme/ml; glucose, 103 unitslml; sucrose, 45 units/ml) the higher was the amount of aamylase produced. Cells grown at the same rate on sucrose and fructose had different amounts of enzyme produced. Cells grown on sucrose had 45 units/ml, whereas cells grown on fructose produced only 0 to 4 units/ml (Welkei,and Campbell, 1963a). Phenyl-, methyl-, and ethyl-a-D-glucoside and methyl-P-D-maltoside were good inducers of a-amylase. These conipounds could not, however, serve as a carbon source in a chemically defined medium with 0.1% casein hydrolyzate (Welker and Campbell, 196313). Maltose, maltotriose, maltohexaose, maltopentaose, and maltotetraose (all at10-4M) produced a stimulation in the differential rate of a-amylase production (in chemically defined medium) by 1.2, 1.6, 1.9,2.3, and 3.0 times that of the sucrose control (Welker and Campbell, 1963~). Not only did Welker and Campbell (196313) study a-amylase production with actively growing cells of B. stearothermophilus but also washed-cell suspensions. They found that the addition of maltose methyl-P-D-maltoside, or phenyl-a-D-glucoside at lOP3M induced the cells to produce a-amylase at a constant rate for 60 min and then level off. Washed cells without the inducer formed only small quantities of a-amylase. Glucose (2 X 10-3M), sucrose (10-3M), and glycerol (4 X 10-3M) failed to induce enzyme synthesis. If the cells were starved of nitrogen, they failed to produce the enzyme (Welker and Campbell, 1963d). The presence of 5-methyltryptophan did not inhibit the synthesis of a-amylase when induced with pure maltose, nor did it prevent the incorporation of I4C in proline into a-amylase. On the other hand, p-fluorophenylalanine prevented the formation of active a-amylase in a system of washed-cell suspension.

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205

The enzyme glyceraldehyde-3-phosphate dehydrogenase, another enzyme involved in carbohydrate metabolism, was crystallized by Amelunxen (1966) from B . stearothermophilus. This enzyme was thermostable. T. L. Thompson et ul. (1958) studied aldolase, another enzyme in carbohydrate metabolism. This enzyme, too, was thermostable due to disulfide linkages. If this enzyme was treated with sulfhydryl compounds, it was rendered more susceptible to heat inactivation, but the enzyme activity was also increased. Pathways of glucose metabolism by rough and smooth variants of B . steurotiiermophilus were studied by Hill et ul. (1967) using the radiorespirometric method. They found that the Embden-Meyerhof (EM) pathway was more active in the smooth variant than in the rough. The smooth variant participated 4.2% in the monophosphate shunt, whereas participation in the Em pathway was 95.8%. On the other hand, the metabolism of the rough variant was exclusively or in combination with a pathway (other than monophosphate shunt) by the EM pathway. The rough variant was 81.3% efficient in the tricarboxylic acid system, whereas the smooth was 4.9%. Gaughran (1949) studied the temperature of activation of certain respiratory enzymes of stenothermophilic bacteria. The respiratory mechanism functions below the minimum growth temperature of these organisms. H e calculated the activation energies for various enzymes involved in respiration as follows: dehydrogenases 28,000 to 28,500 calories per gram-molecule; cytochrome oxidase and cytochrome b and c (substrate phenylenediamine) 16,800 calories per gram-molecule; cytochrome oxidase and cytochrome c (substrate, hydroquinone) 20,200 calories per gram-molecule; catalase 4,100 calories per gram-molecule; complete aerobic respiratory system (plus added glucose) 29,500 calories per gram-molecule. Electron transport particles from €3. stearothermophilus were isolated and studied by Downey et ul. (1962). These particles could oxidize succinate, malate, diphosphopyridine nucleotide, p-phenylenediamine, and hydroquinone. The restorative effect of coenzyme Q and vitamin K implied that a quinonelike intermediate had a part in electron transport. A vitamin K-like naphthoquinone was isolated from B stearothermophilus. This compound was susceptible to light aiid could be isolated from electron transport particles using organic solvents. In 1964, Downey reported on phosphorylation electron transport particles. The enzyme was partially inactivated by 2,3-dimercaptopropanol or light (360 mp), an inactivation which was partially re-

206

M A R I O N L. FIELDS

stored b y the addition of the soluble fraction (of the isolation of the particle). A proteolytic enzyme of B . stearotherniophilus was produced at 55"C, purified, and studied b y O'Brien and Campbell (1957). At 55"C, maximum activity of the proteolytic enzyme occurred at a pH of 6.9-7.2. For activity on casein, the enzyme required calcium and manganese ions. The enzyme hydrolyzed the following compounds: casein, gelatin, alkaline hemoglobin, alpha soy protein, L-leucylglyclglycine, triglycine, tetraglycine, and glutathione. Sulfhydryl inhibitors ( N - ethylmaleimide and p-chloromercuribenzoate) inhibited the enzyme hydrolysis of casein. Glutathione reversed the inhibitions. A peptidase (an endocellular enzyme) of B . stearothermophilus was partially purified and studied by Matheson and Armstrong (1967). Cobalt ions activated the enzyme. The optimum temperature of the enzyme using L-leucylglycine as the substrate was about 70°C. The optimum pH varied with the substrate. With L-leucylglycine as substrate, the optimum pH was 8.1, whereas the optimum pH with Lalanylglycine was 7.1. Of the 19 substrates tested, the two methionyl peptides were hydrolyzed most rapidly. In a study of nitrate reductase of B . stearotliermophilus, Downey (1966) found that the organism required nitrate to grow in the absence of oxygen. The presence of oxygen tended to retard the synthesis of this enzyme. Cyanide and azide strongly inhibited nitrate reductase: carbon dioxide did not. An inorganic pyrophosphatase of B . stearothermophilus had cofactors of magnesium and cobalt ions although the pH optima for these two ions differed. Cobalt replaced magnesium as the optimal cofactor at temperatures above 80°C. The enzyme catalyzed appreciable pyrophosphate hydrolysis at 95°C (Mathemeier and Morita, 1964). The optimal enzyme activity occurred at 700 atm of pressure when tested from 100 to 1000 atm. This enzyme was more stable at 90°C in the presence of the cofactor than the substrate. H. METABOLISMOF NITROGENCOMPOUNDS The amino acid uptake, protein, and nucleic acid synthesis were studied by Bubela and Holdsworth (1966). At 35"-38"C, the uptake of amino acids was low but increased with increased temperatures to an optimum at 60°C. This low rate of amino acid and uracil uptake was thought to be the reason growth occurred slowly at 40°C. These workers hypothesized that the reason B . stearotliermoi?liilus grows at

THE FLAT SOUR BACTERIA

207

high temperatures is because of the high rate of protein and nucleic acid turnover. For this reason, the cell can repair any heat damage which might occur. Amino acids labeled with I4Cwere used to study the incorporation of these compounds into protoplasts of B . stecirotliermo),hilus. When this was done, the radioactivity appeared in cytoplasmic membranes. Enzymes from lysed cells activated amino acids and transferred them to RNA. Heat at 60"C, however, tended to decrease this activity. Bubela and Holdsworth (1966) listed the following factors contributing to growth of the organism at 60°C:

(1) Energy of enzyme activation is higher (2)Turnover of protein and nucleic acid is more rapid ( 3 ) Organization into membrane type structures can confer a certain amount of heat stability on enzymes. Using a subcellular protein synthesizing system from B . stecirotliermophilus, native RNA directed the incorporation of I4C-tagged phenylalanine, lysine, and proline into protein. The optimal temperature was 55"-60°C. Polyuridylic acid (poly-U) was found to have the greatest activity in directing the incorporating phenylalanine at 65°C at a Mg'+ concentration of 0.018 M . At lower concentrations (0.018) incorporation was optimum at 37°C. The incorporation of leucine by poly-U was not affected by the Mg2' concentration. Polyadenylic acid stimulated lysine incorporation at 65°C more than at 37°C (Friedman and Weinstein, 1966). When ribosomes, ribosomal RNA, and soluble RNA of B . stecirotherniophilus were compared to the same components of E . coli, they were fairly similar with respect to the main physical and chemical properties. However, some differences were found in the nucleotide composition of ribosomal RNA. The responses of the B . stecirotherniophilus ribosomes to heat were more stable than those of E . coli (Mangiantini et cil., 1965). T h e maximum ribonucleic acid concentration per cell increased with increase in growth temperature of B . steciromophilus until the optimum growth temperature was reached. After this, the amount of ribonucleic acid per cell decreased (Wellerson and Tetrault, 1955). Saunders and Campbell (1966)examined the gross amino acid composition of B . stecirothermoplailzcs and found no marked differences from those reported for E . coli ribosomes. T h e y hypothesized that the thermal stability of the ribosomes of B . stearothermophilzis was due to the unusual packing arrangement of the protein to the RNA or differences in the primary structure of the ribosomal protein.

208

MARION L. FIELDS

VIII. RESEARCH NEEDS

Research is needed on the classification of the thermophilic bacteria. This author has isolated spore-forming bacteria which grow at 65°C and also grow on acid proteose-peptone agar. Bacillus stearothermophilus grows at 65"C, B . coagulans does not; B . coagulans grows on acid proteose-peptone agar, B . stearothermophilus does not (Smith et al., 1952).Obviously, more information is needed in order to classify these intermediate thermophilic bacteria. The potential role of the rough variant in spoilage of canned foods needs to be determined. The rough variant occurs with the smooth variant in food ingredients and in soil. The stability of the rough is particularly important. If the rough variant changed into the smooth variant (the more heat-resistant form), possibility of more spoilage might occur. Genetic as well as ecological studies are indicated to determine the significance of variants in spoilage of canned foods. Methodshof reducing bacterial spore resistance to heat are still needed. If methods or materials could germinate the spores prior to processing the canned food, less heat would be needed to preserve the food, thus resulting in a more nutritious and desirable food product. Studies of spores formed in chemically defined media would be most helpful in trying to determine the role of dipicolinic acid, amino acids, and various elements in heat resistance. The rough and smooth variants are excellent tools for such study. ACKNOWLEDGMENT Appreciation is expressed to Cleve Denny and Keith Ito of the National Canners Association for their helpful suggestions concerning this manuscript.

REFERENCES Allen, M . B. 1950. The dynamic nature of thermophily. J. Gen. Physiol. 33,205-214. Allen, M. B. 1953. The thermophilic aerobic spore-forming bacteria. Bacteriol. Reu. 17, 125- 173. Amaha, M., and Ordal, Z. J . 1957. Effect of divalent cations in the sporulation medium on the thermal death rate of Bacillus coagulans var. thermoacidurans. J . Bacteriol. 74,596-604. Amaha, M., Ordal, Z. J., and Touba, A. 1956. Sporulation requirements of Bacillus coagulans var. thermoacidurans in complex media. J. Bacteriol. 72,34-41. Amelunxen, R. E. 1966. Crystallization of thermostable glyceraldehyde-3-phosphate dehydrogenase from Bacillus steorothermophilus. Biochim. Biophys. Acta 122, No. 2,175-181.

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209

Anderson, E. E., Esselen, W. B., and Fellers, C. R. 1949. Effect ofacids, salt, sugar, and Food other food ingredients on thermal resistance of Bucillus thermocrcid~r~uris. Res. 14,499-510. A.O.A.C. 1945. “Official Methods of Analysis,” 6th ed. Assoc. Offic. Agr. Chemists, Washington, D.C. Ball, C. 0. 1923. Thermal process time for canned foods. Bull. Natl. Res. Council ( U . S . ) 7, No. 37, 1-76. Barlow, B. 1913. A spoilage of canned corn d u e to a thermophilic bacterium. M.S. Thesis, University of Illinois, Urbana, Illinois. Bartholomew, J. W., and Rittenberg, S. C. 1949. Thermophilic bacteria from d eep oceaii bottom cores. j . Bucteriol. 57,658. Berlin, E., Curran, H. R., and Pallansch, M. J. 1963. Physical surface features and chemical density of dry bacterial spores.]. Bacteriol. 86, 1030- 1036. Berry, R. N. 1933. Some new heat resistant, acid tolerant organisms causing spoilage in tomato juice.]. Racteriol. 25,72-73. Bigelow, W. D. 1921. T h e logarithmic nature of thermal death time curves. / . Zufect. Diseases 29,528-536. Bigelow, W. D., and Esty, J. R. 1920. T h e thermal death point in relation to time of typical thermophilic organisms.]. Infect. Diseuses 27,602-617. Billerbeck, F. W. 1959. Effect of equal lethal radiation and thermal treatments on bacterial spore germination. Ph.D. Thesis, Purdue University, Lafayette, Indiana. Bisset, K. A. 1955. “The Cytology and Life-History of Bacteria.” Livingstone, Edinburgh and London. Bohrer, C. W. 1955. Sugar for canners use. A review of some properties of t h e n o p h i l i c bacteria encountered in sugar. “Fourth Technical Session o n Bone Char Research,” Mater. Testing Lab. Natl. Bur. Std., Washington, D.C. Bohrer, C. W., and Reed, J. M. 1949. Tomato juice spoilage studies in 1948. Nutl. Canners’ Assoc., Inform. Letter Conu. Issue 1219,80433. Brachfeld, B. A. 1955. Studies on media composition and heat activation for the demonstration of viability of spores of Bacillus ster!rothermophilus. Ph.D. Thesis, University of Illinois, Urbana, Illinois. Breed, R. S., Prickett, P. S., and Yale, M. W. 1929. T h e significance of thermophilic spore-forming bacteria in pasteurized milk.]. Bucteriol. 17,37-38. Brown, M. R. W., Brown, M. W., and Porter, G. S. 1968. Activation of Bacillus stearothermophilus spores and release of dipicolinic acid after hydrochloric acid treatment. J . Pharm. Pharmacol. 20,80. Bubela, B., and Holdsworth, E. S. 1966. Protein synthesis in Bucillus stearothermophilus. Biochim. Biophys. Actu 123, No. 2,376-389. Busta, F. F., and Ordal, Z. J. 1964. Use of calcium dipicolinate for enumeration of total viable endospore populations without heat activation. A p p l . Micro6iol. 12, 106-1 10. Calton, F. R. 1936. Thermophilic contamination within the sugar factory. Ind. Eng. Chem. 28,1235-1238. Cameron, E. J. 1936. Report on methods for detecting and estimating numbers of thermophilic bacteria in sugar.]. Assoc. Ofic.Agr. Chemists 19,438-440. Cameron, E. J. 1937. Sugar, starch and spoilage. Food Ind. 9, 182-183. Cameron, E. J., and Bigelow, W. D . 1931. Elimination of thermophilic bacteria from sugar. Ind.Eng. Chem. 23,1330-1333. Cameron, E. J., and Esty,J. R. 1926. T h e examination of spoiled canned foods. 2. Classification of flat sour spoilage organisms from nonacid foods. ]. Infect. Diseases 39, 89-105.

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Cameron, E. J., and Yesair, J. 1931. Canning tests prove presence of thermophiles i n sugar. Food Itid. 3, 265. Campbell, L. L. 1954. T h e growth of an “ol,ligate” theimophilic lxwterium at 36°C. J . Hacteriol. 68,505-507. C:iniphell, L. L., arid Cleveland, P. D . 1961. Thermostable alpha-amylase o f Bacillus stemrot/ierfnophi/u,~. IV. Amino-terminal and carboxyl-terminal amino acid analysis.]. Ri(i1. Clierri. 236, No. 11,2966-2969. Campbell, L. L., and Manning, G. B. 1961. Thermostable alpha-amylase of Rncillus .Ptemrot/zcrt,iop~zilu,~. 111. Amino acid composition. J . R i o l . Chem. 236, No. 11, 2962-2965. Campbell, L. L., and Williams, 0. B. 1953. T h e effect of temperature on the nutritional requirements of facultative and obligate thermophilic bacteria. J . Racteriol. 65, 141-145. Clark, F. M . , and Tanner, F. W. 1937. Thermophilic canned-food spoilage organisms in sugar and starch. Food Hes. 2, 27-39. Cleverdon, R. C., Pelczar, M . J., Jr.. and Doetsch, R. N . 1949. Vitamin requirements of H d l 1 n . s coagulati.s.J . Racteriol. 58, 113-1 14. Collier, C. P., and Townsend, C. T . 1956. T h e resistance of bacterial spores to superheated steam. Food Zechnol. 10,477-481. Cook, A. M . , and Brown, M . R . W. 1964. T h e relation hetween heat activation and colony formation for the spores of Bncilltts stecirotherniop/iiZus. J . Pharni. P h r I ~ Z C ~ C 16, O ~ .725-732. Cook, A . M . , and Brown, M . R. W. 1965. Effect of storage on the heat resistance of Iiac. 17, Suppl., 7s-11s. terial spore papers.J. P h a r n ~Pl~urnicicol. Cook, A. M., and Gilhert, R. J. 1965. The effect o f yeast cells in the heating mediiiin on the heat resistance of Bacillus .sfecirollic,rniophilus spores. J . Phurrn. Phrrrmacol. 17. Silppl., 20s-21s. Cook, A. M . , and Gilbert, R. J. 1968a. T h e effect of storage conditions on the heat resistspores. ]. Pharnt. iince and heat activation response of Bucillus stecrrot/iernzo)~/~ilu.s P/lcirrn(tcol. 20, 626-629. Cook, A. M . , and Gilbert, R . J . 1968b. Factors affecting the heat resistance of H u c i / / u s ~ f r ~ i r ~ ~ t l i e r r spores. ~ i ~ ~ ~ I.l iThe i l i ~effect ~ of recovery conditions on colony count of i~nheatedand heated spores.]. F ~ i o dT t ~ l t r i o l3, . 285-293. Cook, A. M., and Gilbert, R. J. 1968~.Factors affecting the heat resistance of Hucilltts stectrothernio~ihillrsspores. 11. T h e effect of spornlating conditions and nature of the heating medium.]. Food Trc/rtto/.3, 295-302. Cook. D. W., Brown, L. R., and Tischer, R. C.. 1964. A carbon dioxide requirement for the initiation of growth of geimiinated spores of two thermopliilic bacilli. D e c c l o p Ifid. Microbial. 5, 326-333. Cordcs, W. A. 1928. Bacterial action in the coagulation of evaporated milk. J . Zhir!/ Ski. 11.46-51. Curran, H. R . , a i d Evans, F. R. 1944. Heat activation inducing gemlination in the spores of tlierniopliilic aero1)ic Ixicteria. J . Hncteriol. 47,437. Curran, H. R., and Evans, F. R. 1945. Heat activation inducing germination in the spores of therniotolerant and thcrniophilic aerobic lxicteria. J . Hoctcriol. 49, 335-346. Curran, H. R . , and Pallansch, M. J. 1963. Incipient germination in heavy suspensions of spores of Bacillus strtir~it/ir,mo~,hilu.s at subminimal growth temperatures. J . Rncfcriol. 86. 911-918.

T H E FLAT SOUR BACTERIA

21 1

Curran, H. R., Brunstetter, B. C., and Myers, A. T. 1943. Spectrochemical analysis of vegatative cells and spores of bacteria. J . Bacteriol. 45,485-494. deKruyff, E. 1910. Les bactkries thermophiles dans les tropiques. Centr. Rukteriol., Puruistenk., Abt. 11 2 6 , 6 5 7 4 . Denny, C. B. 1969. Personal communication. Natl. Canners Assoc., Washington, D.C. Denny, C. B., and Bohrer, C. W. 1959. Effect of antibiotics on the thermal death rate of spores of food spoilage organisms. Food Res. 24,247-252. Denny, C. B., Reed, J. M . , and Bohrer, C. W. 1961. Effect of tylosin and heat on spoilage bacteria in canned corn and canned mushrooms. Food Techno/. 15,338-340. Desrosier, N. W., and Heilignian, F. 1956a. Some factors affecting growth of Bacillus couguluiis in tomato juice. Food Res. 21, 47-53. Desrosier, N. W., and Heiligman, F. 19.561). Heat activation of hacterial spores. F o o d Res. 21, 54-62. Donk, P. J. 1920. A highly resistant thermophilic organism. J . Bucteriol. 5,373-374. Downey, R. J. 1962. Naphthoquinone intermediate in the respiration of Bacillus steorotherinophilus.J . Bucteriol. 84, No. 5,953-960. Downey, R. J. 1964. Phosphorylation in a partially restored bacterial system. Proc. Soc. E x l > f l .B i o l . Med. 115, No. 2,328-331. Downey, R. J . 1966. Nitrate reditctase and respiratory adaption in Rncillus sfeurofhernioj)/iilir,s.J . Bucteriol. 91, No. 2, 634-641. Downey, R. J., Georgi, C. E., and Militzer, W. E . 1962. Electron transport particles from B a c i l l u s , s f e c i r o t / i e r m o ~ ~ / i i l tBucferiol. ~.s.~. 83, No. 5 , 1140- 1146. Doyle, E. S., Mercer, W. A,, Denisey, J. N., Bohrer, C . W.. and Yesair, J. 1958. Sanitation of canning equiptnent. Nutl. Cari/ic,,s’Assoc..,l n f o r n i . Letter pp. 56-61. El-Bisi, H. M., and Ordal, Z. J. 1956. The effect o f sporulation temperature on the thermal resistance of Bacillus cougrr1trtr.s var. t/ieri,iocrciduratis. J. Ructeriol. El-Bisi, H. M., Ordal, Z. J., and Nelson, A. I. 1955. T h e effect of certain fungicides o n the thermal death rate of spores. F o o d Res. 20, 554-558. El-Bisi, H. M., Lechowich, R. V . , Amaha, M., :ind Ortld. Z. J. 1962. Chemical events during death of bacterial endospores by moist heat./. Food Sci. 27, 219-231. Esty, J. R., and Stevenson, A. E . 1925. T h e examination of spoiled canned foods. I. Methods and diagnosis. J . Ztifict. Ui.ut,crscs36, 486-500. Fields, M. L. 1962. T h e effect of O i d i i r / r r I u c t i s on the sporulation of Bocillus couguluiis in tomato juice. A r ~ j dMicrol)iol. . 10, 70-73. Fields, M. L. 1963. Effect of heat on spores of rough ;ind smooth variants of Bacillus stccrrotherrrio/>/~i~t~,s. Appl. M i c r o b i o l . 11, 1 0 0 - 104. Fields. M. L. 1964. Environmental stresses on spore populations of Rocillus stearotlrern i o p h i l u s . A ? > ) )M / . i c r o 6 i o l . 12, 407-41 1. Fields, M. L. 1966. Growth and sporulntion of smooth and rough variants of Bacillus s t t ~ c i r o f l i c ~ r r ~ i c ~in ~ ~pea / ~ i lextract ns and on pea agar. J . Food Sci. 31, 615-619. Fields, M . L. 1968a. Effect of the refining of sugar on the number of rough and smooth bacteria i n sugar. Unpuhlished data. University o f Missouri, Columbia, Missouri. Fields, M . L. 19681). Thermophilic aerobic spore-forming bacteria in Ilawaiian soils. IJnpuhlished data. University of Missouri, Columhia, Missouri. Fields, M . L.. and Finley, N. 1962. Studies on heat responses of 1)acterial spores causing flat sour spoilage in canned foods. Ill. Effect of storage time on activation and therm;il destruction responses. Missouri Uiiic., Agr. E x p t . Stu., Re.%.B u l l . 807. Fields, M. L., and Finley, N. 1963. Effect of carbohydrates in phosphate buffer on gerspores. Appl. M i c r o h i o l . 11, 453-457. mination of Btrcillirs stecirotl~ernco~~hilus

212

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Fields, M. L., and Finley, N. 1964. The effect of selected carbohydrates and plant extracts on the heat activation of Bacillus stearothermophilus spores. j . Food Sci. 29, 635-640. Fields, M. L., and Frank, H. A. 1969. Dipicolinate-induced germination of Bacillus steurothermophilus spores. j . Bacteriol. 97,464-465. Fields, M. L., and Jenne, R. C. 1962a. Studies of the heat responses of bacterial spores causing flat sour spoilage in canned foods. I. Effects of heating menstrua, spore age, and suspension preparation on the heat activation of Bucillus coagulans spores. Missouri, Unio.,Agr. E r p t . Sta., Res. Bull. 805. Fields, M. L., and Jenne, R. C. 1962b. Studies on the heat responses of bacterial spores causing flat sour spoilage in canned foods. 11. Effect of Oidium lactis on thermal activation and recovery of Bacillus coagulans spores. Missouri Univ., Agr. Expt. Stu., Res. Bull. 806. Fields, M. L., and Rotman, Y. 1968. Amino acid content of rough and smooth variants of Bacillus stearothermophilus. A p p l . Microbiol. 16,960. Finley, N., and Fields, M. L. 1962. Heat activation and heat-induced dormancy of Bocillus stearothermophilus spores. Appl. Microbiol. 10,231-236. Frank, H. A. 1955. The influence of cationic environments on the thermal resistance of Bacillus coagulans. Food Res. 20,315-321. Frank, H. A., and Campbell, L. L., Jr. 1957. The nonlogarithmic rate of thermal destruction of spores of Bacillus coagulans. Appl. Microbiol. 5,243-248. Frazier, W. C., and Foster, E. M. 1959. “Laboratory Manual Food Microbiology.” Burgess, Minneapolis. Minnesota. Friedman, S. M., and Weinstein, I. B. 1966. Protein synthesis in a subcellular system from Bacillus stearothermophilus. Biochim. Biophys. Acta 114, No. 3,593-605. Caughran, E. R. L. 1947. The thermophilic microorganisms. Bacteriol. Reo. 11, 189-225. Gaughran, E. R. L. 1949. Temperature activation of certain respiratory enzymes of stenothermophilic bacteria. j . Gen. Physiol. 32,313-327. Hall, H. H. 1938. Survival of thermophilic food-spoilage organisms in stored white beet sugar. j . Bacteriol. 35,75. Hall, H. H. 1939. Survival of thermophilic food-spoilage organisms in stored white beet sugar. Food Res. 4,259-267. Hammer, B. W. 1915. Bacteriological studies on the coagulation of evaporated milk. Iowa State Coll. Agr. Expt Sta., Res. Bull. 19. Hansen, P. A. 1932. The public significance of the growth of thermophilic bacteria in pasteurized milk. N.Y. State Agr. E r p t . Sta. (Geneoa,N.Y.), Tech. Bull. 196. Hartman, P. A., Wellerson, R., Jr., and Tetrault, P. A. 1955. Bacillus stearothermophilus. I. Thermal and pH stability of the amylase. A p p l . Microbiol. 3,7-10. Heiligman, F., Desrosier, N. W., and Broumand, H. 1956. Spore gemination. I . Activators. Food Res. 21,63-69. Henshelwood, C. N. 1947. “The Chemical Kinetics of the Bacterial Cell.” Oxford Univ. Press, London and New York. Hill, W. M., and Fields, M. L. 1967a. Factors affecting the growth and interaction of rough and smooth variants of Bacillus steurothermophilus. I. Oxygen tension and temperature. j . Food Sci. 32,458-462. Hill, W. M., and Fields, M. L. 1967b. Factors affecting the growth and interaction of the rough and smooth variants of Bacillus stearothermophilus. 11. Media and pH. j . Food Sci. 32,463-467.

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213

Hill, W. M., Fields, M. L., and Tweedy, B. G. 1967. Pathways of glucose metabolism by A p p l . Microbiol. 15, rough and smooth variants of Racillus stearotl~ernio~~hilrrs. 556-560. Hussong, R. V., and Hammer, B. W. 1928. A thermophile coagulating milk under practical conditions.J. Bacteriol. 15, 179-188. Hutchings, I. J. 1949. Presterilization of tomato juice application in in-plant bacteriological control. N ~ t lCatitiers’ . Assoc., Itiforni. Letter Cotiti. Issue 1219,83-86. Ingersoll, C. D. 1930. Thermophiles in sugar. Food Znd. 2,325. Johnson, K. 1959. Some nutritional aspects of irradiated and non-irradiated spores of Bncillus coagulmtis. Ph.D. Thesis, Purdue University, Lafayette, Indiana. Jones, W. L., Jezeski, J. J., and Geddes, W. F. 1953. T h e heat resistance of the thermophilic spores present in wheat and wheat products. Cerecil Clieni. 30, 12-21. Kaufman, 0. W., Ordal, Z. J., and El-Bisi, H. M. 1954. T h e action of several antibiotics on the spores of Bocillus tlierniocicidurcitis in a tomato juice medium. Food Res. 19, 488-493. Kim, J . , and Naylor, H. B. 1966. Spore production by Bacillus stecirotliernophilus. A p p l . Microbiol. 14, 690-691. Knock, G. G. 1954. A technique for the approximate quantitative prediction of flat souring in canned peas. J . Sci. Food Agr. 5, 113. Koffler, H. 1957. Protoplasmic differences between mesophiles and thermophiles. Bncteriol. Reti. 21,227-240. Kosker, O., Esselen, W. B., Jr., and Fellers, C. R. 1951. Effect of allylisothiocyanate and related substances on the thermal resistance of Aspergillus tiiger, Sacc/icironi!ices ellipsoideus, and Bacillus tliertrioacicirtratis. Food Res. 16,510-514. Kuhn, G. D. 1963. Radiation protection of bacterial spores and spore germination. Ph.D. Thesis, Purdue University, Lafayette, Indiana. LaBaw, G. D., and Desrosier, N. W. 1954. T h e effect of synthetic plant auxins on the heat resistance of bacterial spores. Food Res. 19,98-105. LaBree, T. R., Fields, M. L., and Desrosier, N. W. 1960. Effect of chlorine on spores of Bncillus coagulans. Food Techtiid. 14, 632-634. Lamanna, C. 1940. Relation between growth range and size in the genus Bacillus. J . Bncteriol. 39,593-596. Lategan, P. M. 1965. T h e occurrence of photoreactivation and the effect of different salt and sugar concentrations in the recovery medium on ultraviolet irradiated spores of Bacillus stearothernio),/ii/us.S . AfricntiJ.Agr. Sci. 8, 1015-1019. Lategan, P. M. 1966. T h e effect of ultraviolet irradiation followed by heat treatment on the survival of spores of Bacillus stearotliermophiliis. S. African J. Agr. Sci. 9, 71-76. Lechowich, R. V., and Ordal, Z. J. 1962. T h e influence of the sporulation temperature on the heat resistance and chemical composition of bacterial spores. Can. J . Microbiol. 8, 287-295. Lewis, J. C., Snell, N . S., and Alderton, G. 1965. Dormancy and activation of bacterial spores. I n “Spores 111” (L. L. Campbell and H. 0. Halvorson, eds.), pp. 47-54. Am. Soc. Microbiol., Ann Arbor, Michigan. Long, S. K., and Williams, 0. B. 1959. Growth of obligate thermophiles at 37°C. as a function of the cultural conditions employed.]. Bacferiol.77, No. 5,545-547. Long, S. K., and Williams, 0. B. 1960. Factors affecting growth and spore formation of Bacillus stearothermophilus.J . Bocteriol. 79,625-628. McArdle, F. J. 1955. Effects of ionizing radiation on some protein components of foods. Ph.D. Thesis, Purdue University, Lafayette, Indiana.

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McBee, R. H., and McBee, V. H. 1956. T h e incidence of thermophilic bacteria in arctic soils and waters.]. Bucteriol. 71, 182-187. MacFadgen, A,, and Blaxall, F. R. 1894. Thermophilic .bacteria. J . Pathol. Bucteriol. 3, 87-99; Trans.Jenner Inst. Preoentioe Med. 2,162-187 (1899). Mangiantini, M. T., Tecce, G., Toschi, G., and Trentalance, A. 1965. A study of ribosomes and of ribonucleic acid from a thermophilic organism. Biochim. Biophys. Acta 103, NO. 2,252-274. Manning, G . B., and Campbell, L. L. 1961. Thermostable alpha amylase of Rocillus stecrrothermophilus. I. Crystallization and some general properties. /. H i d . CAeni. 236, No. 11,2952-2957. Marsh, C. L., and Larsen, D. H. 1953. Characterization of some thermophilic bacteria froin the hot springs of Yellowstone National Park.]. Bacterird. 65, 193-197. Marshall, R., and Beers, R. J. 1967. Growth of Rocillus conguluns in chemically defined media.]. Ructeriol. 94, No. 3,517-521. Mathemeier, P. F., and Morita, R. Y. 1964. Influence of substrate-cofactor ratios on partially purified inorganic pyrophosphate activity at elevated temperatures. /. Btrcter i d . 88, No. 6 , 1661-1666. Matheson, A. T., and Amistrong, J. 1967. Partial purification and properties of a peptidase fraction from Bacillus stecrrotliemiopltilus. Curt. J . Riochem. 45, No. 10, 1644- 1647. Mefferd, R. B., Jr., and C:unpbell, L. L. 1952. Influence of temperature upon radiation sensitivity of thermophilic and mesophilic bacteria. Proc. SOC. Exptl. H i o l . Med. 79, 12-16. Mercer, W. A,, and Olson, N. A. 1969. Tomato infield washing station study. N n t l . C u r l ner.s’Assoc., Res. Lab. Repit. D-2167. Mercer, W. A,, Rose, W. W., Regier, L. W., and Chapman, J. E. 1960. Better washing of asparagus to improve quality and prevent spoilage. Nrrtl. Cunners’ Assoc., Res. L ~ I . Rejjt. 60-W-46. Michener, H. D. 1953. T h e bactericidal action of suhtilin on Hncillus steuroflierntophilus. AplpIt!.MiC?‘O/JiOl.1, 215-217. Michener, H. D. 1955. T h e action o f suhtilin on heated 1)acterial spores.]. Hwteriol. 70, 192-200. Militzer, W., and Burns, L. 1952. Thermal stability of a pyruvic oxidase. Federation Proc. 11,260. Militzer, W., Sonderegger, T . B., Tuttle, L. C., and Georgi, C . E. 1949. Thermal ellzymes. Arch. Riocltem. 2 4 , 7 5 4 2 . Militzer, W., Tuttle, L. C., and Georgi, C. E. 1951. Thermal enzymes. 111. Apyrnse from a thermophilic bacterium. Arcli. Riocliern. Hiop/i!ys. 31, 416-423. Miquel, P. 1888. Monographie d’un bacille vivant audeli d e 70” centigrades. Aftti. M i crogruphie l, 3-10. Murrell, W. F. 1955. “The Bacterial Endospore.” University o f Sydney, Sydney, Ailstralia. Murrell, W. G . , and Warth, A. D. 1965. Composition and heat resistance o f hacterial spores. 111 “Spores 111” (L. L. Campbell and H . 0. Halvorson, eds.), p. 1-24. Am. Soc. Microhiol., Ann Arbor, Michigan. National Canners Association (in cooperation with the Association of Food Industry Sanitarians, Inc.) 1952. “Sanitation for the Food Preservation Industries.” McGrawHill, New York. Nbgre, L. 1918. Contribution a 1’Ctude des microbes therniophiles. Etude hiologiclue d e la flore hactCrienne thermophilr du Sahara. Thesis, Paris (cited in Allen, 1953).

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215

M .F., and Campl)cll, J. J. R. 1959. Growth studies on Rrrcillus stearothemio),liilus. Ctrn.J . Microhiol. 5, No. 3, 293-297. O’Brien, R. T., and Campbell, L. L. 1957.The nutritional requirements foi- germination Neilson, N. E., MacQuillan,

and outgrowth of‘ spores and vegetative cell growth of some aerobic spore-forming 1xwteria.J. Brrcteriol. 73,522-525. Ordal, Z. J. 1957. T h e effect of nutritional and environmental conditions of sporulation. I n “Spores” 11. (0.Halvorson, ed.),p. 18-25. Am. Inst. Biol. Sci., Washington, D.C. Pearce, W. E., and Wheaton, E. 1952. Autosterilizatioii of therniophilic spores in canned foods. Food Res. 17, 487-494. Pederson, C . S., and Becker, M . E. 1949. Flat sour spoilage of tomato juice. N.Y.S t a t e Agr. E . q ) t . S t u . (Geneoci,N.Y.), Tech. H u l l . 287. Poshveiler, J. E., and Caldwell, E . F. 1961. Survival of Hacilltts s t e c r , . o f / i ~ , r ) t i c ) ~ ) / r i / t ~ ~ ~ spores a s a means of measuring effective electron I)eani dosage. J . Food Sci. 26, No.

2,204-207. Prescott. S. C., a i r d Underwood. W. L. 1897. Coritril)utions to o u r knowledge of niicroorganisms and sterilizing processes i n the c m n i n g industries. 11. T h e souring of canned sweet corn. Tw/itio/. Qtrcrrt. 10, 183-199. Prickett, P. S. 1928. Themiophilic and tliernioduric niicroorg~uiisniswith special reference to species isolated from milk. V. Description ofsi~orefortiiii)~ types. N.Y.Stirtc, Agr. E x ) ) t . Stci. (Getiecci,h’.)’.), Tech. B i t / / . 147, 5-58. Reed, J. M., Bohrer, C. W., and Canieron,E. J. 1951. Spore destruction rate studies on organisms of significance i n the processing of ciinned foods. J . Food Rrs. 16,

383-407.

c.

Rice, A. C., ;ind Pederson, S. 1954. Factors influencing growth of /jaci//rtscocrg:cr/ote.s in canned toniato juice. 11. Acidic constituents of toniato juice and specific organic acids. F o o d R c s . 19, 124-133. Richmond, B., and Fields, M. L. 1966. Distril,iition of therniophilic aerobic sporeforming Ixicteriii in food ingredients. Aj)/d. A f i c r o / > i o l .14, 623-626. Rotman, Y. 1967. SporuI:ition, chemical coniposition antl heat resistance of H(rci//rrs s t c c i r o t l c e r ) , i o ) ~ / c ispores. / ~ ~ . ~ P1i.D. Thesis, University of Missouri, Coliinibia, hlissouri. Rotinan, Y., and Fields, M. L. 1966. Structure of sp o r es of rough and smooth viiriants o f Htrcilliis .~te~rr[Jt/ierttii)))/ii/tr.s with special reference to their heat resistance. / . Food Sci. 31,437-440. Saiinders, G. F., and Cnmpl,ell, L. L. 1966. Hil)oniicleic ncid antl ril)osomes of Hocillrrs .~tc,rirot/ierttiol,llilrrs.l. H~ictc,riol.9 No. I ,

Sniith, N. R., Gordon, R. E., and Clark, F. E. 1952. Aerobic sporeforming 1)iicteri;i. I i . S . Dcljt. Agr.,Agr. ~ f < J i l O ~ ~ / NO. ! / ~ / 16. l Sot-enson, C. M. 1938. Detecting therinophilic containin~itioni n skim milk powder.

Foot/ RCY.3, 421-427. Stark, E., ;ind Tetrault, P. A. l9Sl. 1sol;ttion o f I ) x t e i - i u l , cell-frcc, skirch s;icchritying enzymes from the medium at 70”C.J.Hrrc,tc,rio/. 62, 247-248. Stark, E., and Tetroult, P. A. 1952. 0l)hcXrvatioiis o n ainylolytic 1)ucteria. 111. Cultrird conditions inHiiencing the 1)re;iktlowti of stiirc.11 b y steiiotliermopIiilic 1);icteri;i Ijelonging to R r r c i l l r t s .vtc~trrcitlcc,rrte~)~)/iilir.s. C t r t r . /. Hotntr!/ 30, 360-370. Stark. E., ;uid Tetraiilt. P. A. 19521). A tleterniin;itive strttly of ;iniylolytic steiiothermophilic Iincteriii isolated froni soil. S c i . A g r . 32, 81 -92. Stern, J . A., m t l Proctor, 13. E. 1954. A micromethod antl apparatus for the multiple deterniination of rates ofdestructioii of biictcriii i i n d Ixicterial spores sol)jectctl to heat. FOOd ~ C L ’ / l t I 0 / 8, .

135)- 143.

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MARION L. FIELDS

Stern, R. M., Hegarty, C. P., and Williams, 0. B. 1942. Detection of Bacillus thernioucidurans (Berry) in tomato juice and successful cultivation of the organism in the laboratory. Food Res. 7 , 186-191. Taylor, R. B. 1953. A study of proteose peptone acid agar as a plating medium for the routine enumeration of Bacillus tliernioacidurrrtis (Berry) in tomato juice. Food Res. 18,516-521. Thatcher, F. S., Coutu, C., and Stevens, F. 1953. The sanitation o f Canadian flour mills and its relationship to the microbial content of flour. Cereul Chent. 30,71-102. Thompson, P. J., and Thames, 0. A. 1967. Sporulation of Bacillus steurotherniophilus. AppI. Microbiol. 15, No. 5,975-979. Thompson, T. L., Militzer, W. E., and Ceorgi, C. E. 1958. Partial denaturation of a bacterial aldolase without loss of activity.]. Bucteriol. 76, No. 4,337-341. Thorpe, R. H. 1960. The action of nisin of spoilage bacteria. I. The effect of nisin on the spores. J . A p p l . Bucteriol. 23, No. 1, heat resistance of Bacillus sterirot/ierrnop/iilu~s 136-143. Titus, D. S. 1957. Studies on the germination characteristics of spores of Bacillus steurothermophilua. Ph.D. Thesis, University of Illinois, Urbana, Illinois. Townsend, C. T. 1932. Source of thermophilic bacteria causing spoilage in canned asparagus.]. Irifect. Diseases 51, 129- 136. Townsend, C. T., and Esty, J. R. 1939. The role of microorganisms in canning. Western Cunner Packer, 31.1-8. Tramer, J. 1964. The inhibitory action of nisin on Bacillus stearothermo~,/iilus.4 t h I n tern. S y m p . Food Microbiol., Goteborg, Swed., 1964 pp. 25-33 Almqrist & Wiksell. Troy, V. S., and Schenck, A. M. 1960. “Flat Sour Spoilage of Tomato Juice,” Continental Can Co., Chicago, Illinois. Welker, N. E., and Campbell, L. L. 1963a. Effect of carbon sources on formation of alpha-amylase by Bucillus steurothermophi~us.].Bucteriol. 86, No. 4,68 1-686. Welker, N. E., and Campbell, L. L. 19631,. Induced biosynthesis of alpha-amylase by growing cultures of Bacillus steurothermophilus.]. Bucteriol. 86, No. 6,1196- 1201. Welker, N. E., and Campbell, L. L. 1963c. Induction of alpha-amylase of Bucillus stemotherrnophilus by maltodextrins.]. Bucteriol. 86, No. 4,687-691. Welker, N. E., and Campbell, L. L. 1963d. Denovosynthesis of alpha-amylase by Bucillus stectrotherniopltilus. ]. Bucteriol. 86, No. 6, 1202- 1210. Wellerson, R., and Tetrault, P. A. 1955. The effect of various incubation temperatures on the ribonucleic acid production of a mesophilic and thermophilic bacterium. J . Rueteriol. 69,449-454. Wilder, C. J., and Nordan, H . C. 1957. A micro-method and apparatus for the determination of rates of destruction of bacterial spores subjected to heat and bactericidal agents. Food Res. 22,462-467. Williams, C. C., Merrill, C. M., and Cameron, E. J. 1937. Apparatus for determination of spore destruction rates. Food Res. 2,369-375. Williams, 0. B. 1930. Letter to the Editor. Food Ind. 2,563. Williams, 0 . B. 1936. Tryptone medium for the detection of flat sour spores. Food Res. 1,217-221. Williams, 0. B., and Campbell, L. L. 1951. Effect of subtilin on thermophilic flat sour bacteria. Food Res. 16,347-352. Williams, 0. B., and Hennessee, A. P. 1956. Studies on heat resistance. VII. The effect of phosphate on the apparent heat resistance of spores of Bacillus stectrotherniophilus. Food Res. 21, 112-116.

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217

Williams, 0. B., and Robertson, W:J. 1954. Studies on heat resistance. VI. Effect of temperature of incubation at which formed on heat resistance of aerobic thermophilic spores.]. Bacteriol. 67,377-378. Windle, J. J., and Sacks, L. E. 1963. Electron paramagnetic resonance of manganese (11) and copper (11) in spores. Biochini. Biop/i!/s. Actcr 66, 173-179. Yesair, J., and Williams, 0. B. 1942. Spice contamination and its control. Food Res. 7, 118-126. Youland, G . C., and Stunibo, C. R. 1953. Resistance values reflecting t h e order of death of spores of Bncillrrs comguln~issiilijected to moist heat. Food Techiiol. 7, 286-288.

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FOOD PROCESSING WITH ADDED ASCORBIC ACID BY J . C. BAUERNFEINDAND D. M . PINKERT Chemical Research Department, Hoffniunn-LaRoche Znc., Nutley, New Jersey

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Fruit Juices and Fruit Drinks . .

V. Synergist in Fat Protection . . . . . . . . . . . .

A. Frozen Fruit

. . . . . . . . . . 256 B. Fresh, Iced, and Salted F i s h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Canned Fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Fish Processing Precautions.. . ....... IX. Stabilizer of Meat C o lo r . . . . . . . . . . ............................... A. Fresh Meat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

261 265 265 266 266

D. Meat Curing Precautions X. Flour o r Bread Improver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 XI. Oxygen Acceptor in Beer Processing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,277 XII. Reducing Agent in Wine ................ 283 XIII. Oxidation Inhibition in D ducts. . . . . . . . . . . . 287 A. Milk 29 1 B. Biitter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292

219

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J. C. BAUERNFEIND AND D. M. PINKERT

c. Yogurt . . . . . . . . . . . . . . . . . . . . . .

...

D. Cheese . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . 294

294 XIV. Miscellaneous Uses ...................... . . . . . . . . . . . . . . . . . . .295 XV. Regulations on the Use of Ascorbic Acid in Foodstuffs. . . . . . . . . . . . . . . . . . . 296 . A. United States.. . . . . . . . . ....................... 296 B. Canada and Other Countr ...................... 297 XVI. [.-Ascorbic Acid vs. Erythorbi ...................... 297 ...................................... 304 References . . . . . . . .

I. INTRODUCTION The use of L-ascorbic acid in processing food was reviewed by Bauernfeind (29) in 1953. Since that time, several hundred papers have been published on studies of various applications of this compound in food processing. L-Ascorbic acid, a 6-carbon compound and a water-soluble vitamin also known as vitamin C (for cevitamic acid) exists in both the reduced and the oxidized forms (dehydroascorbic acid) in nature. It is found in all living tissue. Citrus fruits, tomatoes, potatoes, and cabbage are the important natural dietary sources for man, but content varies depending on variety, environmental factors, and processing (119, 155, 361,385). Ascorbic acid is structurally similar to carbohydrates and it is therefore not surprising that it gives some sugar reactions. The unusual properties of the molecule are due to the enediol grouping. L-Ascorbic acid is a moderately strong reducing compound, is acidic in nature, and forms neutral salts with bases. In solution, its oxidation by oxygen is catalyzed by oxidases and by traces of certain metals, the rate increasing with increasing pH (321).The first stage of oxidation to dehydroascorbic acid is reversible and the biological activity is retained; however, the oxidation of dehydroascorbic acid to 2,3-diketogulonic acid is not reversible, and biological activity is lost. Ascorbic acid, added to a particular media, lowers the oxidation potential. This reaction frequently protects other sensitive oxidizable compounds present, by preferential oxidation of the ascorbic acid. L-Ascorbic acid is commercially available in crystalline form as the acid and as the sodium salt in a variety of mesh sizes to meet the requirements of various food products. These crystalline compounds can be stored under cool, dry conditions in closed containers for as long as 2 years with little change in activity. In aqueous solution and in the complete absence of oxygen, ascorbic acid is a fairly heat-stable

22 1

FOOD PROCESSING WITH ADDED ASCORBIC ACID

compound. The solubilities of ascorbic acid and sodium ascorbate in water are given below:

Ascorbic acid, crystalline Sodium ascorbate, powder

(g/100ml) 32.0 87.8

Solubility 25°C (% wt/wt) (o/r wt/vol)

24.2 46.7

26.7 58.1

The daily requirement of vitamin C for man lies between 30 to 150 mg. A daily intake of 75 mg should be sufficient for an optimal supply. While vitamin C deficiency or avitaminosis is now seldom observed, there is a wide range between complete deficiency and optimal supply. In tropical countries, daily intakes from natural foods approximate several hundred milligrams. Man’s metabolism and physiological need for vitamin C is reviewed elsewhere (14,22,57,186,396).

II. METHODS OF ADDITION TO FOOD L-Ascorbic acid (vitamin C) is added to processed fruit, vegetables, meat, fish, milk, fats, oils, flour, soft drinks, malt beverages, wine, confections, and synthetic foods. It may be added to enhance nutritional value, or to improve keeping qualities, color, stability, palatability, clarity, or baking quality (107). Four basic methods have been developed (281)for adding ascorbic acid to foods. These are:

(1) Tablets or wafers. Compressed tables containing ascorbic acid, along with inert edible fillers, in sufficient amount to fortify a container or given quantity of food. The tablet added prior to sealing the container dissolves immediately during container filling and sealing of a liquid or, in a dry product mixture at a later food preparation stage. (2) Dry premixes. A uniform mixture of ascorbic acid with a dry carrier, usually a constituent of the food. The premix blended with the dry food product gives greater assurance of product uniformity since the quantity of the pure vitamin added is small. ( 3 ) Liquid sprays. Sprays of ascorbic acid solutions or suspensions. These may be considered liquid variations of premixes. The sprays are directed onto the surface of a food or injected into liquid food products as a means of getting around difficult or continuous processing conditions. For example, toasted ready-to-eat breakfast cereals are fortified by spraying a solution onto the hot flakes just after toasting.

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J. C. BAUERNFEIND AND D. M. PINKERT

(4) Pure compound. Direct addition of ascorbic acid or sodium ascorbate to the food is a widely used approach. Weighed amounts are added to the food with sufficient mixing to insure uniformity. Preweighed packets for ready use are a variation of this method. The point in the food process at which ascorbic acid is added by any method is important. Ideally, it is added as close to the end of the processing stage as possible without the loss of uniform distribution or decomposition of the vitamin (29).

111. ASCORBIC ACID AS AN ADDED NUTRIENT A fundamental concept of nutrition is that food in order to be right for human consumption should contain the biologically available essential nutrients in adequate amounts. With the exception of mother’s milk, for instance, there is no single food in nature designed for this purpose, and even the adequacy of human milk depends largely on the diet of the mother. Hence, the challenge to daily food preparation and consumption involves selections (1) from natural whole foods fresh fruit, vegetables, milk, etc., (2) from frozen or canned foods, (3) from fractionated and processed foods, (4) from processed foods with added nutrients, and ( 5 ) from the “so-called” “replacement or imitation” foods with added nutrients. Processing can never make a food product more complete than the original fresh product nor can it compensate for nature’s idiosyncracies. However, the preservation, processing, and storage of food are necessary to provide palatability, safety to health, variety of selection, and provision for future use. In the production, handling, preserving, processing, and storage of food, some nutrients are lost or significantly lowered (13, 15, 59, 63, 87, 93, 146, 226, 385). Not only does the nutrient content vary in the natural whole-plant food- because of variety, climate, handling, storage, etc. -but processing (because of exposure to heat, oxygen, metals, etc., and other food-fractionation processes) modifies the nutrient content. One nutrient especially sensitive to the above factors is vitamin C, or L-ascorbic acid. The primary purpose for the addition of L-ascorbic acid to any food is to make that food more beneficial to the consumer by enhancing its nutritive value and/or by improving its taste, texture, and color. This addition can be considered from four approaches: (1) restoration, (2) fortification, (3) standardization, and (4) “preservation or enhancement” of the food’s characteristic (87, 107, 112, 146). Restoration is

FOOD PROCESSING WITH A D D E D ASCORBIC ACID

223

adding back the essential vitamin in the amount lost during processing. Fortification is adding the vitamin to certain widely used foods which, although they may be important in the diet, are not good “ sources of the vitamin. “Standardization” or uniformization” is adding the vitamin to a food to equalize the varying amounts that occur (a) throughout the harvest and processing season and/or (b) within a class of food products used interchangeably within the diet. Standardization is particularly applicable to fruit beverages. The long, expensive, and sometimes nonconclusive efforts to change food habits through an educational program are avoided. Standardization of the vitamin C content of fruit juices benefits the consumer by providing a significant vitamin C intake regardless of the choice of single fruit juice, blended juice, or fruit drink. Since L-ascorbic acid undergoes a reversible oxidation-reduction phenomenon, it has become an acceptable food additive for retarding changes in foods as a result of oxidation, reduction, or enzymic action; thus, “preserving or enhancing” the product (107).This aspect of the vitamin’s use will be discussed later. Mere restoration of the nutrients in a processed food to the levels of the unprocessed natural food has merit, but it must be recognized that natural food was not originally developed for man. For example, the wheat seed has a nutrient composition necessary to grow a stalk of wheat under favorable environmental and cultural practices. Hence, the following factors may be considered when adding nutrients to food: (1)availability of the food, ( 2 )convenience of use, ( 3 )association of the nutrient to a food selection pattern or a replacement or supplemental food product, (4)stability of the nutrient in the food during market life and home preparation, and (5) special food needs (infant, geriatric, and military foods). Hence, food consumption patterns, economics, food technology, marketing, meal preparation practices, psychology, and nutritional requirements also become important in the inatter of nutrient addition (87, 146).

A. FRUITJUICES

AND

FHUITDRINKS

The pattern of the consumption of fruit products in the U.S. has undergone a gradual change over the years. The decline of whole fresh fruit and vegetable consumption is correlated with the increased use of canned fruits, vegetables, and juices and more recently frozen products. These trends in the consumption of fruit and vegetable products are shown in Tables I and 11. The overall per capita consumption of all fruit as measured by market disappearance has held

224

J . C. BAUERNFEIND AND D. M. PINKERT TABLE I CHANGING PATTERNOF FRUIT PRODUCTCONSUWTION"

Year

Fresh fruits

Canned fruits

1915 1925 1935 1945 1955 1965

154.5 132.2 133.2 139.9 99.5 81.0

5.6 11.1 13.4 14.4 22.5 23.4

Canned juices

Frozen frnits

Frozen juices"

(Pounds per capita per year) 0.6 0.2 2.0 10.9 2.3 13.1 3.8 15.8 10.8 3.7 15.4

Chilled juices

All fruir

0.9 2.0

180.7 169.1 174.2 206.1 199.5 173.8

"USDA Econ. Res. Serv., Fruit Situation, TFS-164. U.S. Dept. Agr., Washington, D.C., 1967. bSingle strength. cA1l fruit equivalent.

IhSTRIBUTION OF

Food

TABLE I1 ASCORBIC ACID FROM kkUIT ( I N PERCENT)

1909- 1913 97.8 0.9

Fresh fruit Canned fruit Frozen fruit Dried fruit

1.3 100 96.9 3.1 ~

Fresh vegetables Canned vegetables Frozen vegetables ~

AND

VEGETABLE PRODUCTS"

1947-1949

1967

(percent) 73.5 20.6 5.9 0.4 100 89.8 9.0 1.2

40.5 27.0 32.4 0.05 100 80.0 12.3 7.7

~

~

100

~

~

100

100

"U.S. Dept. Agr., Econ. Res. Serv., "National Food Situation," NFS-122. U.S. Dept. Agr., Washington, D.C. 1967.

fairly constant over the years. From 1909 to 1913, fresh fruits and vegetables accounted for over 95% of the vitamin C distribution in each product class. This dropped in 1967 to 80% for fresh vegetables and 40% for fresh fruits while the frozen and canned percentages have risen. Fruit juices and drinks are currently consumed as thirst quenchers as well as meal-time cocktails or beverages at breakfast, luncheon, and dinner. A household-use profile of various juices and drinks in the United States for 1959 indicates: for breakfast, 9.7-69.4%; for

225

FOOD PROCESSING WITH ADDED ASCORBIC ACID

luncheon-dinner, 7.2-29.70/, and as thirst satisfiers, 21.5-62.7%),as shown in Table 111. The variety and amount of single, blended, and concentrated fruit juices and drinks consumed by the American public provide a basis for the rationale of ascorbic acid unifonnization of these fruit products. The natural vitamin C content of different fruit juices varies widely according to the type of fruit (93, 166). In addition, a wide variation in the vitamin C content of different processed fruit juices exists as shown in Table IV. Since the average consumer tends to use fruit TABLE 111 USE PROFILE“ OF FRUITJUICES A N D DRINKS

(IN PERCENT) Food

Breakfast

Luncheon-dinner

Thirst satisfier

Fruit juices: Home-prepared Chilled Concentrate Single-strength

69.4 58.3 57.4 53.7

7.2 9.2 10.3 14.8

23.4 32.5 32.3 31.5

Vegetable juices

48.7

29.7

21.6

Fruit drinks: Ready-to-serve Concentrate

25.4 9.7

20.1 27.6

54.5 62.7

““Summer Servings in Urban American Homes,” Canco Marketing Div., American Can Company, 1959. TABLE IV RANGE OF ASCORBIC ACID CONTENT OF CANNEDFRUITJUICES Juice Apple“ Cranberry“ Grape“ Grapefruit“ Orange“ Pineapple‘ Raspberry’ TomatoP

mg/IOO g

0.2-3.6 7.5-10.5 0 - 4.7 10.0-49.0 9.7-70.0 6.6- 9.3 7.5- 8.3 1.8-29.3

“U.S. Dept. Agr. data (1951). ”N.Y. Stiite Agr. Expt. S t o . (Geneva, N.Y.) Bull. 743 (1950). ‘Darroch and Gortner (89). “Unio. Muss., Agr. E x p t . S t o . , Bull. 481 (1955). ‘Anderson el ul. (5).

226

J. C. BAUERNFEIND AND D. M . PINKERT

juices interchangeably to provide variety in the diet, it can be assumed that the ascorbic acid intake would vary considerably if not standardized. To comply with United States labeling regul at'ions, a serving of the average daily consumption of a food with claims for vitamin content must contain a substantial amount of the vitamin speci-

fied. Many of the fruit drinks or ades contain 30 mg of vitamin C per 6 or 8 fluid ounces (55, 56). Fruitades or drinks are usually prepared from concentrated fruit juice, flavor emulsion, color, acidulant, sugar, and water. Vitamin C standardization has been carried on commercially with some juice varieties and thus the literature contains reports concerning techniques used for fruit juice, and the stability of ascorbic acid in various fruit products (15, 37, 113, 142, 192,214,233,279,289, 299, 301, 302, 317, 344, 371, 375). On the basis of experience it is usually anticipated that juices, to which 40 to 50 mg of ascorbic acid per fluid volume are present after packaging and processing, will contain 30 mg after the usual market-life storage. For example, a claim of 35 mg per 100 ml in apple juice requires that an initial level of 50 mg be present. More than 90% of canned apple juice marketed in Canada is standardized with added ascorbic acid (279). In the United States ascorbic acid is added to commercially produced apple juice, apricot nectar, apricot-pineapple nectar, cranberry juice, grape juice, grapefruit juice, orange-apricot, orange-apple baby food, pineapple juice, prune juice, prune-orange-apple baby food, and vegetable juice, and to the following concentrates: grape juice, blended fruit juice, orange drink, pineapple juice, pineapple-grapefruit juice, and pineapple-orange juice. It is also added to the following fruit juice drinks: breakfast cocktail, fruit punch, grape, orange, orange-apricot, pineapple-grapefruit, and pineapple-orange. Table V summarizes the results of conimercial trial runs on number of juice and beverage products containing added ascorbic acid. It must be recognized that these were run with the prevailing equipment and under the processing variables in the particular plant at the time and, hence, can only serve as a guide to expected performance. Meticulous attention to control of processing details is imperative for good performance (93, 158, 289). In general, ascorbic acid is quite stable in frozen juice concentrates and somewhat less stable in canned single strength juices and drinks. Stability varies somewhat among the different fruits. Experience in plant trial runs with the particular juice or drink in question dictates the overages needed in commercial practice to meet label claim after processing and storage. See Table VI as an initial guide.

FOOD PROCESSING WITH ADDED ASCORBIC ACID

227

€3. VEGETABLEJUICE

Tomato varieties (a fruit commonly regarded as a vegetable) vary considerably in color, flavor, and vitamin C content. Certain varieties of tomatoes, which are well adapted to large growing areas, yield a juice low in vitamin C, although its flavor may be highly desirable. Other large growing areas yield tomatoes of higher vitamin C content but of different flavor characteristics. There are published data to show that canned tomato juice as purchased varies greatly in vitamin C content (5). In a study of 240 samples (40 leading brands) of commercially canned tomato juice tested at the Massachusetts Agricultural Experiment Station, analysts found a vitamin C content in a range of 1.8to 29.3 mg/lOO g (about 3 1/2 oz) ofjuice (5). A minimum of 30 mg of vitamin C per 4 fluid ounces or any other similar minimum can easily be maintained. When vitamin C was added to tomato juice obtained from both U.S. Eastern and Western growing areas, more than 30 mg were still present in the fortified (or standardized) samples after they had been stored for a year at room temperature (82, 335). Zubeckis (406), in investigating the stability of ascorbic acid standardized tomato juice (40-50 mg/100 g) in 48 fl oz cans, observed losses of 4-10% after 12 months’ storage at 25°C. No significant difference in color and flavor could be detected when the stored samples were compared to the control samples (15 mg ascorbic acid/100 g). Other values are cited in Table V. Siege1 (335) reports favorably on the stability of ascorbic acid standardized tomato juice. In trials with ascorbic acid added to tomato pulp, Hesse obtained a retention figure of 83%)after 561 days of storage (152). The current variable vitamin C content of canned tomato juice (335) could be corrected i n the U.S. by setting u p industry standards and by modifying Food and Drug Administration Standards of Identity to permit specified levels of vitamin C to b e added. Ascorbic acid is not currently added to tomato juice in Canada. Ascorbic acid can also be added to vegetable juice blends. See Table V for plant trial data. C. JUICE AND DRINKPROCESSINGPRECAUTIONS Kropp et d.(196) considers the following important factors in the stability of ascorbic acid in fruit juices: oxidative enzymes, pasteurization methods, light- and heavy-metal contamination, deaeration (or vacuum treatment), and sulfur dioxide content. The processing equipment is of definite importance when ascorbic acid is added to food.

TABLE V STABILITY OF ADDED ASCORBIC ACID I N FOOD

PRODUCTS Storage, 70"-75"F (23°C)

After processing Product Apple juice Apple juice Apple juice Apple juice Apple juice Apple juice Apple juice Apple juice Apple-cherry Apple-orange Apple-orange Apple-orange Applesauce Apricot nectar Apricot nectar Apricot nectar Apricot-pineapple Apricot drink Apricot drink Apricot-orange Cereals, dry Cereals, dry Cereals, dry Cocoa powders Cocoa powders Cocoa powders Cocoa powders

Packaging Class, 1 qt Class, 1 qt Class, 1 qt Class, 1 qt Class, 24 oz Can, 20 oz Can, 18 oz Glass, 1 qt Glass, 1 qt Glass, 4 oz Class, amber Can Can, 1 lb Can, 4 oz Can, 18 oz Can, 4 oz Can, 6 oz Can, 12 oz Can, 12 oz Box, liner Box, liner Box, liner Envelope Foil bags Box, 1 Ib Can, M lb

Coal 30 mg/4 fl oz 30 mg/4 fl oz 30 mg/lO fl oz 30 mg/4 fl oz 30 mg/8 fl oz 35 mg/lOO ml 30 mg/4 fl oz 30 mg/4 fl oz 30 mg/4 fl oz 30 mg/4 fl oz 40 mg/100 ml 40 mg/100 ml 30 mg/lOO g 30 mg/4 fl oz 30 mg/4 fl oz 30 mg/4 fl oz 30 mg/4 fl oz 30 mg/6 fl oz 30 mg/4 fl oz 30 mg/4 fl oz 10 mgloz 10 mg/oz 10 mg/oz 25 mg/lOO g 120 mgllb 75 mg/20 g 15 mg/% oz

Ascorbic acid

Vitamina C

40 38 38 38 35 35

44 41 49 43 47 41 45 49 54 63 62 74 59 40

38 42 53 66 62 61 69 57 35 39 69 47 49 47 10.3 11.4 10.5 34 180 78 14.1

-

70 48 53 49 48 2 14 80 -

6 months Ascorbic acid

12 months

Vitamin C

Ascorbic acid

Vitamin C

-

-

40 (so)* 33 (80)

31 29 -

-

29 31 35 31 28 35 56 (85) 46 49

33 (77) 34 (72) 33 (73) 30 (61) 44 (81) 48 (76) 53 (85) 62 (W 62 (100) 46 (100) 56 (80) 46 (95) 49 (92) 43 (88) 9.7 12.5 10.7 45 (94) 194 (90) 87 (100) 14.0

59 61 31 37 (95) 53 40 48 43 8.3 (80) 8.0 (76) 34 166 76 13.0 (92)

25

-

28 26 26 25 31 54 (82) 37 39

56 45 29 32 (82) 52 29

37

-

7.2 (70) 9.9 (87) 7.0 (67) 28 137 77 11.4 (81)

37 (76) 32 (68) 31 (76) 26 (58) 31 (63) 34 (63) 40 (64) 48 (77) 56 (76) 50 (84) 32 (80) 59 (84) 40 (83) 41 (77) 9.9 10.3 9.1 39 (81) 171 (80) 82 (loo) 12.9

Cocoa powders Cranberry juice Cranberry juice Cranberry juice Cranberry juice Cranberry-orange Cranberry-apricot Fruit punch Fruit punch Fruit carbonated beverages : Grape Orange Root Beer Fruit powder mixes: Orange Orange Orange Orange Lemon Fruit gelatin Fruit gelatin Fruit lollipops: Lemon Pineapple Grape juice C‘ Grape drink Grape drink Grape drink Grape drink Grape drink Grape drink Grapefruit juice Grapefruit juice

67

39 61 49 41 61 49 48 68

35 31 54

30 mg112 fl oz 30 mg/12 fl QZ 30 mg/12 fl oz

42 44 40

46 46 50

30 30 31

Package, 3 oz Envelope Glass, 7 oz Can, 4 oz Class, 7 oz Can, 24 oz Can, 24 oz

30 mg/3 oz 30 mg/4 fl oz 136 mgloz 75 mg/4 fl oz 60 mg/25 g 15 mg/3 oz 15 mg/4 oz

82 78 136 144 75 17.5 63

87 79 122 18.2 68

Can, 6 oz Can Can, 12 oz Can, 46 oz Can, 1 qt Glass, Yz gal Glass, Y2 gal Glass, 17 oz Can, 18 oz

30 mglpop 30 mglpop 15 mg/l oz 30 mg/8 fl oz 30 mg/6 fl oz 30 mg/6 fl oz 30 mg/6 fl oz 30 mg/8 fl oz 30 mg/8 fl oz 30 mg/4 fl oz 30 mg/4 fl oz

29 30 13d 31 44 43 36 26 30

14d 33 48 46 40 35 41 71 58

Package Glass, 16 oz Glass, 16 oz Class, 16 oz Class, 16 oz Class, 1 qt Glass, 1 qt Can, 46 oz Class, Y2 gal

15 mg/oz 30 mg/4 fl oz 30 mg/4 fl oz 30 mg/4 fl oz 30 mg/6 fl oz 30 mg/6 fl oz 30 mg/6 fl oz 30 mg/8 fl oz 30 mg/lO fl oz

18 39 60 42 38 59 38

Can, 12 oz Can, 12 oz Can, 12 oz

44

68 56

15.8 (88) 30

51 7 33 47

75 71 139 (100) 144 71 (95) 16.8 62 12d 31 33 37 35 23 28 48

35 (90) 57 (94) 39 (80) 35 (86) 53 (87) 40 (82) 35 (73)

16.5 (91)

-

32 32

38

58 (85) 35 (76) 35 (76) 41 (82) 80 (92) 77 (97) 121 (99) 17.9 (98) 68 (100)

14d (100) 34 (100) 39 (81) 39 (85) 41 (100) 27 (80) 31 (76)

-

51 (88)

-

26 22 28

73 68 166 (85) 106 71 (95) 15.5 61 30 (100) 30 (100) 1I d 18 26 24 51 46

38 (78) 34 (83) 40 (82) -

28 (61) 25 (54) 32 (64) 79 (91) 76 (96)

-

119 (97) 16.6 (91) 66 (97)

12d (86) 31 (94)

-

32 (70) 26 (65)

-

52 (73) 48 (83)

TABLE V-Continued Storage, 70"-75"F (23°C) After processing Product Grapefruit juice Grapefruit juice Grapefruit juice Grapefruit juice c' Lemonade drink Low-calorie drink powders : Vanilla Chocolate Milk products: Liquid formula Liquid formula Evap. milk Dry formula Dry formula Dry milk, whole Dry milk, whole Dry milk, whole Orange drink Orange drink Orange drink Orange drink Orange drink Orange drink Orange drink Orange drink Orange drink C'

Packaging

Goal

Vitamin" C

Ascorbic acid

63 57 74 44d 76

69 57 76 47d 78

56 50 (88) 68 43d 69

Can, 18 oz Can, 18 oz Can, 6 oz Can, 6 oz Can, 46 oz

30 mg/4 fl 30 mg/4 fl 30 mgl4 fl 30 mg/4 fl 30 mg/8 fl

Can, 8 oz Can, 8 oz

100 mg/qt 100 mg/qt

148 140

188 180

Can, 13 oz Can, 13 oz Can, 13 oz Canlg, gas Canlg, vacuum Can, air Can, air Can, gas Can, 46 oz Can, 12 oz Can, 12 oz Glass, 1 qt Glass, 1 qt Glass, 1 qt Can, 46 oz Glass, Yz gal Can, 6 oz

50 mglcan 50 mglcan

54 99 94 57 63 136 157 184 47 33 67 28 53 40 37 79 536

56 63 79 200 188 200 50 39 88 28 56 44 44 82 57d

oz

oz oz oz oz

60 mglll2 g 60 mglll2 g 100 mgllb 100 mgllb 100 mgllb 30 mgI8 fl oz 30 mg/6 fl oz 60 mgll2 fl oz 15 mgl4 fl oz 30 mgl8 fl oz 30 mgl8 fl oz 30 mg/8 fl oz 30 mgll0 fl oz 30 mg/4 fl oz

12 months

6 months

Ascorbic acid

141 136 27 49 (50) 62 (64) -

147 115 184 37 27 62 25 50 36 31 71 55d

Vitamin C

61 (90) 69 (90) 48d (100) 77 (99)

Ascorbic acid

53 49 60 60

Vitamin C

57 (83) 49 (86) 63 (83) 67 (86)

168 (90) 160 (90)

100 129

121 (64) 143 (80)

34 (60) 195 (98) 158 (84) 189 (95) 41 (82) 30 (77) 81 (92) 27 (99) 54 (96) 40 (91) 37 (84) 73 (89) 57d (100)

49 (50) 58 63 138 133 188 16 32 31 27 66 -

61 (97) 76 (96) 168 (84) 122 (65) 186 (93) 23 (82) 46 (82) 33 (75) 34 (77) 68 (83) -

Orange drink C' Orange drink C' Pineapple juice Pineapple juice Pineapple juice Pineapple juice Pineapple juice Pineapple juice C' Pineapple-grapefruit Pineapple-grapefruit Pineapple-grapefruit Pineapple-orange Pineapple-orange Pineapple-orange Potato Hakes Potato Hakes Potato Hakes Potato sticks Soybean products: Liquid Formula Dry Powder Tomato juice Tomato juice Tomato juice Tomato juice Tomato juice Tomato juice Vegetable juice Vegetable juice Vegetable juice

Can, 6 oz Can, 12 oz Can, 46 oz Can, 46 oz Can, 46 oz Can, 46 oz Can, 12 oz Can Can, air Can, gas Frozen-film pack

30 mg/4 H oz 30 m g / 8 H oz 30 mg/4 H oz 30 mgi4 fl oz 30 mg/4 H oz 30 mg/4 H oz 30 mg/4 H oz 30 mg/4 H oz 30 mg/6 H oz 30 mg/6 fl oz 30 mg/8 H oz 30 mg/8 H oz 30 mg/6 fl oz 30 mg/6 fl oz 60 mg/3 oz 60 mgil00 g 60 mg/lOO g 60 ingil00 g

68" 39" 47 44 43 39 69 79" 40 59 39 36 43 42 48 51 70 84"

71" 51" 48 44 47 39 88" 42 60 40 37 45 49 59 65 79 -

62" 40" 43 38 42 36 63 (91) 78" 35 50 36 32 38 36 30 40 68 61" (73)

66d (94) 476 (92) 43 (90) 45 (100) 42 (89) 36 (92) 82" (93) 39 (93) 54 (90) 38 (95) 32 (82) 41 (91) 41 (84) 43 (73) 60 (92) 71 (90) -

Can, 13 oz Can, 1 lb Can, 18 oz Can, 46 oz Can, 18 oz Can, 46 oz Glass jar Can, 46 oz Can, 46 oz Can, 6 oz Glass jar

50 mgican 60 Illg/4 oz 30/100 nil 30/100 in1 30 mg/4 H oz 30 mg/4 H oz 30 mg/6 fl oz 30 mg/6 fl oz 30 mg/6 H oz 30 mg/4 H oz 30 mg/4 H oz

45 62 52 39 40 41 55 72 53 36 45

62 54

47 (100) 49 45 35 (90) 37 (93) 41 51 57 40 32 42 (93)

60 (97) 45 (83) 50 (100) 56 (95) 63 (84) 43 (74) 34 (81) -

Can, 6 oz Can, 6 oz Can, 11 oz Can, 18 oz Can, 46 oz Can, 46 oz

"Vitamin C equals ascorbic acid plus dehydroascorbic acid. bValues in ( )are percent retention during storage. 'Concentrate. "0" F.

-

41 44 59 75 58 42 -

-

61" 41" 38 (81) 35 35 55 (80) 38 -

65" (91) 46" (90)

-

36 (82) 35 (74) -

-

-

46 (92) -

43 (95) 48 44 27 (70) 34 32 39 45 32 29 41 (91)

61 (98) 44 (81) 30 37 (93) 35 (80) 47 (80) 48 (64) 38 (66) 29 (69) -

-

232

J. C. BAUERNFEIND AND D. M. PINKERT TABLE VI A s c o ~ s i cACID AIIDITION"TO JUICES DUHINC: PHOCESSING Crystalline ascorbic acid to add

Vitamin C god

30 30 30 30

ing/8 fl oz ingl200 ml (cc) mg/4 fl oz mg/100 in1 (cc)

oz/100 gallons

glhectoliter

3 6

25 50

"Added at last stage (mix) and rapidly pasteurized, packaged, and cooled. Higher additions are needed for prune juice.

Stainless steel, aluminum, or plastic equipment should be used. Bronze, brass, copper, monel, cold rolled steel, and black iron equipment should be avoided to minimize dissolved copper, iron, and nickel. Deaeration (vacuum) procedures and inert gas treatment during processing are recommended whenever feasible. Fill containers at a uniform rate and to maximum fill. Employ flash-heat processing and cool containers promptly (11, 93). It should be remembered that ascorbic acid is not a preservative against the growth of microorganisms, nor will it upgrade fruit quality. See Table VI. Many of the deep-red, blue, and purple water-soluble colors of berries and certain fruits are due to glycoside pigments, anthocyanins and their aglycones (anthocyanidins), which are less soluble and stable compounds. The anthocyanins together with L-ascorbic acid, when in solution under anaerobic conditions, do not show increased deterioration; however, in the presence of ascorbic acid and excessive oxygen, the anthocyanins can be more rapidly decolorized. To preserve color and also to lessen ascorbic acid losses, short-time, high-temperature processing should be used and dissolved oxygen, copper, and iron should be kept at a practical minimum (145,369).

D. CARBONATED BEVERAGES, BEVERAGE POWDERS, AND FRUIT ITEMS Carbonated beverages can also be fortified with ascorbic acid. (See Table V for data on processing trials.) The preferred method of addition is to dissolve the ascorbic acid in the sugar syrup. As an antioxidant, ascorbic acid protects flavor in certain carbonated drinks packed in glass; destruction of flavor by dissolved and headspace atmospheric oxygen during storage, distribution, and display is thereby avoided. Mechanical air removal devices are recommended where applicable

FOOD PROCESSING WITH A D D E D ASCORBIC ACID

233

to minimize air levels ( 1 1,220,311). Ascorbic acid is a desirable addition to canned soft drinks because it removes or neutralizes the air corrosion of the container, thus reducing air-caused perforations and extending shelf life. Atmospheric oxygen dissolved in an aqueous acid medium such as carbonated beverages (pH 2.5-4.0) acts as a depolarizer and promotes metal corrosion which in turn causes flavor changes, discoloration, and other objectionable effects (165).(See Fig.

1.) Decolorization of FD&C colors (the so-called azo or coal tar dyes) can occur in carbonated beverages in the presence of ascorbic acid depending on (1) the specific FD&C color’s reaction to reducing agents, (2)the oxygen and dissolved metal content, and (3)exposure of the bottled beverages to sunlight (248). Orange drinks are popular in carbonated form as well as in heatpasteurized form. In orange juice the orange color results from the presence of natural carotenoids. Many chemically synthesized carotenoids, such as /3-carotene, P-apo-8’-carotenal. P-apo-8’-carotenoic acid ethyl ester,’ canthaxanthin, etc., are available and can be used in emulsion or colloidal form to yield yellow-orange, orange, or red colors in beverage formulations. Beverages which are colored with pcarotene and which contain added ascorbic acid are very resistant to ‘Not currently approved in the U.S.A.

2.7

c

1.8

a,

t

c

0 0

c

2

- 0.9

I .5 3.0 4.5 6.0 Room temperature storage (months) FIG,1. Effect of ascorbic acid on iron pickup of a soft drink packed in 12-oz Hat-top cans. Curve A, 9.7 c c air; B, 2.4 cc air; C, 10.2cc air 36 mg ascorbic acid; D, 2.0 cc air 36 mg ascorbic acid (165).

+

+

234

J. C. BAUERNFEIND AND D. M . PINKERT

color fading when exposed to sunlight (31,55,360). It is interesting to note that about 300 8-fl oz servings of carbonated beverages were consumed per capita in the United States in 1967. Dry fruit-flavored beverage powders are easily fortified with ascorbic acid b y dry blending with the other ingredients. Stability of ascorbic acid in low-moisture, dry blends is excellent as long as the product is protected from humidity during storage (Table V). Applesauce, like apple juice, can be fortified with ascorbic acid to give a uniform vitamin C value by adding the compound prior to filling, sterilization, and cooling (25).Fruit gelatin granular products are fortified by dry blending with crystalline ascorbic acid. Retention data are shown i n Table V. European workers (193, 255) report that addition of ascorbic acid to berries and jam enhances the retention of the original vitamin C of the berries. Fruit-flavored hard candy is a good vehicle for L-ascorbic acid addition (163); the compound is either added to the candy mass or liquid melt on the cooling slab or folded into the batch before pulling.

E. MILK PRODUCTS Freshly drawn cow’s milk contains as much a s 30 mg of ascorbic acid in reduced form per quart or liter, mature mother’s milk as much a s 100 mg (221, 395). In the United States several evaporated milk-base infant foods are marketed containing 50 mg of added ascorbic acid per 13-fl oz can which is diluted to a quart before consumption, McColluni in 1944 was one of the original advocates of vitamin-supplemented evaporated milk (219). Some dry-formulated infant foods are likewise fortified with vitamin C. A new group of dry, low-calorie diet foods intended for the adult market also contain added ascorbic acid; these products are to be reconstituted in water or milk before use. In Canada, ascorbic acid fortification of evaporated milk has been recommended to protect infants and others against ascorbutic conditions since evaporated milk is both widely used and economical (132, 235). Standards established b y the Food and Drug Directorate set the minimum level of vitamin C at 40 mg/quart or 14 mg/100 ml ofreconstituted evaporated milk. It is economically and nutritionally sound to fortify evaporated milk with 50 to 100 mg of ascorbic acid per 13 fl oz (or reconstituted quart) in vacuum-sealed containers according to Pennsylvania State University researchers (168).The sodium salt is preferred to avoid a potential destabilization effect on the milk proteins (a flaking of the protein

FOOD PROCESSING WITH ADDED ASCORBIC ACID

235

during the sterilization process). When 50 and 100 mg were added, 35 and 71 mg of ascorbic acid per liter, respectively, were present on a reconstituted basis after processing and 12 months' storage at room temperature (95).More recently, in Canada, Singh of the University of Guelph reported that commercially manufactured evaporated milk, initially containing 17 to 22 mg of vitamin C, retained 14 to 17 mg after 12 months' storage (35,54).A study of infant formula preparation practices (including various sterilizing techniques) demonstrated that, regardless of the preparation technique and a possible 24-hour refrigerated storage period, the final formula would supply the 20 mg per day of vitamin C required for infants, based on an intake of 1 oz of evaporated milk per pound of body weight (293). In another trial, designed to simulate home handling of the fortified product preparatory to use, ascorbic acid losses were as follows: after 18-hours holding at 40°F (4"C), 8%; after boiling 3 minutes and holding 18 hours at 40"F, 18%; and after 20 minutes heating in a water bath and refrigeration, 15%. The authors conclude that evaporated milk is an effective vehicle for added ascorbic acid (288). Vitamins in fluid milk (and reconstituted milk products) are adversely effected by fluorescent light and sunlight. Losses of ascorbic acid, riboflavin, and vitamin A are especially noticeable if the milk is not packaged properly. If exposure is sufficiently long, undesirable flavors will develop. Protection is achieved by packaging in opaque containers, in brown glass, or foil-laminated cartons, or, naturally, b y avoiding light exposure (41). Bagdanova and Selivanova (18) and others (40) reported that cow's milk is a suitable medium for enrichment with ascorbic acid at 5 to 15 mg percent concentration. The vitamin may be added before heat treatment, while the milk is still hot, or after cooling to 40°F. Ascorbic acid losses are slight during boiling and storage in the dark. Ascorbic acid retention figures of 76 to 97% were reported b y Russian investigators for vitamin fortified milk and kefir after 1 to 2 days' storage (121). SedlBCPk and Rybin (323) added 200 to 250 mg ascorbic acid and 100 mg of sodium citrate per liter of whole milk before spray drying during which process 10 percent of the vitamin was lost. However, after prolonged storage and reconstitution no change in milk fats or taste were detectable. Yogurt is enriched with vitamin C prior to inoculation at the rate of 10 to 100 mg ascorbic acid per 8 oz of yogurt. The finished product generally has a minimum of 30 mg/8 oz. Japanese workers (256) report adding 20 to 100 mg/100 g yogurt after fermentation and recovering 70 to 88% of it after a 20-hour storage period at 4°C. Sulc (348, 349) re-

236

J . C. BAUERNFEIND AND D. M . PINKERT

ports adding 125 mg ascorbic acid per liter immediately before inoculation; 67% of this amount was still present after distribution. Baumann and others (33) claim that the consumer receives 97 percent of the 200 mg of ascorbic acid added per liter of yogurt one day after its production.

F. POTATOPRODUCTS Average ascorbic acid values for varieties of potatoes that are commercially important range from about 19 to 33 mg per 100 g of the freshly dug tubers, with an overall value of 26 mg. Losses during storage approximate 1/4 of the ascorbic acid content after 1 month, 1/2 after 3 months, and 3/4 after 9 months (209). A further degradation of about 40% occurs during washing and cooking, a loss which may increase if the cooked product is held on steamtables before serving. Hence, the stability of natural ascorbic acid in potatoes, as well as the ascorbic acid added to potato products, has been of interest since potatoes are a significant dietary source of vitamin C in many countries

(274). An ever-increasing percentage of the potato crop is processed into dehydrated potato flakes, granules, chips, frozen sticks, etc., and it is predicted that this trend will continue. However, dehydration of potatoes to flakes, granules, slices, etc., induces losses of 30 to 60% of the natural ascorbic acid content. In one study, raw potatoes containing 29 mg of ascorbic acid per 100 g were converted into flakes; after reconstitution the product contained 8 mg (48). Frozen shoe-string potatoes retain about 80%ofraw-potato ascorbic acid content when stored, and when heated to serve they retain about 50% (47). Ascorbic acid has been added to cooked potato mash, which is then drum-dried into flakes, packaged into cans, sealed in air or nitrogen gas. Storage retention values in the product after 7 months at 70°F approximated 75%for air-packed containers and 90%for the nitrogenpacked (85). See Table VII. In another study (365) ascorbic acid was dry blended with dehydrated potato granules (45 mg/oz) and packaged in air, in pouches lined with a polyethylene-foil laminate, or under nitrogen in cans. Ascorbic acid retention after 12 months at 70°F approximated 80% for the air pack and 90% for the nitrogen pack. Moisture in the product was 5 to 670. Since this is a dry blending operation, little or no processing loss would be expected. For maximum vitamin and flavor retention, moisture levels should b e kept to a minimum (189,274). Thus, as the trend away from the consumption of fresh potatoes con-

FOOD PROCESSING WITH A D D E D ASCORBIC ACID

237

TABLE VII

C COWI'ENI'OF F ~ H T I F I E PwrAro L) FLAKES. ALTER STOHAGEAT DIFFERENT TELIPEHATUHES" (85).

\'ITAMIN

Storage (weeks) 0

4 10 12 16 28

N, pack

Air pack

75" F

98" F

113" F

75" F

98" F

113" F

80" 69

80

80

80

80

80

80

42 58 60

50 54

76 75

44

78 70

81

80

"Results ofa given trial. Initial value of80 Illg/3 oz represents ii recovery of44 mg/3 oz of'added ascorliic acid; o r 59r/, o f 75.0mg/3 oz added before drying. "Asmilligrams/ 3 oz.

tinues, it is possible to restore ascorbic acid to processed potato products, so that this vegetable can continue to contribute significant amounts of vitamin C to the diets in which potatoes have been relied upon as a vitamin source (261). Very little ascorbic acid restoration to potato products has been done in the U.S.

G. CABBAGEPRODUCTS Acid fermentation is a very ancient art of preserving and storing foods such as cabbage and pickles, while retaining their nutritive value. Although cabbage has been known for centuries as a valuable antiscorbutic vegetable, sauerkraut manufacturing, packaging, and storage can affect ascorbic acid levels. During active acid fermentation, there is little loss but in tank storage and the canning process, losses of vitamin C can occur. In a study of 217 samples of canned kraut, variations of 1 to 25 mg/100 g were found. The canned product could be modified to contain a given added amount of ascorbic acid producing a more uniform product (286).

H. MISCELLANEOUSPRODUCTS Ascorbic acid, alone or with other vitamins, is sprayed on some breakfast cereal products after the toasting process or before the final drying stage prior to packaging. Cocoa powder and instant, reconstitutable chocolate drink powders are fortified by a dry blending process. Condensed soybean milk and dried milk products, used to replace cow's milk in the infant diet, are fortified with ascorbic acid.

238

J. C. BAUEHNFEIND AND D. M . PINKERT

Ascorbic acid added at a level of 37.5 mg/oz to chocolate bars packed either in flexible or rigid containers and stored at 70°F for 1 year a s well as ascorbic acid added to the icing of filled cookies at a level 45 mg/0.5 oz icing, packaged and stored for a year were stable (363,364). Data on the stability performance in these products are shown in Table V. Ascorbic acid fortification of dehydrated and canned soups has been reported by the U.S. Department of Commerce (362) and by USSR nutritionists (326). Confections such as caramels and chocolates have been fortified with ascorbic acid (92,218)at a level of 10 mg/g of caramel. The stability of the vitamin is dependent on increased acidity of the medium at the time of ascorbic acid addition, on decreased surface exposure, and storage at low temperature. Natural vitamin C in honey was reported to be preserved by thermal deactivation of the enzymes followed by supplemental addition of ascorbic acid (91)to attain desired levels. Ascorbic acid added for vitamin fortification to sugar, proved to be stable after l-year storage when 200 to 400 g were added to 100 kg of sugar although a slightly yellow tinge and weakly acid taste developed (3,77,213). Table or kitchen salt has been fortified with 0.5-lq ascorbic acid which was stabilized by the addition of 5% potato starch; losses of ascorbic acid in fortified salt were greater in the presence of magnesium chloride and/or magnesium sulfate (3,39). Japanese workers have claimed that it is practical to fortify tea by spraying the leaves with a 20 to 50% solution of ascorbic acid and drying them at 100" to 110" for 15 minutes. The 80% of applied ascorbic acid which remained after heating proved to be quite stable. N o undesirable taste developed with concentrations up to 20 mg o/r, of ascorbic acid (174).

IV. ASCORBIC ACID REACTIONS

The use of L-ascorbic acid to inhibit or prevent undesirable oxidative changes in the processing of foodstuffs has been largely empirical. It cannot be said that the addition or presence of ascorbic acid in a foodstuff follows a particular pathway in its own destructive oxidation with the formation of definitely identified reaction products. The particular reaction pathway followed during the oxidation of ascorbic acid in a foodstuff is dependent upon many factors:

FOOD PROCESSING WITH ADDED ASCORBIC ACID

239

oxidation-reduction potential of the system; pII; aerobic or anaerobic environment; presence or absence of other reductants or oxidants; presence or absence of trace metals (particularly copper and iron); quantity of L-ascorbic acid in relation to quantities of other reactants in the oxidation process; enzymes; and time. Herrniann and Andrae (1Sl) isolated b y paper chromatography, 17 migrating spots from L-ascorbic acid oxidized in acid medium. Column chromatography enabled them to identify dehydro- and monodehydro-L-ascorbic acids, 2,3-keto-~-guloiiicacid, and threonic acid. The formation of monodehydroascorl~icacid a s an intermediate in the autoxidation of ascorbic acid to dehydroascorbic acid was confirmed by other workers (208) who used ''C-labeled ascorbate in a chromatographic study and who believe it may form a complex with ascorbic acid. Spanyar et (12. (345)reported that in the Cu-catalyzed decomposition of ascorbic acid, H,Op develops in determinable quantities, the quantity, which varies during the reaction, being related in some way to the reaction and the amount of Cu available. They believe monodehydroascorbic acid is formed and is decomposed by the H 2 0 2to form dehydroascorbic acid. In fruit juices the anthocyanins present take up the H 2 0 2 ,are decolorized, and inhibit the further decomposition of ascorbic acid. It is possible that the mechanism of ascorbic acid stabilization by the anthocyanins also involves other polyphenols. Taeufel and Voigt (3.52) reported that ascorbic acid-inhibited browning o f fruit corresponds to the amount of ascorbic acid used in the reduction of the quinoid derived from phenols. I n a model system, they demonstrated that ascorbic acid acts through phenoloxidase. In the apple, resorcinol competes for the phenoloxidase and the enzyme is irreversibly inactivated. While studying color formation in aqueous solutions of ascorbic acid, Otani (277) found that such solutions when heated at 40°C and having a pH 1 to 4, acquired furfural a s a result of ascorbic acid decomposition. Anaerobic conditions are conducive to different reaction pathways and the development of different products. Finholt and co-workers (110)found that in the hydrogen-ion-catalyzed anaerobic degradation of undissociated ascorbic acid, furfural is formed at a rate equal to the disappearance of ascorbic acid. In a noncatalyzed anaerobic cleavage, furfural is formed more slowly than ascorbic acid disappears. They believe that parallel reactions are involved, one leading to furfural and the other leading to other degradation products. In 1960, Whiting and Coggins (394) isolated and identified ~ - x y l o -

240

J. C. RAUERNFEIND AND D. M . PINKERT

sone from apple cider, which was prepared by milling and pressing prior to adding SO2,a process during which the ascorbic acid was autoxidized to dehydroascorbic acid. The formation of L-xylosone from dehydroascorbic acid in the presence of SO, is of interest because of the high proportion of dehydroascorbic acid to ascorbic acid in apples, pears, and cherries and because of the use of SO, as a preservative in other food products. In order to better understand the many factors influencing ascorbic acid stability in foodstuffs, Spanyar and Kevei (343) studied the effect of metals (Cu, Fe), air, inert gas, pH, temperature, concentration, dehydroascorbic acid, isoascorbic acid, sulfur-containing amino acids, polyphenol derivatives (anthocyans), sugar, light, alcohol, and some combinations of these on the decomposition of ascorbic acid. They found that Cu is very destructive to ascorbic acid in air but has almost no effect in a nitrogen atmosphere. Iron is somewhat of a prooxidant. Together Fe and Cu accelerate decomposition to a smaller degree than the sum of their effects per se. In 1967 Khan and Martell presented a theoretical mechanism for the metal-catalyzed oxidation of ascorbic acid (177). In the pH range 2-5, ascorbic acid diminishes with increasing pH. It has good stability up to 1O"C, the deconiposition rate is strongly accelerated between 20"and 50"C, and above 50°C the rate of destruction decelerates and reaches a maximum of 80°C. It is more stable in concentrated than in dilute solution. T h e y also found that the protective effect of anthocyanins and reduction is dependent on pH and temperature. Finally, these studies showed that alcohol and sugar may be either prooxidants or antioxidants of ascorbic acid depending on their concentration and of the presence of natural substances in food such as the anthocyanins. The physical chemist has tried to fathom the fate of the ascorbic acid molecule as it undergoes oxidation under various conditions. Electron spin resonance (ESR) studies of the autoxidation of ascorbic acid have been reported by several investigators. Free radicals result from the reaction between ascorbic acid and H,O, in the presence of peroxidase and from the reaction between ascorbic acid and molecular oxygen in the presence of ascorbic acid oxidase. ESR studies by Piette and co-workers (294,399, 400) at p H 4.8, the p H optimum for the enzymes, revealed a doublet structure with a splitting constant at 1.7 gauss which was thought to be due to an interaction of the unpaired electron with the proton located on the &carbon atom with respect to the double bond (carbon-5). Lagercrantz (205) further resolved the spectrum of the radicals b y

FOOD PROCESSING WITH ADDED ASCORBIC ACID

24 1

0 0 I

I

51 HO-C-H

I

6 CHZOH

oxidizing a 0.1 M solution of ascorbic acid with dissolved molecular oxygen over a range of pH values. Free radicals, which persisted for several hours, were found between pH 6.6 and 9.6. Interpretation of the ESR spectrum led to the belief that there is an interaction involving two equivalent protons, most likely those attached to carbon6. This is considered to be consistent with Piettes’ interpretation that the doublet splitting is probably due to the proton on carhon-5. No splittings due to the carbon-4 proton were observed. In Fig. 2 can be seen some of the pathways and reaction products postulated for the oxidation of ascorbic acid. The function of ascorbic acid as an antioxidant in food systems is (1)to scavenge oxygen and thereby prevent oxidation of oxygen-sensitive food constituents, (2) to shift the redox potential of the system to a reducing range, ( 3 )to regenerate phenolic or fat soluble antioxidants, (4)to maintain sulfhydryl groups in -SH form, ( 5 ) to act synergistically with chelating agents, and/or (6)to reduce undesirable oxidation products.

V. SYNERGIST IN FAT PROTECTION When oxygen attacks the unsaturated, double-bond linkages of the fatty acids contained in fats and oils, autoxidation is said to occur. The energy required for this reaction to proceed is obtained from sunlight, heat, or from chemical components in the food. There are two phases in the development of oxidative rancidity. Phase 1 is the “induction period,” characterized by the slow accumulation of peroxide radicals which only slightly affect odor and taste. These very active radicals remove hydrogen from active methylene groups of other molecules to form new radicals and hydroperoxides, thus initiating a chain reaction whose duration is determined by the supply of energy available. A sharp increase in the concentration of peroxides and the rate at which they progressively decompose heralds the onset of phase 2. During

J. C. RAUERNFEIND AND D. M . PINKERT

242

c"o

I 1 + HO-$:I HO-$

$7+

FOOH HO-$

2 e-+ 2 H+ mild -oxidation

H-$:

'2

CH3 Loctone of 2,3,4trihydroxypent2-enoic acid

Dehydro- L ascorbic acid

0

ocid

F-

F=O

:=0

: