Advances in Food Research, Volume 21

ADVANCES IN FOOD RESEARCH VOLUME 21

Contributors to This Volume

R. G. CASSENS C. L. DAVEY G. EIKELENBOOM D. P. HAUGHEY S. J. JADHAV SHANTHAKRISHNAMURTHY N. H. LAW SAMUELLEPKOVSKY R. H. LOCKER D. N. MARPLE P. M. NOTTINGHAM H. A. B. PARPIA D. K. SALUNKHE H. SUBRAMANYAM

ADVANCES IN FOOD RESEARCH VOLUME 21

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

G. F. STEWART University of Califomio Davis, California

E. M. MRAK University of California Davis, California

Editorial Board J. F. KEFFORD S. LEPKOVSKY EDWARD SELTZER W. M. URBAIN

E. C. BATE-SMITH W. H. COOK J. HAWTHORN M. A. JOSLYN J. R. VICKERY

1975 ACADEMIC PRESS

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COPYRIGHT 0 1975, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. N O PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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CONTENTS CONTRIBUTORS TO VOLUME21

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vii

Regulation of Food Intake

SAMUELLEPKOVSKY

I. Introduction ..................................................... I1. Early Concepts in the Regulation of Food Intake ...................... I11. Appetite. Hunger. Drive. Urges for Food .............................

. . .

IV The Gastrointestinal Tract and Regulation of Food Intake .............. V Role of Adipose Tissues in Regulation of Food Intake .................. VI Alimentary Behavior .............................................. VII.Processing of Food: Flavors ........................................ VIII. Obesity: Impairment of Regulation of Food Intake .................... IX Diet and Span of Life ............................................. X.Overview ........................................................ References .......................................................

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3 5 9 17 25 34 46 51 53 55 58

Animal Physiology and Meat Quality

.

R . G CASSENS. D . N . MARPLE.AND G. EIKELENBOOM

I. Introduction ..................................................... I1.Animal Physiology and Stress Susceptibility .......................... I11.Endocrine Interrelationships ........................................

IV. Muscle Biochemistry .............................................. V Morphology and Histochemistry .................................... VI Importance in the Retail Product ................................... VII Possible Solutions to the Problem ................................... VIII Future Research Needs ............................................ =.Summary ........................................................ References .......................................................

. . . .

72 74 87 99 113 121 129 138 138 139

New Concepts in Meat Processing

R . H . LOCKER.C. L . DAVEY.P. M . NOTTINGHAM. D. P. HAUGHEY. AND N . H . LAW

I. Introduction ..................................................... I1.Tenderness. Contraction. and Cold ..................................

158 159 V

vi

CONTENTS

I11. Application to Processing .......................................... IV.Unconventiona1 Techniques ........................................ V Microbiology ..................................................... VI.Physics of Meat Chilling .......................................... VII.Research Needs .................................................. VIII. Conclusion ....................................................... References .......................................................

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175 191 199 206 213 215 217

Physiology and Biochemistry of Mango Fruit

H . SUBRAMANYAM.

SHANTHA h S H N A M U R T H Y . AND

H . A . B. PARPIA

I. Introduction ..................................................... I1. Physiology of Fruit Growth and Development ........................ I11 Physiology of Ripening ............................................ 1V.Storage and Transport ............................................ V.Economic Aspects ................................................ VI. Research Needs .................................................. References .......................................................

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224 231 249 271 288 294 296

Formation and Control of Chlorophyll and Glycoalkaloids in Tubers of Solanum tuberosum L. and Evaluation of Glycoalkaloid Toxicity S . J . JADHAV

AND

D . K . SALUNKHE

LIntroduction ..................................................... I1.Distribution of Chlorophyll and Glycoalkaloids ........................ I11. Biosynthesis of Chlorophyll and Glycoalkaloids ........................ IV . Factors Affecting Chlorophyll and Glycoalkaloid Formation ............. V. Control of Chlorophyll and Glycoalkaloid Formation ................... VI.Pharmacology and Toxicology of Glycoalkaloids ....................... VII.Summary ....................................................... References ......................................................

308 310 312 316 331

SUBJECTINDEX ...................................................

355

342 348

349

CONTRIBUTORS TO VOLUME 21 Numbers in parentheses indicate the pages on which the authors’ contributions begin.

R. G. CASSENS, Muscle Biology Laboratory, University of Wisconsin,Madison, Wisconsin (71) Meat Industry Research Institute of New Zealand, Hamilton, C. L. DAVEY, New Zealand (157) G. EIKELENBOOM,~ Muscle Biology Laboratory, University of Wisconsin, Madison, Wisconsin (71) D. P. HAUGHEY, Meat Industry Research Institute of New Zealand, Hamilton, New Zealand (157) S. J.

JADHAV, Department of Food Science, University of Alberta, Edmonton, Alberta, Canada (307) KRISHNAMURTHY,Central Food Technological Research Institute, SHANTHA Mysore, India (223)

N. H. LAW,Meat Industry Research Institute of New Zealand, Hamilton, New Zealand (157) SAMUEL LEPKOVSKY, Department of Poultry Husbandry, University of California, Berkeley, California

R. H. LOCKER, Meat Industry Research Institute of New Zealand, Hamilton, New Zealand (157’) D. N. MARPLE,~Muscle Biology Laboratory, University of Wisconsin, Madison, Wisconsin (71) hleat Industry Research lnstitute of New Zealand, P. M. NOTTINGHAM, Hamilton, New Zealand (157) H. A. B. PARPIA,$ Central Food Technological Research Institute, Mysore, India (223) D. K. SALUNKHE,Department of Nutrition and Food Science, Utah State University, Logan, Utah (307) H. SUBRAMANYAM, Central Food Technological Research Institute, M ysore, India (223) “Present address: Research Institute for Animal Husbandry, “Schoonoord” Zeist, The Netherlands. +Present address: Department of Animal and Dairy Science, Auburn University, Auburn, Alabama. 4Present address: Food and -4gricnlture Industries Service, Agricultural Services Division, Food and Agriculture Organization of the United Nations, Via Delle Terme Di Caracalla, Rome, Italy. vii

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REGULATION OF FOOD INTAKE

BY SAMUELLEPKOVSKY Department of Poultry Husbandry. Uniuersity of California. Berkeley. Cakjomia

I. Introduction ................................................... I1. Early Concepts in the Regulation of Food Intake ..................... A . Nutrition: In the Beginning ...................................

B. The Cell .................................................... C. Machinery That Maintains the Composition of the “Fixed” Internal Environment ........................................ 1. Kidney ................................................. 2. Homeostasis of the Internal Environment .................... 3. Nutrition of the Cell ......................................

I11. Appetite, Hunger, Drive, Urges for Food .......................... A. Causes of Appetite and Hunger ...............................

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7 7 8 8 9 9 9 9 10 10 10 11 11 11 12 12 13 13 13 14 14 14 14

1. Depletion ............................................... 2. Appetite as a Mechanism To Prevent Depletion of Food Reserves . 3. Appetite and Future Needs ................................. B. Appetite for Specific Nutrients ................................ 1. Nutritional Deficiencies: Calcium and Vitamin A ............. 2 . Altered Appetite in Thiamine-Deficient Rats .................. 3 Alterations in Hormone Balance ............................ 4 . Appetite for Stinging Cells ................................ 5 . Appetite for Food Containing Heart Poisons .................. 6. Work and Appetite ....................................... 7. Appetites in Hot or Cold Environments ...................... 8. Age and Appetite ........................................ 9. Starvation and Appetite ................................... 10. Estrous Cycle and Appetite ................................ 11. Smoking and Appetite .................................... 12. Nonspecific Neural Stimuli and Appetite ..................... C. Cessation of Eating-Satiety .................................. 15 1. Satiety in Animals and Man ............................... 15 2 . Monotony-Flagging Appetite .............................. 16

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IV. The Gastrointestinal Tract and Regulation of Food Intake . . . . . . . . . . . . 17 A. The Oral Cavity: Oropharyngeal Factors ........................ B. Gastric Factors . . . . . . . . . . . . . ............................. C Intestinal Factors ............................................ 1. Results with Crossover Rats ................................ 2 . Results with Gastrectomized Rats ........................... D . Intestinal Motility ........................................... 1. Pancreas and Gastrointestinal Motility ....................... 2. “Total” Pancreatectomy, Diabetes in Chickens. and Gastrointestinal Function .................................. 3 . Hypothalamus. Pituitary. and Intestinal Motility . . . . . . . . . . . . . . . E . Processed Foods and the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . Role of Adipose Tissues in Regulation of Food Intake . . . . . . . . . . . . . . . . A. A New Nutritional Concept: The Set Point in the Adipose Tissues . . . B. Lipolysis and Lipogenesis: Reciprocal Inhibition . . . . . . . . . . . . . . . . . Hormone-Sensitive Lipase versus Lipoprotein Lipase . . . . . . . . . . . . . . C. Change in Set Point in Adipose Tissue: The Hypothalamus . . . . . . . 1. Hypothalamus and Set Point in Control System of Adipose Tissues 2 . Reversal of Change in Set Point and Decrease in Body Fat . . . . . D . Diet and Set Point in Adipose Tissues .......................... E The Pituitary and Set Point ................................... F. Circadian Rhythm in the Adipose Tissues and the Regulation of Food Intake in the Rat .................................... G. The Set Point in Adipose Tissues of Migratory Birds . . . . . . . . . . . . . . H . The Set Point in the Adipose Tissues of Hibernating Animals . . . . . . I. Regulation of Food Intake Centrally: The Hypothalamus . . . . . . . J . Lipogenesis in the Liver and/or Adipose Tissues . . . . . . . . . . . . . . . . . . K . Feedback from the Adipose Tissues to the Food-Regulating Centers of the Hypothalamus ................................. Alimentary Behavior . . . . . . . . . . ......... ................ A. Food-Seeking Behavior: Moti n ............................ B. Chemical Senses and Recognition of Food ...................... C. Choice of a Nutritionally Adequate Diet ........................ D . Taste and Body Biochemistry ................................. Flavors and Digestion of Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Feeding Pathways . . . . . . . ................................. 1. Electroencephalograms and the Evaluation of Flavors . . . . . . . . . . 2 . Direct Pathway to the Brain ............................... F . Sensations and Aversions to Food and Drink ..................... G. Is It Possible To Deceive or Confuse the Cerebral Cortex in Relation to Appetite for Food? ................................ H . Sensory Stimuli and Choice of Food by Man .................... I. Flavorful Food: Taste and Smell .............................. J . Deprivation in Many Forms .................................. 1. Physical Deprivation of Sensory Stimuli .................. 2. Self-Denial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K . Decrease in the Intake of a Specific Nutrient . . . . . . . . . . . . . . . . . . 1 Decrease in Intake of Protein .............................. 2 . Salt Deficit . . . . . . . . . . ............................... 3. “Fat Hunger” ........................ ...............

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V.

.

VI .

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17 18 19 20 21 21 22 23 23 24 25 25 27 27 28 30 30 31 31 31 32 32 33 33 33 34 34 34 35 35 36 37 37 38 38 39 39 42 43 43 44 44 44 45 45

REGULATION OF FOOD INTAKE VII. Processing of Food: Flavors ..................................... A. Meat ...................................................... B. Bread ..................................................... C. Meat and Vegetable Stew .................................... D. Brining and Fermentation .................................... E. Food Additives ............................................. F. Modern Processing of Food ................................... G. Effect of Processing and Storage ( Shelf-life) on the Nutritional Value of Food .............................................. H. Man, His Chemical Senses and Food Processing ................. VIII. Obesity: Impairment of Regulation of Food Intake .................. Lessons from Wars ............................................. IX. Diet and Span of Life ........................................... A. Undernutrition and Work ..................................... B. Undernutrition and Psychological Responses ..................... C. Is There An Optimum Level of Food Intake? ..................... X. Overview ..................................................... References ....................................................

1.

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46 46 46 47 47 47 49 49 50 51 51 53 54 54 55 55 58

INTRODUCTION

Progress in the knowledge of nutrition during the first half of this century has been impressive. The vitamins were discovered, isolated, and characterized chemically. Many of them are currently being synthesized. Much has been learned about the functions of these vitamins in the physiology of different animals. The animals’ requirements for most of the vitamins have been established. Similar progress has been made on amino acids, proteins, minerals, and micronutrients. Geneticists have applied the newer knowledge of nutrition to select farm animals that use feed more efficiently and yield meat, eggs, and wool more economically ( Brodie, 1945). Antibiotics have reduced mortality and increased the yield of animal products. The food industry has kept pace with the geneticists and nutritionists. The industry provides nutritionally adequate diets for different animals at minimum costs. Forty to fifty years ago chicken was a luxury meat, and politicians promised “a chicken in every pot.” Compared with the price of beef, the price of chicken for the housewife is so low that it is no longer considered a luxury. This was made possible not only by the industry but also by nutritional investigators. Before the discovery of vitamin D, for example, there was no broiler industry. It was not possible, because during the winter the chickens developed rickets-”leg weakness”-and died. Before vitamin D became available, the only defense chickens had against rickets was the ultraviolet light of sunshine (Hart et al., 1923),and that was available only during the summer. Nutritional disease in the human population from vitamin deficiencies

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became scarce, but new nutritional diseases appeared-diseases from the intake of toxic levels of vitamins A and D. Among sources of information of this era are: “Newer Knowledge of Nutrition” (McCollum and Simmonds, 1929); “Handbook of Nutrition” ( 1951); “Bioenergetics and Growth” (Brodie, 1945); “Modern Nutrition in Health and Disease” (Wohl and Goodhard, 1960); “The Vitamins” (Sebrell and Harris, 1954); and “The Pathology of Nutrition” (Follis, 1948). The Nutrition Foundation, Inc., publishes ”Nutrition Reviews” which contains monthly summaries of the current literature on the progress of appropriate nutritional subjects. Periodically, it contributes exhaustive and enlightening surveys on the current status of the whole field of nutrition in a publication entitled “Present Knowledge in Nutrition.” And, not the least, the Nutrition Foundation promotes symposia of timely and pertinent fields of nutrition, such as the chemical senses and nutritional processes. We are now in a new era in nutrition. Nutritionists are currently working on the regulation of food intake. Psychologists have joined the nutritionists in the investigation of the complicated machinery that regulates food intake. The brain in the human being regulates all bodily processes through some 13 billions of neurons (Ottoson, 1963), many of which are used to regulate nutritional processes. Electrolytic lesions in the hypothalamus have provided much information on the role of the brain in the regulation of food intake; this information has been summarized by Anand (1961) and Brobeck (1946). It appears that the lateral hypothalamus (appetite center) elicits urges for food at all times, irrespective of caloric need, and the ventromedial nucleus of the hypothalamus (satiety center) inhibits the lateral hypothalamus and elicits satiety. These two opposing centers are involved in the regulation of food intake. Much information has been obtained by chemical stimulation of the brain. Biologically active compounds have been introduced into various loci of the brain, and alimentary responses recorded. Initially, adrenergic and cholinergic compounds were introduced into the lateral hypothalamus through cannulas (Grossman, 1960). Subsequently, the hypothalamus and other areas of the brain were treated with various biologically active compounds ( Grossman, 1969, 1972; Epstein, 1959, 1960; Miller, 1957, 1965; Myers et al., 1972; Yaksh and Myers, 1972; Leibowitz, 1970). Excellent reviews of the vast literature on the alimentary responses to lesions and chemical and electrical stimulation of various loci of the brain have been published by Hoebel(l971) and by Morgane and Jacobs (1969). The brain regulates body temperature in homeotherms (Myers and Yaksh, 1969; Feldberg and Myers, 1965; Bligh and Moore, 1972). The brain adjusts heat production to heat loss so that normal body tempera-

REGULATION OF FOOD INTAKE

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tures are maintained. Regulation of body temperature requires adjustments in food intake: In hot climates food intake is decreased, and in cold climates food intake is increased. Reproductive processes are regulated by the hypothalamic gonadotrophic releasing factors (Sawyer et al., 1959; Everett, 1964; McCann, 1971; Flerk6, 1963). These factors elicit the release of the gonadotropic hormones from the anterior pituitary, which in turn act upon the target glands, the ovaries and testes. Reproductive processes alter food intake. Water metabolism is regulated by the posterior pituitary through the kidney. It is also regulated by thirst and loss of water by evaporation (Wolf, 1958; Wayner, 1964; Pitts, 1959). Food intake and water metabolism are closely interrelated. Activity plays an important role in the regulation of food intake. Activity increases food intake, and inactivity in general decreases food intake. Food intake, water intake, maintenance of body temperature, activity, and reproductive functions are all regulated and integrated by the brain; the machinery that operates these processes is located in the periphery. Much of the machinery that controls food intake and body weight is located largely in the pituitary, adipose tissues, gastrointestinal tract, liver, adrenals, gonads, thyroid, and pancreas. During the evolutionary development of animals, different mechanisms that regulate food intake arose in different animals. It is postulated that these mechanisms have not been replaced by the brain, but instead the brain regulates them and integrates them with bodily needs. This review will concentrate on the control machinery in some of these organs and their relationship with regulatory centers of the brain.

II.

EARLY CONCEPTS IN THE REGULATION OF FOOD INTAKE

For centuries, human beings have been concerned with food customs, appetite, and satiety. In 1897 Sir William Roberts (see Drummond, 1934) wrote: “The generalized food customs of mankind are not to be viewed as random practices, adapted to please the palate or gratify an idle or vicious appetite. These customs must be regarded as the outcome of profound instincts which correspond to certain wants of the human economy. They are the fruit of a colossal experience accumulated by countless millions of men through successive generations.” Mursell (19W), who examined the literature of comparative and historical dietetics, wrote: “When we find widely sundered races, with vast differences in tastes and preferences, in food opportunities, and in

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habitat, still keeping to pretty much the same general balance of rationing, it is difficult to avoid the impression that we are in the presence of a very powerful autoregulative mechanism which, given certain external conditions, strongly favors a certain proportion of intake from among the basic nutrient substances.” Richter (1942) postulated than an organism is sensitive to many of its physiological needs and will develop motivated behavior appropriate to the fuEllment of these needs. According to Kubie (1948), bodily needs have a “biochemical core,” and alimentary behavior deals with the translation of bodily needs to behavior. Cannon (1932) wrote: “If the requirements of the body are not met, hunger pangs and thirst arise as powerful, persistent and tormenting stimuli which imperiously demand the ingestion of food before they cease their goading.” Zamcheck (1960) appreciated the complicated nature of the biological machinery that underlies nutritional processes and their dynamic interrelationships. He quoted Bean: “The essence of biological and medical problems is that they are enmeshed in a jungle of variables and in the continual flux of dynamic aspects of living matter. Tests must go on in a milieu with multiple coordinates which may shift dimensions, directions, or locations in a bewildering way.”

A. NUTRITION: IN THE BEGINNING Life began in the primordial sea (Oparin, 1938), and the composition of the sea water set the conditions of life under which, and only under which life can continue. It has been postulated that the primordial sea contained prefabricated organic compounds ( Horowitz, 1945) such as glucose, amino acids, fatty acids, vitamins, electrolytes, cations such as calcium, sodium, potassium, magnesium, iron, copper, and manganese, and anions such as chlorides, phosphates, bicarbonate, and sulfate. “This suggests that conditions under which cell life is possible are very restricted indeed and have not changed substantially since life began” ( Baldwin, 1949). “The living elements of the body, therefore, are water inhabitants, or inhabitants of water which has been modified by the addition of salts and thickened by an albuminous or colloidal material” ( Cannon, 1932). When animals left the primordial sea, the external environment changed, but the conditions under which life began did not change. To survive, living systems had to carry their environment with them ( Baldwin, 1949). The external environment, the archaic sea, became the internal environment, commonly referred to as the extracellular

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fluids of animals. The living elements of animals are the cells, and they live in “an internal environment of their own . . . with a controlled unchanging atmosphere which [makes] the organism independent of outside conditions” (Smith, 1953). As Bernard ( 1859) pointed out, “All vital mechanisms, however varied they be, have only one object, that of preserving constant the conditions of life in the internal environment” ( Bayliss, 1924). €3.

THECELL

The cell is considered the unit of living systems. Sherrington (Lord Cohen, 1958) described the dynamic nature of the cell: It is energy-cycles, suites of oxidation and reduction, concatenated fermentactions. It is like a magic hive, the walls of whose chambered spongework are shifting veils of ordered molecules, and rent and renew as operations rise and cease. A world of surfaces and streams. We seem to watch battalions of specific catalysts, like Maxwell‘s demons, lined up, each waiting, stop-watch in hand, for its moment to play the part assigned to it. Yet each step is understandable chemistry. In the spongework of the cell foci coexist for different operations, so that a thousand different processes go forward at the same time within it. The foci wax and wane as they are wanted. Let that catastrophe befall which is death, and these catalysts become a disorderly mob and pull the very fabric of the cell to pieces.

Cells are assembled to form tissues or organs which are in turn connected anatomically, forming a living animal. The basis of life is the enzyme systems which “wax and wane” as they are needed; this is accomplished through the endocrine and nervous systems. All living processes are integrated by the brain; again quoting Shemngton: “it is nervous reaction which par excellence integrates it, welds it together from its components, and constitutes from a mere collection of organs an animal individual.” ( Sherrington, 1947).

THAT MAINTAINS THE COMPOSITION OF THE C. MACHINERY “FIXED” INTERNAL ENVIRONMENT Elaborate machinery was developed to maintain the composition of the internal environment: 1. A vascular system was set up to contain the internal environment and circulate it among the cells. 2. A heart was developed to pump the circulatory fluids through the body. 3. Lungs were developed for the exchange of gases between the internal and external environments.

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4. Red cells were developed to carry oxygen to the cells and remove carbon dioxide from cells. 5. Kidneys were developed to get rid of unwanted chemical compounds and wastes. 6. A skeletal system was developed for supporting structures. 7. A gastrointestinal tract was developed to digest the food and to deliver the compounds needed for the internal environment. 8. The brain was developed to coordinate the mechanisms in these tissues; it does so through the nervous and endocrine systems. 9. A skin was developed which wrapped itself around the organism to protect the machinery in the body (Houssay, 1951). 1. K i d m y The kidney plays a key role in the maintenance of the composition of the internal environment. Compounds, especially minerals, delivered in excess of needs to the cells through the mouth must be eliminated; the kidneys excrete the excess in the urine. Some nutrients ingested in excess of needs are not eliminated. Glucose ingested in excess of need is converted to fat and deposited in the fat depots. Intakes of excessively large amounts of vitamin A and D become toxic because ingestion of these vitamins in excess of need are not eliminated.

2. Homeostasis of the Internal Environment The maintenance of the composition of the internal environment is essential for the survival of animals. Regulators, glands of internal secretion, maintain the “fixity” of the internal environment, and the work of these glands is coordinated by the brain. The posterior pituitary regulates the amount of water in the internal environment. The adrenal cortex regulates the concentartion of sodium; the parathyroid, the levels of calcium and phosphorous; and the pancreas, the level of glucose. Not only does the brain regulate these endocrine glands, it also acts to correct metabolic errors such as those that stem from functional failure or loss of the regulators. In the event of the loss of the posterior pituitary, the kidney nephron cannot reabsorb sufficient amounts of water from the plasma ultrafiltrate for lack of the antidiuretic hormone, and dehydration ensues. The brain institutes appropriate behavior to correct this condition; that is, the animal drinks more water (Richter, 1935). Loss of the adrenals causes the loss of excessive amounts of sodium in the urine. The brain alters appetitive behavior for salt; the

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animal increases its salt intake and maintains itself in fairly good condition (Richter, 1936). When the animal loses its parathyroid gland, blood calcium drops, tetany develops, and many animals die (Richter and Eckert, 1937). Appropriate appetitive behavior is instituted; more calcium is ingested, and only sufficient phosphorus is consumed to maintain a normal calcium-to-phosphorus ratio in the internal environment ( Shelling, 1932).

3. Nutrition of the Cell It appears that in nutrition, we are not feeding the animal. Instead, we are feeding cells. Most of the structures of animals are, so to speak, the vehicle that was developed to provide the cells with the nutrients needed for (1) maintenance of homeostasis of the internal environment; ( 2 ) energy; ( 3 ) growth; (4)repair of damaged tissues; ( 5 ) reproduction; and ( 6 ) lactation.

111.

APPETITE, HUNGER, DRIVE, URGES FOR FOOD

Hunger and appetite are sensations and may be beyond definition, since “. . . each animal, including the human being, lives in a world outlined by his own senses” (Kare, 1960). The usages of the terms hunger and appetite have been so varied that “many have advocated their banishment for the sake of linguistic peace” (Janowitz, 1958). The terms “urges for f o o d and “drive” are frequently used as expressions for the need for food. Freud (see Stunkard, 1961) defined appetite or drive as a specific somatic process in an organ or part of the body from which there results a stimulus which gives rise to behavior called “aim” which “seeks to abolish the condition of stimulaton.” This definition, originally applied to appette for sex, is equally valid as a definition of appetite for food. Hunger and thirst are two of the most powerful motivational drives known. The terms are used to express intense desires, such as “appetite for wealth,” “hunger for power,” “thirst for vengeance,” “insatiable desire for riches.”

A. CAUSES OF APPETITE AND HUNGER 1 . Depbtwn

Physiologists ( Janowitz, 1958; Grossman, 1955) suggest that appetite or hunger is caused by depletion, and repletion is followed by satiety.

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It is difficult to understand why corpulent animals that fast for as much as 24 hours get hungry even though the amount of energy that is depleted from their fat depots is an insignificant fraction of the total energy stored there. Of even greater interest is loss of hunger by people who consume zero calories for periods of 24 or 48 hours (Lundbaek, 1964; Keys, 1964). 2. Appetite as a Mechanism to Prevent Depletion of Food Reserves

Ugolev and Kassil (1961) look upon hunger as a means to prevent depletion of food reserves: “The appetite has been formed not as a reaction to the depletion already arising of the food reserves, but as a mechanism that had prevented such a depletion far in advance . . . Consequently, the theories connecting hunger and appetite with the exhaustion of the reserves, despite their bribing simplicity, should be rejected. The appetite does not follow the disappearance! of the deposits of foods, but precedes it and does not take it for granted.” 3. Appetite and Future Needs

a. Hibernation. Hibernation leads to excessive eating; the woodchuck, ground squirrel, and most rodents are stimulated to prepare for hibernation by becoming extremely obese. An unusual expression of hunger is hoarding; the golden hamster does not fatten prior to hibernation but is stimulated to store food. This food is used during the periodic arousals in the hibernating period, and without such storage the animal would perish (Lyman, 1954). b. Preflight Hyperphagia: Migratory Birds. Before flight, migratory birds become hyperphagic and become obese. Enough fat is stored in the depots to complete the flight. If too little fat is stored, insufficient energy will be available to the bird to complete the flight. Excessive deposits of fat increase the weight of the bird and hamper successful flights in other ways (King and Farner, 1933; Odum, 1960). B. APPETITEFOR SPECIFICNUTRIENTS The complex machinery of the body operates continuously to ensure the survival of the animal. Defects developing in bodily composition, hormone balance, or abnormal biochemistry evoke alterations in alimentary behavior to correct the defects.

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1. Nutritional Deficiencies: Calcium and Vitamin A Drummond (1934) lucidly described the behavior of pigs allowed out for exercise. Well-fed animals usually lay down or contentedly strolled about the yard. Those suffering from a deficiency of calcium spent a large parts of their time attempting to lick whitewash from the walls or to root out a fragment of cement or mortar from the brickwork, while the shortage of vitamin A stimulated the animals to search for the smallest blade of grass or a chance weed which might be growing in the cracks of the floor. 2. Altered Appetite in Thiamine-Deficient Rats

A deficiency of an essential nutrient may alter a specific appetite for certain foods as a consequence of changes in internal biochemistry. Richter et al. (1938) found that rats deficient in thiamine showed a marked decrease in their appetite for carbohydrate and a concomiant increase in appetite for fat. In all probability the decrease in appetite for carbohydrate resulted from a disturbed metabolism in carbohydrate as a consequence of thiamine deficiency (Peters, 1936). 3. Alterations in Hormone Balance Injections of protamine zinc insulin into rats increased their appetite, and they became obese. Cessation of injection was accompanied by a decrease in appetite ( MacKay et al., 1940). Whereas mammals responded to injections of protamine zinc insulin with increases in appetite, the opposite occurred in chickens (Lepkovsky et al., 1965). Mice kept under the influence of insulin injections for about 10 hours show sharply increased appetite for starchy food, and caloric intake increases. After the effect of insulin has worn off, animals eat a correspondingly smaller amount of starchy food, and the caloric intake decreases (Donhoffer, 1960). Similar results were obtained when rats were given a choice of 10% or 35% solutions of glucose. They preferred the 10% glucose solution. After injections of insulin, appetites shifted and the rats preferred the 35% solution of glucose (Jacobs, 1958). It has been commonly assumed that hypoglycemia in response to the injection of insulin caused the increase in appetite. This is disputed by Soulairac (1963), who showed experimentally that the increase in appetite is due to the increase in cholinergic activity elicited by insulin.

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Not all animals have the same appetite for sugar (sweet tooth) as do rats and human beings. Chickens are indifferent to sucrose, irrespective of whether it is offered as part of the food (Jukes, 1938) or as a liquid (Kare and Medway, 1959). Other animals that are indifferent to the sweet taste of sugar are red-winged blackbirds (Rogers and Maller, 1973), porcupine (Bloom et al., 1973), and armadillo (Maller and Kare, 1967). Administration of growth hormone increases appetite for food (Lee and Schaffer, 1934). Andik et al. (1966) showed that the appetite for the starchy diet is largely increased in response to the injection of growth hormone. While it has been known that appetite increases in response to the injection of thyroxin because of the increase in the rate of metabolism, Donhoffer and Vonotzky (1947) showed that in rats and mice the increased caloric requirements were met almost entirely from the highstarch diet. The reverse occurred when the animals were treated with methylthiouracil; caloric intake decreased, and the decrease occurred exclusively at the expense of the starchy food (Andik and Donhoffer, 1948-1949).

4. Appetite for Stinging Cells The roundworm, microstoma, uses nematocysts ( Lashley, 1938, cited by Stellar, 1960) or stinging cells to capture prey, but it must acquire these stinging cells from hydra. After the stinging cells have been depleted from the body of microstoma, a physiological need for these cells arises in microstoma and it develops appropriate appetitive behavior and ingests hydra voraciously. After microstoma obtains a full complement of stinging cells, it ceases to ingest hydra even in the complete absence of other sources of food (Stellar, 1960).

5. Appetite for Food Containing Heart Poisons The larvae of the monarch butterfly feed on milkweed (Asclepias curassavica ) which contains a digitalis-like compound. This compound compound is poisonous to bluejays, and they avoid eating the larvae. Larvae were raised, with difficulty, upon cabbage plants and offered to bluejays. The birds refused these larvae. The bluejays were deprived of food until they ate some of the cabbage-fed monarch larvae. Two birds died of starvation rather than eat the larvae. Finally, some of the birds

REGULATION OF FOOD INTAKE

13

accepted the cabbage-fed monarch larvae. Once the birds overcame their initial hesitancy, they continued to eat without any sign of unpalatability. The bluejays were then again fed the milkweed-fed larvae, and they became sick; they showed violent retching and vomited the partially digested insects. Thereafter the bluejays again rejected the larvae of the monarch butterflies (Brower et al., 1967). 6. Work and Appetite

Appetite generally decreases in animals while they are engaged in excessive physical activity. A compensatory increase in appetite does not occur on the day of exercise, but it appears on the first or second day following the physical activity. This was shown for man by Edholm et al. (1955) and for the rat by Stevenson et al. (1966), Thomas and Miller (1958), and Premack and Premack (1963). Stevenson et al. (1966) suggested that “there may be a slow acting but discrete mechanism of adjustment of food intake to energy expenditure.” In laboratory experiments, the selection of food changed markedly in the rat during exercise; the intake of diets high in fat and protein increased (Andik et al., 1954). Termination of exercise was followed by restoration of the pre-exercise level of the intake of all three varieties of food used in the tests. Although the caloric intake increased during exercise, there was little change in the bulk, suggesting that constancy of bulk is a major factor in the regulation of food intake and selection.

7 . Appetites in Hot or Cold Environments Brobeck (1948) emphasized the relation of food intake to body temperature. At a high temperture where loss of heat is difficult, appetite should be low, lest by eating the body acquire more heat than it can dispose of and become hyperthermic. At a low temperature appetite should be high because the body can use the extra heat in defending itself against hypothermia. Rats and mice kept in the cold increased their caloric intakes, and the increased caloric requirements were met with an increased appetite for the high-starch food. The reverse occurred in a hot environment; the reduction in appetite was confined to this type of food (Donhoffer, 1960). 8. Age and Appetite

Young rats have appetites for carbohydrates. Old rats show decreases

14

SAMUEL LEPKOVSKY

in appetite for carbohydrates with compensatory increases in appetite for fat (Sz. Donhoffer, personal communication). 9. Starvation and Appetite

After a five-day fast, rats sharply increased their intake of high-fat diet, and the increase was balanced by a compensatory decrease in the high carbohydrate diet (Andik et al., 1951). 10. Estrous Cycle and Appetite Estrus and diestrus in rats are associated with major rearrangements in regulation of food intake. During estrus, rats are hyperactive; they show decrease in appetite and eat less. During diestrus, the rats are less active, but they have an increase in appetite and eat more ( Brobeck et al., 1947).

11. Smoking and Appetite Smoking decreases appetite. Cessation of smoking increases appetite (Larson et al., 1961). It appears that in starving human beings, smoking often evokes an exaggerated sensation of satiety as indicated by the increase in the desire for tobacco. So much so, that the desire for tobacco appeared to be stronger than the desire for food “since the men would barter even the little food they had for a small amount of tobacco” (Keys et al., 1950). Smoking or nicotine administration causes mobilization of free fatty acids, increases the concentration of plasma FFA, increases the supply of energy to the cellular mass, and may be a factor in the decrease of the hunger sensation. Nicotine or smoking stimulates the sympathetic nervous system causing an increase in the secretion of catecholamines which decrease appetite ( Kershbaum et al., 1967). Another possible factor may be the increase in smokers of serum somatotropin which also increases lipolysis ( Sandberg et al., 1973). 12. Nonspecific Neural Stimuli and Appetite Animals show an increased appetite for food in response to external stimuli (optic, acoustic, tactile, or olfactory). It has been frequently noted in laboratories that animals eat less on weekends when there is little disturbance or activity in the animal rooms (Lat, 1959).

REGULATION OF FOOD INTAKE

15

C . CESSATION OF EATING-SATIETY Satiety may be achieved metabolically when food is eaten, digested, absorbed, and delivered to the tissues. Nutrient deficits incurred during the fasting periods are restored, and food intake ceases. This is best illustrated with thirst: Dehydrated dogs with a known water deficit drink water in amounts equal to the amount of water deficit. If water equal to the amount of water deficit is administered intragastrically and the dog is alowed to drink immediately, it will drink water equal in amount to its water deficit. When water is offered 20 minutes after the intragastric water load, the dog does not drink. During those 20 minutes, the water given intragastrically has been absorbed and has rehydrated the tissues of the dog, and it is no longer thirsty (Bellows, 1939; Adolph, 1939). In most animals cessation of eating occurs long before the food has been digested and metabolized. After a 24-hour fast, the dog meters in 10 to 20 minutes the amount of food containing energy equal to that depleted during the previous 24-hour fast ( Adolph, 1947; Grossman, 1958; Janowitz, 1958; Janowitz and Hollander, 1955). The gastrointestinal tract plays an important role in this process.

I. Satiety in Animals and Man Man experiences hunger, and it may be assumed that animals also experience hunger. Pain often accompanies hunger. Eating abolishes hunger and, with it, hunger pains. It has been an ancient view and one represented by Schopenhauer, that pleasure is the absence of pain (Sherrington, 1947) . When satiety is achieved, animals stop eating, but human beings often continue to eat. Brillat-Savarin (1960) aptly noted that it is a “privilege of the human species to eat without hunger and to drink without thirst.” This privilege is of doubtful value if it leads to obesity. This difference in eating behavior between human beings and animals is due, in all probability, to the more highly developed cerebrum of human beings. This view is supported by the eating behavior of one human being whose cerebrum did not interfere with his hypothalamus in his choice of food (Katz, 1953). “A young man, 24 years old, had gas poisoning. Subsequently, he was unable to remember anything for longer than two seconds. From this time his feeding was entirely governed by his bodily needs: conscious memories could no longer play any part. He appeared for meals when hunger and thirst made him. He

16

SAMUEL LEPKOVSKY

never over-ate, but stopped eating when satisfied. Once satisfied, nothing on the table attracted him any longer. He knew what he should eat and drink-of course he did not really know, but so his organism directed.” 2. Monotony-Flagging

Appetite

Some foods can be eaten continuously without loss of appetite for them; bread is a good example. Acceptability for some foods that are consumed too often decreases progressively, and ultimately aversions for them may develop. The army had such an experience with Spam in the Pacific Southwest area of operations in World War 11. Spam was served too often, and the aversion to it became so great that it was eliminated from army rations. After a while requests for Spam came from the same area of operations. Monotony may result from neural “fatigue.” Animals show progressive decreases in response to stimuli when the same stimulus (including sensory stimuli such as flavors) is repeatedly applied. This phenomenon is variously referred to as “habituation,” “sensory accommodation,” “effector fatigue,” “negative adaptation,” and “internal inhibition.” For example: If a drop of water falls on the surface of the sea just over the flowerlike sea anemone, the animal contracts vigorously. If a second drop falls within a few minutes, there is less contraction, and the response disappears after the third or fourth drop. Sharpless and Jasper (1956) state: “This is one of the most pervasive phenomena of the animal kingdom. The ubiquity of the phenomenon is one of the most fundamental properties of the animal.” This phenomenon is seen in olfaction. The smell of coffee in the morning is very pronounced before eating. During eating, the odor of coffee is much less pronounced. While neural “fatigue” may apply to monotony in foods, it cannot be the whole explanation. It is known that mildly toxic compounds will decrease appetite for foods containing them. Small amounts of toxic substances in foods may not affect appetite. When such foods are eaten continuously, sufficient amounts of toxic compounds may accumulate to “toxic” levels, and food intake will decrease (monotony). Foods that are marginally deficient in essential nutrients may induce progressively greater nutritional deficiency, which would result in progressively decreased food intake (monotony). The effect of repetitive or monotonous diets has been studied with human subjects (Siegel, 1957; Siegel and Pilgrim, 1958). The results emphasized the problem, as indicated by the following observations: (1)

REGULATION OF FOOD INTAKE

17

monotony is positively related to the number of times a given food item has been consumed ( 2 ) Highly palatable foods slow the growth of monotony. ( 3 ) Foods that are highly palatable for some individuals are not necessarily palatable for others. ( 4 ) The course of monotony is affected by personality factors. ( 5 ) The environment is a factor in the acceptability of food. ( 6 ) Monotony is overtly expressed in lowered food acceptance. ( 7 ) Populations, entire ethnic groups, subsist essentially on repetitive or monotonous diets.

IV.

THE GASTROINTESTINAL TRACT AND REGULATION OF FOOD INTAKE

Food as it exists in the external environment cannot be used by the cells, the living elements in the body. In nutrition, therefore, we are not feeding the animal; we are feeding the cells. Before the food can be used by the cells, it has to be digested and reduced to its unit structures which can be used by the cells. A. THEORALCAVITY:OROPHARYNGEAL FAC~ORS The mouth is the gateway through which food must pass. As World War I1 soldiers put it, “Food is not food until it is eaten.” Seeing, smelling, tasting, chewing, and swallowing food play an important role in eating. During chewing, receptors are activated in the teeth, in the chewing muscles, and in the tongue during its gyrations. All this makes possible stereognosis: consciousness of the size and shape of the bolus without sight ( Sharon, 1965). Stimuli from the central nervous system, memory and anticipation, add to the sensory stimuli of the food. The saliva acts on food to produce new sensory stimuli, adding to the pleasure of eating. Chewing is more important for good nutrition than is generally realized, since during this process many new flavors (sensory stimuli) are formed, producing new and changing flavor profiles in the food as it is being chewed and worked over in the mouth (Sharon, 1965). The importance of oropharyngeal factors in a human being may be illustrated by a patient who had to be fed through a fistula in the stomach. He was fed by his mother, but he did poorly and gained little weight. After his mother died, he had to feed himself. He learned that if he took the food into his mouth and worked it over before it was introduced into the stomach, his appetite was satisfied. Furthermore,

18

SAMUEL LEPKOVSKY

he felt better and gained weight satisfactorily. Presumably, taking the food into the mouth permitted the sensory stimuli of the food to activate the receptors in his oral cavity and helped evoke the feeling of satiety (Wolf and Wolff, 1943). These stimuli may have prompted better food utilization, since oropharyngeal stimuli increase the flow of gastric, pancreatic, and intestinal secretions. Another example (Janowitz et al., 1950) is that of a woman without an esophagus who was fed intragastrically through a tube. The amount of gastric juice secreted into the stomach was measured by withdrawing the gastric juice from the stomach through the same tube. Oatmeal gruel was presented to the patient, who merely chewed it and then spat it out. This constitutes sham-feeding. Little gastric juice was secreted. When the patient was given a meal of her own choice, a large amount of gastric juice was secreted. While food is being chewed in the mouth, signals are forwarded over the nerve tracts to the brain where they are appreciated. The control machinery of the brain is set in motion, activating machinery in the salivary glands, stomach, intestines, pancreas, and gall bladder. The flow of saliva into the mouth is increased (Wolf and Wolff, 1943), and the enzymes in saliva initiate the digestive process and facilitate the swallowing of the food. Concomitantly, the alimentary tract is prepared to receive and digest the food. The flow of gastric juice (Janowitz et al., 1950), pancreatic juice( Behrman and Kare, 1968), and intestinal juices is augmented, and the flow of blood in the gastrointestinal tract is increased. Dogs fitted with esophageal fistulae in the neck, so that the ingested food does not reach the stomach, eat excessively large amounts of food, cease eating for short periods of time, and then continue to eat. Oropharyngeal factors have some satiety value but play a relatively unimportant role in achieving satiety (Janowitz and Hollander, 1955; Grossman, 1958). Rats regulate food intake without the use of oropharyngeal factors. They have been taught to feed themselves intragastrically by bar-pressing, thus bypassing oropharyngeal factors (Epstein, 1967). B. GASTRICFACTORS

Gastric factors, especially gastric distention, have been considered more important than oropharyngeal factors in inducing satiety (Janowitz, 1958; Grossman, 1958). Gastric distention with nutritionally inert material such as celluflour or with water-filled balloons depressed food

REGULATION OF FOOD INTAKE

19

intake, but not to the same extent as an equal volume of food. Intragastric feeding of dogs (Share et al., 1952; Janowitz and Grossman, 1949; Janowitz and Hollander, 1955) and of rats (Miller and Kessen, 1952) depressed food intake. Dogs fed intragastrically required weeks to adjust their ad libitum food intake to their bodily needs (Janowitz and Hollander, 1955). Gastrectomized human beings ( Wangensteen and Carlson, 1931) and gastrectomized rats (Tsang, 1938) regulate food intake, indicating that the stomach is not of decisive importance in the regulation of food intake. Snowden (1970) showed that food intake is influenced by the presence of food in the stomach and by the amount of food therein. When rats are satiated by a meal, the removal of gastric contents through a fistula induces the rats to resume eating long before they normally would if the gastric contents were left undisturbed. This could be due to a decrease in gastric distention or to diversion of food from the duodenum. The work of Balagura and Coscina (1969) suggests that the intestines also play an important role in the satiety mechanism. C. INTESTINAL FACTORS

Less attention has been given to the role of the upper intestine in the regulation of food intake. (Hill et al. (1952) introduced nonnutritive material into the jejunum of the dog through a fistula, which caused distention and temporarily dulled the desire to eat, but the inhibition was not of great magnitude. While the introduction of 5.4% glucose solution into the jejunum had no effect on food intake, glucose in sufficient quantity or hypertonic concentration of glucose had an inhibitory effect (Hill et al., 1952). Similar results have been reported by Ehman et al. ( 1971). Much more impressive was the demonstration that chronic jejunal feeding caused anorexia in the dog (Holinger et al., 1932; Ivy, 1935), but following vagotomy the animals became hyperphagic ( Holinger et al., 1932). These experiments suggest that inhibition of food intake was mediated through neural mechanisms. This possibility is supported by the observation that distention of isolated loops of the intestine caused anorexia in the dog, but the dog did eat after vagal and sympathetic denervation ( Herrin and Meek, 1945) . The upper intestine may also inhibit food intake through humoral mechanisms; enterogastrone administration inhibited food intake in the mouse (Schally et al., 1967). Similar results were obtained with injections of cholecystokinin in rats (Gibbs et al., 1973). In contrast, in-

20

SAMUEL LEPKOVSKY

jections of secretin and cholecystokinin-pancreozymin had no effect on food intake (Click et al., 1971). Very pertinent in this connection are the findings of Balagura and Coscina (1969), who showed that, while distention of the intestine with water moderately decreased food intake, by comparison the presence of nutrients in the intestine had a tremendous inhibitory effect on eating.

1. Results with Crossover Rats A new technique has been devised that permits the extension of studies on the role of the upper intestine in the regulation of food intake (Lepkovsky et al., 1971). The intestines of parabiotic rats were “crossed surgically; the proximal end of the duodenum of one parabiont was anastomosed to the pylorus of its partner. Food eaten by one parabiont entered its stomach but, instead of entering its own duodenum, entered the duodenum of its counterpart partner; in effect, the rats fed each other duodenally. It was possible to study the oropharyngeal and gastric factors separately and uninfluenced by intestinal factors. Likewise, intestinal factors could be studied uninfluenced by oropharyngeal and gastric factors. Many crossover pairs developed an unusual pattern of eating; one parabiont ate, and its partner ate little or nothing. X-rays showed that the stomachs of both rats of crossover pairs did not always empty at the same time. The rat whose stomach emptied first was the “eater,” and the recipient of its gastric chyme did not eat. Autopsy records supported the x-ray findings. Crossover pairs were force-fed the same amount of food at the same time, and 5 hours later they were autopsied. The stomachs of the “eater” rats were practically empty, whereas those of the noneating partners were full of food. It appears that the gastric chyme in the recipient’s upper intestine inhibited gastric emptying and food intake. This suggests that neural and/or humoral signals from the upper intestine containing the gastric chyme inhibited food intake. On the other hand, the gastric chyme of the eating rat did not enter its duodenum; without gastric chyme in its upper intestine, there were no inhibitory signals from its upper intestine to its stomach and brain, and the rat continued to eat. When fed ad libitum, the “eater” rat was not satiated and ate continuously, and undigested intestinal materia1 passed from the recipient noneating partner. The maintenance of these rats was made possible only by several daily intragastric feedings of equal amounts of food at the same time, each feeding separated by 3 to 4 hours.

REGULATION OF FOOD INTAKE

21

2. Results with Gastrectomized Rats Through its control of gastric emptying, the upper intestine of the normal rat prevents overloading of the intestine. When deprived of its stomach, the upper intestine of the gastrectomized rat nevertheless regulates entry of food into the duodenum, and it does so by the regulation of food intake (Tsang, 1938). De Muelenaere (1964) showed that gastric emptying in rats fed raw soybean ( R S ) diet was slower than that in similar rats fed heated soybean (HS) diet. This was due, presumably, to the presence in RS of a nonspecific irritant which inhibits gastric emptying ( Quigley and Louckes, 1962). De Muelenaere suggested that the decreased intake of RS diet, frequently observed in studies of the nutritional value of HS and RS diets in rats, was due in part to the decrease in the rate of gastric emptying. Gastrectomized and normal rats were force-fed equal amounts of HS or RS diets. On autopsy, the intestines of the gastrectomized rats fed HS diet appeared normal; in contrast, the intestines of those fed RS diet were markedly distended. Analysis of the contents of the upper halves of the small intestine revealed twice as much nitrogen in the contents of the gastrectomized rats as in those of normal rats fed HS diet; when RS diet was fed, the difference was sixfold. These differences disappeared in the contents of the lower halves of the small intestines; the nitrogen in the contents of gastrectomized rats was twice that of control rats fed HS or RS diet. The action of the irritant of RS diet had dissipated (S. Lepkovsky, M. K. Dimick, F. Furuta, and R. Park, unpublished). D. INTESTINAL MOTILITY

The ingestion of a nutritionally adequate diet does not ensure good nutrition until the essential nutrients of the food are delivered to the cells, where they are utilized. The food must pass through the alimentary canal where the essential nutrients are released by the digestive processes, absorbed from the small intentines, and transferred by the blood to the cellular mass. Indispensible in this process is the normal propulsion of food through the intestinal lumen by peristalsis, contraction, and relaxation, constituting a wave which sweeps the intestinal contents before it. The overall process of intestinal peristalsis has been clearly stated by Bayliss and Starling (1899) and is referred to as the “law of the intestine” and as a “stimulus applied to a given point in the intestinal

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wall which‘initiates a band of constriction on the proximal side and relaxation on the distal side of the stimulated point” leading to the peristaltic wave in the bowel (Best and Taylor, 1940). The machinery that propels food through the intestinal canal is very complicated. Unlike the case with the adipose tissues, which have been neglected for such a long time, the “study of gastrointestinal motility has had a long and distinguished history, and many of the basic features have not yet been elucidated (Bortoff, 1972). “On no subject in physiology do we meet with so many discrepancies in fact and opinion as in that of the physiology of the intestinal movements” (Bayliss and Starling, 1899). Additionally, “the field of gastric and intestinal motility is very confusing and most papers contribute little to its elucidation” ( Obrink, 1958) . The intestine is innervated with extrinsic and intrinsic cholinergic and adrenergic nerves. However, “rhythmic contraction for any segment of the small intestine of the dog is constant and is unaffected by extrinsic nerve section” (Douglass, 1960). In general, acetylcholine causes contraction, and adrenaline and noradrenaline cause relaxation. The situation is complicated by the action of 5-OH-tryptamine ( 5-HT) . “5-HT stimulates peristalsis, but the gastrointestinal function of 5-HT has long been an enigma” (Daniel, 1968, 1969) . Prostraglandins released from nervous tissue, muscle, adrenal and adipose tissue cause intestinal contraction, but their action remains to be elucidated (Daniel, 1969). Insulin is a good stimulant for intestinal motility and propulsion (Lish and Peters, 1957), and glucagon inhibits motility of the stomach and colon ( Sporn and Necheles, 1956).

1. Pancreas and Gastrointestinal Motility

Pancreatectomy in chickens caused dilation of the gastrointestinal tract (Lepkovsky and Dimick, 1969). Although pancreatectomy did not disturb the extrinsic and intrinsic innervation of the gastorintestinal tract, pancreatectomy nevertheless caused a deficit of acetylcholine in the gastrointestinal tract, and contraction of the bowel decreased, causing dilation. The deficit of acetylcholine was shown by the injection of the cholinesterase inhibitor, eserine, into the wing vein; the eserine prevented the inactivation of the acetylcholine produced in the intestine and caused prompt contraction of the intestine (Lepkovsky and Dimick,

REGULATION OF FOOD INTAKE

23

1969). While it appeared that pancreatectomy was “total,” later work showed that this was not achieved. Gutkin (1963) showed that the pancreas possesses a cholinergic component by demonstrating that pancreatectomy in frogs decreased the rate of release of the acetylcholine from the nerve endings. It is postulated that the dilation of the intestine of depancreatized chickens is caused by a deficit of cholinergic activity in the intestine. The decrease in cholinergic activity in the intestine may be due to the decrease in the release of acetylcholine ( Gutkin, 1963) ; alternatively, pancreatectomy deprives the chicken of insulin which elicits cholinergic activity (Soulairac, 1963). Both mechanisms may operate in the motor activity of the intestine. 2. “Total” Pancreatectomy, Diabetes in Chickens, and

Gastrointestinal Function Preliminary results with chickens show that diabetes produced by “total” pancreatectomy is accompanied by collapse of gastrointestinal function (S. Lepkovsky et al., unpublished). The chickens do not eat. Food forced into the crop through a tube does not leave the crop. Injections of insulin promptly restores normal gastrointestinal function and food normally moves through the gastrointestinal tract. The nature of the disturbance in the gut has not been established.

3. Hypothalamus, Pituitary, and Intestinal Motility Contractions of portions of the intestines have been observed in some chickens with lesions in the hypothalamus. Such chickens refused to eat and some of them were force-fed, but they died of inanition within 7 or 8 eight days, even though their crops were greatly distended with food. Autopsy showed a marked constriction of a section of the intestine ( Lepkovsky and Dimick, 1969). Contractions of segments of the intestine were also observed in hypophysectomized chickens. When the fowl refused food they were force-fed, but little of the food passed out of the crop. At autopsy the crops were distended with food, and segments of the small intestine were contracted. Increase in pressure forced the water through a segment of the intestine, showing that the constriction was functional

SAMUEL LEPKOVSKY

24

rather than a mechanical block. Injection of hydrocortisone frequently corrected this condition. When it did, eating was resumed and normal feces were passed. The use of hydrocortisone reduced the mortality of hypophysectomized chickens. Fowler and Cleghorn (1942) also reported marked constriction of the intestinal tracts of cats dying in adrenal insufficiency and showed that the constricted intestine of animals dying in adrenal insufficiency was not caused by inadequate food intake but, rather, was due to deficit of adrenocortical hormones. Wurtman and Axelrod (1965) showed that the biosynthesis of adrenalin from nonadrenalin is stimulated by the adrenal glucocorticoids, and it is suggested tentatively that the hypothalam-pituitary-adrenocortical axis plays a role in intestinal function by increasing the available supply of adrenalin which appears to inhibit gastrointestinal motility more effectively than does norepinephrine (Kuntz, 1951; Kock, 1959).

E. PROCESSED FOODS AND

THZ

GASTROINTESTINAL TRACT

Crude fiber in wheat bran (Borgstrom, 1941 ) and in breakfast cereals (Murlin et al., 1938) increases bulk and nitrogen in stools of human beings. Heat treatment further increases bulk and nitrogen in stools ( Borgstrom, 1941 ). The data of these investigators indicate that the nitrogen of feces is largely of metabolic origin. This is shown for breakfast cereals in the table adapted from Murlin et al. (1938). Flavors or desirable sensory stimuli in breakfast cereals may also contribute bulk and nitrogen to stools, since sensory stimuli in food increase secretions into the intestinal tract. It is postulated that crude fiber, roasting or toasting and desirable food flavors act in concert in stimulating the secretion of endogenous nitrogen into the gastrointestinal tract, and much of it is not reabsorbed and becomes part of the stools. ~

~~~~~~~~~~~~~~

StooIs Weight (gm) Nitrogen (gm) Nitrogen (%)

~~

Toasted “whole” wheat 772

Inflated wheat 682

Precooked Toasted oats endospem 562

340

Egg

315

10.08

9.26

6.72

4.46

4.28

1.30

1.35

1.19

1.31

1.35

REGULATION OF FOOD INTAKE

V.

25

ROLE OF ADIPOSE TISSUES IN REGULATION OF FOOD INTAKE

The adipose tissues of animals have been regarded as inert. Although physiologists have known that the adipose tissue cells consist of specialized connective tissues and possess cytoplasm and nuclei, and therefore are metabolically active, nevertheless the adipose tissue mass was considered relatively inert. Wells (1940) called attention to this situation in a paper entitled “Adipose Tissue, A Neglected Subject,” The adipose tissues have not been completely neglected, however. Rony ( 1940) noted that “obesity ordinarily develops spontaneously; some intrinsic abnormality seems to induce the body to establish positive caloric balance leading to fat accumulation.” He called the weightgaining stage “dynamic,” and the period when weight gain and food intake stabilized was referred to as “static.” Even before this work, clinicians were concerned with the problem that obesity may be a precursor of diabetes ( Adams, 1929). This situation was largely corrected in 1948 by the review on the adipose tissue by Wertheimer and Shapiro ( 1948). Schoenheimer and his colleagues ( Schoenheimer, 1942; Schoenheimer and Rittenberg, 1935 ) showed that the adipose tissues are active metabolically: “The fatty acids of the depot fat are to be regarded as being constantly transported, in the form of fats or phosphatides to and from the organs, where fatty acids are temporarily liberated by rupture of ester linkages. When fat is absorbed, the acids of dietary origin merge with those from the depot, thereby forming a mixture indistinguishable as to origin. Parts of the liberated acids are converted to others, while new ones are steadily formed by condensation of small molecules derived from other substances. Some of this pool of acids is degraded, and some of it re-enters ester linkages to regenerate fat, which is transported back to the depots. All these complex reactions are so balanced that the total amount and structure of the fat mixture in depot, blood, and organs remain constant.”

A.

A NEWNUTRITIONAL CONCEPT: THESETPOINTIN THE ADIPOSE TISSUES

The body weight of adult animals is maintained over long periods of time under varying conditions of food intake, activity, and different climatic environments. This led Stettin (1957) to consider it “one of the more baffling problems in biochemistry, namely, why does the composition of the adult organism tend to remain constant?” The chief variable

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SAMUEL LEPKOVSKY

in the body of the adult organism is the level of fat in the depots. This level of fat in the depots is maintained and is referred to as the set point. Nisbett (1972) reviewed the literature dealing with the set point in the adipose tissues and concluded: “Recent evidence suggests that the adipose tissue mass is defended by the central nervous system. The classic hypothalamic feeding centers appear to adjust food intake so as to maintain fat stores at the base line ‘set point’ level.” A test was made of the set-point hypothesis. Obesity was induced in White Leghorn cockerels without hypothalamic lesions and presumably without change in the set point in the control system in the adipose tissues. After the cockerels were force-fed approximately twice their ad libitum food intake, they soon ceased to eat (Lepkovsky and Furuta, 1971). The food introduced into the crops was well digested and efficiently utilized, and food calories ingested in excess of current need were deposited as fat in the depots. With the cessation of forcefeeding, the birds did not eat for 7 to 10 days. They lived on endogenous fat calories which were released from the adipose tissues by accelerated lipolysis and lost weight rapidly. When the fat in the adipose tissues decreased toward normal, the birds began to eat, and food intake reached normal when the fat in the adipose tissues decreased to the level required by the set point. It appears that the hypothalamus does not differentiate between carbohydrate and fat calories ( Lepkovsky and Furuta, 1971) . A similar experiment was carried out with human beings. Medical students agreed to consume 5000 calories daily. Great care was taken to maintain the balance of carbohydrate, fat, and protein; otherwise no change was made in their daily life. The students readily consumed the extra calories for about a week. At the end of this period, they were so nauseated by the extra food that they were unabIe to continue ( A . E. Kellie, in Brobeck, 1960). These results parallel those reported for rats. Rats made obese without change in set point by overfeeding (Cohn and Joseph, 1962), by electrical stimulation of the lateral hypothalamus ( Steinbaum and Miller, 1965), or by injection of insulin ( MacKay et al., 1940) ate sparingly after cessation of treatment until the weights of the rats returned to normal, and normal food intake was resumed. It may be concluded from these experiments that the adipose tissues play an important role in the regulation of food intake. The level of fat in the adipose tissue mass of each adult animal is maintained at the level set by the set point. When the amount of fat in the depots is reduced below the set-point level, as in fasting, restoration of ad libitum feeding

REGULATION OF FOOD INTAKE

27

accelerates lipogenesis and elicits hyperphagia until the body weight and body fat increase to prefasting level, and food intake returns to normal. Conversely, when body weight and the amount of fat in the depots rise without a change in the set point, as in overfeeding, cessation of overfeeding results in hypophagia or aphagia. Lipolysis is accelerated, and endogenous fuel is supplied to the lean body mass; concomitantly, there is a compensatory decrease in food intake (hypophagia). When body weight and body fat decrease to the set-point level, food intake returns to normal.

B. LIPOLYSIS AND LIPOGENESIS : RECIPROCALINHIBITION Sherrington (1947) used the term “reciprocal inhibition” for biological processes that are effected by two opposing processes. For example, reciprocal inhibition makes breathing possible. During inhalation, exhalation is inhibited, and conversely, during exhalation, inhalation is inhibited. Many nutritional processes are made possible by reciprocal inhibition. During eating, anabolic processes are activated, and concomitantly catabolic processes are inhibited. Conversely, during fasting, catabolic processes are activated, while concomitantly anabolic processes are inhibited. This is especially true for the adipose tissues.

Hormone-Sensitive Lipase versus Lipoprotein Lipase The level of fat in the adipose tissue is maintained at its set point by two opposing enzymes-namely, hormone-sensitive lipase and lipoprotein lipase. During fasting, hormone-sensitive lipase is activated and lipolysis is accelerated. Concomitantly, lipoprotein lipase is inhibited, and lipogenesis and increase in fat in the depots are inhibited. During eating, the reverse occurs. Lipoprotein lipase is activated, and lipogenesis and the amount of fat in the depots are enhanced; concomitantly, hormone-sensitive lipase is inhibited and lipolysis falls. Hormone-sensitive lipase is activated by cyclic AMP in fat cells. Cyclic AMP is synthesized by the enzyme adenyl cyclase from ATP. Adenyl cyclase is activated by the hormones of the pituitary and those of its target glands-catecholamines, glucagon, secretin, vasopressin, and others. Conversely, cyclic AMP in fat cells is decreased by insulin, which depresses the activity of adenyl cyclase. Cyclic AMP is a ubiquitous nucleotide, termed “second messenger,” and plays a very important role

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in nutritional processes in general and more especially in the adipose tissues (Butcher et al., 1969; Butcher and Baird, 1968; Sutherland, 1970; Rizack, 1964; Illiano and Cuatrecasas, 1972; Fain, 1968; Hollenberg et al., 1970; Rodbell, 1970). The level of cyclic AMP in the fat cells is also decreased by the prostaglandins (PGE, ), which depress the activity of adenyl cyclase and/ or activate phosphodiesterase, which inactivates cyclic AMP (Butcher and Baird, 1968; Bergstrom, 1967; Christ and Nugteren, 1970; Westermann and Stock, 1969). Fasting or eating evokes cascades of reactions, beginning with lipolysis and lipogenesis, respectively, in the adipose tissues. During fasting, fat becomes the fuel of the lean body mass. Utilization of carbohydrates by the muscles is inhibited by the decrease in oxidation of glucose and by the inhibition of entry of glucose into the muscle cells (Grodsky, 1969). Utilization of glucose is depressed in liver by the decrease in oxidation of glucose, and the synthesis of glycogen is depressed. Fats are utilized as fuel and in addition are converted to ketone bodies which are used as fuel by the muscles and brain. Gluconeogenesis and glycogenolysis are enhanced to supply glucose for the brain. After eating, these reactions are reversed. Glucose enters the muscle cells and is used for fuel and for synthesis of glycogen. Fat utilization is decreased. In the liver, utilization of glucose for fuel is enhanced, glycogen synthesis proceeds, and gluconeogenesis and glycogenolysis are inhibited. Fats are no longer utilized for the formation of ketone bodies.

IN SET POINTIN ADIPOSE TISS~JE: THE HYPOTHALAMUS C. CHANGE

Kennedy (1953) has suggested that the primary function of the ventromedial nucleus of the hypothalamus is to stabilize fat stores. Mu et al. (1968) elaborated this concept as foIIows: “The hypothalamus adjusts food intake so as to regulate the size of the fat depots. After ventromedial lesions the food intake is increased because the control system is given a new set point that requires a greater amount of fat in the body.” According to this hypothesis, the hypothalamus acts peripherally and gives the control system of the adipose tissues a new set point which requires more fat in the depots. This causes a mandatory diversion of food calories to the fat depots, and concomitantly, there is a compensatory increase in food intake, causing hyperphagia. Lean White Leghorn cockerels were given electrolytic lesions in the ventromedial hypothalamus, and their adipose tissues were given a new set point requiring more fat. Hyperphagia and obesity proceeded in a reproducible and characteristic manner; obesity increased progressively,

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while, concomitantly, food intake progressively decreased until body weight and the level of body fat plateaued and food intake returned to normal (Snapir et al., 1969). Of unusual interest are the results reported by Hoebel and Teitelbaum (1966) with rats with lesions in the ventromedial nucleus of the hypothalamus after they had stabilized their obese weight required by the new set point in their adipose tissues. When these rats were force-fed excessive amounts of food, they gained body weight, and voluntary food intake decreased. On cessation of force-feeding, subnormal amounts of food were eaten; but when their weights decreased to that required by the new set point, food intake returned to normal. These findings suggest that the hypothalamus regulates the amount of fat in the adipose tissues, not by regulating food intake centrally, but by acting peripherally through its control of the set points in the adipose tissues. The work of Hoebel and Teitelbaum (1966) supports this hypothesis with another experiment. Before lesions were introduced into the ventromedial nucleus, the rats were made obese by the injection of protamine zinc insulin. After the placement of the lesions, the rats gained little weight because the rats were already obese; the amount of fat in the adipose tissues was already adjusted to the new set point, and little change in food intake was needed to produce the obese weight required by the new set point. Hence, hypothalamic lesions do not cause hyperphagia under these conditions. The work of Liebelt and his co-workers (1965) with mice shows that the adipose tissues play an unsuspectedly important role in the regulation of food intake: The “adipose tissue mass exerts regulatory influences on neural pathways involved in appetite regulation, primarily in the hypothalamus. Further, the total adipose tissue mass is quantitatively determined in part by the intrinsic level of activity of the appetite regulatory ‘centers’ in the nervous system and in part by the intrinsic levels of activity of the fat cells.” These conclusions were based on the following experimental evidence: (1) There is a quantitative relationship between the degree of damage to the ventromedial region of the hypothalamus caused by injection of goldthioglucose, and increases in food intake and body weight. After the weight of the goldthioglucose-injected mice plateaued, food intake returned to normal. The body lipid content (weight) of the obese mice was reduced by starvation. When the animals were again given free access to food, there was a transient increase in food intake; food intake returned to normal when the prestarvation weight was restored. 2. When the same amount of goldthioglucose was injected, causing the same amount of damage to the ventromedial region of the hypo-

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thalamus of obese (yellow) and lean (brown) mice, there was a greater increase in weight, food intake, and deposition of fat in the lean than in the obese mice. This indicates that the shift in set point in the adipose tissues in response to the same brain damage was similar, irrespective of the amount of fat in the adipose tissues at the time of the administration of the goldthioglucose. Consequently, the increased body fat content of yellow obese mice necessitated the consumption of less food to reach the level of fat in the adipose tissues required by the new set point given to the adipose tissues. 3. The total body fat is regulated; removal of one fat organ (gonadal fat organ) caused compensatory hypertrophy of other fat organs (inguinal fat organ, as an example), keeping the adipose tissue mass unchanged.

1 . Hypothalamus and Set Point in Control System of Adipose Tissues The hypothalamus appears to use many mechanisms in regulating set points and thereby the size of the fat depots. The data of Snapir et al. (1969) suggest one mechanism which involves the use of testosterone. The hypothalamus controls the production of testosterone through its gonadotrophic-releasing factors ( McCann, 1971; Flerk6, 1963) which are needed for the normal function of the pituitary-testis system. Lesions in the hypothalamic area dorsal to the stalk caused obesity and functional castration (Snapir et al., 1969); the lesions reduced blood testosterone. Combs and testes atrophied, and the roosters became obese. 2. Reversal of Change in Set Point and Decrease in Body Fat

If elimination of testosterone by the hypothalamic lesions was responsible for the new set point requiring more fat in the depots, restitution of testosterone should reverse the change in set point and decrease body fat. Daily injections of testosterone intramuscularly ( 2 mg/kg) evoked prompt decreases in food intake; although there was a marked decrease in body fat, the cockerels demained obese, albeit at a lower level. These findings suggest that the hypothalamic lesions evoked multiple changes involving the new set point that was given to the adipose tissues, and hence testosterone-injected cockerels remained obese. Surgical castration eliminated testicular testosterone and gave the adipose tissues a new set point requiring more body fat. The cockerels gained weight and body fat. Injections of testosterone reversed the change in set point in the adipose tissues. Body weight and body fat decreased

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and the lean body condition of the birds was restored (Snapir et al., 1974). The autonomic nervous system appears to influence the setting of the set point in the adipose tissues. Destruction of the noradrenergic bundle nearby the ventromedial nucleus of the hypothalamus elicits hyperphagia and obesity (Gold, 1973). In contrast, subdiaphragmatic vagotomy completely reverses the obesity caused by electrolytic lesions in the ventromedial hypothalamus ( Powley and Opsahl, 1974).

D. DIETAND SETPOINTIN ADIPOSETISSUES Rats fed a low-protein diet increase their body fat, but do not get obese (Meyer and Hargus, 1959). The rats eat less and grow more slowly than do rats fed a normal diet. Presumably, the set point in the adipose tissue is changed little by the low-protein diet. In contrast, dogs (beagle) fed a wheat gluten diet (25%wheat gluten) -which is the equivalent of a low-protein diet, since it is deficient in lysine-nevertheless ate and grew as well as did those fed an egg protein (25%) diet. The dogs fed the wheat gluten diet became obese, presumably owing to changes in set point in the adipose tissues which required more fat. The dogs were inactive and did not respond to stimuli as did lean dogs fed egg protein, which were very active and barked energetically when exposed to stimuli such as visitors. When the dogs fed the wheat gluten were changed to the egg protein diet, food intake and weight decreased. Body fat decreased and body protein increased, and the dogs acted normally (Allison, 1949). The set point in the adipose tissues may be shifted by other changes in the composition of diets, such as a high-fat diet in the rat (Mu et al., 1968). AND SETPOINT E. THEPITUITARY

Hypophysectomy without brain damage elicits marked obesity in the chicken (Nalbandov and Card, 1943), but, in contrast, it does not cause obesity in the rat and dog (Samuels, 1947). It may be anticipated that gastrectomy and adrenalectomy may also change set points in some animals, but not in others. It appears that the control of the set point in the adipose tissue is very complicated and little understood at this time. RHYTHM IN THE ADIPOSETISSUES AND F. CIRCADIAN OF FOOD INTAKE IN THE RAT

THE

REGULATION

Although the daily food intake and body weight of the adult rat changes very little, the rat consumes most of its food during the dark

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period when it is most active and eats little during the light period when it is inactive or asleep. Le Magnen and Devos (1970) showed that during the dark period active lipogenesis goes on, and, in contrast, there is lipolysis during the light period. It is suggested that during the dark period the adipose tissues of the rat receive a set point that requires more fat, causing diversion of food calories to the adipose tissues with concomitant increase in food intake. During the light period the adipose tissues receive a set point that requires less fat in the depots, evoking accelerated lipolysis. Endogenous fuel is used by the lean body mass, causing concomitantly a compensatory decrease in food intake. This hypothesis was supported by the use of propanolol, the adrenergic blocking agent ( Le Magnen and Devos, 1970). Administration of propanolol inhibited lipolysis during the light period and decreased the supply of endogenous fat calories. Concomitantly, there was a compensatory increase in food intake. Like propanolol, insulin also inhibited lipolysis and increased food intake during the light period.

G. THESETPOINTIN ADIPOSETISSUES OF MIGRATORY BIRDS Migratory birds, in response to some factor in the season of the year, become hyperphagic before flight and accumulate enough body fat to supply the energy needed for the flight (Odum, 1960; King and Farner, 1933). In inducing hyperphagia, the hypothalamus could act centrally, increasing the appetite, and the calories consumed in excess of current need would be converted to fat and deposited in the depots. Or the hypothalamus could act peripherally, giving the adipose tissues a new set point that requires more fat in the depots, thus inducing hyperphagia. In deciding which of these two mechanisms is operant, it is essential to keep in mind that hyperphagia or obesity is not possible without a change in the set point in the adipose tissues, since the level of body fat or set point is defended against change. It is therefore postulated that preflight hyperphagia is induced by a change in set point.

H. THESET POINTIN

THE ADIPOSETISSUES OF

HIBERNATING ANIMALS

Hibernating animals also become hyperphagic and obese before they go into hibernation. It is postulated that before hibernation the adipose tissues receive a new set point which requires more body fat and induces hyperphagia. Golden hamsters hibernate but do not become obese before hibernation. It is postulated that there is little or no change in the set point of the adipose tissues of hamsters before hibernation; hence, they cannot

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store enough fat in their depots to supply their energy needs during hibernation. Instead, they hoard food which takes the place of body fat ( Lyman, 1954).

I. REGULATION OF FOOD INTAKE CENTRALLY: THEHYPOTHALAMUS Observations with White Leghorn cockerels suggest that the hypothalamus not only acts peripherally, but may also act centrally in the regulation of food intake. Unlike normal cockerels whose crops are empty or almost empty most of the time, the crops of some of the lesioned birds were always full and at times were distended as though the amount of food eaten was greater than current needs. IN THE LIVER AND/OR ADIPOSETISSUES J. LIPOGENESIS

Lipogenesis in the bird goes on largely in the liver, as observed by Goodridge and Ball (1967) in pigeon and by Leveille et al. (1968) in chicken. This is supported anatomically by data obtained with the White Leghorn cockerel; the liver hypertrophied (five times normal) in response to the increase in the work load imposed on the liver by the force-feeding of twice the normal food intake (Lepkovsky and Furuta, 1971). In contrast, in the pig when glucose is the substrate, virtually all the newly synthesized fatty acids are formed in the adipose tissues, whereas there is appreciable lipogenesis in the liver when acetate is the substrate (OHea and Leveille, 1969). Lipogenesis in the mouse (Favarger, 1965; Jansen et al., 1966) and rat (Jansen et al., 1966; Leveille, 1967) proceeds in both the liver and adipose tissues. FROM THE ADIPOSE TISSUES TO THE FOOD-REGULATING K . FEEDBACK CENTERS OF THE HYPOTHALAMUS

Feedback from the adipose tissues to the nervous system has been postulated by Magoun ( 1963) : “A negative feedback appears operant in alimentary behavior, by which peripheral stores relay information concerning their inventory to hypothalamic regulatory mechanisms.” The following hypothesis is suggested for such a feedback system when the amount of fat in the adipose tissues is less than that required by the set point in the control system and more fat is required in the depots. ( 1) Lipoprotein lipase, which hydrolyzes triglycerides, is activated by insulin and glucose (Hollenberg et al., 1970), thus facilitating the entry of fat into the fat cell. ( 2 ) Lipogenesis is enhanced by insulin, and the newly formed fat (triglycerides) in the liver is transported by the blood

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to the adipose tissues and deposited there. ( 3 ) Energy absorbed from the small intestine is diverted from the lean body mass to the fat depots, resulting in an increase in the rate of absorption of food from the small intestine (S. Lepkovsky et al., unpublished) to replace the diverted energy. This hypothesis is supported by the work of Soulairac ( 1947), who showed that an increase in appetite raises the rate of absorption of glucose from the small intestine. ( 4 ) An increase in the rate of absorption of food from the small intestines accelerates gastric emptying and leads to an empty stomach and to an increase in food intake. Conversely, when the amount of fat in the adipose tissues is in excess of that required by the set point, lipolysis is accelerated. Lipoprotein lipase is inhibited (Hollenberg et al., 1970) and lipogenesis is inhibited, concomitantly, the activity of hormone-sensitive lipase is stimulated. Lipolysis increases the amount of endogenous energy (fatty acids) for the lean body mass; this increase is accompanied by a compensatory decrease .in the absorption of food from the small intestine. Evacuation of food from the stomach falls off, food accumulates in the stomach, and food intake decreases ( Lepkovsky and Furuta, 1971 ).

VI.

ALIMENTARY BEHAVIOR

A. FOOD-SEEKING BEHAVIOR: MOTIVATION Since there are no restaurants in nature, the animal must find its food. “Drive” sets the animal to searching, which requires locomotion, exploration, and examination. Food may require chasing, since food is often on the hoof. The search may also require fighting and fleeing so that the searcher does not become food for other animals.

B. CHEMICAL SENSESAND RECOGNITION OF FOOD All animals must possess chemical senses in order to recognize food and to be able to select a nutritionally adequate diet from the available food supply. The amoeba possess chemical knowledge; without chemical senses, the organism would be unable to distinguish between a grain of sand and an alga or some other digestible matter. Little is known of the taste mechanism in the amoeba. More is known about the food-sensing mechanism in the coelenterate, Hydra littoralis, which lives on the water flea, Daphniu. Hydra will not eat dead water fleas; they will eat wounded fleas after puncturing the flea with their nematocysts. The juice that oozes out contains reduced glutathione which acts as a “marker” or “feeding hormone.” This is

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“recognized by Hydra, and the wounded flea becomes acceptable as food (Loomis, 1955). Another example is the sea anemone, Anthopleura ebgantissima, which employs two “markers” or “feeding hormones.” Asparagine induces bending of the tentacles which bring its prey to the mouth, but it is not eaten until reduced glutathione reacts with the mouth when prey is swallowed (Lindstedt, 1971). It is more complicated in higher forms.

C. CHOICE OF A NUTRITIONALLY ADEQUATEDIET Feeding is detemiined not only by the chemical properties of food, but also by need. Rats are indifferent to flavors such as diacetyl, monosodium glutamate, anise, or butyric acid; they will respond to these compounds only if the stimuli are meaningful. For example, rats deficient in pantothenic acid do not choose diets containing pantothenic acid in preference to diets that do not contain the vitamin. But if the diet containing pantothenic acid is flavored with anise, rats deficient in pantothenic acid will prefer the diet containing the pantothenic acid. The sense of well-being that follows the ingestion of the pantothenic acidcontaining diet is associated with the anise which serves as the “marker,” since rats do not seem to be able to “recognize” pantothenic acid as such (Scott and Quint, 1946). Similarly, when normal rats were offered two diets, one containing thiamine and the other deficient in the vitamin, the rats ate these diets at random. When the rats became deficient in thiamine, they ate only the diet containing thiamine. After cure of their deficiency, the rats again ate these diets at random (Harris et al., 1933). The flavor of thiamine had meaning to the rat only when the rat was deficient in thiamine. Similar results were obtained with riboflavin and pyridoxine (Scott and Quint, 1946). Thus, in rats, flavor is not the sole determinant of acceptability. Bodily needs also play a role in the choice of food.

D. TASTEAND BODYBIOCHEMISTRY The chemical compounds suspected of endowing food with sensory stimuli can be separated by gas chromatography, column chromatography, paper chromatography, electrophoresis, and other classical chemical and physical procedures. The chemical structure and physical properties of the pure compounds can be determined. These, however, are not sensory stimuli until they are evaluated by the brain. Feeding is determined by the sensory properties of food. When nutrients excite sensory cells in the mouth, the nerve fibers with which

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they are associated forward the excitations to the brain as electrical impulses and elicit sensations that are expressed as taste. “The sensory world of every being is sealed to him. Nobody can reveal his sensations to others” (von Buddenbrock, 1958). The sensory properties of nutrients may be pleasant, unpleasant, or neutral. Taste is determined not only by the chemical properties of food but also by body biochemistry. Human subjects were asked to choose between 5% and 30% sucrose for a long drink. They chose 5% sucrose; 30% sucrose was sickeningly sweet. After insulin injection, the subjects’ blood sugar levels decreased to 40 to 50 mg%; they then chose 30% sucrose because it no longer tasted sickeningly sweet. The taste of sucrose was altered by the action of insulin (Mayer-Gross and Walker, 1946). Normal fasting human subjects found the taste of sucrose solutions pleasant at all concentrations. After ingestion of 200 ml of 2.5% glucose, the sweet sensation became unpleasant ( Cabanac, 1971). After a reduction of body weight by lo%, the intragastric load of glucose no longer rendered the sweet sensation unpleasant. Return of body weight to previous weight restored the subjects’ former responses to sweet stimuli; an intragastric load of glucose again rendered the sweet sensation unpleasant. Fasting obese subjects responded as do normal subjects to the sweet stimulus. In contrast to normal subjects, after an intragastric glucose load, the sweet sensation of sucrose remained pleasant (Cabanac and Duclaux, 1970). The responses of the underweight subjects paralleled those of the obese subjects (Cabanac, 1971.

Flavors and Digestion of Food There is a large factor of safety in the secretion of the digestive enzymes in the gastrointestinal tract, as shown by the work of Smith et al. (1920), who studied the effect of palatability of food and the digestive process. Food served to human subjects was rendered unpalatable by the mixing together of meat, biscuits, jelly, and other meal components. The plate on which the food was served was smeared with animal charcoal, and the table was dirty and strewn with dirty dishes. A little indole was sprinkled about under the table. The food was so unpalatable that one subject vomited shortly after eating. Yet there was little difference in digestibility as measured by nitrogen retention of the palatable and unpalatable foods. Smith et al. (1920) stated: “This short test indicates that flavor is not the outstanding dietetic asset that some people would have us believe.”

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Lacking further investigation, this conclusion should be accepted cautiously. However, it does emphasize the large safety factor in the production of digestive enzymes.

E. FEEDING PATHWAYS Specific bodily nutritional needs are communicated to the brain, where they are appreciated and where they lead to motivated behavior appropriate for the selection of the specific nutrients needed. When a stimulus, evoked by taste or smell, excites a sensory cell, the nerve fiber with which it is associated forwards the excitation as electrical impulses to the brain. The nerve does not transmit the stimulus in its original form. The nerve is an indifferent conductor, like a telephone wire. For example, the sensation “ r e d has nothing to do with light or a wavelength of about 800 millimicrons; the odor sensation of camphor has nothing to do with its chemical composition (von Buddenbrock, 1958). The taste of food is communicated to the brain over cranial nerves 5, 7, 9, and 10. Olfaction or smell of food is communicated over the first cranial nerve. The number of neurons conveying olfactory signals to the brain of the rabbit is about 100 million (Ottoson, 1963). The reticular activating system monitors the sensory information about the taste, smell, and other sensory properties of the food by its sensoryfiltering function and limits the amount of input at any one time; only sensory information pertinent to bodily needs reaches the brain. The food that meets bodily needs is accepted (French, 1960).

I . Electroencephalograms and the Evaluation of Flavors The sensory stimuli acting upon the receptors in the mouth are transmitted over the nerve tracts as electrical impulses to the brain, where they are appreciated as sensations, but no information is available about the nature of the sensations resulting from food flavors (Rosenblith, 1961). Electroencephalograms ( EEG) are providing results on alimentary behavior. Recordings from the hypothalamus and frontal lobes of hungry cats show marked desynchronization, characterized by low-voltage highfrequency potentials. Intravenous injection of glucose, or tube-feeding directly into the stomach, converts the EEG from desynchronization to synchronization, characterized by spindlelike clusters of regular high-

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voltage slow waves, suggesting satiety ( Anokhin, 1961). Th’is is even more striking if injections and insertion of food into the stomach are given together. Hungry cats were trained to press a bar to receive 1/4 milliliter of milkbroth. The EEG showed low-voltage high-frequency potentials suggesting hunger. When the cats drank the milk-broth, slow-wave synchronous activity in the EEG replaced the hungry EEG and suggested satiety. A water reward did not change the EEG. After each ration of the milk-broth was consumed, the EEG reverted from the slow-wave synchronous activity to the low-voltage fast activity, suggesting hunger. The sequence of the EEG change proceeded cyclically, with hunger following satiety recurrently, until about 40 milliliters of milk-broth were consumed. Then slow-wave synchronous activity persisted, suggesting lasting satiety EEG patterns (Clemente et al., 1964). Satiety was achieved after sufficient milk was absorbed and metabolized. 2. Direct Pathway to the Brain

It is generally accepted that sensory information is communicated from the mouth to the brain over nerve tracts. Recent work (Kare et al., 1969) suggests that there is a direct pathway from the mouth to the brain. Introduction of isotopically labeled glucose into the esophagus -ligated oropharyngeal cavity of the rat produced a greater concentration of radioactivity in the brain than did gastric or intestinal administration. The anatomical pathway from the oropharynx to the intracranial cavity remains to be elucidated. F. SENSATIONS AND AVERSION TO FOOD AND DRINK

In general, foods that are beneficial are preferred, and harmful foods are avoided. Association of illness with foods or liquids leads to aversion to these products. Illness in rats following the ingestion of food or drink containing a poison leads to bait-shyness (Garcia et al., 1972; Kalat and Rozin, 1970) or aversion to the vehicle used for the poison (Rzoska, 1953). Similarly, rats rendered ill by x-irradiation shortly after injection of sodium saccharin avoid saccharin, since intravenously injected saccharin is tasted and is associated with the taste of oral saccharin (Bradley and Mistretta, 1971). An overindulgence may lead to aversions. As an example, von Buddenbrock (1958) relates: “In my student days, I hap-

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pened to dip my cup too freely in a bowl of delightful strawberry punch, and for the next five or six years I felt the most violent loathing for that particular fruit.” Another case of an aversion comes from Booth Tarkington: Aged 10, returning famished from skating, I found in the pantry four full-sized pumpkin pies unattended. Morally undermined, I ate all of the first pie. Fairly excited, I ate the second pie. Happy, I could not give up remaining so, and began the third. Enthusiasm departed; but out of a sense of duty to myself, I ate all of the third pie and a section of the fourth before a creeping illness seized me. Aged 35, I again tried to eat pumpkin pie. Failed.

Aversion after overindulgence of nontoxic food and drink are not necessarily the usual responses. Too little information is available on this subject at this time.

To DECEIVEOR CONFUSE THE CEREBRAL CORTEX IN G . Is IT POSSIBLE RELATION TO APPETITE FOR FOOD? The brain of the rat may be deceived. Harris et al. (1933) provides such an example. Thiamine-deficient rats chose diets containing thiamine, because thiamine has a sensory stimulus and was associated with the diet. If the diet contained thiamine and Bovril (meat extract), the thiamine-deficient rats associated benefits derived from thiamine with Bovril. When Bovril was transferred to a diet that lacked thiamine, such conditioned thiamine-deficient rats ate the Bovril-containing diet and died of thiamine deficiency. An unusual contradiction in the ability of deficient animals to choose the needed nutrient involves magnesium. Magnesium-deficient rats avoided diets containing magnesium, and many of them died of magnesium deficiency. Scott (1948) gives a reason for this aversion: “Magnesium is a narcotic and magnesium-deficient animals are highly nervous and high excitable. The apparent explanation of these results was that the high excitable state characteristic of magnesium deficiency was pleasurable to the rat-he actually felt intoxicated. Consequently, he avoided the diet which contained magnesium, and which detracted from his pleasure in that intoxication.”

STIMULIAND CHOICEOF FOOD BY MAN H. SENSORY When animals, such as the rat, are given choices of foods, they will frequently select foods that meet their physiological needs-they select

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a nutritionally adequate diet (Richter, 1942). In contrast, human beings are not so discriminating in their choice of foods and frequently eat diets that are nutritionally inadequate. Basic hypothalamic mechanisms are obscured by habits, customs, prejudices, social pressures, and sales promotion, which act primarily through the neocortex. Kennedy (1960) summarized the situation thus: The hypothalamus does it for lower animals, but the higher centers interfere in human beings. As an example, most housewives in England know that whole wheat bread is better for them than white bread, but they buy white bread (Brown et al., 1963). Similarly, human beings in the orient subsisting on white rice develop beriberi, even though brown rice, which prevents beriberi, is available. Here social status is involved. Relgion may interfere with the choice of an adequate diet. Some Russian priests in Siberia were attended by a small Russian boy. Religious vows prevented the priests from eating fresh meat; they subsisted on salt fish, and during the winter vegetables were unavailable. The following May the little boy was found to be the only surviving member of the party: He had buried his late masters in the snow, but he had survived because he ate fresh meat (Medical Research Council, 1932). The sensory properties of food are important for the human being, and they often play a greater role in the acceptability of food than do the essential nutrients. This was foreshadowed in ancient times by Hippocrates with his aphorism 38: “An article of food or drink which is slightly worse but more palatable is to be preferred to such as are better but less palatable” (Lusk, 1922). In the same vein, McCollum and Simmonds (1929) wrote: “The artificially established liking for white flour and white cornmeal is an illustration of the failure of the instinct of man to serve as a safe guide in the selection of food. The esthetic sense is appealed to in greatest measure in this case by products of lowest biological values.” This is so today because the nutritional value of food has no meaning unless the food is eaten (Krieger, 1963; Mrak, 1973 ; Schweigert, 1969). The highly developed brain of Homo sapiens is at odds with the hypothalamus. This is due in part to the importance of sensory stimuli for normal mental processes. Schultz (1965) introduced his book entitled “Sensory Restriction” with the following statement: “A changing sensory environment seems essential for human beings. Without it, the brain ceases to function in an adequate way and abnormalities of behavior develop. In fact, as Christopher Burney observed in his remarkable account of his stay in solitary confinement: ‘Variety is not the spice of life; it is the very stuff of it.”’

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Despite customs, social pressures, and prejudices, human beings do choose some nutrients correctly in accordance with their needs. Human beings deficient in sodium respond as do rats with correct choices. A child whose adrenal cortex was diseased craved salt, consumed large amounts of it when he could, and survived as long as he had access to it. He was put in the hospital and fed a hospital diet; without access to salt, he soon died ( Wilkins and Richter, 1940). Drummond (1934) wrote: “In man we can detect definite traces of the survival of a protective instinct, but even in primitive races it is seldom marked except for certain minerals. In East Africa the search for edible earths rich in calcium was frequently a cause of tribal raids, and the evidence points to these products being instinctively consumed to make good the lack of lime in the customary diet.” Evidence, assuredly scanty, indicates that the “protective instinct” has protected man against deficiencies of many essential nutrients other than minerals and may have been a factor in the survival of man. In the rural areas of northern China mixed cereals were used. These cereals were fed singly and in mixtures to rats, and the results showed that the proportions of the mixed cereals chosen and used by the population had the highest nutritional value (Adolph, 1944). Soybeans contain heat-labile trypsin ( inhibitors ) and other inhibitors which decrease their nutritional value. Osborne and Mendel (1917) showed that the autoclave (heat) destroyed the inhibitors and increased the nutritional value of the soybean. In ancient China soybeans were processed differently; the beans were thoroughly ground and the insolubles settled out, giving soybean milk, The milk was heated, and the proteins were precipitated with calcium or magnesium salts. After the curd (tofu) was collected, the residual liquid, which contained the inhibitors, was discarded (Smith, 1961). The soybean proteins, freed of inhibitors, were used as food. In northern Sweden, potatoes, which are the chief source of vitamin C , were eaten boiled and not mashed. Boiling does not destroy much of the vitamin C in the potato, whereas mashing introduces air into the potato, and the oxygen destroys the vitamin C (S. Lepkovsky, unpublished). This culinary practice protected the population against scurvy. Culinary practices often proved to be of critical value in the survival of populations. In the near past, pellagra was common on the American side of the Rio Grande River but was seldom seen on the Mexican side. In preparing corn to make tortillas, Mexicans used slaked lime, which makes the nicotinic acid, normally incompletely available in corn, more

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available. When Americans made corn bread, they did not use the slaked lime, and many developed pellagra (Leguna and Carpenter, 1951 ) . Davis (1928) found that when growing babies were allowed to choose their food they selected a nutritionally adequate diet. Sweet (1936) also obtained evidence to indicate that children allowed a free choice of food and a choice of the time of eating chose a superior diet. In World War 11, American soldiers in a German prison camp were subsisting on a ration 200 calories short of their basal needs. They were ultimately saved by Red Cross food parcels. Trading of the parcels’ contents went on among the men, and a point system took form which pretty well reflected the nutritional value of the food. A can of powdered whole milk topped the list at 150 points. Meat followed at 120. There was no point listing for cheese, jam, sugar, and chocolate bars (Englander, 1945). As prisoners, profound changes had taken place in the soldiers’ internal chemistry. Because of these changes in body chemistry, the previously unacceptable powdered milk became highly acceptable. It now received the same high acceptance among the prisoners of war as it did with rats. Englander (1945) remembered that milk powder had low acceptability among these prisoners when they were soldiers. After inquiry, he found that the powdered milk was the most acceptable item because “it satisfied-even more than the chocolate-the prisoners’ craving for something rich to eat.” I. FLAVORFUL FOOD: TASTEAND SMELL

Our senses react to an unlimited number of chemical compounds that have an odor or taste, but very few are classed as flavors in the sense that they play a role in nutrition. Odors such as coffee evoke sensations in the “pit of the stomach,” excite the flow of digestive secretions, and are associated with food, whereas other equally pleasant odors such as perfumes have no such effect and are not classed as food flavors. A Marine Corps chef was frying a couple of onions while preparing food for about a thousand men. When asked how he proposed to divide a couple of onions among a thousand men, he answered that he had no intention of feeding the onions to the men, but he found that when the odor from these onions permeated the mess hall the men ate better ( E . Burdick, personal communication). Flavorful foods such as onions, garlic, leeks, and spices have played a key role in human nutrition and have been highly prized by man from the earliest times. The first such record (to the writer) is described in the Bible (Numbers l l : l ) ,when

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the Israelites were in the Sinai desert subsisting on manna, which apparently lacked desirable flavors. Among the six food items missed most, three were chiefly sources of flavor: onions, garlic, and leeks. The others were fish, melons, and cucumbers. Dana (1840) wrote of the sailors’ craving for onions after a long sea voyage during which they subsisted on preserved foods. When onions were made available, they were a “glorious treat. We were perfectly ravenous for them. It was like a scent of blood to a hound.” In a German prison camp during Warld Wax 11, American soldiers lived on a monotonous diet and the onion was highly prized. “A gravy, made of onions carefully hoarded from a small crop grown in the camp garden during the summer, was served as a special treat on Sunday nights and helped take the curse off the potatoes” (Englander, 1945). During World War 11, General Wingate raided the Japanese behind their lines in Burma, and food for him and his men had to be dropped from the air.. Usually K-rations were dropped to them. Once a week, a “holiday” package was dropped to the troops, and among other food items, a raw onion was included for each man. This the men appreciated very much, nibbling the onions and making them last for two or three days.

J. DEPRIVATION IN MANYFORMS 1 . Physical Deprivation of Sensory Stimuli

As men moved toward battle areas in World War 11, they became separated from many of their sources of sensory stimuli. Isker (1944) wrote: Despite the efforts of entertainers and Red Cross workers who devoted their time and risked their lives contributing to the morale of the troops, there could be no sports, movies, libraries, or other pleasures for men fighting in foxholes. Sometimes labels on ration containers were the only reading matter at hand. To a civilian the idea that a manufacturer’s label could hold any interest for a man having temporary respite between killing or being killed may seem slightly ridiculous. This, however, was apparently not the way men actually in the situation saw it. The things they took for granted at home were precisely what they missed in the field . . . To a man in combat or waiting to go into combat, food can assume an importance amazing to people in ordinary circumstances. Rations have proved to have a psychological as well as a physiological effect. They can raise or lower morale.

These men looked to food for sensory stimuli to compensate for those they were deprived of. During World War 11, soldiers in the battle

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areas were given rations that lacked desirable flavors. These rations were deficient in pyridoxine (S. Lepkovsky, unpublished data) but they were not rejected because they were nutritionally deficient; the rations were rejected long before malnutrition could develop nor were there any signs of malnutrition in the men subsisting on these rations. The soldiers complained that the rations lacked “belly-filling” properties and did not give them the feeling of having had a good meal. The rations contained too many hashes; they wanted food they could “sink their teeth into” The judgment of the soldiers of these rations was expressed thus: “We undoubtedly could survive on these rations a lot longer than we’d care to live” (Lepkovsky, 1959).

2. Self-Denial Many who are devoutly religious denounce the pleasures of the flesh. Brillat-Savarin ( 1960) describes such pleasures: “Things unequivocally damnable and never to be countenanced, such as dancing, the theatre, gaming and other pastimes of a like nature . . . Here enters gourmandism, very guilefully and with most theological features. By divine right man is king of nature, and for him was all the produce of the earth created. For him the quail grows fat, for him is mocha coffee fragrant . . . we refer to the chevaliers and the abbb. What gourmands they were . . . It was impossible to mistake those widespread nostrils, staring eyes, glistening lips, and salient tongues; they handled every mouthful with dignity.” IN THE INTAKE OF A SPECIFIC NUTRIENT K. DECREASE

The sense of taste of human beings who subsist on diets deficient in a nutrient is altered, leading to aversions and preferences in food.

1. Decrease in Intake of Protein In tests at Iowa State College during World War 11, volunteers subsisted on high-carbohydrate and practically protein-free diets for 16 to 18 days. These subjects developed aversions to anything that tasted sweet, especially sugar. They developed cravings that were more or less nonspecific. Some craved pickles, some lettuce. One subject craved eggs to a point where hydrogen sulfide smelled good to him (P. Swanson, personal communication).

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An unusual case of specific hunger was reported by Hoelzel (1944), who subsisted on a low-protein diet for a long period of time. He craved meat so much that he went on “meat sprees;” he rapidly devoured raw steak without tasting it. After a day or two the impulse to swallow the raw meat began to lag. It then became apparent that the raw steak had a disagreeable slimy taste and an objectionable (bloody) odor.

2. Salt Deficit During the Civil War, Union soldiers in Confederate prison camps lived largely on corn mush and were short of salt. “We traded buttons to guards for red peppers, and made our mush or bread or dumplings hot with the fiery pods, in hopes that this would make up for the lack of salt, but it was a failure. One pinch of salt was worth all the pepper pods in the Southern Confederacy. The old adage says that ‘hunger is the best sauce for poor food,’ but hunger failed to render this detestable stuff palatable” ( McElory, 1957).

3. “Fat Hunger’’ Before glycerol was produced biologically, the only source of glycerol for gunpowder was fat. During the war, fat became scarce and “fat hunger” was common in the population. Fat became delicious in taste. After fat became available, it was consumed in excessive amounts. However, after bodily deficits of fat were repleted, it lost its delicious taste and at times became aversive. It appears that in the early history of the human race, fat was a scarce commodity in the diet in some areas; this is reflected in a passage in the Bible. God enjoyed food, but only through smell. After the flood abated and Noah, his family, and all the animals left the ark, “Noah builded an altar unto the Lord . . . and burned offerings on the altar. And the Lord smelled a sweet savour, and the Lord said in his heart, I will not again curse the ground anymore for man’s sake” (Genesis 8:20-22). In burnt offerings, the “principle was laid down in Mosaic law that to the Lord belongs all the fat of sacrificed animals” (Westminister Dictionary of the Bible, p. 181). In the peace offering “the fat that covereth the inwards . , . and the two kidneys and the fat that is on them which is by the flanks . . . it is an offering made by fire, of a sweet savour unto the L o r d (Leviticus 3:3-5).

SAMUEL LEPKOVSKY

VII.

PROCESSING OF FOOD: FLAVORS

In prehistoric times, early man discovered that meat roasted with fire lost its slimy, bloody taste and odor and acquired very desirable flavors. Cooking and roasting give the meat high palatability in two ways: The roasted outside has very distinctive flavors, and the “outside cut” is often preferred. The rest of the meat has other flavors, also making the meat very palatable. The flavors of meat cannot be tasted in the raw state because the flavors are chemically bound. During cooking, the meat flavors are progressively released ( R. Bouthilet, unpublished ) for many hours until, finally, all the flavors are released. When such cooked meat is freeze-dried in a high vacuum, the flavors are lost, and the meat acquires a taste ‘that has no resemblance to the taste of properly cooked food and becomes inedible ( S. Lepkovsky, unpublished). B. BREAD The use of fermentation to make bread began early in the history of Egypt. In the baking process, the heated outside, the crust, becomes very palatable, and the crumb is equally palatable. Additionally, slices of the crumb are roasted to make toast, thus adding another parameter to the flavor of the bread. During World War 11, biscuits made of flour were part of most rations; they were edible but not highly acceptable. In contrast, bread made from the same flour was considered so palatable that no amount of bread given to the soldiers was considered too much. They referred to the bread as a “morale builder” ( S. Lepkovsky, unpublished). The heat used to bake bread and to cook or roast meat decreases in varying degrees the nutritional value of the flour and meat. However, the loss of nutritional value is more than balanced by the increase in palatability. As Kare (1972) put it, we “separate sensation from nutrition . . . At a modest expense to a balanced diet, and with a minimum of hazard, we should devote some of our energy to adding to the quality of life.” Soybeans are not very palatable. Heating soybeans renders them more palatable and in addition, increases the nutritional value of the bean (Osborne and Mendel, 1917). Trypsin inhibitors and other toxicants (Mickelsen and Yang, 1966) in the soybeans are destroyed, but the heat

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may reduce the l e ~ofl some nutrients in the bean. However, the positive factors outweigh the negative factors.

C. MEATAND VEGETABLESTEW One of the food items used in the ration of soldiers in the Southwest Pacific area during World War I1 was canned meat and vegetable stew. It had very low acceptability. The soldiers said that the stew was too highly spiced, yet requests for spices came from the same area. Another comment was that the meat and vegetable stew was monotonous. This criticism was taken seriously, and a greater variety of components was used in preparing the stew. When the meats and vegetables were prepared in separate cans, the meats were very palatable and so were the vegetables (S. Lepkovsky, unpublished), but these were not tried in the field. The new stew with a greater variety of components had no greater acceptability than the old stews. Indeed, the taste of the new meat and vegetable stew was no different from that of the former stews. Apparently the flavor (spices) of the mixture of the meat and vegetables changed during processing so that it was different from that of the meat and vegetables canned separately. It was concluded (S. Lepkovsky, unpublished) that, when the soldiers said the meat and vegetable stew was monotonous or too highly spiced, all they meant was that the stew was inedible.

D. BRININGAND FERMENTATION Few people today realize the extent to which our ancestors used fermented liquors for all manner of domestic purposes (Larson, 1943). The brining or salting of vegetables, meats, and fish has been in worldwide use since prehistoric times ( Sanders, 1966; Fellers, 1960). Some of the brined vegetables were also fermented. Outstanding examples are cucumbers and sauerkraut. During fermentation, lactic acid is formed, as well as small amounts of acetic acid. Although considered condiments, cucumbers and sauerkraut furnish nutrients, and claims have been made that the products of the lactic fermentations improve intestinal tone and reduce putrefactive action in the colon ( Fellers, 1960).

E. FOOD ADDITIVES To secure maximum sensory stimuli and nutritional values, the processing industry resorted to food additives, among them acids, alkalis, buffers, bread improvers, emulsifiers, stabilizers, thickening agents,

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flavors, colors, preservatives, antioxidants, nonnutrient sweeteners, sequestering agents, humectants, anticaking agents, firming agents, whipping agents, glazes, polishes, and nutrient supplements such as vitamins and minerals. These food additives make possible desirable volume, viscosity, crumb, palatability and color in cakes, ice creams, and various mixes. The mixes are stabilized, and separation and settling out of particles is prevented. Antioxidants prevent rancidity and off-flavors. Humectants prevent loss of moisture in products such as marshmallows. Sequestering agents prevent adverse effects of metallic ions, such as copper and iron, which may cause discoloration and clouding of liquids (Food Protection Committee of the Food and Nutrition Board, 1961; Mrak, 1973; Sanders, 1966). Insecticides may be classed as food additives even though they are not added directly into food. I n our struggle for survival, we are at war for space and food with weeds, viruses, fungi, bacteria, insects, and rodents. Man’s struggle with insects has been summarized by Winteringham and Barnes (1955): “Among his most serious competitors are many members of the insect world spreading diseases to both man and his domestic animals and consuming or destroying growing crops and stored food . . . He must face the problem of killing the insects without concurrently injuring the well-being of himself and those other living things upon which his continued existence depends.” The weight of insects produced in our pastures and meadows frequently exceeds the weight gains of the domestic animals consuming them (Food Protection Committee of the Food and Nutrition Board, 1961). One way to deal with this problem is to harvest the insects. “From ancient times to the present day, insects have been consumed in many societies throughout the w o r l d ( Meyer-Rochow, 1973). It is only fairly recently that there has been almost total absence of insects from European diets ( Bodenheimer, 1951). The greatest variety of insects used as human food is found in the area of highest population density and greatest shortage of protein. The hazards of malnutrition could be reduced by more extensive use of insects as human food ( Meyer-Rochow, 1973). Knowledge of insect resistance is of crucial importance to our struggle with insects for survival (Brown, 1959; Jackson, 1972). It appears that insects inherit resistance against insecticides. It is probable that in some insects the gene for resistance to insecticides arises by mutation and is preserved by differential survival of the exposed insect ( Metcalf, 1955). In an attempt to avoid the application of insecticides on crops, biological methods, the use of sex attractants, and chemical sterility pro-

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cedures are under intensive study (Jacobson and Beroza, 1963; Food Protection Committee of the Food and Nutrition Board, 1961). Every phase of the use of agricultural control chemicals and their effects, as well as the literature on this subject has been covered very thoroughly by the Symposia on Agricultural Control Chemicals (St. John, 1950) and theReport of the Secretary’s Commission on Pesticides and Their Relationship to Environmental Health ( Mrak, 1969).

F. MODERNPROCESSING OF FOOD TO be successful, the food-processing company must meet the following objectives: (1) The food must have acceptable sensory stimuli such as taste, smell, texture, tactile properties, and appearance. ( 2 ) The food must be stable. This involves canning, dehydration, refrigeration, radiation sterilization, fermentation, and the use of preservative agents such as smoke, acids, antibiotics, and chemicals that antagonize fungal and microbial spoilage. ( 3 ) The food must be wholesome, have high nutritional value, and not have harmful levels of toxicants. ( 4 ) The food must be “convenient” requiring minimal time and effort to prepare the meal. The achievement of these objectives is beset with many complex and perplexing problems. The food processing and chemical industries are active in meeting the challenges raised by these problems. Many of these industries have research laboratories staffed by scholarly and well-trained personnel who carry on basic research work that deals with the numerous problems inherent in processing food. Some industries go so far as to study soil fertilization to produce crops of maximum yields and highest nutritional value (Schmidt, 1963). The effect of processing on the nutritional values of plant and animal foods is covered by Harris and von Leosecke (1960). Their volume includes sections on agricultural practices, harvesting, washing, bleaching, trimming, pasteurization, sterilization, canning, ionizing radiations, freezing, dehydration, milling practices, storage, insecticides, additives, and losses and gains of nutrients. A comprehensive summary of the effects of food processing on nutritional values (Darby and Chichester, 1973) emphasizes gains and losses of nutrients.

G . EFFECT OF PROCESSING AND STORAGE ( SHELF-LIFE) ON THE NUTRITIONAL VALUEOF FOOD Much work has been done on the effect of processing on the nutritional value of food (Harris and von Loesecke, 1960; Darby and

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Chichester, 1973). Less has been done on the losses of nutritional values in food during storage, a subject that needs to be studied more completely (Harris and von Loesecke, 1960; Darby and Chichester, 1973). Much information has been obtained upon the effect of processing plus storage on the nutritional value of military rations used during World War 11. C and K rations consisted of food items which, before processing, provided optimum nutrition. When these rations were fed to rats, little growth occurred ( Silber, 1945, personal communication). These results with rats were confirmed by Sporn and Elvehjem (1948). Likewise, the rations fed to chicks elicited poor growth (Lepkovsky, 1946-1949; Scott et al., 1951). Similar results were obtained with monkeys (Sporn et al., 1948). Results obtained in these studies vaned widely; pyridoxine deficiency in the rations characterized most of the studies. Folic acid deficiency was frequently encountered in the rations. Deficiencies of other members of the vitamin B complex also occurred. Soldiers subsisting on these rations, especially on K rations responded in a manner similar to that of rats, chicks, or monkeys. After a few days, the consumption of K ration decreased and body weight decreased. Supplementation of the K ration with fresh food increased the food intake of the soldiers ( Lepkovsky, unpublished). Since consumption of K rations decreased in as little as 3 or 4 days, it was concluded that the rejection of K ration was not due to the nutritional qualities of the food since the ration was rejected before the possibility of the development of nutritional deficiencies in the subjects. It was concluded that the rations were rejected because they did not contain desirable sensory properties. H. MAN,HIS CHEMICAL SENSESAND FOOD PROCESSING In dealing with the complex problems that are inherent in food processing, it is important to keep in mind that there is a continuous battle between the neocortex and biology. In general, the highly developed neocortex of man determines the choice of the food, and frequently the choice has little to do with need. The neocortex overwhelms the exquisite machinery of the hypothalamus and interferes with its function. The sensory stimuli of food often exert a greater role in the choice of food than does the nutritional value of the food. In short, the chemical senses play an unusually important role in human nutrition. The realization, long in coming, of the importance of sensory stimuli to human beings led to the establishment of the Monell Chemical Senses Institute at the University of Pennsylvania. The research at this institute is basic and includes biochemical, physiological, and neurological studies

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and their interrelationships as they affect the nutrition, physiology, and behavior of human beings.

VIII.

OBESITY: IMPAIRMENT OF REGULATION OF FOOD INTAKE

Some animals are lean, others are fat. Some workers hold that obesity is due to gluttony. In this vein, Rynearson has been quoted: “The only glands involved in obesity are the salivary glands” (Brosin, 1953). It has also been suggested that obese animals are biologically programmed to be fat (Nisbett, 1972). Rony (1940) suggested that “some intrinsic abnormality seems to establish caloric balance leading to fat accumulation.” Much research has been directed toward establishing the nature of the “intrinsic abnormality.” Basal metabolism is not one of them, since the basal metabolism of the obese is often normal (Newburgh, 1942). The data of the Danish workers (Hagedorn et al., 1927; Krogh and Lindhard, 1920) indicate that obesity is due to a “qualitative anomaly in metabolism; i.e., an abnormally increased transformation of carbohydrate to fat.” Decreased physical activity has been suggested as an anomaly that leads to obesity (Mayer, 1955). Obesity is considered the most serious nutritional disorder in the United States (Goldsmith, 1952; Mayer, 1953; Wilson, 1969). “Obesity predisposes to diabetes, increases the tendency to hypertension, favors the development of atheroscelerosis and contributes to heart disease” (Barr, 1953). “Death from coronary heart disease remains the major medical problem of the United States” (Hodges and Krehl, 1965). “It is therefore not surprising that there is an almost overwhelming amount of research activity directed toward the solution of the alarming increases in death from ischemic heart disease” (Krehl, 1960). Despite the tremendous amount of work on obesity, little progress has been made in its control. LESSONS FROM WARS The role of nutrition in atherosclerosis and ischemic heart disease was highlighted by information obtained during wartime when food supplies became curtailed. The incidence of vascular coronary heart disease dramatically decreased during World Wars I and 11. Paton (1933) concluded that during World War I the incidence of athero-

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sclerosis and diabetes decreased, due to a curtailed consumption of sugar. With the end of the war, sugar became available and obesity, atherosclerosis, and diabetes increased. In contrast, Aschoff ( 1930), surveying the same situation, concluded that the decrease in dietary fat intake accounted for the decrease in deaths from atherosclerotic and degenerative heart disease. Additionally, Himsworth (1935) reported a decrease in the incidence of diabetes mellitus with the ingestion of a low-fat diet. During World War 11, Malmros (1950) associated the decreased incidence of atherosclerosis, cardiosclerosis, and diabetes in the Scandinavian countries with their reduced consumption of cholesterol and animal fats. Dedichen et al. (1951) compared the mortality from circulatory disease in Oslo, Norway, during the years 1940-1945 with that of the periods preceding and following them. These years were chosen because of the rigid restrictions in food intake imposed on the population during the war. Shortly after the beginning of the deprivation there was a definite reduction in mortality from circulatory disease, which reached a minimum at about the end of the third year and continued without much change until the restrictions were removed. Mortality then rose again, finally attaining a level observed before the beginning of the deprivation. Brozek et al. (1946) reported that during the Leningrad siege malnutrition was probably the main factor in decreasing the incidence of cardiovascular disease. The nature of this malnutrition was not defined. Wars have provided nutritionists with a wealth of epidemiological information. The incidence of diabetes, atherosclerosis, and cardiovascular disease was dramatically reduced in all countries, neutral or combatant, where food shortages became severe ( s e e review, Ahrens, 1974). There was no agreement on the cause or causes for the decrease in these diseases; some held that it was due to a decrease in the intake of carbohydrates, especially sucrose, while others insisted that it was due to the decrease in the intake of fat, especially animal fat. “The controversy over dietary fat and sucrose as possible causative agents in arteriosclerotic and degenerative heart disease was already joined in 1935” ( Ahrens, 1974). The controversy still continues, because the intakes of sucrose and fat within population groups are closely and positively correlated with each other (Yudkin, 1964). McGandy et al. (1967), after a review of the literature, concluded that atherosclerosis may be decreased by “a lowering of the proportion of dietary saturated fatty acids. Increasing the proportion of polyunsaturated acids and reducing the level of dietary cholesterol are the dietary changes most likely to be of benefit.”

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In particular, the role of saturated fat in the induction of coronary heart disease received a great deal of attention (Keys et al., 1974; Reiser, 1973). In contrast, Yudkin (1972) contended that sucrose is the villain promoting the development of atherosclerosis and heart disease. Overlooked in this controversy is the effect of activity, intake of crude fiber and of total calories. The information obtained during the war years has led to the realization that malnutrition is caused not only by nutritionally inadequate diets rendered so by deficiencies or excesses of essential nutrients or their improper balance, but also by the excessive intake of diets of optimum nutrition causing obesity, which increases the incidence of diseases such as diabetes, atherosclerosis, and ischemic heart disease.

IX.

DIET AND SPAN OF LIFE

The information obtained during the war years has raised the question: Is there an optimum level of food intake for good health and maximum longevity? While insurance statistics show that overnutrition decreases longevity by an increase in the incidence of degenerative diseases such as diabetes, renal diseases, hypertension, atherosclerosis, and ischemic heart disease (Barr, 1953; Dublin, 1953), it is well to bear in mind that “. , . pneumonia and tuberculosis exact a higher death toll among the lighter-weight persons than among overweights” ( Dublin, 1953). Overnutrition is common among human beings, especially among the elderly. “The prevalence of obesity increases to about the age 40 for men . . . and declines after age 60. The decline is due not only to loss of fat, but also to the loss of fat people” (Sebrell, 1953). Much of the work on the span of life was done with rats. Diet restriction so severe that growth and sexual maturity were retarded nevertheless increased the life span (McKay, et al., 1943). Diet restriction that caused little retardation of growth and permitted normal reproduction also increased the span of life (Berg and Simms, 1961; Nolen, 1972). Ad libitum feeding for three months, followed by restriction of food intake to 60 per cent of the controls was most conducive to a long life in the rat. All research work on obesity and incidence of degenerative diseases and life span i s complicated by genetic, aging, and environmental factors. Kennedy (1955) studied the role of obesity uncomplicated by genetic and other factors. He produced obesity by damaging the ventromedial nucleus of the hypothalamus and showed that uncomplicated obesity also hastened senescence. Renal disease was the major disease in these rats.

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Similarly, the life span of genetically obese mice was lower than that of the thin siblings (Lane and Dickie, 1958). This work and that of Kennedy (1955) suggest that excess body fat is one villian in shortening the life span of rats and mice. The literature on diet and life span has been summarized by Silberberg and Silberberg ( 1955). A. UNDERNUTRITION AND WORK

While we can measure food intake, body weight, and the course of degenerative diseases in rats, we cannot follow productivity or psychological reactions in animals. This can be done only in human beings. Underfed laborers show low productivity. In highway work, the productivity of the workers was increased 5-fold with improved nutrition (Condliffe, 1952); “Morale begins only at 1800 calories” (0.Guttentag, see Condliffe, 1952). Haggard and Greenberg ( 1935; 1939) studied between-meal feeding in industry. The data demonstrated an improvement in the output of certain piece-work operations and showed a lower rate of absenteeism than among those that had not received between-meal feeding.

B. UNDERNUTRITION AND PSYCHOLOGICAL RESPONSES Human volunteers subsisting on severely restricted food intake, fatigue easily and do not have the energy to participate in sport and social activities. “I’m hungry. I’m always hungry . . I’m cold . . . I’m weak . . . social graces take second place to concerns of food. I lick my plate unashamedly at each meal . . . If we see a show, the most interesting part of it is contained in scenes where people are eating” ( Keys et al., 1950). American soldiers in a German prison camp during World War I1 were subsisting on a starvation diet. Food was the passionate preoccupation of most “Kriegies.” No one ever tired of talking about food during the day or dreaming of it at night. “Sometimes the dreams took odd twists.” A young lieutenant produced a nightmare in which he was imprisoned in a can of meat (Englander, 1945). Food is a primary necessity. Mahatma Gandhi referred to hunger: “I may place before the dog over there the message of God as before those hungry millions who have no lustre in their eyes. . . . How am I to talk of God to the millions who have to go without two meals a day? To them God can only appear as bread and butter.”

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C. Is THERE AN OPTIMUM LEVELOF FOOD INTAKE? In using calorically restricted diets such as those that are used to treat obesity, a decision has to be made as to which is worse, the obese state or a state of chronic undernutrition. It is doubtful that such a decision can be made and the decision will likely be different for each individual. The information drawn from life insurance statistics that the death rate is lower from suicides among the obese deserve serious consideration and cannot, by any means, be ignored. Fat people have been characterized as happy, and Caesar preferred them (Barr, 1953; Berman, 1932). Let me have men about me who are fat; Sleek-headed men and such as sleep o’nights; Yond Cassius has a lean and hungry look; He thinks too much: such men are dangerous.

This question recalls the remarks of the soldier who subsisted upon Army K rations: “We could undoubtedly survive on these rations a lot longer than we’d care to live.”

X.

OVERVIEW

Life began in the primordial sea (Oparin, 1938), and the composition of the primordial sea set the conditions of life. The nutrition of the living systems was ensured, since presumably the primordial sea contained all the needed nutrients (Horowitz, 1945) such as glucose, amino acids, vitamins, electrolytes, and micronutrients. When animals left the primordial sea, the external environment changed but the conditions under which life began did not: “. . . the conditions under which cell life is possible are very restricted indeed and have not changed substantially since life began” (Baldwin, 1949). To survive, living systems had to take their environment with them ( Baldwin, 1949). During the evolutionary period of life, animals developed along different lines-anatomically, physiologically, biochemically, and behaviorally, but in one respect there was little or no change: . . the living elements of the body, therefore, are water inhabitants, or inhabitants of water which has been modified by the addition of salts and thickened by an albuminous or colloidal material” (Cannon, 1932), now called internal environment or extracellular fluids.

“.

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In nutrition, we are not feeding the animal; instead we are feeding the living elements, the cells of the animal. This concept leads to the realization of the extensive and numerous mechanisms that are involved in nutritional processes. Appropriate machinery, anatomical and biological, has been developed to ensure nutrition. Food exists in the external environment. To obtain food requires a sense of need or appetite. Appetite or “drive” sets an animal in search for food. This requires locomotion, exploration, and examination. Recognition of food is made possible by the chemical senses. The animal must choose an adequate diet in accordance with its needs. Information about food in the external environment is integrated in the brain with information on bodily needs, leading to a choice of food best suited to the survival of the animal. Food as it exists in the external environment cannot be used by the cells. The food has to be digested and reduced to unit structures which can be utilized by the cells. These unit structures (essential nutrients) must be delivered to the cells in proper amounts and in proper balance. Excesses and cellular wastes must be eliminated. A new era in nutrition is in the making, with several major new developments. It is now recognized that malnutrition is possible on diets of optimum nutrition. Food ingested chronically in excess of current need evokes obesity. Obesity increases the incidence of diabetes, hypertension, atherosclerosis, and ischemic heart disease. Obesity is due to an impairment in the regulation of food intake, caused by an “intrinsic anomaly” that leads to the accumulation of fat in the depots. Little is known of the “abnormal anomaly” except that it is not necessarily due to an altered basal metabolism, since there is no difference in basal metabolism between many obese and lean subjects. Although the hypothalamus regulates food intake, it does so indirectly. During the evolution of animals, food regulatory mechanisms were developed in the pituitary, adipose tissues, gastrointestinal tract, liver, thyroid, pancreas, and elsewhere; these mechanisms are integrated in the hypothalamus. The gastrointestinal tract regulates food intake in accordance with the capacity of the intestinal tract to digest food. Overloading of the intestinal tract leads to diarrhea and other disturbances. The adipose tissues appear to play a key role in the regulation of food. The weight of adult animals changes little over long periods of time.This is due largely to maintenance of the level of fat in the body at a “preferred level,” referred to as the set point in adipose tissues. This

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level of fat in the body is defended against change. Fasting reduces body weight and depletes body fat without change in set point in the adipose tissues. Restoration of ad libitum food intake elicits an increase in food intake and when body weight and body fat has been restored to prefasting level, food intake returns to normal. When body fat is increased without change in the set point in the adipose tissues as by force-feeding, cessation of force-feeding is followed by refusal of food or by eating sparingly until body weight and body fat fall approximately to the preexperimental level and food intake increases to normal. A change in set point in the fat depots is effected by damage to the ventromedial nucleus, and hyperphagia and obesity follow. When body weight and body fat plateau, food intake returns to normal. The new level of body fat of the obese animal is defended against change by fasting or force-feeding. There can be no understanding of the cause of obesity nor of the control of the amount of fat in the body until more information is obtained on the mechanisms that set the set point or preferred level of fat in the adipose tissues. Lipolysis and lipogenesis are key processes in the regulation of food intake. Fasting accelerates lipolysis and mobilization of fatty acids from the depots. Concomitantly, lipogenesis and deposition of fat in the depots are suppressed. Lipolysis evokes cascades of metabolic reactions. Fat becomes the fuel of the lean body mass (cells). Utilization of carbohydrates by the muscles is suppressed; entry of glucose into the muscle cells is inhibited. Utilization of glucose by the liver is depressed; oxidation of glucose and synthesis of glycogen are decreased. Production of glucose is enhanced by increases in glycogenolysis and gluconeogenesis. Fats are utilized as fuel and in addition are converted to ketone bodies which serve as fuel for the muscles and brain. After eating, lipolysis is inhibited and lipogenesis and deposition of fat in the depot are enhanced. Cascades of metabolic reactions are evoked, reciprocally inhibiting many of the reactions that were elicited during fasting. Glucose enters the muscle cell and is used for fuel and for synthesis of glycogen. Fat utilization is decreased. In the liver, utilization of glucose for fuel is enhanced, glycogen synthesis proceeds, and gluconeogenesis, the production of ketone bodies, and glycogenolysis are inhibited. It appears to be increasingly evident that nutrition consists of many biochemical, physiological, and behavioral processes, all of them interlocked and geared to the specific function which is the survival of each animal.

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REFERENCES Adams, S. F. 1929. Obesity as a precursor of diabetes. 1. Nutr. 1, 339-342. Adolph, E. F. 1939. Measurements of water drinking in dogs. Amer. J. Physiol. 125, 75-86. Adolph, E. F. 1947. Urges to eat and drink in rats. Amer. 1. Physiol. 151, 11C125. Adolph, W. H. 1944. The protein problem in China. Science 100, 14. Ahrens, R. A. 1974. Sucrose, hypertension, and heart disease: An historical perspective. Amer. I. Clin. Nutr. 27, 403-422. Allison, J. B. 1949. Biological evaluation of proteins. Adoan. Protein Chem. 5, 155200. Anand, B. K. 1961. Nervous regulation of food intake. Physiol. Rev. 41, 677-708. Andik, I., and Donhoffer, Sz. 1948-1949. The effect of methylthiouracyl on food intake and selection. Z. Vitam. Hormon- Fermentforschung. 2, Suppl., Nos. 3 4 . Andik, I., Donhoffer, Sz., Moring, I., and Szentes, J. 1951. The effect of starvation on food intake and selection. A d a Physiol. 2, 363-368. Andik, I., Bank, J., Moring, I., and Szegvari, G. Y. 1954. The effect of exercise on the intake and selection of food in the rat. Acta Physiol. 4, 457461. Andik, I., Sardi, F., and Schmidt, P. 1966. The effect of growth hormone on food intake and food selection. Acta Physiol. 29, 177-182. Anokhin, P. K. 1961. The multiple ascending influences of the subcortical centers on the cerebral cortex. In “Brain and Behavior” ( M . A. B. Brazier, ed.), Vol. 1, pp. 139-170. Amer. Inst. Biol. Sci., Washington, D.C. Aschoff, L. 1930. Die Arteriosklerose (arteriopathia deformans) : Ein erniilirungs und abnutzungs-problem. Beih. Z. Med. Klin. 26, 1-20. Balagura, S., and Coscina, D. V. 1969. Influence of gastrointestinal loads on mealeating patterns. 1. Comp. Physiol. Psychol. 69, 101-106. Baldwin, E. 1949. “An Introduction to Comparative Biochemistry.” Cambridge Univ. Press, London and New York. Barr, D. P. 1953. Health and obesity. N . Engl. 1. Med. 248, 967-970. Bayliss, W. M. 1924. “Principles of General Physiology.” Longmans, Green, New York. Bayliss, W. M., and Starling, E. H. 1899. The movements and inneration of the small intestine. 1. Physiol. (London) 24, 99-143. Behrman, H. R., and Kare, M. R. 1968. Canine pancreatic secretion in response to acceptable and aversive taste stimuli. €‘roc. Soc. Erp. Biol. Med. 129, 343-346. Bellows, R. T. 1939. Time factors in water drinking in dog. Amer. 1. Physiol. 125, 87-97. Berg, B. N., and Simms, H. S. 1961. Nutrition and Longevity in the Rat. 111. Food Restriction Beyond 800 days. 1. Nutr. 74, 23-32. Bergstrom, S. 1967. Prostaglandins: Members of a new liornional system. Science 157, 382-391. Bennan, L. 1932. “Food and Character.” Houghton, New York. Bernard, C. 1859. “LeCons sur les propri6tAs physiologiques et les alterations pathologiques des liquides de l’organisme.” Bailliere et Fils, Paris. Best, C . H., and Taylor, N. B. 1940. “The Physiological Basis of Medical Practice,” 2nd ed. William & Wilkins, Baltimore, Maryland. Bligh, J., and Moore, R. E., eds. 1972. “Essays on Temperature Regulation.” Amer. Elsevier, New York.

REGULATION OF FOOD INTAKE

59

Bloom, J. C., Rogers, J. G., Jr., and Maller, 0. 1973. Taste responses of North American Porcupline ( Erethizon Dorsatum ). Physiol. G Behau. 11, 95-98. Bodenheimer, F. S. 1951. “Insects as Human Food.” Junk, The Hague. Borgstrom, S. 1941. Studien iiber nahrungsphysiologischen wert der Weizenkleie under besonderer beriicksichtigung der bedeutung der erhitsung. Acta Physiol. Scand. 2, Suppl. VII, 1-128. Bortoff, A. 1972. Digestion: Motility. Annu. Reu. Physiol. 34, 261-290. Bradley, R. M., and Mistretta, C. M. 1971. Intravascular taste in rats as demonstrated by conditioned aversion to sodium saccharin. J. Comp. Physiol. Psychol. 75, 186-1 89. Brillat-Savarin, J. A. 1960. “The Physiology of Taste.” Dover, New York (original publication in 1825). Brobeck, J. R. 1946. Mechanism of the development of obesity in animals with hypothalamic lesions. Physiol. Reu. 26, 541-558. Brobeck, J. R. 1948. Food intake as a mechanism of temperature regulation. Yale J . Biol. Med. 20, 545552. Brobeck, J. R. 1960. Food and temperature. Recent Progr. Horn. Res. 16, 439-466. Brobeck, J. R., Wheatland, M., and Strominger, J. L. 1947. Variations in regulation of energy exchange associated with estrous, diestrous and pseudopregnancy in rats. Endocrinology 40, 65-72. Brodie, S. 1945. “Bioenergetics and Growth.” Van Nostrand-Reinhold, Princeton, New Jersey. Brosin, H. W. 1953. The psychology of overeating. In “Overeating, Overweight and Obesity,” Nutr. Symp. Ser. No. 6, pp. 52-72. Nat. Vitamin Found., New York. Brower, L. P., Brower, J. V. Z., and Corvino, J. M. 1967. Plant poisons in a terrestrial food chain. Proc. Nut. Acad. Sci. U S . 57, 893-898. Brown, A. M., McKenzie, J. C., and Yudkin, J. 1963. Knowledge of nutrition amongst housewives in a London suburb. Nutrition (London) 17, 15. Brown, A. W. A. 1959. Insecticide resistance as a world problem. Can. J. Biochem. Physiol. 37, 1091-1097. Brozek, J., Wells, S., and Keys, A. 1946. Medical aspects of semi-starvation in Leningrad (Siege 1941-1942). Amer. Reu. Sou. Med. 4, 70-86. Butcher, R. W., and Baird, C. E. 1968. Effects of prostaglandins on adenosine 3’, 5’-monophosphate levels in fat and other tissues. J. Biol. Chem. 243, 1713-1717. Butcher, R. W., Baird, C. E., and Sutherland, E. W. 1969. Effects of lipolytic and antilipolytic substances on adenosine 3’, 5’-monophosphate in isolated fat cells. J . BioZ. Chem. 243, 1705-1712. Cabanac, M. 1971. Physiological role of pleasure. Science 173, 1103-1107. Cabanac, M., and Duclaux, R., 1970. Obesity: Absence of satiety aversion to sucrose. Science 168, 496-497. Cannon, W. B. 1932. “The Wisdom of the Body.” Norton, New York. Christ, E. J., and Nugteren, D. H. 1970. The biosynthesis and possible function of prostaglandins in adipose tissue. Biochim. Biophys. Acta 218, 296-307. Clemente, C . D., Sterman, M. B., and Wyrwicka, W. 1964. Post-reinforcement EEC synchronization during alimentary behavior. Electroencephalogr. Clin. Neurophysiol. 16, 355-369. Cohen, Lord, of Birkenhead. 1958. “Sherrington: Physiologist, Philosopher and Poet.” Thomas, Springfield, Illinois. Cohn, C., and Joseph, D. 1962. Influence of body weight and body fat on appetite of “normal” lean and obese rats. Yale J . Biol. Med. 34, 598-607.

60

SAMUEL LEPKOVSKY

Condliffe, J. B. 1952. Some International Economic Aspects of Nutrition. In “Nutrition in the Practice of Medicine.” ( R. S. Goodhart, ed.). Nutr. Monograph Ser. No. 4, National Vitamin Foundation, New York, pp. 137-162. Dana, R. H. 1840. “Two Years Before the Mast: A Personal Narrative of Life at Sea,” pp. 442443. Harper, New York. Daniel, E. E. 1968. Pharmacology of the gastrointestinal tract. In “Handbook of Physiology” (Amer. Physiol. SOC.,J. Field, ed.), Sect. 6, Vol. IV, Chapter 108, pp. 2267-2324. Williams & Wilkens, Baltimore, Maryland. Daniel, E. E. 1969. Digestion: Motor function. Annu. Reti. Physiol. 31, 203-226. Darby, W. J., and Chichester, C. 0. 1973. “The Effects of Food Processing on Nutritional Values.” Nutr. Found., New York. Davis, C. M. 1928. Self-selection of diet by newly-weaned infants. Amer. J. Dis. Child. 36, 651-655. Dedichen, J., Strom, R., Adelstrom-Jensen, and Closs, K., 1951. Incidence of atherosclerosis during war years. In “Transactions of the Fifth Conference on Factors Regulating Blood Pressure” (B. W. Zweifach, and E. Shorr, eds.), pp. 117-123. Josiah Macy, Jr. Found., New York. de Muelenaere, H. J. H. 1964. Studies on the digestion of soybeans. J. Nutr. 82, 197-205. Donhoffer, Sz. 1960. Spontaneous selection of food. Triangle 4, 233-239. Donhoffer, Sz., and Vonotsky, J. 1947. The affect of thyroxine on food intake and selection. Amer. J. Physiol. 150, 334439. Douglass, D. M. 1960. Transmission of excitation in the wall of the intestine. Amer. J. Dig. Dis. [N.S.] 5, 339-347. Drunimond, J. C. 1934. “Lane Medical Lectures,” 2nd ed. Stanford Univ. Press, Stanford, California. Dublin, L. I. 1953. Fat People Who Lose Weight Live Longer. In “Overeating, Overweight and Obesity.” ( R . S . Goodhart, ed.) Nutr. Monograph Ser. No. 6., pp. 106-122. National Vitamin Foundation, New York. Edholm, 0. C., Fletcher, J. G., Widdowson, E. M., and McCance, R. A., 1955. The energy expenditure and food intake of individual men. Brit. J. N u t r . 9, 286-300. Ehman, G. K., Albert, D. J., and Jamieson, J. L. 1971. Injections into the duodenum and the induction of satiety in the rat. Can. J. Psychol. 25, 147-166. Englander, D. A. 1945. They dreamed of food. Liberty Mag. July 7. Epstein, A. N. 1959. Suppression of eating and drinking by amphetamine and other drugs in normal and hyperphagic rats. J. Comp. Physiol. Psychol. 52, 37-45. Epstein, A. N. 1960. Reciprocal changes in feeding behavior produced by intrahypothalamic chemical injections. Amer. J. Physiol. 199, 969-974. Epstein, A. N. 1967. Feeding without oropharyngeal sensations. In “Chemical Senses and Nutrition” In ( M . R. Kare and 0. Maller, eds.), pp. 263-280. Johns Hopkins Press, Baltimore, Maryland. Everett, J. W. 1964. Central neural control of reproductive functions of the adenohypophysis. Physiol. Reti. 44, 373431. Fain, J. N. 1968. Effect of dibutyryl-3’, 5’-AMP, theophylline and norepinephrine on lipolytic action of growth hormone and glucocorticoid in white fat cells. Endocrinology 82, 825-830. Favarger, P. 1965. Relative importance of different tissues in the synthesis of fatty acids. In “Handbook of Physiology” (Amer. Physiol. SOC.,J. Field, ed.), Sect. 5, p. 19. Williams & Wilkins, Baltimore, Maryland.

REGULATION O F FOOD lNTAKE

61

Feldberg, W., and Myers, R. D. 1965. Changes in temperature produced by microinjections of aniines in the anterior hypothalamus of cats. j . Physiol. (London) 177, 239-245. Fellers, C. R. 1960. Effects of fermentation on food nutrients. In “Nutritional Evaluation of Food Processing” (R. S. Harris and H. von Loesecke, eds.), pp. 161165. Wiley, New York. Flerk6, B. 1963. The central nervous system and the secretion and release of luteinizing hormone and follicle stimulating hormone. In “Advances in Neuroendocrinology” (A. V. Nalbandov, ed.), pp. 211-237. Univ. of Illinois Press, Urbana. Follis, R. H. 1948. “The Pathology of Nutritional Disease.” Thomas, Springfield, Illinois. Food Protection Committee of the Food and Nutrition Board. 1961. The use of chemicals in food production, processing, storage and distribution. Nat. Acad. Sci.-Nat. Res. Cormc., Publ. 887, 8. Fowler, J. L. A., and Cleghorn, R. A. 1942. The response of splanchic blood vessels and of the small intestine to vasoconstrictor influences in adrenal insufficiency in the cat. Amer. I. Physiol. 137, 371-379. French, J. D. 1960. Brain physiology and modern medicine. Postgrad. Med. 27, 559568. Garcia, J., Hankins, W. G . , Robinson, J. H., and Vogt, J. L. 1972. Bait shyness; tests of CS-US mediation, Physiol. d7 Behau. 8, 807-810. Gibbs, J., Young, R. C., and Smith, G. P. 1973. Cholecystokinin decreases food intake in rats. 1. Comp. Physiol. Psychol. 84, 488-495. Glick, Z., Thomas, D. W., and Mayer, J. 1971. Absence of effect of injections of the intestinal hormones secretin and cholecystokinin-pancreozymin upon feeding behavior. Physiol 62 Behau. 6, 5-8. Gold, R. M. 1973. Hypothalamic obesity: the myth of the ventromedial nucleus. Science 182, 488-490. Goldsmith, G. A. 1952. Recent advances in nutrition and metabolism. Arch. Intern. Med. 90, 513-516. Goodridge, A. G., and Ball, E. G. 1967. Lipogenesis in the pigeon: In vivo studies. Amer. j . Physiol. 213, 245-249. Grodsky, G. M. 1969. Metabolic considerations in obesity. In “Obesity” ( N . L. Wilson, ed. ) , pp. 67-76. Davis, Philadelphia, Pennsylvania. Grossman, M. I. 1955. Integration of current views on the regulation of hunger and appetite. Ann. N. Y . Acad. Sci. 63, 76-91. Grossman, M. I. 1958. Regulation of food intake. Amer. j . Dig. Dis. [N.S.] 3, 659668. Grossman, S. P. 1960. Eating or drinking elicited by direct adrenergic or cholinergic stimulation, respectively, of the lateral hypothalamus. Science 132, 301-302. Grossman, S. P. 1969. A neuropharmacological analysis of hypothalamic and extrahypothalamic mechanisms concerned with the regulation of food and water intake. Ann. N.Y. Acad. Sci. 157, 902-917. Grossman, S. P. 1972. Neurophysiological aspects; extrahypothalamic factors in the regulation of food intake. Aduan. Psychosom. Med. 7, 49-72. Gutkin, V. I. 1963. Effect of depancreatization on the state of the chemical components of the cholinergic system. Fed. Proc., Fed. Amer. Soc. E x p . Biol. 22, T537-T539.

62

SAMUEL LEPKOVS,KY

Haggard, H. N., and Greenberg, L. A. 1935. “Diet and Physical Efficiency.” Yale Univ. Press, New Haven, Connecticut. Haggard, H. N., and Greenberg, L. A. 1939. Between-meal Feeding in Industry: Effects on the Absenteeism and Attitude of Clerical Employees. J. Amer. Diet., Ass. 15, 435439. Hagedorn, H. C., Holten, C., and Johansen, A. H. 1927. The pathology of metabolism in obesity. Arch. Intern. Med. 40, 30-37. Handbook of Nutrition. 1951. American Medical Association, Chicago, Illinois. Harris, L. J., Clay, J., Hargreaves, F. J., and Ward, A. 1933. Appetite and choice of diet. The ability of the vitamin B deficient rat to discriminate between diets containing and lacking the vitamin. Proc. Roy. Soc. Ser. B 113, 161-190. Harris, R. S., and von Loesecke, H., eds. 1960. “Nutritional Evaluation of Food Processing.” Wiley, New York. Hart, E. B., Steenbock, H., Lepkovsky, S., and Halpin, J. G. 1923. The nutritional requirements of baby chicks, 111, The relation of light to the growth of chickens, J . Biol. Chem. 58, 3 3 4 1 . Herrin, R. C., and Meek, W. J. 1945. Afferent nerves excited by intestinal distention. Amer. J. Physiol. 144, 720-723. Hill, R. G., Ison, E. C., Jones, W. W., and Archdeacon, J. W. 1952. The small intestine as a factor in regulation of eating. Amer. J. Physiol. 170, 201-205. Himsworth, H. P. 1935. Diet and incidence of diabetes mellitus. Clin. Sci. 2, 117. Hodges, R. E., and Krehl, W. A. 1965. The role of carbohydrates in lipid metabolism. Amer. 1. Clin. Nutr. 17, 334-346. Hoebel, B. G. 1971. Feeding: Neural control of intake. Annu. Rev. Physiol. 33, 533568. Hoebel, B. G., and Teitelbaum, P. 1966. Weight regulation in normal and hypothalamic hyperphagic rats. J. Comp. Physiol. Psychol. 61, 189-193. Hoelzel, F. 1944. An explanation of appetite. Amer. J. Dig. Dis. 11, 71-76. Holinger, P. H., Kelley, E. H., and Ivy, A. C. 1932. The vagi and appetite. Proc. Soc. E x p . Biol. Med. 29, 884-885. Hollenberg, C. H., Vost, A., and Patten, R. L. 1970. Regulation of adipose mass: Control of fat cell development and lipid content. Recent Progr. Horm. Res. 26, 463-503. Horowitz, N. H. 1945. On the evolution of biochemical synthesis. Proc. Nut. Acud. Sci. US.31, 153-157. Houssay, B. A. 1951. “Human Physiology.” McGraw-Hill, New York. Illiano, G., and Cuatrecasas, P. 1972. Modulation of adenylate cyclase activity in liver and fat cell membranes by insulin. Science 175,906-908. Isker, R. A. 1944. “U. S. Armed Forces Operation Studies,” No. 1. Office U. S. QM General, Washington, D. C. Ivy, A. C. 1935. The applied physiology of the gastrointestinal innervation. Amer. 1. Dig. Dis. Nutr. 1, 845-853. Jackson, W. H. 1972. Rodenticide resistance reviewed. Pup., Urban Rut Contr. Proj. Conf., New Orleans, Louisiana, Jan. 31-Feb. 4, 1972. Jacobs, H. L. 1958. Studies on sugar preference. 1. The preference for glucose sohtions and its modifications by injections of insulin, J. Comp. Physiol. Psychol. 51, 304-310. Jacobson, M., and Beroza, M. 1963. Chemical insect attractants. Science 140, 13671373.

REGULATION OF FOOD INTAKE

63

Janowitz, H. D. 1958. Editorial: Hunger and appetite. Amer. I. Med. 25, 327-332. Janowitz, H. D., and Grossman, M. I. 1949. Hunger and appetite: Some definitions and concepts. 1.Mt. Sinai Hosp., New York 16, 231-247. Janowitz, H. D., and Hollander, F. 1955. The time factor in the adjustment of food intake to vatied caloric requirement in the dog: A study of the precision of appetite regulation. Ann. N.Y. Acad. Sci. 63, 56-67. Janowitz, H. D., Hollander, F., Orringer, D., Levy, M. H., Winklestein, A., Kaufman, M. R., and Margolin, S. G. 1950. A quantitative study of the gastric secretory response to Sham feeding in a human subject. Gastroenterology, 16, 104-116. Jansen, G. R., Hutchison, C. F., and Zanetti, M. E. 1966. Studies on lipogenesis in vivo. Effect of dietary fat or starvation on conversion of 14C glucose into fat and turnover of newly synthesized fat. Biochem. 1. 99, 323-332. Jukes, C. L. 1938. Selection of diet in chicks as influenced by vitamins and other factors. J. Comp. Psychol. 26, 135-156. Kalat, J. W., and Rozin, P. 1970. “Salience:” A factor which can override temporal continguity in taste-aversion learning. 1. Comp. Physiol. Psychol. 71, 192-197. Kare, M. R. 1960. Animals “taste” senses differ from man’s. Nuclaid News October, pp. 22-23. Kare,.. M. R., and Medway, W. 1959. Discrimination between carbohydrates by the fowl. Poultry Sci. 38, 1119-1126. Kare, M. R., Schechter, P. J., Grossman, S. P., and Rothe, L. J. 1969. Direct pathway to the brain. Science 163, 952-953. Kare, M. R. 1972. Concluding remarks. In “Health and F o o d (G. G. Birch, L. F. Green, and L. G. Plaskett, eds. ), Wiley, New York. Katz, D. 1953. “Animals and Men.” Penguin Books, Baltimore, Maryland. Kellie, A. E. In J. R. Brobeck 1960. Food and Temperature. Recent Progr. Horn. Res. 16, 439-466. Kennedy, G. C. 1953. The role of depot fat in the hypothalamic control of food intake in the rat. Proc. Roy. SOC., Ser. B 140,578-592. Kennedy, G. C. 1960. Appetite and satiety. Med. Press 243, 211-214. Kennedy, G. C., Obesity, Disease and Life Expectancy. 1955. Voeding 16, 16-27. Kershbaum, A., Osada, H., Scriabine, A., Billet, S., and Pappajohn, D. J. 1967. Influence of nicotine on the mobilization of free fatty acids from rat adipose tissue in vitro and in the isolated perfused dog limb. Circulation Suppl. 11, 36, 20. Keys, A., Brozek, J., Henschel, A., Mickelsen, O., and Taylor, H. L. 1950. “The Biology of Human Starvation,” Vol. 2. Univ. of Minnesota Press, Minneapolis. Keys, A. 1964. Influence of diet on body composition. In “Occurrence, Causes and Prevention of Overnutrition” (G. Blix, ed.), Symp. Swed. Nutr. Found., pp. 85-94. Almqvist & Wiksell, Stockholm. Keys, A., Grande, F., and Anderson, J. T. 1974. Bias and misrepresentation revisited: Perspective on “saturated fat.” Amer. J . Clin. Nutr. 27, 18S212. King, J . R., and Farner, D. S. 1933. The relationship of fat deposition to Zugunruhe and migration. Condor 65,200-223. Kock, N. G. 1959. An experimental analysis of mechanisms engaged in reflex inhibition of intestinal motility. Acta Physiol. Scand. 47, Suppl., 5 5 4 . Krehl, W. A. 1960. Highlights of the Fifth International Congress on Nutrition. Borden Rev. Nutr. Res. 21, 75-89. Krieger, C. H. 1963. “The Foods of the Future,” Paper presented at the American Dietetic Association meeting in Philadelphia.

64

SAMUEL LEPKOVS,KY

Krogh, A., and Lindhard, J. 1920. The relative value of fat and carbohydrate as sources of muscular energy. Biochem. J. 14, 290463. Kubie, L. S. 1948. Instincts and homeostasis. Psychosom. Med. 10, 15-30. Kuntz, A. 1951. “Visceral Innervation and its Relation to Personality.” Thomas, Springfield, Illinois. Laguna, J., and Carpenter, K. J. 1951. Raw vs corn in niacin-deficient diets. J. Nutr. 45, 21-28. Lane, P. W. and Dickie, M. M. 1958. The Effect of Restricted Food Intake on the Life Span of Genetically Obese Mice. J. Nutr. 64, 549-554. Larson, P. S., Haag, H. B., and Silvette, H. 1961. “Tobacco: Experimental and Clinical Studies,” p. 533. Williams & Wilkins, Baltimore, Maryland. Lat, J. 1959. The relationship between excitability of the central nervous system, food intake and body growth. Proc. Nutr. Soc. 18, xxiv (abstr.). Lee, M. O., and Schaffer, N. K. 1934. Anterior pituitary growth hormone and composition and growth. J. Nutr. 7, 337-363. Leibowitz, S. F. 1970. Reciprocal hunger-regulatiing circuits involving a- and p-adrenergic receptors located respectively in the ventromedial and lateral hypothalamus. Proc. Nat. Acad. Sci. U.S. 67, 1063-1070. Le Magnen, J., and Devos, M. 1970. Metabolic correlates of the meal onset in the free food intake of rats. Physiol. G Behau. 5, 805-814. Lepkovsky, S. 1946-1949. Nutritional value of combat rations. Project Report No. 7-84-12-06. Quartermaster Food and Container Inst. Armed Forces, Chicago, Illinois. Lepkovsky, S. 1959. Potential pathways in nutritional progress. Food Technol. 13, 421-424. Lepkovsky, S., and Dimick, M. K. 1969. The hypothalamus and pancreas in intestinal function. Ann. N.Y. Acad. Sci. 157, 1062-1068. Lepkovsky, S., and Furuta, F. 1971. The role of homeostasis in adipose tissues upon the regulation of food intake of White Leghorn Cockerels. Poultry Sci. 50, 573-577. Lepkovsky, S., Len, R., Koike, T., and Bouthilet, R. 1965. Effects of protamine zinc insulin on chickens. Amer. J. Physiol. 208, 589-592. Lepkovsky, S., Bortfeld, P., Dimick, M. K., Feldman, S. E., Furuta, F., Sharon, I. M., and Park, R., 1971. Role of upper intestines in the regulation of food intake in parabiotic rats with their intestines “crossed surgically. Isr. J. Mecl. Sci. 7 , 639-646. Leveille, G. A. 1967. In vivo fatty acid synthesis in adipose tissues and liver of meal-fed rats. Proc. Soc. E x p . B i d . Med. 125, 85-88. Leveille, G. A., O’Hea, E. K., and Chakrabarty, K. 1968. In vivo lipogenesis in the domestic chicken. Proc. Soc. E x p . Biol. Med. 128, 398-401. Liebelt, R. A., Ichinoe, S., and Nicholson, N. 1965. Regulatory influences of adipose tissue on food intake and body weight. Ann. N.Y. Acad. Sci. 131, 559582. Lindstedt, K. J, 1971. Biphasic feeding response in a sea anemone: Control by aspargine and glutathione. Science 173, 333-334. Lish, P. M., and Peters, E. L. 1957. Antagonism of insulin-induced gastrointestinal hypermotility in the rat. Proc. Soc. E x p . Biol. Med. 94, 664-668. Loomis, W. F. 1955. Glutathione control of the specific feeding reactions of hydra. Ann. N.Y. Acad. Sci. 62, 209-228. Lundbaek, K. 1964. Cited in ‘Discussion’ (under Keys, 1964).

REGULATION O F FOOD INTAKE

65

Lusk, G . 1922. A history of metabolism. In “Endocrinology and Metabolism.” ( L . F. Barker, ed.), Vol. 3, pp. 3-78. Appleton, New York. Lyman, G . P. 1954. Activity, food consumption and hoarding in hibernators. J. Mammal. 35, 545-552. McCann, S. M. 1971. Mechanism of action of hypothalamic-hypophyseal, stimulating and inhibiting factors. In “Frontiers in Neuroendocrinology” ( L. Martini and W. F. Ganong, eds.), pp. 209-235. Oxford Univ. Press, London and New York. McCollum, E. V., and Simmonds, N. 1929. “The Newer Knowledge of Nutrition.” Macmillan, New York. McElroy, J. 1957. “This Was Andersonville.” McDowell, Obolensky, New York. McGandy, R. B., Hegsted, D. M., and Stare, F. J. 1967. Dietary fats, carbohydrates and atherosclerotic vascular disease. N . Engl. J. Med. 277, 186-192 and 242-247. McKay, C. M., Sperling, G. and Barnes, L. L. 1943. Growth, Aging, Chronic Diseases and Life Span of Rats. Arch. Biochem. 2, 469-479. Mackay, E. M., Callaway, J. W., and Barnes, R. H. 1940. Hyperalimentation in normal animals produced by protamine insulin. J. Nutr. 20, 5 9 4 6 . Magoun, H. W. 1963. “The Waking Brain,” 2nd ed. Thomas, Springfield, Illinois. Maller, O., and Kare, M. R. 1967. Observations on the sense of taste in the armadillo (Dasypus Novemcinctus) Anim. Behao. 15, 8-10. Malmros, H. 1950. The relation of nutrition to health. A statistical study of the effect of wartime on arteriosclerosis, cardiosclerosis, tuberculosis and diabetes. Acta Med. Scand., Suppl. 246, 137. Mayer, J. 1953. Genetic, traumatic and environmental factors in the etiology of obesity. Physiol. Rev. 33, 472-508. Mayer, J. 1955. The role of exercise and activity in weight control. In “Weight Control” (E. s. Eppright, P. Swanson, and C . ,4.Iverson, eds.), pp. 199-210. Iowa State Press, Ames. Mayer-Gross, W., and Walker, J. W. 1946. Taste and selection of food in hypoglycemia. Brit. 1. Erp. Pathol. 27, 297-298. Medical Research Council. 1932. Vitamins: A survey of present knowledge. Med. Res. Counc. (Gt. Brit.) Spec. Rep. Ser. 167. Metcalf, R. L. 1955. Physiological basis for insect resistance to insecticides. Physiol. Rev. 35, 197-232. Meyer, J. H., and Hargus, W. A. 1959. Factors influencing food intake of rats fed low-protein rations. Amer. J. Physiol. 197, 1350-1352. Meyer-Rochow, V. P. 1973. Edible insects in three different ethnic groups of Papua and New Guinea. Amer. 1. Clin. Nutr. 26, 673-677. Mickelsen, O., and Yang, M. G. 1966. Naturally occurring toxicants in foods. Fed. Proc., Fed. Amer. Soc. Exp. Biol. 25, 104-123. Miller, N. E. 1957. Experiments on motivation. Science 126, 1271-1278. Miller, N. E. 1965. Chemical coding of behavior in the brain. Science 148, 328438. Miller, N. E., and Kessen, M. L. 1952. Reward effects of food via stomach fistula compared with those of food via mouth. 1. Comp. Physiol. Psychol. 45, 555564. Morgane, P. J., and Jacobs, H. L. 1969. Hunger and satiety. World Rev. Nutr. Diet. 10, 100-21.3. Mrak, E. M., chairman. 1969. “Report of the Secretary’s Commission on Pesticides and their Relationship to Environmental Health.” U.S. Dept. of Health, Education, and Welfare, Washington, D.C.

66

SAMUEL LEPKDVSXY

Mrak, E. M. 1973. “Food Additives,” Address, Franklin Institute Lecture Series. Mu, J. Y., Yin, T. H., Hamilton, C. L. and Brobeck, J. R. 1968. Variability of body fat in hyperphagic rats. Yale J. Biol. Med. 41, 133-142. Murlin, J. R., Nasset, E. S., and Marsh, M. E. 1938. The egg replacement value of the proteins of cereal breakfast foods with a consideration of heat injury. I . Nutr. 16, 249-269. Mursell, J. L. 1925. Contributions to the psychology of nutrition: Hunger and appetite. Psychol. Reo. 32, 317-333. Myers, R. D., and Yaksh, T. L. 1969. Control of body temperature in unanaesthetized monkeys by cholinergic and aminergic systems in the hypothalamus. J. Physiol. (London) 202, 483-500. Myers, R. D., Bender, E. A. Krstic, M. K., and Brophy, P. I). 1972. Feeding produced in the satiated rat by elevating the concentration of calcium in the brain. Science 176, 1124-1125. Nalbandov, A. V., and Card, L. E. 1943. Effect of hypophysectomy of growing chicks. J. E x p . 2001.94, 387409. Newburgh, L. H. 1942. Obesity. Arch. Intern. Med. 70, 1033-1096. Nisbett, R. E. 1972. Hunger, obesity and the ventromedial hypothalamus. Psychol. Reu. 79, 433453. Nolen, G. A. 1972. Effect of Various Restricted Dietary Regimens on the Growth, Health and Longevity of Albino Rats. J. Nutr. 102, 1477-1494. Obrink, K. J. 1958. Digestion. Annu. Rev. Physiol. 20, 377404. Odum, E. P. 1960. Premigratory hyperphagia in birds. Am. J. Clin. Nutr. 8, 621-629. OHea, E. K., and Leveille, G. A. 1969. Significance of adipose tissue and liver as sites of fatty acid synthesis in the pig and the efficiency of utilization of various substrates for lipogenesis. J. Nwtr. 99, 338-344. Oparin, A. I. 1938. “The Origin of Life.” Macmillan, New York. Osborne, T. B., and Mendel, L. B. 1917. The use of soybean as a food. J. Biol. Chem. 32, 369-387. Ottoson, D. 1963. Some aspects of the functions of the olfactory system. Pharmucol. Reu. 15, 1 4 2 . Paton, J. H. P. 1933. Relation of excessive carbohydrate ingestion to catarrhs and other diseases. Brit. Med. J. 1, 738-740. Peters, R. A. 1936. The biochemical lesion in vitamin Bi deficiency. Lancet 0, 1161. Pitts, R. F. 1959. “The Physiological Basis of Diuretic Therapy.” Thomas, Springfield, Illinois. Powley, T. L., and Opsahl, C. A. 1974. Ventromedial hypothalamic obesity abolished by subdiaphragniatic vagotomy. Am. J. Physiol. 226, 25-33. Premack, D., and Premack, A. J. 1963. Increased eating in rats deprived of running. J . E x p . Anal. Behau. 6, 209-212. Quigley, J. P., and Louckes, H. S. 1962. Gastric emptying, editorial. Amer. J. Dig. Dis.[N.S.] 7, 672-676. Reiser, R. 1973. Saturated fat in the diet and serum cholesterol concentration: A critical examination of the literature. Amer. J. Clin. Nutr. 26, 524-555. Richter, C. P. 1935. The primacy of polyurea in diabetes insipidus. Amer. J. Physiol. 112, 481-487. Richter, C. P. 1936. Increased salt appetite in adrenalectomized animals. Amer. J. Physiol. 115, 155-167. Richter, C . P. 1942. Physiological psychology. Annu. Reo. Physiol. 4, 561-574.

REGULATION OF FOOD INTAKE

67

Richter, C. P., and Eckert, J. F. 1937. Increased calcium appetite in parathyroidectomized rats. Endocrinology 21, 50-54. Richter, C. P., Holt, L. E., Jr., Barelare, B., Jr., and Hawkes, C. D. 1938. Changes in fat, carbohydrate and protein appetite in vitamin B deficiency. Amer. 1. Physiol. 124, 596-602. Rizack, M. A. 1964. Activation of an epinephrine-sensitive lipolytic activity from adipose tissue by adenosine 3‘, 5’-phosphate. J. Biol. Chem. 239, 392495. Rodbell, M. 1970. The fat cell in mid-term: Its past and future. In “Adipose Tissue: Regulation and Metabolic Functions” (B. Jeanrenaud and D. Hepp, eds.), pp. 14. Academic Press, New York. Rogers, J. G., Jr., and Maller, 0. 1973. Effect of salt on the response of birds to sucrose. Physiol. Psychol. 1, 199-200. Rony, H. R. 1940. “Obesity and Leanness.” Lea & Febiger, Philadelphia, Pennsylvania. Rosenblith, W. A. 1961. “Sensory Communication.” Wiley, New York. Rzoska, J. 1953. Bait-Shyness: A study in rat behavior. Brit. J. Anim. Behavior 1, 128-135. St. John, J. L., ed. 1950. “Symposia, Agricultural Control Chemicals,” Advan. Chem. Ser. 1. Amer. Chem. SOC.,Washington, D.C. Samuels, L. T. 1947. The relation of the anterior pituitary hormones to nutrition. Recent Progr. Horm. Res. 1, 147-176. Sanders, H. J. 1966. Food additives. Chem. Eng. News Oct. 10, 100-120; Oct. 17, 108-128. Sandberg, S., Roman, L., Zavodnick, J., and Kupers, N. 1973. The effect of smoking on serum somatotropin, immunoreactive insulin and blood glucose levels of young adult males. J. Pharmacol. E x p . Ther. 184, 787-791. Sarson, H. S. 1943. Fermented liquors in old-time cooking. Nature (London) 152, 386. Sawyer, C. H., Kawakami, M., Markes, J. E., and Everett, J. W. 1959. Physiological studies on some interactions between the brain and the pituitary-gonad axis in the rabbit. Endocrinology 65, 614-688. Schally, A. V., Redding, T. W., Lucien, H. W., and Meyer, J. 1967. Enterogastrone inhibits eating by fasted mice. Science 157, 210-211. Schmidt, W. A. 1963. Influence of zinc on the performance of vegetables in the Bajio region of Mexico. Proc. Carib. Reg., Amer. SOC. H o e . Sci. 7, 17-27. Schoenheimer, R. 1942. “The Dynamic State of Body Constituents.” p. 23. Harvard Univ. Press, Cambridge, Massachusetts. Schoenheimer, R., and Rittenberg, D. 1935. Deuterium as an indicator in the study of intermediary metabolism. 111. The role of the fat tissues 1. Biol. Chem. 111, 175-181. Schultz, D. P. 1965. “Sensory Restriction: Effects on Behavior.” Academic Press, New York. Schweigert, B. S. 1969. Technological advances and their effect on food habits and food uses. In “Food, Science and Society,” p. 18. Nutr. Found., New York. Scott, E. M. 1948. Self selection of diet. Trans. Amer. Ass. Cereal Chem. 6, 126-133. Scott, E. M., and Quint, E. 1946. Self-selection of diet. 111. Appetites for B vitamins. J . Nutr. 32, 285-292. Scott, L. M., Maynard, L. A., and Norris, L. C. 1951. Report to Quartermaster Food and Container Institute on the nutritive value of army rations as determined by growing chicks, For period covering Jan. 2, 1951 to Dec. 31, 1951.

68

SAMUEL LEPKOVSKY

Sebrell, W. H. 1953. Nutrition Research-Potentialities in Chronic Disease. Pub. Health Rep. 68, 737-741. Sebrell, W. H., Jr., and Harris, R. S., eds. 1954. “The Vitamins,” 1st ed., Vols. 1 and 2. Academic Press, New York. Share, I., Martyniuk, E.; and Grossman, M. I. 1952. Effect of prolonged intragastric feeding on oral food intake in dogs. Amer. I. Physiol. 169, 229-235. Sharon, I. M. 1965. Sensory properties of food and their function during eating. Food Technol. 19, 35-36. Sharpless, S., and Jasper, H. 1956. Habituation of the arousal reaction. Brain 79, 655-680. Shelling, D. H. 1932. Calcium and phosphorus studies. I. The effect of calcium and phosphorus on tetany, serum calcium and food intake of parathyroidectomized rats. I. Biol. Chern. 96, 195-214. Sherrington, C. 1947. “The Integrative Action of the Nervous System.” Yale Univ. Press, New Haven, Connecticut. Siegel, P. S. 1957. Repetitive element in the diet. Amer. J. Clin. Nutr. 5, 162-164. Siegel, P. S., and Pilgrim, F. J. 1958. The effect of monotony on acceptance of food. Amer. J . Psychol. 71, 756-759. Silberberg, M., and Silberberg, R. 1955. Diet and Life Span. Physiol. Rev. 35, 347362. Smith, A. K. 1961. “Oriental Methods of Using Soybeans as a Food.” Agr. Res. Sew., US Dep. Agr., Washington, D.C. Smith, C. A., Holden, R. C., and Hawk, P. B. 1920. Is unpalatable food properly digested? Proc. Soc. E x p . Biol. Med. 17, 98-99. Smith, H. W. 1953. Comparative physiology of the kidney. J. Amer. Med. Ass. 153, 1512-1514. Snapir, N., Nir, I., Furuta, F., and Lepkovsky, S. 1969. Effect of administered testosterone propionate on cocks functionally castrated by hypothalamic lesions. Endocrinology 84, 611-618. Snapir, N., Nir, I., Furuta, F., and Lepkovsky, S. 1974. Effects of functional and surgical castration of White Leghorn cockerels, and replacement therapy, on food intake, obesity, reproductive traits, and certain components of blood, liver, muscle and bone. Gen. Comp. Endocrinol. 24, 53-64. Snowden, C. T. 1970. Gastrointestinal sensory and motor control of food intake. J. Comp. Physiol. Psychol. 71, 68-76. Soulairac, A. 1947. Importance de l’absorption intestinale dans la rkgulation de l’appktit glucidique. C . R. Acad. Sci. 224, 961-963. Soulairac, A. 1963. Neurological factors in the control of appetite. Int. Reo. Neurobiol. 5, 303-346. Sporn, E. M., and Elvehjem, C. A. 1948. Growth and reproduction of rats fed army combat rations. J. Nutr. 35, 549-558. Sporn, E. M., Ruegamer, W. R., and Elvehjem, C. A. 1948. Studies with monkeys fed army combat rations. 1. Nutr. 35, 559-575. Sporn, J., and Necheles, H. 1956. Effect of glucagon on gastrointestinal motility. Amer. J. Physiol. 187, 634 (abstr.). Steinbaum, E. A., and Miller, N. E. 1965. Obesity from eating elicited by daily stimulation of hypothalamus. Amer. J. Physiol. 208, 1-5. Stellar, E. 1960. Drive and motivation. In “Handbook of Physiology” ( Amer. Physiol. Soc., J. Field, ed.), Sect. 1, Vol. 111, p. 1501. Williams & Wilkins, Baltimore, Maryland.

REGULATION O F FOOD INTAKE

69

Stettin, De W. 1957. Certain aspects of hormonal regulation of carbohydrate metabolism. In “Hormonal Regulation of Energy Metabolism” ( S . W. Kinsell, ed.), pp. 3-44. Thomas, Springfield, Illinois. Stevenson, J. A. F., Box, B. M., Feleki, V., and Beaton, J. R. 1966. Bouts of exercise and food intake in the rat. J. Appl. Physiol. 21, 118-122. Stunkard, M. D. 1961. Hunger and satiety. Amer. 1. Psychiat. 118, 211-217. Sutherland, E. W. 1970. On the biological role of cyclic AMP. 1. Amer. Med. Ass. 214, 1281-1288. Sweet, C. 1936. Voluntary food habits of normal children. I . Amer. Med. Ass. 107, 765-768. Thomas, B. M., and Miller, A. T., Jr. 1958. Adaptation to forced exercise in the rat. Amer. I. Physiol. 193, 350-354. Tsang, Y. C. 1938. Hunger motivation in gastrectomized rats. J. Comp. Psychol. 26, 1-17. Ugolev, A. M., and Kassil, V. G. 1961. Physiology of the appetite. Usp. Sovren. Biol. 51, 352-368. (translated from Russian by Translating Unit, Library Branch, Division of Research Activities, No. 9-23-63 ) . von Buddenbrock, W. 1958. “The Senses.” Univ. of Michigan Press, Ann Arbor. Wangensteen, 0. H., and Carlson, H. A. 1931. Hunger sensations in a patient after total gastrectomy. Proc. Soc. EX^. Biol. Med. 28, 545-547. Wayner, M. J., ed. 1964. “Thirst.” Pergamon, Oxford. Wells, H. G. 1940. Adipose tissue, a neglected subject. J. Amer. Med. Ass. 114, 2177-2183 and 2284-2289. Wertheimer, E., and Shapiro, B. 1948. The physiology of adipose tissue. Physiol. Rev. 2 8 , 4 5 1 4 6 4 . Westermann, E., and Stock, K. 1969. Effects of adrenergic blocking agents on FFA mobilization. In “Drugs Affecting Lipid Metabolism,” pp. 45-61. Plenum, New York. Wilkins, L., and Richter, C. P. 1940. A great craving for salt by a child with corticoadrenal insufficiency. I. Amer. Med. Ass. 114, 866-868. Wilson, N. L. 1969. The development and perpetuation of obesity: an overview. In “Obesity” ( N. L. Wilson, ed. ), pp. 3-12. Davis, Philadelphia, Pennsylvania. Winteringham, F. P. W., and Barnes, J. M. 1955. Comparative response of insects and mammals to certain halogenated hydrocarbons used as insecticides. Physiol. Rev. 35, 701-739. Wohl, M. G., and Goodhard, R. S. 1960. “Modern Nutrition in Health and Disease.” Lee & Febiger, Philadelphia, Pennsylvania. Wolf, A. V. 1958. “Thirst.” Thomas, Springfield, Illinois. Wolf, S. G . , and Wolff, H. G. 1943. “Human Gastric Function: An Experimental Study of a Man and His Stomach.” Oxford Univ. Press, London and New York. Wurtman, R. J., and Axelrod, J. 1965. Adrenaline synthesis: Control by the pituitary gland and adrenal glucocorticoids. Science, 150, 1464-1465. Yaksh, T. L., and Myers, R. D. 1972. Neurohumoral substances released from the hypothalamus of the monkey during hunger and satiety. Amer. 1. Physiol. 222, 503-515. Yudkin, J. 1964. Patterns and trends in carbohydrate consumption and their relation to disease. Proc. Nutr. Soc. 23, 149-162. Yudkin, J. 1972. “Sweet and Dangerous.” Peter H. Wyden, Inc., New York. Zamcheck, N. 1960. Dynamic interaction among body nutrition, gut-mucosal metabolism and morphology and transport across the mucosa. Fed. PTOC.,Fed. Amer. SOC.E r p . Biol. 19, 855-864.

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ANIMAL PHYSIOLOGY AND MEAT QUALITY *

BY R . G . CASSENS. D . N . MAFWLE. f AND G . EIKELENBOOM$ Muscle Biology Laboratory. University of Wisconsin. Madison. Wisconsin

I. Introduction ................................................... I1. Animal Physiology and Stress Susceptibility ........................

A . Environmental Interactions .................................... B. Effects of Adrenergic Blocking or Stimulating Agents ............. C. Rigor Mortis ................................................ D . Membrane Characteristics .................................... E . Malignant Hyperthermia ...................................... I11. Endocrine Interrelationships ...................................... A. Pituitary Function ........................................... B . Adrenal Cortex .............................................. C . Adrenal Medulla ............................................ D . Thyroid Function ............................................ IV Muscle Biochemistry ............................................ A Anaerobic Metabolism ........................................ B. Aerobic Metabolism .......................................... C . Other Biochemical Considerations .............................. V . Morphology and Histochemistry ................................... A. Structure ................................................... B. Histochemistry .............................................. VI . Importance in the Retail Product ................................. A. Occurrence of PSE ..........................................

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* Contribution from the College of Agricultural and Life Sciences. This review was prepared during the term of support by Public Health Service Research Grant FD-00107-13 and by Agricultural Research Service Cooperative Agreement 12-14100-11 214 (44). D.N.M. was a Public Health Service Postdoctoral Fellow . Muscle Biology Manuscript No . 51 . f Present address: Department of Animal and Dairy Science, Auburn University. Auburn. Alabama . $ Present address: Research Institute for Animal Husbandry. “Schoonoord.” Zeist. The Netherlands . 71

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B. Processing Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Palatability Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Possible Solutions to the Problem .......................... .. A. Genetics .......................... .................. B. Detection Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... .......... C. Control Methods ...................... VIII. Future Research Needs .................... .... .......... IX. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . ..................................

124 126 129 129 131 134 138

I. INTRODUCTION The title of this review is broad and general; the writing follows the same pattern. This was done not only to make it more palatable reading for those with peripheral interest, but also to emphasize the important role that animal physiology plays generally in controlling the changes that occur in the postmortem conversion of muscle to meat, thereby affecting the meat supply for the human population. While the majority of literature citations will be drawn from work that has been directed at the problem of pale, soft, exudative (PSE) muscle and the porcine stress syndrome (PSS) merely because of the abundance and critical nature of that material, it is our goal to relate this as a general situation with application to other domestic species used for meat. The early classical papers of Moran and Smith (1929) and BateSmith (1948) set forth very clearly the changes that occur in postmortem muscle. Much has been written since then about the details of postmortem change (Bendall, 1961; Briskey, 1964; Lawrie, 1966; Goll, 1968; Cassens, 1966; Buttkus and Tomlinson, 1966; DeFremery, 1966; Newbold, 1966; Marsh, 1970). Enormous effort has been expended on such studies, with the result that minute details about the biochemical processes and morphological alterations are understood. One senses through the years of work, though, the developing concern of researchers with the important role that the antemortem state of the animal plays in controlling postmortem change in the muscle and the conviction of some that the physiology of the animal is an integral part of the entire complex that dictates meat quality. A problem of great practical concern to the meat industry, and which prompted an overwhelming share of the research on postmortem muscle, is that of PSE muscle. This subject was reviewed by Briskey in 1964 and considered also by Bray in 1966. Today, PSE is still a problem of considerable monetary significance, as is illustrated by recent estimates

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of Hall (1972) *that the adverse economic impact of PSS and PSE is between 230 and 320 million dollars per year in the United States. A number of symposia have been called on the subject of PSE and PSS (Topel, 1968 [Iowa State University]; Sybesma et aZ., 1969; Hessel-de Heer et al., 1971 [the Netherlands]; Cassens et al., 1972 [University of Wisconsin] ). Each has resulted in an interesting and informative Proceedings, and we have quoted frequently from them. The Proceedings edited by Sybesma et al. (1969) and by Hessel-de Heer et al. (1971) have been particularly useful, as they have provided access to European work which we might have otherwise omitted because it was published in a foreign language or as a laboratory report. Since the review by Briskey (1964), the rate of publication of papers on the subject of postmortem biochemistry has slowed, but a number of very accomplished experiments have been published on the effect of animal physiology and endocrine interrelationships; discussion of these two subjects will constitute major sections in the present review. It is clear that an abnormal in vivo situation exists in these animals which predisposes them to an abnormal postmortem metabolism; but it is still not known whether the effect of PSS and associated problems on quality of the postmortem meat is due to a defect in the muscle itself or is central in the hormonal or neurological systems of the animal. Several other problems should be kept in mind as this review is read. There is a general belief, documented by some results reported in the literature, that selection for muscling in the live animal has been successful; but along with the desirable improvement in muscle mass, undesirable characteristics in the live animal (stress susceptibility) and in the meat (poor quality) have also been realized. This is a serious consequence that must be considered by those in animal breeding work, particularly in other species where great effort is being made to improve the production of muscle mass from an individual animal, but where a problem such as PSS is not yet clearly apparent. An obvious aspect of research effort has been to relate live animal parameters to judgment of the quality of postmortem meat. This procedure has grown to be almost standard routine, but it is not wihout some dangerous consequences. A typical assessment of the PSE condition, for example, involves a judgment of color and structure score on a numerical scale (Wisconsin system) running from 1 being pale, soft, and exudative (PSE), to 3 being normal, and to 5 being dark, firm, and dry (DFD). It is now known that meat from stress-susceptible animals may be PSE, DFD, or even normal in appearance, depending on the handling of the animal before, during, and after slaughter. Simple correlations between

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parameters for PSS in the live animal and postmortem meat quality will therefore remain of limited value (Eikelenboom et uZ., 1974). Much recent effort has been directed at development of nondestructive detection methods for use on the live animal to identify stress SUSceptibility. Success in that area, coupled with knowledge of genetics and selection procedures, offers promise for elimination or at least minimization of the problem from the pig population. An interesting aspect, which will be reviewed in the section on animal physiology, is the apparent similarity of PSS to the malignant hyperthermia syndrome (MH). MH is a serious complication of anesthesia experienced in certain human patients. The medical profession has interest in using PSS pigs as models for experimental work on MH. It is our aim, then, to establish that a problem first recognized as a low-quality condition in meat at 24 hours postmortem can in fact be traced to a condition in the live animal. Relevance to the food industry lies in the broad implications of a possible link between attempts to improve the production of muscle mass in the live animal on the one hand, and the subsequent problem of lower quality meat from such animals on the other hand.

II. ANIMAL PHYSIOLOGY AND STRESS SUSCEPTIBILITY A. ENVIRONMENTAL INTERACTIONS

I. Physiological Responses The observation that many pigs have poor tolerance to heat and succumb when exposed to the stress of a warm or hot environment has prompted studies to characterize various physiological parameters of such animals under carefully controlled environmental conditions. Forrest et al. (1968) exposed stress-susceptible (poor heat tolerance) and normal swine to a hot environment (42°C) in a small, single-pig dark chamber. Without prior anesthesia, the stress-susceptible Poland China pigs showed a significant increase in heart and respiration rates during the first 10 minutes of treatment. Venous blood pC0, increased significantly while PO, and pH decreased significantly in the stresssusceptible pigs. Pretreatment anesthesia removed much of the response noted earlier, and heart and respiration rates of stress-susceptible pigs did not differ signscantly from those of control Chester White pigs. On the basis of differences in cardiac output, arterial and venous blood

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pressure, body temperature, and blood pCO,, PO,, and pH data, the authors concluded that the stress-resistant pigs were able to maintain homeostasis during exposure to the high-temperature conditions both with and without anesthesia. The stress-susceptible pigs apparently suffered from a tissue hypoxia due to exposure to the stress and hightemperature environment. Kastenschmidt et aZ. ( 1965) employed preslaughter treatments of warm air, cold air, and cold air with an ice-water spray to pigs in order to study the effect on muscle properties. In general, a change from a warm air to a cold air environment, even if only of short duration, altered glycolytic rate and associated properties and improved meat quality. Tope1 et d. ( 1971) and Galloway et aZ. (1973) used large environmentally controlled chambers to subject Poland China and Chester White pigs to four different environments (WOC, 6"C, 6" to 43°C fluctuating six times daily, and 37°C) for 2 days each. Control animals were maintained at 25°C during the study. Blood PO, and pCQ, levels varied with the environment but were not significantly different between breeds. Blood pH and plasma lactate were not influenced significantly by treatment or breed, but daily changes in heart and respiration rate indicated a more rapid adaptation by the Chester White pigs. Similar work on short-term exposure to climatic stress by Kallweit and Haase ( 1971) revealed that stress-susceptible pigs could be identified by characteristic physiological differences at rest and following exposure to carefully controlled environmental conditions. High blood pC0, values were found among stress-susceptible pigs at rest, and the blood oxygen saturation was significantly lower among pigs exposed to conditions of 37°C and 1 0 0 ~ R.H. o for 3 hours. The authors suggested that stress-susceptible pigs may have a higher metabolic rate at rest and cautioned that the above method for identification of stress-susceptible pigs would be useful only under carefully controlled laboratory conditions. Kallweit (1969) studied earlier the effect of heat stress and exercise on blood pH, pCO,, and base excess. The results indicated that heat stress and exercise reduced base excess, buffer excess, standard and actual bicarbonate, and total and actual pCO,, while blood pH increased in response to heat exposure but decreased to 7.07 when exercise was imposed during the heat treatment. Oxygen metabolism was studied in boars by Haase and Steinhauf ( 1971). Various stresses, such as electric prods or epinephrine injection, were administered to seven German Landrace boars with highly variable results. The authors report that acute stress resulted in hypoxia, acidosis,

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and erythrocytosis. However, the reaction disappeared within 60 minutes after the initiation of stress, even though the stress may have been continued. The acute stress response was followed by slight oxyhemia and alkalosis. The variability of blood pH, lactate, pCOz, percentage of oxygen saturation, and hemoglobin levels was too great to be of value in identifying stress-responsive pigs. Judge et al. (1972) examined the effect of exercise and thermal stress on blood acid-base status and blood oxygen consumption. Three groups of Dutch Landrace pigs were exposed to ( 1) exercise for three 10-minute periods at 25"C, ( 2 ) exercise as in (1) but at 35"C, and (3) exposure to 35°C without exercise. Values for blood pH, pCO,, PO,, actual and standard bicarbonate, and percentage of oxygen saturation were determined on blood samples obtained through surgically implanted catheters in a carotid artery and jugular vein of each pig. Pigs were also grouped as stress-susceptible or stress-resistant on the basis of their rectal temperatures at the conclusion of exposure. Animals with temperatures >42"C were termed stress-susceptible. The stress of exercise and excitement due to treadmill walking was more severe than the stress of exposure only to 35°C as determined by the observed change in rectal temperature. The heat-plus-exercise treatment resulted in respiratory alkalosis followed by metabolic acidosis, while heat treatment alone caused only respiratory alkalosis. Following the stress treatment, both stresssusceptible and stress-resistant pigs had high levels of arterial oxygen, but the stress-susceptible pigs had a low blood pH which reduced the actual and standard bicarbonate in both arterial and venous samples with a lower venous oxygen saturation and, correspondingly, a greater arterial-venous difference in oxygen saturation. The authors suggest that the body temperature increase and acidosis of stress-susceptible pigs are the result of the heat and lactate produced by muscle anaerobic glycolysis in the presence of adequate arterial PO,. We can conclude from the above reports that stress-susceptible pigs undergo a more vigorous physiological response when exposed to high environmental temperatures or forced exercise than do stress-resistant pigs. This response appears to be characterized by a decrease in blood pH, a rise in lactate, a rise in pCO,, a decrease in oxygen saturation, and an increase in body temperature. 2. Postmortem Muscle Response

Thomas et al. (1966) studied postmortem muscle properties of pigs reared in various combinations of temperature and humidity. Pigs

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reared in an alternating temperature environment (18°C and 29°C) yielded longissimus muscles with inferior structure compared with that of muscles from pigs reared at a constant temperature of 18°C or 29"C, if the relative humidity was low (30%). Rearing in high relative humidity ( 85% ) tended to improve muscle structure and tenderness of cooked chops regardless of environmental temperature. Addis et al. (1967a,b) studied the effects of humidity level 85%, 3oyO,or alternating 3Oy0 and 85%) in a warm rearing environment (29°C) on the quantitative and qualitative carcass traits of Landrace animals. Rate of gain, carcass composition, muscle color, and gross morphology were not significantly influenced by humidity level. The authors concluded that a high humidity level in combination with a warm growing environment did not have a detrimental effect on muscle quality of Landrace pigs. The effects of fluctuating environmental temperatures (21°C or 32°C) on postmortem muscle were studied by Howe et al. (1968). Pigs reared in the 3-day periods of fluctuating environment yielded carcasses with a more rapid postmortem glycolytic rate, a higher light-to-dark fiber ratio, and a greater light reflectance of the longissimus than did pigs reared at a constant 27°C in a moderate (38 to 42%) relative humidity environment. These temperature effects, however, were masked when the relative humidity was low ( 17 to 23%), since the exposure to low humidity resulted in increased reflectance values and an increased rate of postmortem glycolysis. Aberle et al. (1969) examined the effect of rearing environment on some high-energy phosphate compounds in muscle and found that Poland China pigs reared at 32°C with moderate humidity had significantly higher levels of ATP and creatine phosphate ( C P ) at death with significantly lower levels of glucose-6-phosphate (G-6-P) than did pigs reared at 21°C. The pigs reared at 21°C underwent a more rapid rate of postmortem glycolysis with a more rapid increase in light reflectance values. Environment has also been shown to influence muscle myoglobin content (Thomas and Judge, 1970). Pigs reared in a constant temperature environment had a higher myoglobin content in skeletal muscle than did pigs reared in alternating temperature environments, when the humidity was moderate or high. However, when humidity levels were low, these differences did not exist. The authors proposed that pigs reared in an alternating temperature environment experienced intermittent and persistent development of tissue hypoxia and, consequently, had reduced aerobic efficiency compared to pigs reared in a constant environment.

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3. Effect of Exercise Sybesma and van Logtestijn (1966) found higher body temperatures in Pietrain pigs after short-term exercise than in Large White pigs and suggested that environmental stress, such as transport and preslaughter handling, increased body temperature in certain pigs. Body temperature measured just prior to slaughter in Dutch Landrace pigs was found to be highly related to meat temperature (measured at 40 minutes postmortem) and an accelerated glycolysis postmortem. Briskey et al. (1959) found that pigs adjusted rapidly to a repeated exercise. A single severe exercise, however, depleted muscle glycogen and produced postmortem characteristics of high pH and dark color. Sayre et al. (1963) observed that short-term exercise and excitement immediately prior to slaughter resulted in a rapid p H decline and pale muscle with poor water-binding properties. Fitts et al. (1973) exercised miniature pigs for 7 months and found that isometric contractile properties did not change, but the training did produce an increased endurance in skeletal muscle. Judge et al. (1967) studied the effect of duration of antemortem restraint on postmortem muscle of stress-susceptible Poland China and stress-resistant Chester White pigs. As the duration of restraint was increased among stress-susceptible pigs, their muscles became more severely affected in terms of the rate and extent of glycolysis, color, and gross muscle morphology. The muscle of the stress-resistant pigs responded in the same way initially, but with prolonged restraint they adapted to the stress as shown by the return of their glycolytic rate and morphological state to levels similar to those observed in unrestrained controls. These results may indicate a relative ability or inability of pigs to withstand a psychological type of stress and an increased aerobic capacity of stress-resistant pigs. Barton (1971) exposed pigs with low stress-resistance to short periods of stress which resulted in the development of PSE in the longissimus and to a lesser extent in the biceps femoris. However, when the period of preslaughter stress was increased, the incidence of PSE declined in both muscles and the muscle pH values increased. These results are similar to those cited by Tope1 (1972), who summarized observations on slaughtering stress-susceptible pigs immediately after arrival at the slaughter facility or holding them overnight after a long transport. Pigs slaughtered soon after a short transport had an average ultimate pH in longissimus of 5.31 and were PSE; pigs held overnight before slaughter had an average muscle p H of 6.85 and a dark, firm, and dry texture

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(DFD). The mechanisms involved in the glycogen depletion of the DFD carcasses have been investigated by Hedrick et al. (1964) and will be explored further in the section on function of the adrenal medulla. The effect of type of housing system during rearing was examined by Addis et a2. (1965) and Jones et al. (1971). The first study utilized 289 pigs in four housing systems; in the second study a total of 548 pigs in three housing systems was used. No consistent significant effect of housing system on carcass quality or other carcass traits was observed in either study. Jones et al. (1971), however, did note that pigs that took longer to reach market weight, in an ad libitum feeding system, were more susceptible to the development of PSE muscle. For further information on the effects of environmental stress on meat quality, the reader is referred to the review by Judge (1969). OF ADRENERGIC BLOCKING OR STIMULATING AGENTS B. EFFECTS

Val der Wal (1969) administered an adrenergic blocking agent to Pietrain pigs and observed a slower decrease in postmortem ATP levels of the semimembranosis than was found in controls from the same breed. The observed response was attributed to the possibility that, after pretreatment with the n-blocking agent, sympathetic stimuli to the muscle were impeded, thereby inhibiting ATP degradation. Postmortem glycolysis was not inhibited by the a-block treatment. These results are similar to those of Lister et al. (1970), who found significantly higher creatine phosphate and ATP levels in the longissimus muscle 1 hour postmortem among pigs receiving an a-blocking agent. The +block apparently inhibited the glycolytic rate somewhat, since O-time and l-hour lactate accumulations were significantly lower among treated pigs. However, when the pigs were anesthetized prior to treatment, there was no significant effect of the a-blocking agent on ATP or creatine phosphate levels. When isoproteranol ( a 8-stimulating agent) was given to anesthetized pigs, a slower postmortem depletion of ATP and creatine phosphate was observed in the longissirnus muscle than in muscle from anesthetized controls. There was no significant effect on the rate of postmortem lactate production due to the isoproteranol. Lister et al. ( 1970) also administered P-histidine to anesthetized pigs and obtained a response similar to that elicited by the a-blocking agent-that is, slower accumulation of lactate and slower depletion of ATP in postmortem muscle. These responses were interpreted as a beneficial effect on the (Y

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depletion rate of high-energy metabolites due to the general vasodilatory or antivasoconstrictive effects of the drugs examined. Weiss et al. (1973) administered a-or /.?-blocking agents to stresssusceptible and stress-resistant pigs and then observed the response of the animal to 5 minutes of forced physical exercise. The blood p H of the stress-susceptible pigs declined when they were stressed, but the pigs receiving the a-blocking agent had a lower average blood p H than did similar pigs receiving the p-blocking agent. The a-blocked stresssusceptible pigs also had higher average blood lactate levels. Both groups, however, had four to five times as much blood lactate after stress as did the stress-resistant pigs that had received blocking agents (263 and 205 mg% versus 54.5 and 46.5 mg%). I n discussion of the above work, Tope1 (1972) noted that the data suggest an extensive adrenergic simulation of the p receptors of the stress-susceptible pigs. Weiss et al. (1973) also noted that stress-susceptible pigs that received the p-blocking agent reacted less severely to forced exercise than did stress-susceptible pigs that received the (a-blocking agent.

C. RIGORMORTIS The concept of rigor mortis was discussed in the 1964 review on PSE by Briskey. Our purpose here is to refer the reader to other major reviews on the topic of rigor mortis and then discuss only a few nianuscripts that have been published since the 1964 review. The most comprehensive coverage of the subject has been published by Go11 (1968), and the reader is referred to that manuscript for a complete review of the chemical basis of the physical changes that occur during the development of rigor mortis; an excellent discussion of the resolution of rigor is also given. Forrest et al. (1966) used electrical stimulation of muscle immediately after exsanguination to predict the time course of rigor mortis, rate of postmortem glycolysis, and ultimate color morphology rating. Excitability threshold (lowest voltage for a contraction) was found to be high in muscle that had a short time course of rigor mortis, a fast postmortem glycolysis, and ultimate PSE characteristics; the opposite characteristics developed in muscle that had a low excitability threshold. Strength of contraction ( a t 5, 10, 25, and 50 volts) was highest in muscle that exhibited a long time course of rigor mortis, a slow glycolytic rate, and normal color morphology. The duration of contractility ( maintained under repeated stimulation at 2 cps) was also noted to be longer in this type of muscle. The authors applied multiple regression analysis to the

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data and found that up to 87% of the variability in color morphology could be predicted by combining various parameters of muscle response to electrical stimulation. They also mentioned breed differences which should be considered when such prediction equations are used. In a subsequent study, Forrest and Briskey (1967) reported that the initial lactic acid concentration, unless present in excessive quantities, appeared to have little influence on responsiveness of the muscle to electrical stimulation. Lactic acid content of the muscle did increase significantly and was accompanied by lowering of the pH during electrical stimulation of the muscle. Electrical stimulation of the spinal cord caused a significant lowering of the color-morphologyrating and of pH in carcasses from Poland China and Yorkshire but not in carcasses from Duroc animals. Electrical stimulation in carcasses from other than Poland China animals did not produce PSE muscle. The effect of electrical stimulation of muscle, in vivo or in &To, on postmortem metabolism is discussed further in the section on biochemistry (see Section I V ) . Schmidt et al. (1970a) reported that muscle from stress-susceptible pigs had a shorter time course of rigor mortis than did muscle from stress-resistant pigs. This difference occurred even when the two groups started with the same level of phosphocreatine and lactic acid postexsanguination, as is the case following magnesium treatment of the animal (see Section IV) . Therefore, even though magnesium treatment can retard glycolysis sufficiently to prevent development of the PSE condition, it does not necessarily standardize the postmortem changes in all skeletal muscles of all pigs. Further consideration of the role of Ca*+binding by the sarcoplasmic reticulum during the development of rigor mortis has been reported by Schmidt et al. ( 1 9 7 0 ~ ) . An interesting application of measurement of rigor mortis has been used by Sybesma and van Logtestijn (1967). They constructed a small portable instrument to measure rigor mortis in the whole carcass soon after slaughter. The instrument is spring-loaded and measures the counterpressure in the exposed semimembranosus muscle of the ham. A higher rigor reading indicates a more rapid development of rigor mortis; this information can be used at commercial levels to measure objectively a rapid onset of rigor mortis after slaughter without interfering with the packing-house operation. Rigor values were significantly related to muscle quality characteristics measured at 40 minutes to 24 hours postmortem. Approximately 60% of the carcasses that were in rigor at 40 minutes postmortem showed abnormal muscle quality at 24 hours postmortem. This percentage could be further increased by combination of the rigor value with a measurement of pH at the same time (Sybesma and van Logtestijn, 1967).

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D. MEMBRANECHARACTERISTICS Schmidt et aE. ( 1972b) studied the electromyographic ( E M G ) properties of stress-resistant Chester White and stress-susceptible Poland China animals before, during, and after slaughter. Transmembrane potentials were determined on another series of pigs 45 minutes postmortem, and samples of longissimus were analyzed for high-energy phosphate compounds, lactic acid, and acetylcholinesterase activity. The longissimus muscle from stress-resistant pigs had a greater electrical activity for the first minute postmortem, a longer time course of rigor mortis, normal color, higher initial levels of phosphocreatine and adenosine triphosphate, and lower initial levels of lactic acid. There was no significant difference between muscles of the two groups in cholinesterase activity. Postmortem muscle from stress-susceptible pigs had a lower resting membrane potential than did that from stress-resistant pigs, and it decreased rapidly to very low levels within 45 mintues of death. The lack of EMG activity, the immediate loss of membrane potential, and the shortening that occurs during development of rigor mortis indicates that the muscle fiber membrane may be one of the important defective sites in the stress-susceptible animal. Strong support for the latter statement is given by the evidence that muscle cell membranes in PSS animals are in fact “leaky” and allow the escape of specific muscle cell enzymes into the blood. Analysis of nonplasma-specific enzymes is being used as an identification or detection method for PSS animals; this consideration is explained more fully in Section IV under The Abnormal in Vivo Situation and in Section VII under Detection Methods.

E. MALIGNANT HYPERTHERMIA Case reports of an abnormal response to anesthesia in humans have appeared in recent literature. The condition, known as malignant hyperthermia ( M H ) or malignant hyperpyrexia, is characterized by a progressive increase in body temperature, severe generalized muscle spasm, and a metabolic acidosis after exposure to one or a combination of several anesthetics. The anesthetic frequently employed is halothane ( Fluothane; ICI), a fluorinated hydrocarbon, with or without the use of a depolarizing muscle relaxant. The incidence of malignant hyperthermia is about 1 in 15,000 cases, 3Ooj, of the human cases do not develop rigidity, and mortality is 70y0 (Britt and Kalow, 1970).

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Hall et al. (1966) observed a similar condition in pigs, which they thought was due to the action of suxamethonium. Later, however, it was reported that the syndrome could be provoked by halothane administration in pigs of the Landrace (Harrison et al., 1968, 1969; Berman et al., 1970), Pietran (Sybesma and Eikelenboom, 1969; Allen et aZ., 1970), Poland China (Jones et al., 1972; Nelson et al., 1972) and Yorkshire ( Christian, 1972) breeds. During the First International Symposium on the Condition and Meat Quality in Pigs in Zeist, May 1968, a fatal case of halothane narcosis in a Pietrain pig was demonstrated. The symptoms provoked by halothane were observed to be similar to those that sometimes occur in the same stress-susceptible breed during stress conditions (Eikelenboom and Sybesma 1969; Sybesma and Eikelenboom, 1969; Allen et al., 1970). During the onset of the syndrome a decrease in muscle ATP content was observed, and a specific effect of halothane on cell respiratory mechanisms has been suggested ( Sybesma and Eikelenboom, 1969). The cause of malignant hyperthermia during halothane anesthesia in humans is unknown, although myopathies of different types, such as myotonia congenita or Thomson’s disease ( Churchill-Davidson, 1968) and Barnes myopathy (Steers et al., 1970), have been suggested as predisposing factors. In view of the number of similar characteristics such as familial occurrence, high serum creatine phosphokinase levels, a pronounced development of the musculature, and a relatively high incidence of sudden and unexplained deaths, which have been reported as specific traits in human patients and their respective families (Denborough et al., 1962; Britt et al., 1969; Isaacs and Barlow, 1970; Steers et al., 1970), as well as in pigs susceptible to malignant hyperthermia ( Sybesma and Eikelenboom, 1969; Allen et al., 1970; Woolf et al., 1970; Christian, 1972; Nelson et al., 1972), the stress-susceptible pig appears to be a useful experimental animal for study of this abnormal condition which exists in humans. Certain results obtained in studies on stress susceptibility may prove to be of direct significance in human medicine. The mode of action of halothane appears to be peculiar to skeletal muscle itself. The basic assumption lies in the fact that malignant hyperthermia can be triggered in pigs pretreated with the nondepolarizing drug curare and that curare has no therapeutic effect in averting an already developed case of M H (Harrison, 1971). Moreover, occlusion of blood supply prevents the occurrence of rigidity in the area supplied by the occluded arteries during the onset of the syndrome in humans ( Satnick, 1968).

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Berman et al. (1970) have described the change in the blood during the development of the syndrome in pigs. These changes include an 0, consumption elevated slightly more than would be expected from the elevated temperature, a shift of water into the intracellular space, increased serum Na+ and total protein, a shift of Ca2+and Mg2+into the extracellular space, a release of Pi in plasma, an increase in blood glucose, and an uncompensated metabolic acidosis. Muscle biopsies taken prior to the administration of halothane showed no difference in initial ATP level between malignant hyperthermia syndrome ( MH)-prone and nonprone swine. However, the samples obtained from the MH-prone swine showed a more rapid ATP decline, which was speeded even more when the samples were exposed to halothane (Harrison et al., 1969; Nelson et al., 1972). During the onset of the syndrome, a decline in ATP levels was observed in Pietrain pigs (Eikelenboom and Sybesma, 1969; Sybesma and Eikelenboom, 1969) but not in Landrace (Berman and Kench, 1973) and Poland China pigs (Nelson et al., 1972). Berman and Kench (1973) observed the accumulation of lactate and fructose-1,6-diphosphate and a rapid decline in the concentration of creatine phosphate; ADP was unaffected, but AMP and more particularly IMP accumulated. It was suggested that activation of an ATPase system was the primary event and that glycogenolysis was accelerated by a mechanism other than depletion of stored ATP. Studies on the in dtro effect of halothane mainly involve whole muscular tissue, or its subcellular fragments. However, liver tissue has also been used, since certain people exposed to halothane have exhibited liver damage (Trey et al., 1968).

1. Efiect of Halothane on Oxidative Metabolism Wilson et al. (1966, 1967) injected dogs with the uncoupler 2,4dinitrophenol (DNP) and observed a response similar to that found in malignant hyperthennia. There was a progressive increase in body temperature, muscle rigidity, and metabolic acidosis. They concluded that the mode of action of halothane must be similar to that of DNP. In an experiment designed to measure the direct effect of halothane on isolated rat liver mitochondria, Snodgrass and Piras (1966) demonstrated that halothane uncoupled oxidative phosphorylation of isolated rat liver mitochondria at all three phosphorylation sites and inhibited the oxidation of NAD-linked substrates. As a result of more recent findings, this work has been criticized for two reasons (Miller and Hunter, 1970). First, the concentrations of halothane were far in excess

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of those used clinically which leaves the relevancy of these findings open to question. Second, the 0, uptake was not measured during the exposure to halothane. Instead the mitochondria were exposed to halothane, washed, and resuspended in medium without halothane, and then 0, uptake was determined. More recent results (Cohen and Marshall, 1968; Harris et al., 1971; Miller and Hunter 1970) show that low concentrations of halothane, supposedly obtained in vivo during anesthesia, produce the following results: ( 1) markedly and reversibly depressed state 3 oxidation of glutamate but not of succinate due to inhibition at the NADH-dehydrogenase step; ( 2 ) marginally affected state 4 respiration with NAD-linked substrates giving signs of limited uncoupling with succinate as a substrate, particularly at elevated concentrations of halothane. Later studies by Miller ( 1972) indicated that halothane cannot be considered a true uncoupler. Results similar to those described by Miller and Hunter (1970) were obtained by Eikelenboom (1972) and Eikelenboom and Sybesma (1974) in experiments on rat skeletal muscle mitochondria. It was suggested that a lower activity of oxidation phosphorylation demonstrated previously in muscle mitochondria isolated from stress-susceptible Pietrain pigs ( Eikelenboom and van der Bergh, 1971, 1973; Eikelenboom, 1972) might make these animals more sensitive to a direct or indirect inhibitory action of halothane on mitochondria1 respiration and ATP synthesis ( Eikelenboom and Venvey, 1971; Eikelenboom and Sybesma, 1974). Denborough et al. (1973) found an inhibition of ADP-stimulated oxidation of glutamate as well as a decreased ADP/O ratio in muscle mitochondria isolated from Australian Landrace pigs. No difference was found in the concentration of halothane necessary to induce a sharp fall in the ADP/O ratios between MH-prone and nonprone Landrace pigs. However, mitochondria obtained from both types of Landrace pigs were more susceptible to halothane than were samples obtained from pigs from the abattoir. Brucker et al. (1973) found that succinate oxidation by liver mitochondria isolated from Poland China swine during the development of the syndrome was not inhibited by halothane, whereas in normal animals marked inhibition was observed. The in situ ultrastructure of skeletal muscle mitochondria was observed to undergo drastic configurational changes at the same time. Britt et a2. (1973) reported that mitochondria isolated from skeletal muscle of three humans who survived anesthetic-induced MH showed no difference in respiratory control or oxygen consumption when compared with mitochondria from normal patients. With halothane exposure

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the mitochondria of both groups of patients showed chiefly an inhibition of electron transport plus a small degree of uncoupling. It was concluded, however, that this effect could not account for the clinical and laboratory changes observed in malignant hyperthermia.

2. Efiect of Halothane on Sarcoplamnic Reticulum Isolated sarcoplasmic reticulum from two of the human MH patients mentioned above exhibited a decreased Caz+uptake when exposed to halothane (Kalow et al., 1970). Berman and Kench (1973) observed that Ca2+-accumulatingability of sarcoplasmic reticulum isolated from MH-prone Landrace pigs was enhanced rather than reduced at 1%halothane. However, Nelson et al. (1972) did not observe differences in Caz+ uptake of sarcoplasmic reticulum between normal and MH-prone Poland China pigs in muscle samples also obtained prior to exposure to halothane. Brucker et al. (1973) observed a decreased Caz+-accumulatingability of sarcoplasmic reticulum isolated from Poland China pigs during the onset of the syndrome when compared with sarcoplasmic reticulum of normal swine. No differences in ATPase activity were observed. However, the value of these observations may be disputed, since the muscle samples were taken during or after the onset of the syndrome. Greaser et al. (1969a) observed a rapid decrease of Ca2+-accumulating ability and Ca*+activatedATPase of isolated sarcoplasmic reticulum in postmortem muscle of Poland China pigs. Later studies (Greaser et al., 1969b) strongly suggested that this observation might be explained by the combined effect of high temperature and low pH observed in Poland China muscle in the early postmortem period. Therefore, these observations may explain part of the results cited above. From the literature reviewed to this point, it is evident that the mechanism of action of halothane in triggering the syndrome is by no means clear. The reason for a primary involvement of the energy-rich phosphates which control the release and uptake of Ca2+by the sarcoplasmic reticulum (Weber et al., 1964) is, in our opinion, that the same syndrome can be provoked by exercise in these pigs. However, it is difficult to assess what is primary and what is secondary during the onset of the syndrome, since a number of changes occur in a rapid and drastic way. The origin of the increased heat production, from which the syndrome takes its name, is still not understood. Various sources, including uncoupling of oxidative phosphorylation, muscle contraction, hydrolysis of

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energy-rich phosphates, anaerobic glycolysis, neutralization of lactic acid, and loss of central heat regulatory mechanisms, have been suggested to be at least partially responsible for the excessive heat response.

3. Treatment and Prevention Once the MH syndrome develops in pigs, induced by either halothane or exercise, the best therapy seems to be to cool the body in an ice bath or cold water. Intravenous infusion of sodium bicarbonate (Muylle et al., 1968) or tris (hydroxymethy1)aminomethane (THAM) (Harrison et al., 1969) may prove to be of some success in correcting the acidotic situation. Procaine hydrochloride has been reported by Harrison ( 1971) as a promising treatment for pigs, but it failed to produce results in English Landrace pigs ( Hall et al., 1972). The first method of prevention can be through identification of potentially susceptible animals. As will be discussed in Sections IV and VII, serum enzyme levels, particularly that of creatine phosphokinase ( CPK ), as well as metabolite levels in muscle biopsies, appear to be useful indications, although there are limitations. Eikelenboom (1972) suggested that the use of halothane as an anesthetic is contraindicated in meaty-type pigs. Surgery performed under barbiturate anesthesia did not reveal any complication in MHprone Pietrain pigs; thiopentone anesthesia also seems to give protection against the initiation of the syndrome (Hall et al., 1972). Various publications suggest that early recognition of susceptible pigs is made possible by subjecting young pigs to a halothane/oxygen mixture delivered through a facemask (Hall et al., 1972; Brucker et al., 1973). As soon as signs of muscle rigidity develop, recovery measures may be taken which have proved to be successful in a reasonable number of cases. These observations are of particular interest for those who study the inheritance of the MH or the related PSE-PSS condition. However, in all these conditions the reactions that develop are in our opinion more qualitative than quantitative in nature, thus making these studies more complicated even when the susceptibility is controlled by a single gene. A certain type of muscle metabolism induced by or associated with muscular hypertrophy in pigs seems to be the primary predisposing factor.

I I I.

EN DOCRI N E I NTE RRELATlONS H I PS

Since the early reports of PSE and its related problems, the endocrine system has been suspected of functioning abnormally ( Ludvigsen, 1953,

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1954, 1957). Although the answers are not yet all available, it is apparent that some of the early hypotheses should be modified slightly. A. PITUITARY FUNCTION Kraeling and Gerrits (1972) examined the effect of hypophysectomy on the postmortem glycolytic rate of porcine longissimus muscle and demonstrated that the pituitary has a regulatory role in muscle metabolism. When the levels of biochemical intermediates of the longissimus muscle of hypophysectomized pigs were compared at various times postmortem with those of sham-operated controls, the authors observed a slower accumulation of lactic acids, a higher muscle pH, a lower muscle temperature, and lower levels of glucose-6-phosphate and fructose-6phosphate with a slower depletion of glycogen stores. These results suggest that through the action of the pituitary hormones (growth hormone [GH] , thyrotropin [TSH] , and adrenocorticotropin [ ACTH] ) the endocrine system has a profound influence on muscle metabolism and very likely on PSE and PSS.

1 . Growth Hormone Baird et al. (1952) reported that the anterior pituitary glands of pigs selected for rapid growth contained more growth hormone than did those from pigs selected for slow growth. Turman and Andrews (1955) injected growth hormone into pigs and reported that the treatment resulted in slightly faster gain, significantly better feed efficiency, and meatier carcasses. Similarly, Wismer-Pedersen ( 1968) suggested that a considerable part of the increased length and meatiness of the Landrace pig from 1950 to 1965 may have been due to an indirect selection for increased production of growth hormone. However, Siers and Hazel ( 1970) and Siers and Swiger (1971) report lower levels of circulating growth hormone in faster-gaining and meatier swine and suggest that these pigs have a more rapid rate of utilization of growth hormone. Also of interest is the observation by Althen et al. (1972) that selection for or against backfat did not influence growth hormone levels, since circulating levels of growth hormone in pigs selected for high or low backfat did not vary significantly at weaning or slaughter. Marple and Aberle ( 1972) compared stress-susceptible Poland China and stress-resistant Chester White gilts and found no significant difference in levels of plasma growth hormone. Stress-susceptible pigs, however, had significantly different saphenous arterial-venous levels, whereas

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the stress-resistant pigs did not-a finding which suggested a greater rate of tissue utilization and metabolism of growth hormone in stress-susceptible pigs. 2. Adrenocorticotropin Ludvigsen (1957) noted that the ACTH content of pituitary glands from seventy PSE pigs was approximately one-half that found in an equal number of pituitaries from normal pigs. Since administration of hydrocortisate allowed his stress-susceptible pigs to survive a period of muscular exercise, Ludvigsen ( 1957) concluded that the stress-susceptible pigs suffered from a lack of ACTH and consequently, a lowered stimulation of the adrenal cortex. Similarly, Henry et al. (1958) reported a deficiency of glucocorticoids in stress-susceptible pigs, but proposed that the disorder may be the result of a pituitary hyposecretion of ACTH which induced an atrophy of the adrenal cortex, rather than a primary adrenal insufficiency. The above hypotheses regarding ACTH in stress-susceptible pigs should be modified somewhat in light of the findings of Marple et al. (1972c), who determined the level of circulating ACTH in normal and stress-susceptible pigs. By using a radioimmunoassay for porcine ACTH, they found significantly higher resting levels of ACTH in plasma from stress-susceptible pigs. All blood samples were obtained with a minimum of stress by utilizing catheters implanted at least one week prior to sample collection, In addition to having higher levels of plasma ACTH at rest, the stress-susceptible pigs were capable of doubling their plasma ACTH level in response to exposure to fluctuating temperature environmentsa finding that indicated no apparent inability of the animals to release pituitary ACTH when exposed to a stressor.

B. ADRENALCORTEX 1. Histology

The earlier hypotheses of adrenal insufficiency and adrenal atrophy provided the basis for morphological studies of the adrenal cortex. Cassens et al. (1965) examined adrenal glands from Poland China and Chester White pigs and found no apparent difference in cell morphology with routine hematoxylin and eosin techniques. Frozen sections stained with Sudan black B, however, revealed a significantly higher incidence of large lipid masses in the zona reticularis of adrenal gland from the Poland China pigs. This finding was interpreted as representing a

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degenerative change in adrenocortical function and was significantly correlated with a rapid postmortem muscle pH decline and a shorter sarcomere length in muscle at 24 hours postmortem. A similar study by Howe et al. [ 1969) revealed that lipid masses could be induced in the adrenal cortices of pigs reared in environments of fluctuating temperatures (13”to 29°C or 21” to 32°C) or in environments with a low relative humidity ( 17y0 R.H. or 21 to 24% R.H.). There was no significant environmental effect on adrenal gland weight, and within-treatment correlation coefficients between adrenal weight and abundance of lipid masses were not statistically significant. Similar results were found by Panaretto and Ferguson (1969a,b), who exposed newly shorn sheep to a cold, wet environment and reported that six of ten untreated sheep died, whereas there were no deaths among ten sheep receiving cortisone. The authors noted heavier adrenal weights and hemorrhagic adrenal cortices were more heavily infiltrated with lipid than were those of the survivors. It was suggested that the enlarged, lipid-laden hemorrhagic adrenals were the result of excessive ACTH stimulation prior to death. Evidence indicating adrenal hyperplasia due to increased ACTH release by stress-susceptible swine has been reported by Ball et al. ( 1971). They suggested that, owing to repeated intermittent episodes of stress, the stress-susceptible pigs may acquire a relative adrenal inadequacy so that they respond to a stressor with lower than normal adrenocortical activity. Rantsios ( 1972) attempted to relate the thickness of the zona glomerulosa of Pietrain adrenal glands with postmortem fall in pH. He reported a marked reduction of width of zona glomerulosa in adrenal glands from Pietrain pigs compared to other breeds. It was suggested that stress-susceptible pigs have an impaired development of the adrenal zona glomerulosa. 2.

Glucocorticoids

The suggestions of adrenal insufficiency in pigs with “muscle degeneration” (PSE) by Ludvigsen ( 1957) and Henry et al. ( 1958) have resulted in many subsequent studies on the adrenal function of swine. Tope1 et al. ( 1967) observed plasma 17-hydroxycorticosteroid ( 17-OHCS) levels in blood samples taken at death from barrows and gilts of four breeds. The results were analyzed on the basis of loin quality. They found lower (nonsignificant) plasma 17-OHCS levels in pigs yielding PSE carcasses than in the DFD group. Judge et al. (1966, 1968) measured urinary 17-ketosteroid ( 17-KS) and 17-hydroxysteroid ( 17-

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OHS) excretion rates from Poland China and Chester White pigs and found no significant differences between steroid excretion rates of pigs yielding PSE carcasses and those of pigs yielding normal quality carcasses. However, the pigs that yielded PSE carcasses tended to have lower 17-KS and 17-OHS excretion rates. The effect of exogenous adrenocortical steroid therapy has been examined by Ludvigsen (1969a). Three Poland China pigs were injected intramuscularly with 100 or 200 mg of Nordisolone 60 minutes prior to a 30-minute exposure to a heat stress of 43" to 45°C. As controls, three Poland China and three Chester White pigs were exposed to the heat stress, and three Chester Whites were slaughtered normally. The Nordisolone injection appeared to slow the rate of postmortem pH decline in the longissirnus in a dose-dependent response. In addition, the postmortem temperature of the loin was lower among injected pigs and resembled that of the heat-stressed Chester White pigs. Marple et d. (1969) induced adrenal atrophy in an effort to examine the effect of adrenal insufficiency on stress response and postmortem muscle characteristics. The injection of 100 mg of Prednisolone daily for 10 days resulted in significant adrenocortical atrophy and increased the susceptibility of the pig to stress as determined by the ability to withstand 5 minutes of forced exercise. The results revealed a significant reduction of average blood pH at death among the adrenal-insufficient group and a significant increase in the plasma lactic acid level due to stress. The glycogen content of the longissimus muscle was significantly increased owing to the Prednisolone injection, but the ultimate level of lactate produced by postmortem glycolysis was not affected. The induced adrenal insufficiency and corresponding low levels of plasma 17-OHCS at death did not significantly increase the rate of postmortem glycolysis as determined by muscle pH decline. Perry et al. (1972) related blood levels of 17-OHCS at death and histamine levels of longissimus muscle at death, 1, and 24 hours postmortem to postmortem muscle pH and ultimate muscle quality. Muscle histamine content at death was significantly related to muscle pH at 3 hours postmortem. It was noted that high plasma 17-OHCS and muscle histamine levels at death appeared to be related to poor meat quality.

3. Mineralocorticoids and Electrolyte Levels Tope1 et al. (1967) found no significant differences in serum or muscle sodium or potassium concentrations between groups of pigs yielding severely PSE, slightly PSE, or DFD carcasses. The DFD group, how-

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ever, had (not statistically significant ) the highest plasma 17-OHCS levels and average adrenal gland weights when the three groups were compared. Dekker ( 1969) compared the sodium, potassium, cortisol, and corticosterone content of slaughter blood from Pietrain pigs and found no significant relationship between electrolyte and steroid levels in the same sample. Also, no clear correlation was noted between meat quality and corticoid levels. However, Passbach et al. (1970) reported that intravenous injection of 1.5 mg of aldosterone 90 minutes antemortem resulted in a significant reduction in muscle quality score (that is, increased PSE ) ; the oral administration of an aldosterone blocking agent resulted in a significant increase in the quality score and a decrease in transmission value (meat quality) index. The treatments, however, did not significantly alter serum or muscle sodium or potassium levels. Later work by D. E. Galloway and B. B. Marsh (unpublished data, 1973) failed to substantiate the claim of aldosterone-induced PSE. Schmidt et at. ( 1970b) slaughtered ten stress-susceptible Poland China and ten stress-resistant Chester White pigs and characterized their serum electrolyte and muscle biochemical properties. The stress-susceptible pigs went into rigor mortis significantly faster and had a lower meat quality score at 24 hours postmortem. A comparison of serum electrolyte levels revealed significantly higher levels of serum calcium, sodium, and inorganic phosphorus and a higher calcium-to-magnesium ratio in the stress-susceptible pigs. Serum levels of cholesterol, total protein, glucose, potassium, and magnesium were not significantly different in the two groups. Similarly, Dekker ( 1971) compared muscle electrolyte levels of Dutch Landrace and Pietrain pigs. No relation was found between electrolyte levels and meat quality (transmission value), but the Pietrains had significantly greater muscle potassium and magnesium and significantly less muscle calcium than the Dutch Landrace. Muscle sodium levels did not differ significantly. The author noted that the higher potassium levels of Pietrain muscles may be indicative of a hypoaldosteronism or adrenal hypofunction, or perhaps the higher potassium and magnesium levels contribute to a more intensive postmortem glycolysis among Pietrains. A related study by Weiss et al. ( 1971b) compared muscle and serum electrolyte levels of two strains of fat and lean pigs at various ages. Data pooled over live weights of 1 to 137 kg revealed that the lean pigs had significantly greater potassium and significantly less zinc and copper in longissimus muscle. Muscle sodium, magnesium, calcium, and iron levels did not differ significantly in the two strains. Also, no significant differ-

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ences were noted for serum sodium, potassium, chloride, magnesium, zinc, or copper when data for fat and lean strains were compared.

4 . Adrenocortical-Pituitary Function References cited in the adrenal histology section noted that stressful environmental conditions can induce alterations in adrenal morphology, thereby indicating that the environment can influence pituitary-adrenal function. Marple et al. (1972a) used stress-resistant crossbred pigs and noted significant endocrine responses after exposure to various environments. Plasma ACTH levels increased significantly after exposure to cold with high humidity or to high temperature, whereas plasma corticoid levels rose in response to cold with low humidity but declined during exposure to cold with high humidity or to high temperture with low or high humidity. These results were interpreted as indicative of an alteration in the turnover rate of plasma corticoids by increasing environmental stress. Plasma glucose and free fatty acid levels were significantly elevated after exposure to cold with high humidity or to a hot environment. A report by Tope1 et al. (1971), however, found significantly lower blood glucose levels in pigs maintained at 6°C for 2 days. Endocrine responses of stress-susceptible and stress-resistant pigs in various environments were also studied by Marple et al. (1972b). Plasma ACTH levels were significantly increased in response to high humidity (97% R.H.) at 21°C or to high temperature (32"C,%yo R.H.). Plasma corticoid levels declined significantly among the stress-susceptible pigs when they were exposed to low (4"C, 46% R.H.), moderate 21"C, 97% R.H.), or high temperatures. A similar plasma corticoid response among the stress-resistant pigs was noted only in the moderate and high temperature treatments. Plasma glucose was increased among the animals exposed to the low temperature conditions but was not influenced by the moderate or high temperature treatments. A similar study by Kallweit and Haase ( 1971) compared various responses of stress-susceptible pigs to a high temperature with high humidity; they found significantly lower blood glucose levels at rest and during exposure to 37°C and 100% R.H. among pigs that died during or immediately after the test. Adrenocortical function and responsiveness in vitro have been examined by Dvorak (1967, 1972). Plasma 17-OHCS levels were highest at birth and decreased to adult levels by the second month of life. Adrenal tissue from piglets at birth was capable of producing three times

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as much 17-OHCS (per milligram of tissue) as was produced by similar tissue from adult sows under identical assay conditions. This decrease in adrenal responsiveness was essentially complete during the second month of life, since adrenal activity assays which compared tissue from 33- to 52day-old animals with that from old sows were not significantly different. A similar decline in plasma 17-OHCS levels with age has been noted by Weiss et al. ( 1971a). These reports of lower circulating levels of adrenocorticoids in the presence of higher plasma ACTH levels among stress-susceptible pigs and during exposure to stressful environments could be related to the decreased adrenal responsiveness with age noted by Dvorak (1967, 1972) and to the adrenal atrophy proposed by Henry et al. (1958). As was noted by Judge and Marple (1971), there is evidence to suggest at least a relative, if not actual, adrenal insufficiency in stress-susceptible pigs. Investigations into the mechanisms responsible for this altered adrenal function revealed some new facts. Sebranek et al. (1973) compared in uiwo adrenocortical response with that of exogenous ACTH in stresssusceptible and stress-resistant pigs. In an effort to inhibit endogenous ACTH release, all pigs were pretreated with dexamethasone. Blood samples were drawn through venous catheters before and 60 minutes after the intravenous infusion of 10 I.U. of porcine ACTH. The net change in plasma corticoid levels indicated a significantly lower response by the stress-susceptible pigs. Similar results were reported by Lister ( 1971). Tope1 (1969) noted a lower increase in plasma 17-OHCS in stresssusceptible pigs exposed to a controlled stress. If there were no difference in the metabolic clearance rate or biological half-life of cortisol in stress-susceptible swine, the above evidence of adrenal insufficiency would be conclusive. However, Marple and Cassens ( 1973) compared the turnover rate and metabolic clearance rate ( M C R ) of cortisol in normal and stress-susceptible pigs. (MCR is defined as the volume of blood that is completely and irreversibly cleared of the test substance per unit of time.) Labeled cortisol was removed from the blood of stress-susceptible pigs significantly faster than in normal pigs. Also, the cortisol production rate, estimated by the cortisol turnover rate, was increased significantly in stress-susceptible pigs. This observation indicates that, instead of an adrenal insufficiency, the stress-susceptible pig has greater than normal adrenocortical secretory activity with increased metabolism of glucocorticoids. It is possible, however, that, although the stress-susceptible pig has a greater production rate of adrenocorticoids at rest, during a severe stress such an animal may not be able to produce adequate levels of corticoids to maintain homeostasis. This

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idea is supported by the observation that treatment with exogenous glucocorticoids prior to an anticipated stress often improves the survival rate of stress-susceptible pigs ( D. N. Marple, unpublished observation).

C. ADRENAL MEDULLA Catecholamines influence cardiovascular physiology and general metabolism. Judge and Stob (1963) studied the effects of daily electrical shocking and epinephrine or saline injection for 92 days on various growth factors in lambs. Epinephrine injection depressed weight gain and fat disposition but did not Significantly alter muscle or bone growth. The epinephrine treatment also resulted in adrenal hypertrophy, reduced muscle glycogen content, increased ultimate muscle pH postmortem, and increased liver glycogen. Investigations on the acute effects of epinephrine injection in pigs were done by Hedrick et al. (1964). Epinephrine (4 mg per 45 kg) injected 24 and 12 hours preslaughter significantly reduced liver and muscle glycogen levels. At 30 minutes postmortem, the pH values of longissimus and semimembranosis muscles were higher in treated pigs than in nontreated pigs, and subsequent changes were less rapid. Also, the ultimate muscle p H from epinephrine-treated pigs was significantly higher. However, when the time between epinephrine injection and slaughter was shortened to 10 minutes, Aberle and Merkel (1968) found no significant treatment effect on rate of postmortem p H fall, ultimate muscle pH, Munsell value, or transmission value. Pigs receiving epinephrine 10 minutes antemortem had significantly greater total muscle phosphorylase activity and slightly greater phophorylase a activity. Adrenal medullary response to environmental stress was examined by Stefanovic et al. (1970). Three pigs were exposed to environments of 20"C, 5"C, and 33°C and daily urinary vanilmandelic acid (VMA) excretion rates were determined. Pigs maintained a t 5°C or 33°C produced two to three times as much VMA per day as did pigs maintained at 20°C. The authors concluded that the stress of exposure to adverse environmental temperatures resulted in increased catecholamine production. Judge et al. (1966, 1968) compared urinary catecholamine excretion rates of pigs yielding PSE and normal carcasses and found no significant relationship between daily catecholamine excretion by pigs being held at 21°C to 24°C and their ultimate meat quality or muscle properties. Plasma epinephrine levels a t rest and after forced exercise were measured by Weiss ( 1971). When stress-susceptible pigs were compared with fat-strain controls, the stress-susceptible pigs had significantly lower

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plasma epinephrine levels both at rest and after exercise. Exercise also resulted in significant declines in circulating epinephrine levels in both strains. Topel ( 1972) (citing preliminary results) presented data suggesting higher catecholamine secretion rates by stress-susceptible pigs. In a comparison of urinary VMA and epinephrine levels from stress-susceptible and stress-resistant Danish Landrace pigs at rest, there was little difference in urinary VMA levels, but tIie urinary epinephrine excretion rate of stress-susceptible pigs was on the average three times as great as the control rate. However, plasma epinephrine levels were lower among the stress-susceptible pigs. Therefore, the data suggest an increased rate of utilization of epinephrine in stress-susceptible pigs at rest, a condition similar to that noted for cortisol metabolism rates by Marple and Cassens (1973). Topel (1972) also reported data on the effect of electrical stimulation stress on plasma and urinary epinephrine and urinary VMA. Plasma levels of epinephrine rose and fell sharply after electrical stimulation in the stress-susceptible pigs but were higher than control levels at 30 and 60 minutes after the stress. Urinary epinephine during the next day was higher among the stress-susceptible pigs, but the increase in urinary epinephrine was greatest among the stress-resistant pigs. Urinary VMA excretion was greatest from the stress-susceptible pigs; this finding, combined with the epinephrine data, indicates that stress-susceptible pigs metabolize, and therefore must produce greater amounts of catecholamines per day at rest or under stressful conditions.

D. THYROID FUNCTION Ludvigsen (1953, 1954) suggested that thyroid hypofunction was related to the development of PSE muscle, since the incidence of PSE was greater in the summer when thyroid activity would be lower. The reports suggest that the PSE condition could be induced by feeding methylthiouracil, whereas feeding iodinated casein improved the muscle quality of pigs predisposed to become PSE. Briskey (1963) found similar results from feeding methylthiouracil, while Topel and Merkel (1966) fed methylthiouracil or tapazole and could find no significant treatment effect on quality of longissimus muscle. Some of the pigs fed methylthiouracil did develop a slightly PSE condition in the gluteus medius muscles, but the pigs fed tapazole had normal-colored, firm, and dry ham muscles. Topel and Merkel (1966) concluded that goitrogenically induced hypothyroidism per se had little effect on ultimate physical and biochemical muscle properties.

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Ludvigsen ( 1969b) made histological comparisons of thyroid tissue from Poland China, Chester White, Hampshire, Duroc-Yorkshire crossbred, Pietrain, and Danish Landrace pigs. He found that the Pietrains had the heaviest thyroid gland weights, and, in general, the thyroids of stress-susceptible pigs appeared to be larger, since the thyroids from eight Danish Landrace pigs that died were almost twice as heavy as the average thyroids of the 33 Danish Landrace that survived. The author noted that the heaviest thyroid glands had the largest alveoli, and most variable alveoli were from thyroids of Pietrains where a stratified epithelium (indicative of thyrotropin stimulation) of three to four layers was often found. Similarly, a stratified epithelium was noted in thyroid of pigs fed methylthiouracil. A comparison of thyroid gland weights of different strains of pigs, expressed in grams of thyroid weight per 100 kg of the theoretical amount of total meat, revealed that the Pietrain has the lowest thyroid and adrenal gland weights per amount of meat (Unshelm et al. 1971). It was suggested that selection for the meat-type pig has altered endocrine function, resulting in various deficiencies. Studies of thyroid function in swine have not yielded conclusive results. Romack et al. (1964) estimated thyroid secretion rate indirectly by measuring the rate of release of l3II from thyroids of pigs receiving daily injections of L-thyroxine while on a diet containing tapazole. When the ambient temperature was less than %OF, Poland China pigs had significantly higher daily thyroid secretion rates than did Hampshires or Yorkshires. When the temperature was greater than %OF, there was no increase in the secretion rate by the Poland China pigs, but Yorkshires doubled their secretion rate to a level not significantly different from that of the Poland China pigs but significantly greater than that for Hampshires and Durocs. This apparent increase in thyroid secretion rate as temperature increased is not consistent with other theories of thyroid function (Collins and Weiner, 1968), but in replicate analyses of the Hampshire pigs at two ages, thyroid secretion rates, as expected, decreased with age, indicating that the method should be reasonably valid. Judge et al. (1966, 1968) examined thyroid function in normal and stress-susceptible pigs and found a trend toward lower thyroid 1311uptake and significantly higher PBI131 levels among stress-susceptible pigs. Also, the PBI levels were significantly more variable among stresssusceptible pigs exposed to a warm environment. The results of the study do not clearly support the hypothesis of hypothyroidism in stresssusceptible pigs. It was suggested that there might be an increased utilization rate in the periphery of stress-susceptible pigs. Lister (1972)

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compared thyroid function in Pietrain, Landrace, and Large White pigs and found evidence suggesting hyperthyroid tendencies among Pietrains. From blood samples taken during anesthesia, Pietrains had “free thyroxine indexes” similar to those of Large Whites; the T, uptake ratios for the Pietrains were noticeably lower, indicating a greater saturation of the thyroid-binding globulin. Eikelenboom and Weiss (1972) compared thyroid function in Pietrain and Dutch Yorkshire barrows and gilts before and after treadmill exercise. At rest, the Pietrains tended to have higher serum T, levels owing to the significant difference between gilts of the two breeds. Exercise resulted in a significant decrease in serum T, levels for gilts of both breeds, while the levels for barrows increased among Pietrains and decreased among the Dutch Yorkshires. In general, the more recent studies have failed to support the early hypothesis of thyroid hypofunction in stress-susceptible swine. In fact, the results suggest that stress-susceptible pigs may have greater than normal thyroid activity or a decreased utilization rate of thyroxine. In an effort to relate the various endocrine observations to some established clinical abnormality, we suggest that many of the symptoms of stress susceptibility are similar to those of hyperthyroidism. For instance, Pittman (1972) noted that plasma ACTH levels and cortisol metabolic clearance rate and turnover rate are increased, while plasma cortisol levels after ACTH stimulation are lower in hyperthyroidism and in stresssusceptible swine. Serum thyroxine levels, PBI levels, and thyroidal radioiodine uptake are elevated in hyperthyroidism, and similar findings have been noted for stress-susceptible pigs with the exception of nonsignificant differences in thyroid 1311 uptake in Poland China pigs. Also, stresssusceptible pigs tend to be more nervous with involuntary muscle twitching or tremors, have lower levels of muscle ATP and creatine phosphate, and are generally leaner individuals with less carcass fat and more carcass protein-all characteristics of hyperthyroidism. Conversely, stresssusceptible pigs usually have elevated levels of serum creatine phosphokinase, lactic dehydrogenase, glutamic-oxalacetic transaminase, and aldolase-a circumstance similar to that found in hypothyroidism. Also, hypothyroidism is characterized by an increased muscle volume which seems to be associated with stress susceptibility. Therefore, it would appear that the disorder is not simply the result of altered thyroid function with its corresponding side effects on pituitary-adrenocortical function, but may also involve some form of a myopathy resulting from the selection for larger muscle conformation.

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MUSCLE BIOCHEMISTRY A. ANAEROBICMETABOLISM

I. Metabolic Intermediates Hallund and Bendall (1965) demonstrated that muscle excised from Danish Landrace pigs and then exposed to a brief tetanus had a lower initial pH and an increased rate of pH fall compared with unstimulated controls kept under anaerobic conditions at 37°C. This effect seems unique for pig muscle and does not occur in rat, rabbit, or frog muscle (Bendall, 1966). Later experiments in which English Large White pigs were pretreated with curare confirmed these results. Pretreatment with curare resulted in high ATP, CP, and initial pH level. Because the very slow pH fall induced in this way was comparable with that found in rabbit muscle pretreated with Myanesin and in muscle from other species, it was concluded that there is essentially little or no difference between breeds or species in the rate or time course of postmortem changes (Bendall, 1966). The cause of extreme variability in rate of pH fall observed in commercial practice must be sought in the intensity of nervous stimuli reaching the muscle before and during slaughter (Bendall, 1966). It was argued that the above observations did not support the hypothesis of Briskey and Wismer-Pedersen (1961) that this variability is due to variability in amounts or activity of one or another of the glycolytic enzymes. It was calculated that the glycolytic enzymes have a potential rate forty times as great as is necessary to explain the most extreme rates of lactic acid production observed in pig muscle. Therefore glycolysis could easily keep in step with the most extreme rates of ATPase activity postmortem. Bendall (1961, 1966) suggested the variability in overall ATPase activity postmortem was primarily responsible for the variability in postmortem pH fall in porcine muscle. However, Sair et a,?. (1970) observed that anesthetized Poland China pigs treated with curare still had a more rapid postmortem metabolism in muscle than did similarly treated Chester White pigs. The same is true for Pietrain pigs as compared with Dutch Yorkshire pigs (Eikelenboom, unpubl. ). It was observed that lactate levels immediately postmortem were 15 pmolesl gm in curarized stress-resistant animals, but almost doubled in untreated control stress-resistant animals and in curarized stresssusceptible animals. The difference in lactate levels between treated and control stress-resistant animals suggests that muscular activity and con-

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traction at the time of death play a role in postmortem metabolism, but they cannot be the main factors involved, as was suggested (Bendall, 1966), when the lactate levels in stress-susceptible pigs are taken into account. These observations also exclude the possibility that the increased lactate levels are caused by vasoconstriction, as has been suggested (Lister, et al., 1970), because the anesthesia and subsequent curare treatment should prevent not only muscular contractions but also anoxia due to vasoconstriction. It was concluded that the low pH in the longissimus muscle of stress-susceptible animals was due to a marked susceptibility of the muscle to anoxia, particularly that associated with exsanguination, although muscular stimulation did contribute partially to the elevated lactic acid levels in the musculature of stress-susceptible pigs (Sair et al., 1970). Besides differences in muscle lactate accumulation between stress-susceptible and stress-resistant pigs, differences were also present in the rate of CP and ATP breakdown. However, of particular interest is the observation that lower CP levels were found in the muscle of curarized stress-susceptible pigs in biopsies taken prior to exsanguination than were found in muscle of curarized stress-resistant pigs. Since estimates of total creatine revealed no difference between the groups, it was concluded that both breeds have the same potential for producing CP, assuming that their energy source is adequate. In all the samples taken in this study, a relationship between CP and lactate levels was found, whether the animals were stress-susceptible or not (Sair et al., 1970.) McLoughlin (1970) observed in Irish Landrace pigs that curare treatment prior to captive bolt stunning resulted in a higher initial pH and lower rate of pH fall than was found in muscle from pigs stunned without pretreatment. While the curare treatment resulted under these conditions in unavoidable antemortem stress and even anoxia as a result of respiratory difficulties induced by the treatment, and the control animals were subjected to a minimum of stress prior to stunning, it seems unlikely that these factors played a role in the induction of a low initial muscle pH in the control group. McLoughlin (1970) suggested that it was unlikely that the release of adrenaline following stunning and exsanguination was a major factor responsible for the low initial pH of muscle because of the marked elevation in muscle pH following curarization. It was concluded, therefore, that neural stimuli entering the muscle at the time of death were the major factors involved in the rapid postmortem glycolysis in porcine muscle. This has also been suggested by Bendall ( 1966). Furthermore, McLoughlin ( 1970) observed that in V ~ U O stimulation of the gastrocnemius muscle via the sciatic nerve provoked a more rapid postmortem glycolysis than was found in the experiments

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with excised muscle specimens in vitro (Hallund and Bendall, 1965; Bendall, 1966; McLoughlin and Tarrant, 1969; Tarrant et al., 1972). The discrepancy in results between the work of Sair et al., (1970) and that of McLoughlin (1970) might be explained by the fact that the former used both stress-susceptible and stress-resistant pigs, whereas the Irish pigs used in the study of the latter are supposed to be more stressresistant, with a low incidence of PSE ( McLoughlin, 1965). Moreover, one group of animals in McLoughlin’s experiment was subjected to halothane but did not develop the malignant hyperthermia syndrome (McLoughlin, 1968, 1970), a condition characteristic of stress-susceptible pigs (see Section 11). The rate of pH fall was slower in muscle from these animals than in muscle taken from curarized but not preanesthetized pigs killed by captive bolt stunning. In the experiments of Bendall (1966) and Sair et al. (1970), the experimental pigs were anesthetized before curarization and compared with control pigs which were stunned electrically or by captive bolt pistol. These methods of stunning cause considerable muscular contraction. However, when anesthetized pigs receiving curare treatment are compared with untreated anesthetized control animals, a favorable effect of curare can be observed on postmortem metabolism in stress-resistant pigs. In stress-susceptible animals, the curare treatment was more effective than in captive-bolt-stunned pigs, but less effective than in anesthetized pigs. The lack of favorable effect may be due to the fact that lactate levels observed at various times postmortem in the musculature of curarized stress-susceptible pigs were three to four times as high as those found in similarly treated stress-resistant pigs ( G. Eikelenboom, unpubl. ) .

2. Effectof Myotomy Koch et al. (1970a) studied the effect of myotomy at the time of exsanguination on pH decline, levels of glycolytic intermediates, and qualitative properties of porcine longissimus muscle. Myotomy at or shortly after exsanguination stimulated contractile activity, increased the rate of postmortem glycolysis, and tended to decrease ultimate qualitative properties of the muscle. The authors compared longissimus muscle incised at the time of exsanguination with muscle not incised until 45 minutes postmortem; the pH values were lower, glycogen, Am, and CP contents were lower, and lactate was higher in the longissimus muscle at all time periods studied through 2 hours postmortem. The effect of myotomy was greater in normal quality muscle. In related work, the same authors (Koch et al., 1970b) reported that rapid chilling of the rectus femoris

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at early postmortem time periods ( 0 to 15 minutes) markedly decreased the effects of the contractile response that accompanied myotomy of the longissimus dorsi. The effect of rapid chilling was greatest in low-quality muscle. 3. Effect of Magnesium Anesthesia induced for 20 minutes or more prior to exsanguination by intravenous injection of MgSO, resulted in higher levels of CP and ATP and lower levels of lactate in muscle than were found in anesthetized curarized stress-susceptible pigs. However, MgSO, treatment had no effect on stress-resistant pigs (Sair et al., 1970). Campion et al. ( 1971) used a whole-body perfusion technique and observed that MgSO, addition in the perfusate resulted in higher initial muscle pH levels, a slower rate of pH fall, and a delay in the onset of rigor mortis in stress-susceptible pigs when they compared these vaIues with values from muscle biopsy samples taken from the same animals prior to the administration of MgSO,. Addition of CaCl, instead of MgSO, did not affect the initial pH or the time necessary for the muscle to reach 50% extensibility but did increase the rate of pH fall. Lister and Ratcliff (1971) injected lethal doses of magnesium sulfate intravenously less than 1 minute prior to exsanguination in pigs of the Landrace, Large White, and Pietrain breeds. A significant retardation of the rate of glycolysis postmortem in terms of changes in ATP, CP, and lactate levels was observed in the treated pigs from all breeds when they were compared with untreated controls stunned electrically. However, the initial pH, CP, and ATP values were lower and the subsequent fall in pH was still more rapid in the muscles from the Pietrain pigs than in muscles of pigs from the other breeds. No effect of treatment was observed in the pale color and two-toning which tends to occur in the Pietrain muscle. Therefore, this observation does not fully support the work cited previously and may be due to breed differences or treatment procedure. Several specific actions of Mg", supported by evidence from the literature, have been suggested as possible mechanisms for the effect on postmortem metabolism. Such suggestions include inhibition of muscle ATPases, decreasing the sensitivity of the motor end-plate region, induction of vasodilation, and a stabilizing effect on the membranes (Sair et al., 1970; Campion et al., 1971) . 4 . Eflect of Anoxia The role of anoxia in the etiology of PSE musculature has been investigated by Lister et al. (1970), who allowed anesthetized stress-susceptible

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and stress-resistant pigs to breath 100% oxygen for 30 minutes or 100% nitrogen for 10 minutes prior to death. The rates of lactate accumulation and the rate of energy-rich phosphate depletion were similar in muscle samples from stress-resistant pigs breathing either nitrogen or oxygen, but were much slower when compared with those from stress-susceptible pigs breathing nitrogen and, to a smaller extent, from those breathing oxygen. It was concluded that the longissimus muscle of stress-susceptible pigs is highly sensitive to the anoxia induced by the administration of 100% nitrogen. Additionally, the greater amounts of lactate in the musculature of stress-susceptible pigs was suggested to be the result of a higher sensitivity to anoxia created by the exsanguination process itself. Sair et al. (1972) subjected muscle sample strips taken from MgS0,treated pigs immediately postmortem to a nitrogen or an oxygen atmosphere. No difference in postmortem lactate accumulation was observed between the two types of pigs when muscle strips were incubated in oxygen, but the strips from stress-susceptible pigs accumulated lactate significantlyfaster when incubated in nitrogen. The use of 'ttu-blocking and p-stimulating agents permitted Lister et al. (1970) to investigate the hypothesis that an anoxic condition may be induced in the musculature of stress-susceptible pigs owing to vasoconstriction as a result of an adrenergic response at the time of death. The use of these drugs was found to be of some benefit in slowing postmortem metabolic rates (see Section 11, B ) . The work of Sair et al. (1970) and Lister et al. (1970) led Lister (1969) to the conclusion that stress-susceptible Poland China pigs are more prone to anoxia resulting from exsanguination, and stress-resistant (Chester White) pigs are more sensitive to muscular stimulation. However, in spite of treatment with 100% oxygen (Lister et al., 1970) or curare (Sair et al., 1970), stress-susceptible pigs still have lower ATP and CP levels and higher lactate levels postmortem in their musculature; even antemortem biopsy samples contain lower CP and higher lactate than do samples from similarly treated stress-resistant pigs. This observation suggests that factors other than anoxia or struggling, and probably located beyond the neuromuscular junction, might be involved.

5. Glycolytic Control Kastenschmidt et al. (1968) examined the glycolytic changes in fastand slow-glycolyzing longissimus muscle samples taken at various times postmortem. Fast-glycolyzing muscles exhibited higher levels of monophosphate compounds ( G-1-P, G-6-P, and F-6-P), pyruvate, and lactate

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and lower levels of intermediates from fructose diphosphate through phosphoenolpyruvate than did slow-glycolyzing muscle. It was concluded that both phosphorylase and phosphofructokinase ( PFK ) were activated in fast-glycolyzing muscle. The cessation of glycolysis appeared to be the result of an inactivation of PFK. In attempts to explain the apparent activation of the glycolytic system in fast-glycolyzing muscle, the concentrations of several activators of glycolysis were measured. Since the level of ATP was markedly decreased, whereas the levels of ADP, AMP, and inorganic phosphate were increased, it was concluded that these altered levels of metabolites were responsible for the activated key enzymes in glycolytic control. Bendall (1961, 1966) suggested that the rate-determining step in muscle metabolism postmortem is the hydrolysis of adenosine triphosphate by an increased overall ATPase activity. Quass and Briskey (1968) evaluated the relationship between a possible increased rate of ATP hydrolysis during the first half-hour after death in the in situ musculature of PSE-prone pigs and the in vitro ATPase activity of myosin isolated from the same animals. The Ca2+activated ATPase activity of myosin extracted from PSE muscle of Poland China pigs was greater than that from Chester White and normal Poland China pigs, but lower than that from rabbit myosin. EDTA-activated ATPase activities were greater in myosin from stress-susceptible Poland China pigs than in that from rabbits and normal Poland China’s, but equal or lower than in myosin from Chester White pigs. In spite of these dissimilarities, the authors concluded that “there does seem to be some direct correlation between the enzymatic activity of myosin in vitro and the ATP splitting in the muscle in situ.” Tsai et al. (1971) used ion exchange chromatography to study nucleotide metabolism in postmortem muscle that was undergoing either fast or slow glycolysis. The four major peaks resolved in prerigor muscle were ATP, ADP, IMP with a trace of AMP, and DPN+ + DPNH. Muscle from stress-susceptible animals entered rigor (as judged by extensibility loss ) between 0.5 and 1.0 hour postmortem, whereas muscle from stress-resistant animals reached a similar stage in 3 to 4 hours. Muscle from stress-susceptible animals had lower ATP and higher IMP levels at 0 hour than did muscle from stress-resistant animals. Between 0 and 2 hours postmortem the concentration of ATP, ADP, and DPN+ + DPNH decreased rapidly, while the concentration of IMP and inosine plus hypoxanthine increased. The changes occurring from 3 hours to 144 hours postmortem were of lesser magnitude and much slower. The

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activity of AMP deaminase was higher in the muscle of stress-resistant animals at 3 hours and later postmortem. Beecher et al. (1965) utilized the light and dark portions of the semitendinosus muscle as a model for study of muscle metabolism. The semitendinosus is unique, as a distinctly red (deep) and a distinctly white (peripheral) portion can be easily separated from the same muscle. Bodwell et uZ. (1966) attempted to induce PSE by exposing carcasses to a 37°C environment immediately following slaughter. The 37°C treatment did not consistently result in PSE muscle; a loss of fibrillar water-holding capacity as a result of low pH and high temperature did, however, confirm earlier investigations. The authors concluded that, even though a loss of fibrillar water-holding capacity occurs as a consequence of low pH and high temperature and is frequently encountered in PSE, the low fibrillar water-holding capacity by itself is not the primary causal factor in making pork muscle appear PSE.

6. Sarcophmic Reticulum Greaser et aZ. (1969a) studied the Ca2+uptake and Ca+2-activated ATPase activities of isolated sarcoplasmic reticulum and the rate of postmortem glycolysis in pig muscle. The oxalate-stimulated Ca2+accumulating ability declined by a factor of 5 to 10 by 24 hours postmortem. The major portion of the decline was noticed during the first hour after death in PSE muscle but was more gradual in the normal muscle. The ATPase activities of fractions from normal and PSE muscle obtained at death did not differ significantly, but increased with time postmortem in most fractions obtained from normal muscle while decreasing in PSE muscle. Schmidt et aZ. (197Oc) investigated the relationship between Ca2+binding ability and loss of extensibility in longissimus muscle. NO significant difference in the ability of the sarcoplasmic reticulum to bind calcium or to develop isometric tension was observed between fast- and slow-glycolyzing muscle at identical stages of change in extensibility, even though the fast-glycolyzing muscle had lower levels of CP and ATP and higher levels of lactate at each point. Although these experiments argue against a primary involvement of the sarcoplasmic reticulum, it is possible that a relationship exists which has been confounded by experimental conditions such as p H or temperature on either Caz+-accumulatingability or ATPase activity. Studies on

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more purified sarcoplasmic reticulum fragments (Greaser et al., 1969a) showed that Caz+uptake in samples taken immediately after slaughter was twenty times as high as that in samples taken 24 hours postmortem. No significant differences in Ca2+-activated sarcoplasmic ATPase were observed between the samples mentioned above. I n vitro incubation of isolated sarcoplasmic reticulum fragments at p H 7.2 and 37°C or at pH 5.6 and 0°C caused negligible inhibition of the Ca2+-accumulating ability (Greaser et al., 1 9 6 9 ~ ) .However, treatment at pH 5.6 and 37°C for 1 hour almost completely abolished Caz+uptake by the sarcoplasmic reticulum. These experiments strongly support the idea that the more rapid loss of Ca2+-accumulating ability and the lower Ca2+-activated ATPase in sarcoplasmic reticulum of PSE muscle (Greaser et al., 1969a) may be caused by the combined effect of high temperature and low muscle pH in the early postmortem period. If there is denaturation, it may result in a release of Ca2+from the sarcoplasmic reticulum, which in turn can stimulate the myosin ATPase. Bendall and Wismer-Pedersen (1962) suggested that the sarcoplasmic proteins were denatured and precipitated on the structural proteins. Changes were not observed in the structural proteins. In view of later work, however (Cassens et al., 1963; Scopes, 1964; Greaser et al., 1969b; Borchert, 1967), it is likely that the structural proteins do participate at least partially in the denaturation process. Heffron and McLoughlin (1971) investigated the ATPase activities of isolated actomyosin, myofibrils, and sarcoplasmic reticulum. They observed that the rate of depletion of ATP in preparations from pigs stunned by captive bolt was four times as great as in anesthetized pigs. No differences were found in the ATPase activities of both natural actomyosin and sarcoplasmic reticulum from both groups of pigs. I n vitro studies demonstrated that decreasing the pH of the incubation medium decreased the Mg2+-stimulated myofibrillar and sarcoplasmic ATPase activity, although the sarcoplasmic ATPase was far more sensitive to the fall in pH than was the myofibrillar fraction. A myofibrillar ATPase was suggested as being the cause of the high rate of ATP decline during the 8 minutes between stunning and acquisition of the muscle samples from the pigs stunned by captive bolt.

7. The Abnormal in Vivo Situation From the literature reviewed to this point, it is evident that the majority of researchers considered the processes leading to the development of PSE to be initiated at death or in the early postmortem period.

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The observation that particularly muscular breeds and strains of swine were prone to PSE was for a long time the only indication of the predisposition of the living animal. Hence it became apparent that the situation in the musculature of the living animal might already be abnormal. Sybesma and van Logtestijn (1966) observed a higher body temperature just prior to killing in Dutch Landrace pigs which showed a more rapid glycolysis postmortem. Furthermore, the higher incidence of PSE in certain strains or breeds of pigs coincides with a higher death rate during stress (PSS ) . Exercise-induced lactacidemia, rise in body temperature, and muscular rigidity were observed in Pietrain pigs prior to spontaneous death ( Sybesma and Eikelenboom, 1969; Eikelenboom, 1972), and the similarity between these symptoms and those occurring postmortem in PSE muscle suggested a strong relationship. These symptoms could also be provoked in vivo when pigs of the same breed were subjected to halothane anesthesia (see Section 11). There are also the observations of increased serum enzyme levels in PSE-prone pigs. Sybesma and Hessel-de Heer (1967) found increased serum lactate dehydrogenase (LDH) levels due to an increased activity of the specific muscle isoenzyme in stress-prone pigs as compared with more stress-resistant pigs. (See Section VII, B for a description of the utilization of this finding as a detection method.) This observation has been confirmed by others (Addis and Kallweit, 1969; Hessel-de Heer, 1969; Merkel, 1971a; Reddy et al., 1971). In addition, serum levels of glutamic-oxalacetic transaminase ( GOT) ( Eikelenboom et al., 1970; Schmidt et al., 1970b; Bickhardt, 1971; Merkel, 1971a), creatine phosphokinase ( CPK) (Allen and Patterson, 1971; Bickhardt, 1971; Merkel, 1971a; Schmidt et al., 1971), malate dehydrogenase (MDH), glutamicpymvic transaminase ( GPT), and 1,6-diphosphofructoaldolase ( ALD ) have been reported to be increased in these animals (Bickhardt, 1971). This research indicates that there is in such pigs an increase in permeability, or more probably a decrease in selective permeablity, of the muscular membranes which allows leakage of these protein molecules. These observations suggest a myopathy in PSE-prone animals, but do not qualify the nature of this myopathic condition. The muscle biopsy samples in the studies of Sair et al. (1970) and Lister et al. (1970) were put into liquid nitrogen immediately after excision. However, since there is a time lapse, the estimated metabolite levels probably do not represent true in vivo levels-a possibility that has also been considered by the authors (Sair et al., 1972). Nevertheless, considerable differences in lactate and CP were found between stresssusceptible and stress-resistant pigs in these studies.

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Schmidt et al, (1971, 1972a) found ATP, G-6-P, and lactate in biopsies taken 3 days antemortem to be significantly related to postmortem muscle quality, as indicated by protein solubility (transmission value; Hart and Sybesma, 1964) of the longissimus muscle. A biopsy technique was designed whereby 200-mg samples of muscle were obtained with Koffler tongs without the use of a general anesthetic. Lactate, but particularly G-6-P, was significantly related to all ultimate quality characteristics. Muscle quality could be detected about as well by level of G-6-P in muscle at 6 or 12 days antemortem as by postmortem pH or rigor mortis measurements. It was concluded that this procedure could be a useful tool in practical prognosis and selection experiments. Sybesma et al. (1972) repeated the experiment on a larger number of pigs and found lower, although significant, relationships between G-6-P or lactate and ultimate quality. At 45 minutes postmortem, pH measurements were a better pedictor than G-6-P or lactate. No substantial difference in predictive value could be shown between G-6-P and lactate. Sampling at 14 and 6 weeks antemortem did not show a significant relationship with ultimate quality. It was suggested that, to be useful in selection, correlations between biopsy anaylsis and muscle quality require improvements which could probably be achieved by further standardization of sampling technique, transport, and slaughter circumstances ( Sybesma et al., 1972). Teeter et al. (1969) used a cryobiopsy technique to demonstrate that freezing prior to excision of the sample resulted in considerably higher levels of energy-rich phosphates and lower levels of lactate than were obtained when the sequence was reversed. Bickhardt (1971) and Bickhardt et al. (1972) also used an in viuo freezing technique and demonstrated higher levels of lactate and lower levels of pyruvate and glycolytic intermediates such as glyceraldehyde-3-phosphate, dehydroxyacetone phosphate, and G-6-P in the longissimus muscle of a strain of German Landrace swine with a high incidence of PSE than were found in a strain from the same breed with a low incidence of PSE. In addition, reduced levels of CPK, GOT, ALD, GPT, and G-6-PDH were found in the muscle samples in the PSE-prone strain. The author suggested that the initial step in the pathogenesis of the myopathic condition, which he calls “exertional myopathy,” is an insufficiency of the mitochondria1 oxidative phosphorylation in white muscle ( Bickhardt, 1971) . One point of interest is the fact that Bickhardt’s PSE-prone strain of pigs consisted of offspring of animals which had suffered from what is known in Europe as “acute back muscle necrosis,” thus suggesting a

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possible relationship between this myopathy and the PSE-PSS syndrome (Bickhardt, 1971; Bickhardt et al., 1972). The inthguing question is what factors caused the in vivo denaturation of part of the longissimus muscle. B. AEROBICMETABOLISM

Sybesma and Eikelenboom ( 1969) suggested that muscle mitochondria from stress-susceptible pigs show an uncoupling of oxidative phosphorylation. If mitochondria become uncoupled in uivo, the energy liberated by the oxidation process is not used to synthesize ATP from ADP and inorganic phosphate, but is released as heat. This will result in an increase in body temperature and muscle rigidity, symptoms that were also observed by these authors in highly stress-susceptible pigs after conditions of stress. Eikelenboom and van den Bergh (1971, 1973) isolated mitochondria from biopsy samples and biceps femoris muscle taken from Dutch Landrace and Pietrain pigs under general anesthesia, and measured polarographically the respiratory rate and the respiratory control ratio (RCR). The respiratory control ratio is the ratio of state 3 to state 4 respiratory rates. State 3 respiration occurs when the mitochondria are actively phosphorylating added ADP (with Pi and substrate in the medium); state 4 is induced after expenditure of ADP. The RCR is a measure of uncoupling, since in uncoupled preparations there is an increased mitochondria1 ATPase activity, and thus a steady supply of ADP, a higher respiratory rate, and consequently a lower RCR. The mitochondria isolated from the biopsies of the Pietrains showed a lower RCR than did mitochondria from the Dutch Landrace pigs. This lower RCR was, according to the authors, not due to an uncoupling of oxidative phosphorylation, since addition of oligomycin, a potent inhibitor of the phosphorylation process, strongly inhibited respiration in all preparations. It was concluded that the lower RCRs in the preparations from the Pietrain pigs were caused by an inhibited state 3 respiratory rate. The fact that this inhibition could be almost completely abolished by adding an uncoupler such as 2,4-dinitrophenol indicated a decreased capacity of the phosphorylation system in these preparations. At slaughter ( 2 to 4 weeks postbiopsy ) all characteristics of average muscle quality were significantly lower in the Pietrain breed. It was suggested that a decreased capacity of oxidative phosphorylation might exist in vivo in the musculature of PSE-prone pigs.

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Brooks and Cassens ( 1973) compared polarographic characteristics of mitochondria isolated from trapezius and longissimus muscles of stresssusceptible and stress-resistant pigs. The preparations from stressresistant pigs had a slightly greater state 3 respiratory capability than did those from stress-susceptible pigs. 2,4-dinitrophenol ( DNP ) -stimulated, and oligomycin-inhibited State 4 respiratory rates were apparently not different in the two types of pigs. Of particular interest was the finding of no gross differences in respiratory functions of mitochondria from muscle of stress-susceptible and stress-resistant pigs in response to incubation at high temperatures (25"C, 37"C, and 43°C). Sair et ul. (1972) found that treatment of animals with MgSO, resulted in higher oxygen consumption ( manometrically) in muscle samples taken immediately postmortem in stress-susceptible pigs but not in stressresistant pigs. If, indeed, there is a decreased rate of ATP synthesis via oxidative phosphorylation in vivo in PSE-prone pigs, then when these pigs are stressed they will be forced more rapidly to rely on additional ATPdelivering systems in their musculature-that is, first by the splitting of CP in the Lohman reaction and, second, via glycolysis. The stimulated glycolysis does not necessarily result in an accumulation of lactic acid. When aerobic pathways are unable to oxidize the excess NADH generated by glycolysis, anaerobic glycolysis will take over this function by converting pyruvate to lactate. A reduced aerobic capacity of oxidative phosphorylation will, therefore, result in a more rapid switch to anaerobic glycolysis in periods of increased energy demand. Eikelenboom (1972) and Eikelenboom and van den Bergh (1973) suggested that a decreased capacity for oxidative phosphorylation may lead to lower levels of energy-rich phosphates at the time of death. A t death, ATP synthesis via oxidative phosphorylation is completely blocked owing to tissue anoxia. Synthesis is then continued by the splitting of CP. As long as the CP supply lasts, ATP is kept at a constant level so that at this stage a balance is maintained in the level of ATP. When the CP supply is depleted, however, the muscle is dependent solely on anaerobic glycolysis for resynthesis of ATP. Since this pathway is much less efficient than aerobic pathways, the ATP level begins to fall. The lower the initial postmortem CP level, the more rapid will be the onset of the decline of ATP concentration. Besides the effect on postmortem pH decline, lower levels of ATP will decrease the efficiency of the sarcoplasmic reticulum to bind calcium and lead to a loss of extensibility and an onset of rigor mortis. The origin of excessive heat production,

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another variable in PSE musculature, has been discussed in regard to malignant hyperthermia (see Section 11). Although this theory explains why the decline of the ATP level might be expected to start more rapidly in these carcasses, the question remains whether it can explain sufficiently the increased rate of ATP decline postmortem. Another point of interest is the possibility that glycolysis is more easily stimulated by a certain low level of ATP in the very early postmortem period than afterward. Although questions still remain to be answered, the advancements made in this field since the earlier review (Briskey, 1964) are considerable. PSE research started out as a characterization of meat 24 hours postmortem; it has moved forward in the past decade toward attempts at the biochemical characterization of PSE- and PSS-prone animals through blood sampling and muscle biopsy techniques. Improvements have been made in understanding the abnormal muscle metabolism in stresssusceptible pigs as well as the effect of factors such as muscular contractions and anoxia, induced by stunning and the death struggle, on ultimate quality of the meat.

C . OTHERBIOCHEMICAL CONSIDERATIONS

1. Myogbbin Briskey (1964) discussed the problem of concentration and properties of myoglobin as contributing factors in the pale appearance of PSE muscle. He concluded that the question of quantity, state, and kind of myoglobin in PSE muscle required clarification. Unpublished work by W. D. Brown (1973) is interesting in view of a possible difference in characteristics of myoglobin isolated from normal muscle and from PSE muscle. Only oxymyoglobin was present in extracts from both types of muscle, and there was no apparent difference in concentration of myoglobin between normal and PSE muscle. He did find, however, a difference in oxidation rate of oxymyoglobin in the extracts; those from PSE muscle oxidized at approximately double the rate of those from normal muscle. A recent paper by Satterlee and Zachariah (1972) was not directed at a study of myoglobin from PSE muscle, but the authors did report some interesting species differences. They found porcine myoglobin to be different from bovine and ovine myoglobins. Porcine myoglobin was much more susceptible to acid denaturation, and the isoelectric point and amino acid composition were significantly different.

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2. Lipid Composition Sink et al. (1967) investigated the lipid content and lipid composition of ten normal and ten PSE longissimus muscles. Total lipid did not differ in the two groups, even though it is generally accepted that PSE muscle shows less visible fat or marbling. Similarly, there was no difference in fatty acid composition, nor did fatty acid composition change from 0 hour to 24 hours postmortem in the two kinds of muscle. 3. Connective Tissue Some interest has been shown in investigation of the connective tissue from PSE muscle; this probably is a result of the general observation that PSE muscle is “loose” and can be forced apart into segments more easily than can normal muscle. Results from the studies described below have shown differences in properties of connective tissue from normal and PSE muscles, but it is still unclear whether these differences are in some way related to the development of PSE or whether they are a result of alteration postmortem. McClain et al. (1969) characterized the chemical properties of epimysial connective tissue from normal and PSE muscle and found a wide difference between the PSE and normal muscle they had selected for study by the turbidity test. Epimysium from PSE muscle contained significantly higher quantities of salt-soluble tropocollagen. This may indicate that the connective tissue of animals that develop PSE is more immature. Likewise, epimysium from PSE had a greater amount of heatlabile collagen than did epimysium from normal muscle. No differences and ,8 subunit composition, plasma hydroxyproline were evident in levels, or amino acid composition. In further work, McClain and Pearson ( 1969) failed to find a difference in conventionally determined shrinkage temperature of connective tissue from the two sources. Differential thermal analysis studies, however, revealed that epimysium from PSE muscle had lower onset and recovery temperatures and contained a higher percentage of components melting at low temperatures than did epimysium from normal tissue. Epimysium from PSE muscle also had a higher initial moisture content and a lower dry matter content. Epimysial connective tissue underwent osmotic swelling in a neutral solution; the preparations from PSE muscle imbibed significantly more water. The authors reported a molecular weight ( Mc ) between cross-links for collagen from PSE muscle of 6.37 x lo4 and a resultant cross-link density of 5.23 per molecule; for collagen from normal muscles the figures were (Y

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4.67 x lo4 and 7.73. These differences were significant at only the 10% level. McClain et al. (1968) also found an altered or decreased ground substance content in the epimysium from PSE muscle. Field et al. (1970) studied the differential thermal analysis properties of epimysial connective tissue from normal and low-quality pork at both 0 hour and 24 hours postmortem. Melting characteristics of intramuscular collagen at 0 hour were determined. Collagen from low-quality muscle consistently gave slightly lower peak melting points than did collagen from normal muscle. Epimysial collagen had a significantly lower peak melting point at 24 hours than at 0 hour postmortem. The thermal behavior of intramuscular collagen at 0 hour postmortem was similar to that of epimysial collagen at 0 hour, but peak melting temperatures were slightly higher for intramuscular connective tissue.

V.

MORPHOLOGY AND HISTOCHEMISTRY

Since Ludvigsen (1954) described the condition of pale, exudative muscle, and referred to it as “muskeldegeneration,” there have been numerous attempts to demonstrate, with microscopy, lesions in PSE muscle and in muscle from PSS-afflicted pigs. The importance of this search for structural evidence of muscle abnormality is clear because the approach to solving the problem could be simplified and concentrated if it was known to originate in a describable muscular defect. There is always the lingering worry that, if real pathological criteria of dystrophy were found, then the use of PSE muscle as a human food could be seriously questioned. 1 . Light Microscopy

Both the Danish (Ludvigsen, 1954) and the French (Henry et al., 1955) descriptions of exudative muscle included histological studies of the muscle, and both reported histological changes. Among other factors, the exudative muscle was characterized by the absence of cross striations. Lawrie ( 1960) reported that the unusual superficial characteristics of white exudative muscle were reflected at the histological level, with the degree of abnormality apparently related to ultimate pH. At a pH of 5.1 the muscle fibers were parallel and showed cross striations, but the inte-

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rior of many was disrupted. At pH 4.9, all the fibers had an abnormal appearance. Some were straight and showed clear longitudinal markings, while others were both twisted and finely corrugated, with frequent breaks and spaces between the fibers. The samples went into rigor while attached to the carcass. Straight fibers did have striations, but the striations were closer and of a finer order than in the corrugated fibers. It was concluded that the corrugated fibers had not shortened but had been passively twisted by shortening of the adjacent straight fibers. This was likened to the circumstance in which there was a precipitate onset of rigor mortis. In a cross section of exudative semimembranosus muscle the intrafibrillar contents had shrunk away from the sarcolemma. Norman (1965) examined a series of longissimus muscles from pig and found a number of abnormalities, but he was unable to relate the observations directly to PSE. The blood vessels appeared essentially normal in all cases, but there were instances of hyaline degeneration and, to a lesser extent, some granular degeneration. “Herztod has been described by Jubb and Kennedy (1970) as sudden death most often seen at the time of breeding and during the hot summer months. The clinical symptoms are stiffness during movement, lassitude, cyanosis, and sudden death. The affected muscles are described as pale gray or nearly white. The histological changes are characterized by cloudy swelling, loss of myoglobin and cross striations, and edema of the supporting stroma. Other workers have reported no histological abnormalities in PSE muscle. Wismer-Pedersen (1959) studied the effect of rapid pH fall in pig muscle and found no systematic difference in the appearance of the muscle fibers and the cell structure. Tope1 et al. (1968) described the porcine stress syndrome and failed to reveal any characteristic alteration of muscIe tissue with light microscopy. Work on malignant hyperthermia in swine has revealed some abnormalities in the muscle. Allen et nl. (1970) reported that many of the skeletal muscles examined from Pietrain pigs suffering from acute stress syndrome contained moderate numbers of rounded hyaline fibers with apparent loss of striation. Occasional fibers had fluctuating cytoplasm with internal nuclei and/or internal macrophages. Harrison et al. (1969) examined brain, liver, kidney, adrenal, and muscle of Landrace pigs in their work on anesthetic-induced malignant hyperpyrexia. Only muscle showed demonstrable changes. The majority of fibers in a section appeared normal. Isolated fibers, however, were shortened and shrunken. At their ends, these fibers were separated from adjacent fibers, and in some instances they appeared to have ruptured transversely. Such short-

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ened fibers were more intensely eosinophilic than normal fibers. Similar fibers were observed in muscle section- from normal pigs, but in the affected pig they were more frequent and readily observable. Recent work by Muir (1970) on muscle from Pietrain pigs has brought forth some new information on regenerating fibers. He examined antemortem and postmortem muscle samples and found evidence for in vivo degeneration and subsequent regeneration with a small proportion of the fibers from all four animals showing regeneration. Necrotic fibers contained eosinophilic masses of myofibrillar material and varying numbers of macrophages with large, pale vesicular nuclei. Some endomysial tubes were filled with cells identified as myoblasts. Myotubes were also identified, and regenerating fibers were scattered throughout the muscle. The author concluded that a real diagnosis could not be made from postmortem samples and that the findings on antemortem material did not reveal any unusual features to account for the rapid postmortem change. The demonstration of an in vivo muscle degeneration and subsequent regeneration localized a true myopathy in a breed susceptible to postmortem degeneration, but a correlation between events could not be established. Earlier reports on histological changes in PSE muscle were viewed with some skepticism, not only because the observed changes were minor and rather nonspecific, but also because in many cases the muscle was examined postmortem, so that in fact the rapid glycolysis may have caused the observed changes. There seems to be little doubt now, however, in view of the work on malignant hyperthermia (see Allen et al., 1970; Harrison et al., 1969) and particularly in view of the report by Muir (1970), that a histological condition affecting only a very few of the fibers is present in certain pigs.

2. Electron Microscopy Greaser et al. (196913) worked with subcellular fractions from normal and PSE muscle and as one phase of the work examined the structure of fractions isolated from muscle at death and at 24 hours postmortem. Myofibrillar, mitochondrial, heavy sarcoplasmic reticulum, and light sarcoplasmic reticulum fractions were isolated from homogenates. No difference was found in myofibrils at death from the two kinds of animals, but myofibrils from PSE muscle at 24 hours postmortem had more granularappearing filaments and wider Z lines than did myofibrils from normal muscle. Myofibrils from PSE muscle at 24 hours postmortem were granular in appearance, which was probably due either to precipitated sarco-

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plasmic proteins (Bendall and Wismer-Pendersen, 1962) or to changes in conformation and properties of the myobrillar proteins themselves. The heavy sarcoplasmic reticulum fraction from PSE muscle at death appeared to have more granular material than that from normal muscle. Most mitochondria isolated from PSE muscle at death appeared normal, but some were markedly swollen and had a decreased matrix density. Mitochondria from 24-hour-postmortem PSE muscle had a highly altered form. The predominant configuration was a gray nonmembranosus matrix surrounded by a variable number of concentric, triple-layered membrane forms. Van Vorstenbosch (1969) found abnormal mitochondria1 structures particularly in the periphery of the cell in samples of the biceps femoris obtained in vivo from a stressed Pietrain pig. Swollen and degenerated mitochondria with vesicles enclosed by the cristae were observed. In filamentous organization, a remarkable similarity to human myotonic dystrophy was observed. It is difficult to say whether the changes were inherent or were imposed on the sample during isolation and preparation procedures. The results of the first study did indicate that composition of the subcellular fraction changed with time postmortem; this is a factor of consideration for biochemical studies on such fractions. 3. Fiber Dimensions

The role of muscle fiber number and size in determining muscle mass and quality has been an intriguing subject for many years. Work by Staun (1963) was based on large numbers of animals and appears to have been critically designed and conducted. He concluded that the meaty races of pigs have the thickest muscle fibers, or, expressed another way, a smaller number of fibers per square centimeter. He wrote that, even though opinions differ, there can be no doubt that the diameter and number of fibers in individual muscles influence the quality of the meat. And meat with a large number of muscle fibers per unit of area in cross section is of a higher quality than meat with a small number of fibers. Staun and Jensen (1972) recently reaffirmed that selection for bigger muscles and more meat in a carcass causes a steady increase in the number of muscle fibers. It was also shown that selection for a high total number of fibers improves quality in the direction of more tender meat with a good water-holding capacity. Hendricks et al. (1971) reported that the muscle fibers from Poland China pigs were larger than those from Hampshires. Dildey et al. (1970)

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found that light fibers with a large diameter and a lack of muscle tenderness were common to animals with PSE musculature as well as to those with muscular carcasses. Swatland and Cassens (1972a) studied muscle from rats that had been selected for either increased or decreased postweaning growth rate, Mean fiber diameters of high-gain rats were larger than those of low-gain rats. The contribution to muscle enlargement in high-gain rats made by histochemical type I1 fiber hypertrophy was greater than that derived from type I and intermediate fibers. The determination of fiber number and size has always presented a troublesome hindrance to progress. A recent paper by Swatland and Cassens (1972b) has provided a new insight into the technical problems involved. They described the occurrence of numerous intrafascicularly terminating fibers in several muscles of bovine and porcine animals. This circumstance could, of course, cause considerable error in estimating fiber number and size from muscle cross sections.

B. HISTOCHEMISTRY

1. Fiber Types The development and use of histochemical techniques for study of muscle during the mid-1960’s opened the path for some interesting work on the association of mucle fiber type with various postmortem properties. However, the lack of uniform terminology proved to be one of the greatest problems in this area of research. It is beyond our objective here to present a historical and present description of terminologies; terms will be used as the authors used them in the original publications. Sair et a2. (1970) were the first to examine the concept of muscle fiber type and its possible association with the PSE problem. They applied Sudan black B, succinic dehydrogenase, and cytochrome oxidase techniques to frozen sections of the longissimus muscle and classified fibers only as red or white. Quantitation showed that muscle from stresssusceptible pigs had a larger area of red fibers than did muscle from normal pigs. The findings were difficult to rationalize because stresssusceptible animals respond to anoxia with a greater production of lactic acid, which would be characteristic of white fibers as would the rapid postmortem glycolysis characteristic of PSE muscle. A report by Quass and Briskey (1968) also revealed that muscle from stress-susceptible animals had a higher myosin ATPase activity than that from stress-resistant animals, Sair et al. (1970) discussed the results in terms of a possible

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problem from fiber contraction and suggested the application of phosphorylase and ATPase techniques to obtain more information. The confusion from these findings was clarified by the work of Cooper et al. ( 1969). They found that muscle from stress-susceptible animals had more intermediate fibers than did muscle from stress-resistant animals. The intermediate fibers were defined on the basis that, even though they reacted positively for DPNH-TR,* they also had a high amylophosphorylase and ATPase activity. The authors postulated that such fibers were important in the drastic postmortem behavior of muscle from stresssusceptible animals and could be the characteristic that caused the muscle to become PSE. The muscle fibers from stress-susceptible animals were also larger than those from normal animals. Some quantitative estimates were given in regard to the proportion of the various types present. The white fiber area was rather constant (for longissimus) at 70 to 757' when the stress-susceptible and stress-resistant groups were comparedthe remainder of the fiber area was composed of red and intermediate fibers referred to collectively as dark fibers. In stress-susceptible animals, only 20 to 35% of the dark fibers were actually red, whereas in muscle from normal animals 60 to 75y0 of the dark fibers were red, based on a negative reaction for phosphorylase and ATPase. De Bruin (1971) removed biopsy samples from the biceps femoris muscle of Pietrain and Dutch Landrace pigs and applied the histochemical test for succinic dehydrogenase. He classified fibers as white or red, with the latter including red and intermediate. A higher mean value of red fiber area was determined for the biceps femoris of Pietrain than for Dutch Landrace. Lactic acid level was also higher in the Pietrain. Merkel (1971a) studied fiber type distribution in gluteus medius and rectus femoris muscles that were either normal or PSE and from several breeds of pigs. He employed succinic dehydrogenase and phosphorylase procedures. Muscle that was PSE had more white and less red fibers than that of normal quality, and no difference was found in content of intermediate fibers. When area of types was expressed as percent of total fiber area, the quality group differences were small. Muscle fibers were significantly larger in PSE muscle. Anderson and Parrish (1972) have reported histochemical work on the muscle of genetically fat, muscular, and muscular stress-susceptible pigs. They used techniques for phosphorylase, NADH-TR, and acid- and basestable ATPase. Fibers were classified as red, white, and intermediate. The fat group had fewer red fibers than did the other two groups for all

* Reduced

diphosphopyridine nucleotide tetrazolium reductase.

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reactions. The fat group also showed more intermediate fibers with all reactions. Muscular stress-susceptible pigs had fewer white and more intermediate fibers as judged with the phosphorylase stain. Dildey et al. (1970) studied forty castrated male pigs representing three levels of muscling and two color structure scores. They used Sudan black B staining and classed intermediate staining fibers in the red fiber group. A low 45-minute muscle pH and slow heat dissipation from the muscle were clearly associated with the PSE condition. A high proportion of light to dark fibers, large diameter of light fibers, and lack of tenderness were common to animals with PSE musculature as well as to those with muscular carcasses. Quantities of muscle pigment were inversely related to muscularity. The authors concluded that the size and number of light fibers are more important in the PSE muscularity correlation than are characteristics of the dark fibers. In a subsequent report by Sair et al. (1972), the muscle from stressresistant and stress-susceptible animals within Poland China and Chester White breeds of pigs was examined. Muscle was clamped during excision in order to minimize contraction; fibers were examined by utilizing DPNH-TR and amylophosphorylase reactions, and red plus intermediate fibers were referred to as dark fibers. There was a trend for restrained muscle samples to have somewhat smaller fibers than did unrestrained samples. In Poland China animals, the area of both dark and white fibers was larger in stress-susceptible than in stress-resistant animals, but it did not differ in Chester White animals. Stress-susceptible animals had a larger number of intermediate and white fibers, both of which stained positively for phosphorylase, indicating their glycolytic capacity. The data indicated that size per se did not predispose muscle fibers to postmortem anoxia and the development of PSE characteristics, but rather that a high glycolytic potential seems to be the chief factor predisposing fibers to the PSE condition. Fiber size appears to be related to the PSE condition in that large fibers will contribute to the severity of the PSE condition provided that the basic requirement of a high glycolytic capability is met. A recent finding by Swatland and Cassens (1973a) indicates the importance of sampling time on histochemical findings. They removed muscle samples from a group of pigs immediately at the time of exsanguination and also at about 15 to 20 minutes postmortem. The histochemical reactivity of phosphorylase and the stainability of glycogen by the PAS reaction appeared to increase postmortem. Bodwell et al. (1965) used histochemical techniques to determine if any abnormalities existed in pork muscle. They examined a large number

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of enzymes in the muscle of pork carcasses, one side of which was placed at -29°C while the other was placed at 37°C for the first 4% to 5 hours postmortem. They detected acid and alkaline phosphatase activity in muscles from seven of thirteen carcasses and therefore suggested that a degenerative condition existed in the muscle. Work by Dutson et al. (1971) was based on the possible interrelationships of PSE and lysosomes. They employed histochemical techniques for esterase, p-glucurondidase, p-galactosidase, aryl sulfatase, succinic dehydrogenase, and acid phosphatase. There was no apparent difference between PSE and normal muscle for any of the lysosomal enzymes investigated. Muscle from Landrace and Poland China pigs (reported as highly stress-susceptible) exhibited a greater acid phosphatase activity than that from Chester White pigs. Three pigs in the PSE group had very high acid phosphatase activity, which was shown to be associated with degenerating fibers. Biochemical studies on myoglobin in PSE muscle are referred to elsewhere in this review (see Section IV, C ) , but a histochemical study of myoglobin in B E muscle should be mentioned at this point, The study of Morita et al. (1970) was, unfortunately, rather inconclusive. They used two groups of pigs (twenty-three in one group and six in the second) and studied the association of histochemically stained myoglobin with 24-hour postmortem color score. In the first group there was a significant correlation of 0.52 between area of myoglobin-positive fibers and color score, whereas when the pigs from the second group were added the correlation dropped to 0.23. The occurrence and histochemical properties of giant fibers were described by Cassens et al. (1969), and a possible association with stress susceptibility was noted. De Bruin (1971) has suggested that giant fibers are a phenomenon produced postmortem. There is a distinct possibility that giant fibers are an artifact; even if they are, the observation that they are found more frequently in PSE muscle or in muscle from PSS animals is undoubtedly significant. Ashmore et al. (1972) have put forth some suggestions about the interrelationships between shifts in fiber type and increased muscularity in domestic animals. They suggest that increasing muscularity in domestic animals is achieved by transformation of IYR to .(YWfibers. They also believe that the rapid and extreme variations in postmortem muscle pH are more likely associated with “white” muscles, since pH reflects the capacity of muscle for anaerobic metabolism. The changes in muscle characteristics of pigs during growth and development have been described by Cooper et al. ( 1970), van den Hende et al. (1972), and Swatland and Cassens (1973b).

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2. Circulatory System Microtechniques have been used in a study of capillary distribution in muscle from normal and stress-susceptible animals. Cooper et al. (1969) used an alkaline phosphatase reaction to visualize capillaries. More capillaries were associated with red than with white fibers, and there was a greater capillary-to-fiber ratio in red muscle. These authors found no difference in capillary-to-fiber ratio between normal and PSE muscle. Merkel (1971a) used an infusion procedure with India ink to study capillary distribution. He found fewer capillaries per square millimeter in PSE muscle; the fibers of PSE muscle were also significantly larger. He concluded that from the standpoint of diffusion dynamics the PSE muscle would be more predisposed to the development of anoxia.

3. Innervation Swatland and Cassens ( 1 9 7 2 ~ )have recently reported on a study of the peripheral innervation of muscle from stress-susceptible pigs. The medial red face and the distal pale face of the peroneus longus from six stress-susceptible Poland China and two stress-resistant Chester White animals ranging in weight from 6.4 to 130 kg constituted the experimental material. There was an increased mean maximum end-plate diameter, increased complexity of the terminal ramifications, increased ultraterminal sprouting, and more double end plates in Poland China red and pale sample and in Chester White red muscle as compared with Chester White pale muscle. Poland China muscle also had more sprouting from terminal axons. The growth of end plates in Poland China muscle was found to be in proportion to muscle fiber cross-sectional area only during the early phases of muscle fiber hypertrophy. The authors suggested a relationship between increased neural growth and muscle fiber hypertrophy related to the condition of stress susceptibility.

VI.

IMPORTANCE IN THE RETAIL PRODUCT

Hall (1972) estimated that the loss at retail due to excess shrinkage and lower yields of PSE meat amounts to approximately 95 million dollars annually in the United States. We quote his estimate here to illustrate one author’s idea of the importance of the problem of PSE in the retail product. While a search of the literature reveals good agreement

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that PSE meat is present, there is disagreement as to the real importance of the problem. The disagreement probably stems in part from the wide variation in incidence reported from day to day and from place to place but, more important, may lie in the apparent scarcity of documented reports of surveys of incidence in the literature. There has undoubtedly been a reluctance to publish such figures in some cases. We believe it is rather clear from the literature, though, that there is a loss in PSE meat due to drip (in terms of both shrinkage and loss of nutrients) and that PSE meat has poorer processing characteristics than normal pork in specific instances such as canned hams. There is marked disagreement in the literature about the palatability of PSE meat. Preslaughter treatment of the animal can markedly affect the postmortem conversion of the muscle to meat. An excellent and complete review on the effect of preslaughter stress on meat palatability has been written by Hedrick (1965). The general conclusion of his review was that, when cattle and lambs were subjected to stress for several hours preslaughter, the muscle usually had a lower glycogen content, a higher postmortem pH, increased water-holding capacity, and improved muscle tenderness and juiciness. When pigs are subjected to an immediate preslaughter stress, however, the situation may be quite different and result ultimately in meat with decreased water-holding capacity that is less tender and juicy. The intensity of the stress and the time prior to slaughter that the stress is experienced are controlling variables. The stress may result in depletion of glycogen and a subsequent high postmortem pH, or if a strong stress is given quite soon before slaughter it may apparently set the glycolytic machinery into fast action so that a more rapid than normal fall in pH postmortem is elicited. Hedrick et al. (1964) tested the effect of adrenalin injection in live pigs on subsequent quality of the meat. The color of loin chops was generally more desirable and stable, and there was little difference in flavor and tenderness of chops from treated animals. Bacterial growth was enhanced by higher pH in meat from the treated animals. The effect of preslaughter stress by electric shock on meat quality has been described by Lewis et al. (1962, 1967). It is also important to note that antemortem treatment of the animal is much more difficult to control than is postmortem handling. The major objective of postmortem handling is to cool the carcass rapidly in order to minimize bacteriological problems. The reader is referred, however, to Section VII, C for a discussion of potential problems inherent in too-rapid cooling.

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A. OCCURRENCE OF PSE An often-quoted survey is that of Forrest et al. ( 1963). They examined hams of two weight classes for the incidence of PSE characteristics over a 12-month period. Eighteen percent of all the hams observed were pale, soft, and watery; the daily incidence vaned form 0 to 75%. There was a greater occurrence of PSE in the 18- to 20-pound hams than in the 14- to 16-pound hams. There was also a greater occurrence of PSE during periods of high or widely fluctuating temperature. The fall season, which showed the greatest fluctuations in temperature, also show the greatest incidence of PSE. I n various European countries, particularly in the exporting countries, there is a tendency not to publish openly data about the incidence of PSE, because of its possible negative effects. Van Logtestijn et al. (1970) concluded, on the basis of survey figures from Belgium, Denmark, France, Germany, ‘and Holland, that the incidence of abnormal muscle quality is between 10 to 20%. However, differences in weather, transport, stunning, and handling conditions, as well as breed differences, do cause considerable variations in these figures. It is well known that the Europeans have selected for leanness and muscularity in pig breeding and have used confinement systems in pig husbandry for a longer period than in the United States. Since these factors are thought to be associated with abnormal muscle quality, it is very likely that the incidence is higher than in the United States, unless the generally mild Western European climate conditions are a sufficient compensation. Dalrymple and Kelly (1969) surveyed the incidence of PSE hams from September through June (twice monthly) in a plant in Virginia. At each survey time, 250 hams were selected, one half from midwestern pigs, and the other half from southern (Tarboro, North Carolina) pigs. The incidence of PSE in hams from both locations was highest in February and March and lowest in May and June. The overall incidence of PSE was 23.7%, with 16.9% from pigs of southern origin and 30.5% from pigs of midwestern origin. They also noted that more PSE hams came from pigs with heavy muscling and that the p H was significantly lower in PSE hams than in normal hams. A conclusion was that PSE muscle was produced more frequently in pigs that had been subjected to greater temperature fluctuations during hauling and holding prior to slaughter. The pigs of midwestern origin were subjected to greater temperature fluctuations and a lower average temperature and produced a higher incidence of PSE.

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A recent experiment by Herring et al. (1971) utilized 100 randomly selected carcasses. The pH and temperature at 45 minutes in the ham and loin and ham rigor score were measured. Twenty-four-hour quality was evaluated by the Wisconsin system ( 1 is very PSE, 3 is normal, and 5 is dark, firm, and dry), and after the third day postmortem, chops were cut and evaluated. The distribution of %-hour quality scores showed 8 carcasses at score 1, 35 carcasses at score 2, 45 carcasses at score 3, 10 carcasses at score 4, and 2 carcasses at score 5. Both low and high &-minute pH values were observed in all categories of color score. The authors concluded that the use of loin color score as a sole criterion of prediction of the ability of pork muscle to bind water was therefore subject to error in practice. The pH at 45 minutes was less related to water-holding capacity than was the final pH. Final pH, which appeared first in the stepwise regression analysis, was the most important factor in prediction of water-holding capacity. A combination of final pH and loin color score was of slightly greater practical value than color score alone in prediction of water-holding capacity. Eikelenboom et al. (1974) evaluated the relationship between several meat quality measurements at 45 minutes and at 24 hours postmortem. Transmission values at 24 hours showed the highest relationship with pH, temperature, and rigor readings at 45 minutes postmortem. In a step wise regression analysis for prediction of transmission values, initial loin pH and temperature accounted for 52% of the variation. The pH at 45 minutes postmortem showed a higher signscance than did the ultimate pH when related to transmission value. Of interest was the high correlation between muscling score and rigor reading which suggested that the visual score of muscling might be influenced by degree of onset of rigor mortis. B. PROCESSING CHARACTERISTICS

There has been concern not only about the organoleptic properties but also about gelatinous cookout loss in PSE hams because the ham is a high-priced, very desirable portion of the carcass; most ham is further processed rather than being sold fresh at retail. There seems to be little doubt that PSE meat is less desirable for certain processing procedures than is normal meat. The following reports illustrate this conclusion. Karmas and Thompson (1964) canned hams representing a wide range of color structure scores. The hams were thermally processed to 66°C internal temperature with constant and differentially changing cooking temperatures that ranged from 68" to 93°C. Hams in the pale color

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groups produced consistently greater quantities of gelatinous cookout ( 2 to 8%) than did dark hams. The cookout difference was markedly greater in the extremely light hams; this difference was even further exaggerated at cooking temperatures above 77"C. Wismer-Pedersen (1960) cooked pale, watery, and normal hams to 64°C or 102°C and then subjected them to taste panel analysis. Those with watery structure cooked to 64°C were inferior in color, taste, and texture to normal hams that had been treated similarly. The differences for color and texture were not significant. There was no difference in color, taste, and texture between watery and normal hams that had been cooked to 102°C. Percent jelly in the can was 29.6% for watery hams and 29.9% for normal hams. Moerman and Krol (1969) have also studied hams prepared from PSE and normal carcasses. Organoleptic values were poorer for PSE hams than for normal hams. The differences in color, taste, and saltiness were, however, small. The shrinkage of PSE hams was greater than that of normal hams. The difference in jelly formation between the two groups was less than expected, and the authors discuss the situation where all muscles in a ham may not be of low pH. The beneficial aspects of polyphosphates are also discussed. Further work by Moerman and Krol (1971) was directed at a study of heat penetration in canned hams of different qualities. They found no differences in terms of heat penetration among normal, PSE, and DFD hams. They did note, nevertheless, that the actual heat treatment is very important. Tope1 et al. (1972) studied the effect of thermal processing on normal and PSE hams. They examined three muscle groups identified as follows: ( I ) quadriceps femoris, rectus femoris, vastus intermedius, vastus lateralis, vastus medialis; ( 11) semimembranosus, gracilis pectineus; (111) semitendinosus, biceps femoris. The product was canned commercially, and purge was defined as the quantity of fluid released from muscles into the container after heat processing. Color grouping (PSE versus normal) had no significant effect on protein, moisture, or fat, and a greater variation existed in percentage of protein, fat, and moisture among ham portions than between individual hams selected for color. The effects of color firmness and of muscle portion on percentage of purge were highly significant. PSE muscle had a higher percentage of purge than did normal muscle. Specific gravity of the purge was higher for PSE than for normal hams, which probably reflects a greater amount of total solids. There was no difference in sodium or potassium levels in purge from normal and PSE hams, but there was a difference among the three muscle portions.

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Merkel (1971b) found that smoked hams prepared from PSE meat had significantly lower processing yields than those prepared from normal quality pork. In overall acceptability, normal was preferred to PSE, but Warner-Bratzler shear force values were lower in ham prepared from PSE meat. In studies on the use of PSE meat in a luncheon meat product, Leest et al. (1971) found that PSE meat underwent a more rapid increase in temperature during chopping; an increase in separated jelly from the lean mix of 5% of the can contents was also observed in product prepared from PSE meat. Merkel (1971b) showed that bologna made with PSE meat had a significantly greater loss during processing than that prepared from normal meat. Emulsion stability tests showed that bologna made from PSE meat had a significantly greater total cookout (water plus fat) than that made from normal meat. I t was emphasized that, even when bologna contained all PSE pork, there was no detectable emulsion breakdown.

C . PALATABILITY CHARACXERISTICS In 1960, Judge et al. selected a group of pork loins representing a range of colors from light to dark and studied various characteristics of the meat including an organoleptic evaluation. As the color scale ranged from light to dark, the juice lost upon heating and centrifugation decreased and p H increased. Firmness and p H were related to tenderness; tenderness increased as the loins became less firm and as p H decreased. In this experiment chops were prepared by broiling. Essentially opposite results were reported by Sayre et al. in 1964. They found that, when rigor onset occurred at a pH less than 5.9 and at a temperature greater than 35"C, the longissimus muscle became pale and exudative. With such meat 40 to 507" of sample weight was lost by evaporation during cooking, which allowed the muscle temperature to rise at about twice the rate found for pale, exudative muscle. The meat in this case was prepared as a roast and cooked at 177°C to an end point of 85°C. Recent work by Merkel (1971b) showed that normal pork loin roasts required a significantly longer cooking time than did PSE roasts. The

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overall panel score showed no difference between PSE and normal roasts. Cooking losses were greater in PSE roasts, and panel juiciness scores were significantly lower. Warner-Bratzler shear force values were significantly lower for PSE muscle. The meat in this case was from roasts cooked at 160°C to an internal temperature of 77°C. Wismer-Pedersen ( 1959) examined the effect of decreased waterholding capacity on general quality of pork. As water-holding capacity decreased, the color of the raw meat was generally paler, whereas taste and texture of the fried chops were not essentially affected. Hedrick et al. (1968) examined the loins and hams from 123 Duroc and 155 Hampshire animals and found that the loins and hams from Durocs were significantly firmer and darker in color. There was some indication within each breed that firmer loins and hams and higher pH values were associated with carcasses that had lower yields of the four lean cuts; firmness and color of loins were not related to rate of gain for either breed. Firmer and darker loin chops from Hampshires were less tender, while softer and lighter chops from the Durocs were less tender, Searcy et al. (1969) studied longissimus roasts that were normal, PSE, or DFD. The meat was roasted at 176.7"C to an internal temperature of 75°C and then subjected to organoleptic and objective evaluation. There was no significant organoleptic preference for any one type. Also, there was no significant difference for roasting time, volume of press fluid, or total moisture (press method). DFD muscle exhibited the smallest roasting loss and greatest total moisture, whereas PSE had the greatest roasting loss and least total moisture. DFD rated highest in pH and lowest in Warner-Bratzler shear value. Buchter and Zeuthen (1971) found that normal and PSE loins had, except for flavor, the same organoleptic qualities one day after slaughter. The difference between the two types increased with aging, and the overall average results showed that the panel preferred cutlets from normal meat over PSE meat for all organoleptic properties (fried color, aroma, flavor, tenderness, juiciness, and overall impression). Deethardt and Tuma (1971) selected loins as being PSE, normal, or dark and firm, and cut each loin into four roasts. All roasts were cooked at 177°C to an internal temperature of 77°C by four procedures as follows: ( 1 ) open pan, ( 2 ) covered pan, ( 3 ) open pan to 52°C then covered with foil, (4)pan covered to 52°C then foil removed. Dark, firm loins had the least desirable texture and tenderness according to the panel, and shear values also indicated that dark, firm loins were the least tender. PSE and normal loins had significantly greater total cooking

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loss and drip loss than did dark, firm loins. The panel scored PSE loins as significantly more tender than the other two types of loins. K a u h a n et al. (1964) reported that increased muscle acidity was associated with pale, soft tissue which yielded a higher percentage of expressible juice. Dark, firm, dry muscle had a relatively high pH, shrank less during curing and cooking, and was more tender and juicy than PSE muscle of low pH. Regardless of muscle acidity, the curing process raised all palatability ratings to a comparable and acceptable level. It was concluded therefore, that the primary importance of a higher pork muscle pH, especially for hams, was its association with less shrinkage during processing. In a study involving twenty normal and twenty PSE hams, Dalrymple and Kelly (1966) found by taste panel evaluation that normal hams were more juicy but that there were no differences in cooked pH, color by Hunter color difference meter, percentage of dry matter, percentage of protein, tenderness, number of chews, texture, or firmness of lean. Meyer et al. (1963) found that PSE muscle showed a greater exudate formation, more expressible juice, and a significantly higher cooking loss than did normal muscle. The higher cooking loss contributed to a significantly higher nutrient loss on a fresh-weight basis. It is certainly difficult to make a generalization about the palatability of PSE meat in view of the reports cited above. The answer will most likely emerge when greater standardization of starting material is realized. Van Logtestijn (1969) studied some characteristics of PSE pork and found that PSE hams lost approximately 1 to 1.5% more weight during curing and maturing and produced about 3 to 5% more jelly in the cans than did normal hams. The differences were much smaller with country-style hams. He also reported that some consumers preferred the PSE meat. This observation has been considered further by Zuidam et al. (1971). They placed pale, normal pink, and dark pork chops in a self-service counter and then interviewed the customers after selections had been made. They found that greatest emphasis was placed on cost per portion of fat-free meat. The consumer wants pork to be light pink in color. Dark red muscle was associated with beef, added color, or partially spoiled meat. The authors concluded that the consumer does not prefer pale pork either. The industry should, therefore, provide pork of a medium color, neither white nor dark red. Color was not a major reason for selecting a certain package. Number of pieces, price, fat, and weight were major reasons in over 70% of the purchases.

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POSSIBLE SOLUTIONS TO THE PROBLEM A. GENETICS

The use of genetic selection may solve the problem of animal stress susceptibility and PSE meat. An examination of the literature provides hope in this regard, but it also reveals the complexity involved in the genetic approach as well as the great difficulty and expenses inherent in obtaining adequate genetic information on sufficient numbers of animals. Minkema (1969) has expressed the problems inherent to this approach by indicating that the living process is an expression of hundreds of chemical reactions which are perhaps all interacting. He believes it will be difficult to find one or more characteristics that are strongly genetically related to stress resistance and meat quality and show a reasonably high heritability. The most recent and comprehensive statements made on the role of genetics in animal stress susceptibility and meat quality are by Christian (1972); the interested reader is referred to that publication for a complete review. The heritability of a number of pork quality traits has been studied by various groups including Allen et al. ( 1966), Jensen et al. ( 1967), Omtvedt (1968), Jonsson et al. (1971), and Walstra et al. (1971). Most of the traits studied have a heritability of about 0.3. Christian (1972) considers that estimates for pork quality traits ranging between 20 and 40% are moderately influenced by heredity. Steinhauf ( 1969) suggests, in view of the work done, that selection for meat quality characteristics is possible. He concludes that meat quality may be improved by providing adequate environmental conditions, but in the future a genetic approach to this criterion may be possible and even necessary. There has been much concern over the possibility that selection for very muscular or very lean pigs has carried with it a lowered meat quality. Recent work has shed some light on this. Flock (1968) found an estimated association of 0.56 between color score and area of the longissimus muscle in German Landrace swine. Charpentier et al. (1971), however, studied a group of 244 Large White pigs and found few significant correlations between meatiness and meat quality. They suggested that, in that breed, good carcass characteristics and good meat quality may be obtained simultaneously. They also emphasized that the difference between total correlation and intraslaughter-day correlation suggests that consideration must be given to the slaughtering conditions if selection is to be applied to maintain good meat quality. German

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results (Steinhauf, 1969) indicate that selection for meat quality would negatively affect the criteria of meat-producing capacity. A consideration of the definition of meat quantity may be paramount to the somewhat confused reports. This has been clarified by Vos and Sybesma (1971). They measured quantitative aspects of backfat thickness and type. Meat quality was assessed in terms of p H and rigor mortis. They found no relation between backfat thickness and meat quality, but the relation between type rating and meat quality was negative. Therefore, higher demands for backfat thickness did not influence meat quality, but a more rigorous selection for meat type may result in a further decrease in meat quality. Increasing meat quantity by decreasing fat thickness probably does not lower meat quality. However, increasing meat quantity by selection for the highest type will result in carcasses with lower meat quality. There has been some controversy about the breed incidence of PSE and stress susceptibility. Researchers have identified certain breeds as having a higher incidence of the problem and have therefore concentrated research effort in that direction. Van d e Pas and Walstra (1971), for example, studied subjective meat quality scores, transmission values, pH, and rigor mortis in Dutch Landrace, Large White, and Pietrain breeds; tSe most desirable traits were found in the Large White, while the least desirable traits were observed in the Pietrain. The Poland China animal has been employed frequently as a stress-susceptible animal in research in the United States; in Europe the Pietrain has been the animal of choice. This action has naturally drawn criticism from breed associations and producers. Even though a higher incidence has been observed in certain breeds or strains, it is safe to say that stress susceptibility is a potential problem to all pig producers. It has probably best been put by Christian (1972), who concluded that stress susceptibility is a current or potential problem within all breeds that have been chosen for carcass improvement programs without consideration of carcass quality characteristics. Clear-cut research on the inheritance of stress susceptibility seems rather elusive. There have been numerous speculations based generally on breed and strain differences as described above. Iowa State University has, however, launched a program designed to provide some unequivocal answers. Although incomplete, the research is described by Christian (1972). The reader is referred thereto for a description of the basis of the herd established and for some preliminary results. Christian suggests that color and marbling scores of sibs are of limited value in predicting the stress-susceptibility status of surviving littermates.

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Elsewhere in this review the problem of malignant hyperthermia syndrome ( M H ) is discussed (Section 11,E). Such work, even though with very limited numbers, has produced information on inheritance that is undoubtedly applicable to the PSS and PSE condition. Hall et al. (1966) suggested that the abnormal reaction of the stress-prone pig to halothane was inherited as an autosomal dominant of variable penetrance and variable expressivity. Christian ( 1972) believes that stress susceptibility is definitely under genetic control, but the exact mode of inheritance is unclear, and variation in the degree of expression is certain to exist. The economic significance of PSS is recognized, since a survey by Livestock Conservation Incorporated ( 1971) estimated that 36% of hog producers in the United States encountered PSS. Affected herds constituted 44% of market hog production. The literature cited in this section on genetics shows that most pork meat quality characteristics are moderately heritable. Stress susceptibility is almost certainly under genetic control. The problem can probably be minimized with little loss of production and meatiness characteristics provided that a carefully controlled selection program is used.

B. DETECTION METHODS A great deal of effort has been expended on trying to develop a simple, reliable, and nondestructive test for identification of stress-susceptible animals. The goal of such work is to establish an inexpensive screening method for use in a selection program against stress susceptibility; the pay-off would be the elimination of the problem from the pig population. A number of methods are now available, but cost of sample collection and analysis are significant; furthermore, the effects of method and time of sampling, animal age, animal sex, stress of sampling, environment, and other factors are not clearly understood. Stress of sample collection may drastically alter certain parameters of the blood even in stressresistant animals, and contamination of blood samples with minute pieces of muscle tissue or tissue juice can drastically alter such measurements as enzyme activities. A combination of methods and observations may be employed to produce the most accurate and reliable results. This approach has been advocated by Judge (1972). He has developed a protocol to determine whether stress susceptibility in an animal is suspect, highly probable, or practically certain. It is easy to see that, if one wishes greater reliability, more extensive observations and tests must be employed. Ob-

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servation of the animal for visual symptoms (such as muscle tremors, general nervousness, and splotchy red areas on the skin) is comparatively simple, but more refined tests are generally required to document the suspicion that an animal is stress-susceptible. This section will be devoted to a description of the basis for certain detection methods presently under consideration.

1. Elevated Temperature An elevated temperature of muscle was recognized early as one of the characteristics in the development of PSE. It was subsequently learned that the temperature of the live stress-susceptible animal rises markedly (to 40" to 43°C) in response to stress. The measurement of rectal temperature is relatively simple, but animals often succumb when such high temperatures are attained. Addis et al. (1967a) suggested that elevated body temperature and respiration rate may be used to predict postmortem muscle properties.

2. Blood Component Tests Blood pH and gas composition may change drastically in response to forced exercise in stress-susceptible animals, as described in the section on animal physiology (Section 11, A ) . The type and extent of stress are difficult to control. The facts that the body functions very strongly to maintain homeostasis and that terminal stress may develop before large changes are noted are negative considerations. Great effort in research has been directed at the study of nonplasmaspecific enzymes as indicators of stress susceptibility. The general premise is that, in stress-susceptible animals, muscle cell enzymes leak into the blood because of changes in permeability of the muscle cell membrane. Increased blood serum or plasma levels of such enzymes can therefore be used as diagnostic aids just as they are in human medicine for some myopathies or to show muscle damage such as in myocardial infarction. These enzymes (see Section IV for reference citations) include CPK, aldolase, GOT, GPT, LDH, MDH, and alkaline phosphatase. In general the highest relationships with ultimate quality were found in various studies with CPK, aldolase, and LDH, (See Sybesma and Hessel-de Heer 1967; Addis and Kallweit, 1969; Addis, 1969; Schmidt et al., 1970b; Reddy et al., 1971). Since CPK is specific for muscular tissue, and is easier to determine than aldolase and LDH,, we believe that CPK is the best choice as a predictor of PSE. In our studies we also observed that two animals with extremely high CPK serum enzyme levels died

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showing terminal traumatic characteristics of the porcine stress syndrome after being exposed to severe experimental stress conditions (G. Eikelenboom, unpublished observations). However, a few comments should be made regarding the possible factors that might influence serum enzyme tests and the significance of a serum enzyme test for the prediction of stress susceptibility or PSE. Bickhardt ( 1971) demonstrated that most of the serum enzymes in both stress-susceptible and stress-resistant pigs are increased after short-term exercise. Therefore, chasing and restraining the pig for blood collection might influence the results. Although the differences in serum enzymes between clearly stress-susceptible and stress-resistant animals are probably large, it is necessary to minimize and standardize the stress provoked by blood collection in order to increase the accuracy of the test. Also, the method of blood sample collection may play an important role. Allen and Patterson (1971) reported that increased levels of CPK and aldolase originating from the muscles of Pietrain pigs may possibly be correlated with increased stress susceptibility. They compared blood collected from the ear vein immediately before slaughter with that collected from the throat wound during exsanguination and found that enzyme activities were two to fourteen times as high in the samples collected at slaughter. Puncture of the vena cava also gave elevated readings, possibly from tissue contamination of the blood sample due to the relatively large-bore needle used in this technique. Experiments have also been conducted on the feasibility of using blood levels of certain hormones as a diagnostic test. These procedures require elaborate sampling techniques, as stress can markedly alter circulating levels of the hormones; assay methods are, moreover, generally quite exacting and laborious. Marple et al. (1972a) reported that the ratio of corticoid to ACTH might be a good indicator of stress susceptibility. Susceptible pigs had higher levels of ACTH under both resting and stress conditions, and the ratio of corticoids to ACTH was lower than that in stress-resistant pigs by a factor of 2 to 4. 3. Muscle Biopsy Some rather accurate detection tests can be made by muscle biopsy. The most promising work in this area has been reported by Schmidt et a2. (1972a), who streamlined the procedure by using liquid nitrogen as a local anesthetic on the skin and by using Koffler tongs to remove a small biopsy sample. They found that the G-6-P level of the biopsy is a good predictor of postmortem muscle quality. They stated that the level of muscle G-6-P at 6 or 12 days antemortem provided as good a

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prediction of muscle quality as did p H or rigor measurements made within 2 hours postmortem. Sybesma et al. (1972) repeated the experiment on a large number of pigs and found lower, although significant, relationships between G-6-P or lactate and ultimate quality. Measurements of p H at 45 minutes postmortem were a better predictor than G-6-P or lactate. No substantial difference in predictive value could be shown between G-6-P and lactate. Sampling at 14 and 6 weeks antemortem did not show a significant relationship with ultimate quality. It was suggested that, to be useful in selection, correlations between biopsy analysis and muscle quality need to be improved; this can probably be achieved by further standardization of sampling technique, transport, and slaughter circumstances ( Sybesma et al., 1972). Eikelenboom et al. (1974) suggested that the relationships of any predictive test to postmortem muscle quality will remain of limited value because of environmental factors which are nearly impossible to control. More emphasis should be placed on relating the results of such tests to the genetic predisposition of the animal rather than to postmortem quality. 4 . Halothane Anesthesia Christian ( 1973) reported that stress-susceptible swine responded to halothane anesthesia with muscle rigidity and hyperthermia. Pigs were exposed to a 6% halothane in oxygen mixture for 2 minutes followed by 3 minutes exposure to 2% % halothane in oxygen. Susceptible pigs often responded to halothane within 30 seconds and usually recovered rapidly if the nose cone was removed at the first signs of muscle rigidity. However, as body weight exceeds 50 kg, the frequency of fatal responses increased among susceptible swine. Pigs responding to halothane appear to be stress-susceptible but unfortunately, not all stress-susceptible swine are halothane sensitive ( D. Marple, unpublished observations).

C. COKTHOL METHODS Although the ultim'itc and complete control of the PSS and PSE problem depends on elucidation of the mechanism of these conditions, several methods or procedures are now available which reduce their occurrence or intensity. Ordinary sound management and proper slaughterhouse procedure are important; other more specific techniques are generally expensive or involve the use of chemicals that would probably not be approved for use in meat-producing animals, particularly since they would be administered quite soon prior to slaughter.

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1. Management and Handling

Producers, truckers, and personnel in livestock holding and slaughter areas should be instructed in procedures that will in every way reduce stress to the animals. Such techniques as cooling devices in warm weather and avoidance of crowding, loud noise, and mixing of strange groups of animals, as well as providing an opportunity for the animals to rest, will minimize death loss and improve subsequent meat quality.

2. Transportation Transportation of animals from the producer to the slaughterhouse is a major stress that often results in death loss of PSS animals. Lendfers ( 1971) studied this problem and concluded that environmental temperature is one of the most important factors causing death of pigs during transport. Other factors that may increase the death rate, especially when the environmental temperature is greater than 10°C, are overcrowding, unloading during midday, and transporting for long distances. Scheper (1971) studied approximately 2200 pigs from 1963 to 1970 and found a close correlation between stress and meat quality which was dependent on transport distance. In some European countries a period of rest for the animals is allowed after transport and before slaughter. Barton (1971) found, however, that giving Danish Landrace pigs a rest period of 1 to 2 hours prior to slaughter did not always improve meat quality. Container transport is also under investigation. 3. Tranquilizers Oldigs and Unshelm (1971) studied the effect of administration of Stresnil ( azaperone ) to pigs before they were transported over relatively short distances (15 k m ) . Such treatment resulted in a distinct improvement in the characteristics of carcass temperature and color, pH, swelling capacity, and water-binding capacity of muscle. Carcass temperature of treated pigs was significantly lower than that of untreated pigs a t 30 minutes postmortem. I t was not known whether Stresnil decreased body temperature directly or whether it reduced the increase of body temperature caused by stress. Devloo et al. (1971) studied a group of over 11,OOO pigs, 4510 of which received an intramuscular injection of Stresnil prior to transport. They found that five times as many untreated pigs died during transport or were emergency slaughtered at the slaughterhouse. Temperature of the

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meat and rigor mortis status, measured at 27 minutes after slaughter, showed significant decreases in the treated group. The authors stated that, if such measurements are reliable criteria of meat quality, then significantly more treated animals had higher quality meat. These authors also placed due consideration on the drug residue problem. They studied the resorption and metabolism of azaperone with a tritium-labeled compound and found that it was rapidly metabolized in the liver and eliminated in the urine and feces. Four hours following injection there was less than 4% of the total injected dose remaining in the body. Diffusion was limited to an area of less than 1 cm3 at the injection site, and only 1 to 5% of the total injected amount was found there after 4 hours. They concluded that the residue of azaperone in meat and organs of a pig treated 4 hours prior to slaughter was pharmacologically and toxicologically inactive to man. Other tranquilizers ( including promazine derivatives) have been used successfully to improve muscle quality.

4. Magnesium Treatment The use of MgSO, as a preventive for PSE meat is intriguing and certainly requires further work not only on basic issues but also on alternative administration methods and levels required for a reasonably consistent improvement in meat quality. A complete account of the effect of magnesium is given in Section IV, A.

5. Stunning Van der Wal (1971) has investigated stunning methods from the viewpoint of PSS and meat quality. He concluded that electric stunning and the use of CO, gas were suitable for industrial use. The captive bolt instrument was not suitable, however, as it produced violent muscle contractions, poor debleeding, and hemorrhages. Sybesma and Groen (1970) compared a group of pigs which were stunned by carbon dioxide with another group of pigs that had gone through the carbon dioxide tunnel (which did not contain CO, at that moment) and were subsequently stunned electrically with 70 volts. The work was conducted at a commercial plant. The carbon dioxide stunning resulted in an increased glycolytic rate and higher incidence of PSE than were found in the electrically stunned group. This observation, therefore, tends to confirm the significance of anoxia as a contributing factor. The reader is referred to the statements by Mcbughlin (1971) for greater detail on several aspects of stunning.

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6. Carcass Cooling

A rather rapid cooling has been considered a desirable treatment to minimize the development of PSE, since the combination of low pH and high temperature in the meat has been shown to be undesirable. Proper refrigeration, therefore, not only lowers the temperature of the muscle as such but also slows the rate of chemical reaction (glycolysis) as the temperature is lowered. The pig carcass represents a formidable problem to the refrigeration engineer because of the thick layer of backfat which acts as an efficient insulator. Hart and Sybesma (1964) studied the possibility of removing the fat layer prior to cooling, thereby achieving a more efficient cooling. Such a procedure did result in improved quality of muscle, The work of Marsh et al. (1972) has shown that, even though immediate exposure of pork muscle to 0°C with subsequent rapid cooling has a toughening effect, it is not nearly as serious as the consequences of cold shortening in muscle of bovine and ovine species. Work with muscle of beef (Marsh and Leet, 1966) and lamb (Marsh et al., 1968) has revealed that a too rapid chilling may have a detrimental effect via cold shortening which results in a drastic decrease in tenderness (Marsh, 1972). Pig muscle is quantitatively very different from beef and lamb muscle in this respect, however. Marsh et al. (1972) found that prerigor excision followed by a rapid cooling produced a statistically significant toughening in several major muscles of the pig; but the extent of toughening was considerably less than that experienced in the other species if they were treated in a similar manner. It can therefore be concluded that a rather rapid chilling postmortem in pig muscle is beneficial, but a similar procedure in beef and lamb muscle requires some caution.

7 . Liquid Nitrogen Cooling As was noted above, the efficient cooling of pork carcasses offers a method to minimize the development of PSE. Borchert and Briskey (1964, 1965) reported a method that uses liquid nitrogen to achieve a rapid cooling. They employed a wide range of immersion rates in liquid nitrogen to determine its effectiveness in controlling not only the development of PSE but also other muscle properties. Liquid nitrogen treatment was extremely effective in preventing the development of PSE muscle regardless of whether subsequent equilibration was at - 18" or 4°C. Only the severe immersion rates produced any evidence of thaw rigor, which would, of course, be undesirable. The color structure score of liquid nitrogen-treated samples was superior to that of untreated sam-

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ples, and the extractability of myofibrillar and sarcoplasmic proteins was also improved in treated samples. Emulsifying properties of the muscle were also improved by liquid nitrogen treatment. Commercial development of the liquid nitrogen procedure may be hampered by cost.

VIII.

FUTURE RESEARCH NEEDS

The problem of animal stress susceptibility and the low-quality meat produced from such animals has been studied extensively, and a voluminous bank of descriptive information has been published. The problem is not solved, however, or even under control. There are two key areas for further research. The first area is strictly fundamental, and that is to determine whether the animal stress susceptibility problem is due to a primary muscle defect or whether it is controlled by the hormonal or nervous system. This requires serious research effort, as the origin of the mechanism must be known before it can be elucidated and before steps can be made to control or alter the defect. Along this line, the developmental aspects of the problem also require attention. Moreover, recent work on malignant hyperthermia has opened the door for close and valuable cooperation with medical researchers. The second area is quite practical. Optimum management, transportation, and stunning procedures should be established by critical research instead of by intuition. The information so gained should be written in understandable fashion together with reasonable recommendation for proper techniques. This approach will minimize the overt effects of the problem. Of prime necessity is continued work on detection methods. A simple, reliable, and inexpensive screening method for animal stress susceptibility is prerequisite to complete elimination of the problem through genetic selection. Important research is also needed on genetic aspects and inheritance of stress susceptibility. A current extensive and critical survey on the incidence and financial implication of PSS and PSE would be very useful. Lastly, consideration should be given to the possible existence of the same problem in other species.

IX.

SUMMARY

Research effort on animal stress susceptibility, ranging from very fundamental to very applied, has been reviewed in this manuscript. Much has been learned in recent years about the endocrinological aspects of

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stress susceptibility in domestic animals. The more classic approach of study of muscle biochemistry and morphology has been pursued with greater refinement. The physiology of stress-susceptible animals has been investigated, and particular interest has centered on the malignant hyperthermia syndrome. The problem of PSS and PSE in pigs has been openly recognized. Efforts are being made, particularly by the live animal producers, to identify the problem in their animals and then to minimize or eliminate it by proper management and genetic selection. Some evidence exists that the problem results from too intensive selection for muscling in animals. Even though the pig is the outstanding example, the same situation may be occurring in other species. The mechanism of the problem is not really understood; indeed, it is not yet clear whether the basis resides in the muscle itself or is central in the hormonal or neurological systems of the animal. It is clear, though, that stress susceptibility in animals is not best studied by consideration of only postmortem quality of meat from such animals.

REFERENCES Aberle, E. D., Thomas, N. W., Howe, J. M., and Arroyo, P. T. 1969. Environmental influence on high energy phosphate metabolites in porcine muscle 3. Food Sci. 34, 600. Aberle, E. D., and Merkel, R. A. 1968. Physical and biochemical properties of porcine muscle as affected by exogenous epinephrine and prednisolone. I . Food Sci. 33, 1. Addis, P. B. 1969. Relation of blood serum lactate dehydrogenase isoenzymes to pale, soft, exudative muscle. Proc. Reciprocal Meat Conf., 22nd, 1969, p. 151. Addis, P. B., and Kallweit, E. 1969. Beziehungen zwischen Isoenzymen der Laktatdehydrogenase ( LDH ) im Blutserum and Merkmalen der Fleischbeschaffenheit beim Schwein. Fleischwirtschaft 21, 218. Addis, P. B., Judge, M. D., Pickett, R. A., and Jones, H. W. 1965. Environmental factors associated with porcine adrenal size and muscle characteristics. J. Anim. Sci. 24, 127. Addis, P. B., Johnson, H. R., Thomas, N. W., and Judge, M. D. 1967a. Effect of temperature acclimation on porcine physiological responses to heat stress and associated properties of muscle. J. Anim. Sci, 26, 466. Addis, P. B., Johnson, H. R., Heidenreich, C. J., Jones, H. W., and Judge, M. D. 1967b. Effect of humidity level in a warm growing environment on porcine carcass composition and quality. J. Anim. Sci. 26, 705. Allen, E., Forest, J. C., Chapman, A. B., First, N., Bray, R. W., and Briskey, E. J. 1966. Phenotypic and genetic associations between porcine muscle properties. J. Anim. Sci. 25, 962. Allen, W. M., and Patterson, D. S. P. 1971. The possible relationship between plasma creatine phosphokinase activity and muscle characteristics in the pig. In “The

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R. G. CASSENS ET AL.

Condition and Meat Quality in Pigs” (J. C. M. Hessel-deHeer et al., eds.), Proc. 2nd Int. Symp., p. 90. Wageningen, The Netherlands. Allen, W. M., Berrett, S., Harding, J. D. J., and Patterson, D. S. P. 1970. Experimentally induced acute stress syndrome in Pietrain pigs. Vet. Rec. 87, 64. Akhen, T. G., Gerrits, R. J., and Hetzer, H. 0. 1972. Serum growth hormone levels at three ages in pigs selected for high- and low-backfat thickness. J. Anim. Sci. 35, 235. Anderson, L. D., and Parrish, F. C. 1972. Histochemical, organoleptic and tenderness properties of porcine muscle. PTOC.Reciprocal Meat Conf., 25th, 1972, p. 176. Ashmore, C. P., Tompkins, G., and Doerr, L. 1972. Postnatal development of muscle fiber types in domestic animals. J . Anim. Sci. 34, 37. Baird, D. M., Nalbandov, A. V., and Norton, H. W. 1952. Some physiological causes of genetically different rates of growth in swine. J. Anim. Sci. 11, 292. Ball, R. A., Topel, D. G., Wagner, W. C., and Annis, C. L. 1971. Porcine stress syn. Med. Ass. 158, 1855 (abstr.). drome. J . A ~ TVet. Barton, P. A. 1971. Some experience on the effect of pre-slaughter treatment on the meat quality of pigs with low stress resistance. I n “The Condition and Meat Quality of Pigs” (J. C. M. Hessel-deHeer et aE., eds.), Proc. 2nd Int. Symp., p. 180. Wageningen, The Netherlands. Bate-Smith, E. C. 1948. The physiology and chemistry of rigor mortis, with special reference to the aging of beef. Aduan Food Res. 1, 1. Beecher, G. R., Briskey, E. J., and Hoekstra, W. G. 1965. A comparison of glycolysis and associated changes in li&t and dark portions of the porcine semitendinosus. J. Food Sci. 30, 477. Bendall, J. R. 1961. Post-mortem changes in muscle. I n “The Structure and Function of Muscle” (G. H. Bourne, ed.), Vol. 3, p. 227. Academic Press, New York. Bendall, J. R. 1966. The effect of pre-treatment of pigs with curare on the postmortem rate of pH fall and onset of rigor mortis in the musculature. J. Sci. Food Agr. 17, 333. Bendall, J. R., and Wismer-Pedersen, J. 1962. Some properties of the fibrillar proteins of normal and watery pork muscle. J. Food Sci. 27, 144. Berman, M. C., and Kench, J. E. 1973. Biochemical features of malignant hyperthermia in Landrace pigs. In “International Symposium on Malignant Hyperthermia” (R. A. Gordon, B. A. Britt, and W. Kalow, eds.), p. 287. Thomas, Springfield, Illinois. Berman, M. C., Harrison, G. G., Bull, A. B., and Kench, J. E. 1970. Changes underlying halothane induced malignant hyperpyrexia in Landrace pigs. Nature (London) 225, 653. Bickhardt, K. 1971. Muscle metabolism and enzyme patterns in Landrace strains with different meat quality. In “The Condition and Meat Quality of Pigs” (J.C. M. Hessel-de Heer et al., eds.), Proc. 2nd Int. Symp., p. 36. Wageningen, The Netherlands. Bickhardt, K., Chevalier, H. J., Giese, W., and Reinhard, H. J. 1972. Akute Rucken Muskelnekrose und Belastrugsmyopathie beim Schwein. Aduan. Vet. Med. 18. Bodwell, C. E., Pearson, A. M., and Fennell, R. A. 1965. Post-mortem changes in muscle. 111. Histochemical observations in beef and pork. J. Food Sci. 30, 944. Bodwell, C. E., Pearson, A. M., Wismer-Pedersen, J., and Bratzler, L. J. 1966. Postmortem changes in muscle. 11. Chemical and physical changes in pork. 1. Food Sci. 31, 1.

ANIMAL PHYSIOLOGY AND MEAT QUALITY

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Borchert, L. L. 1967. Temperature induced post-mortem changes in skeletal muscles. Ph.D. Thesis, University of Wisconsin, Madison. Borchert, L. L., and Briskey, E. J. 1964. Prevention of pale, soft, exudative porcine muscle through partial freezing with liquid nitrogen post-mortem. J. Food Sci. 29, 203. Borchert, L. L., and Briskey, E. J. 1965. Protein solubility and associated properties of porcine muscle as influenced by partial freezing with liquid nitrogen. J. Food Sci. 30, 138. Bray, R. W. 1966. Pork quality-definition, characteristics and significance. J . Anim. Sci. 25, 839. Briskey, E. J. 1963. Influence of ante- and post-mortem handling practices on properties of muscle which are related to tenderness. PTOC.Meat Tenderness Symp., 1963, p. 195. Briskey, E. J. 1964. Etiological status and associated studies of pale, soft, exudative porcine musculature. Aduan. Food Res. 13,89. Briskey, E. J., and Wismer-Pedersen, J. 1961. Biochemistry of pork muscle structure. I. Rate of anaerobic glycolysis and temperature change. J. Food Sci. 26, 297. Briskey, E. J., Bray, R. W., Hoekstra, W. G., Grummer, R. H., and Phillips, P. H. 1959. The effect of various levels of exercise in altering the chemical and physical characteristics of certain pork ham classes. J. Anim. Sci 18, 153. Britt, B. A., and Kalow, W. 1970. Malignant hyperthermia: A statistical review. Can. Anuesth. SOC. J. 17,293. Britt, B. A., Locher, W. G., and Kalow, W. 1969. Hereditary aspects of malignant hyperthermia. Can. Anoesth. Soc. J. 16, 89. Britt, B. A., Kalow, W., and Endrenyi, L. 1973. Malignant hyperthermia and the mitochondria in human patients. In “International Symposium on Malignant Hyperthermia” ( R. A. Gordon, B. A. Britt, and W. Kalow, eds.), p. 387. Thomas, Springfield, Illinois. Brooks, G. A., and Cassens, R. G. 1973. Respiratory functions of mitochondria isolated from stress-susceptible and stress-resistant pigs. J. Anim. Sci. 37, p. 688. Brucker, R. F., Williams, C. H., Popinigis, J., Galvez, T. L., Vail, W. J., and Taylor, C. A. 1973. In uitro studies on liver mitochondria and skeletal muscle sarcoplasmic reticulum fragments isolated from hyperpyrexic swine. In “International Symposium on Malignant Hyperthermia” (R. A. Gordon, B. A. Britt, and W. Kalow, eds.), p. 238. Thomas, Springfield, Illinois. Buchter, L., and Zeuthen, P. 1971. The effect of aging on the organoleptic qualities of PSE and normal pork loins. In “The Condition and Meat Quality of Pigs” (J. C . M. Hessel-de Heer et al., eds.), Proc. 2nd Int. Symp., p. 247. Wageningen, The Netherlands. Buttkus, H., and Tomlinson, N. 1966. Some aspects of postmortem changes in fish muscle. In “The Physiology and Biochemistry of Muscle as a Food” (E. J. Briskey, R. G. Cassens, and J. C. Trautnian, eds.), p. 197. Univ. of Wisconsin Press, Madison. Campion, D. R., Marsh, B. B., Schmidt, G . R., Cassens, R. G . , Kauffman, R. G., and Briskey, E. J. 1971. Use of whole-body perfusion in the study of muscle glycolysis. J. Food Sci. 36,545. Cassens, R. G. 1966. General aspects of post-mortem change. In “The Physiology and Biochemistry of Muscle as a F o o d ( E . J. Briskey, R. G. Cassens, and J. C. Trautman, eds.), p. 181. Univ. of Wisconsin Press, Madison.

142

R. G. CASSENS ET AL.

Cassens, R. G., Briskey, E. J., and Hoekstra, W. G. 1963. Electron microscopy of post-morten changes in porcine muscle. J. Food Sci. 28, 680. Cassens, R. G . , Judge, M. D., Sink, J. D., and Briskey, E. J. 1965. Porcine adrenocortical lipids in relation to striated muscle characteristics. Proc. SOC. Exp. BioZ. Med. 120, 854. Cassens, R. G., Cooper, C. C., and Briskey, E. J. 1969. The occurrence and histochemical characterization of giant fibers in the muscle of growing and adult animals. Acta Neuropathol. 12, 300. Cassens, R. G., Giesler, F., and Kolb, Q., eds. 1972. “Proceedings of the Pork Quality Symposium.” Univ. of Wisconsin Ext., Madison. Charpentier, J., Monin, G., and Ollivier, L. 1971. Correlations between carcass characteristics and meat quality in Large White Pigs. In “The Condition and Meat Quality of Pigs” (J. C. M. Hessel-de Heer et al., eds.), p. 255. Wageningen, The Netherlands. Christian, L. L. 1972. A review of the role of genetics in animal stress susceptibility and meat quality. In “Proceedings of the Pork Quality Symposium” (R. G. Cassens, F. Giesler, and Q. Kolb, eds.), p. 91. Univ. of Wisconsin Ext., Madison. Christian, L. L. 1973. Recent developments in swine stress research. Presented 65th Annu. Meet. Amer. Soc. Anim. Sci., Lincoln, Nebraska. Churchill-Davidson, H. G. 1968. Malignant hyperpyrexia. Brit. Med. J. 3, 69. Cohen, P. J., and Marshall, B. E., 1968. Effects of halothane on respiratory control and oxygen consumption of rat liver mitochondria. In “Toxicity of Anesthetics” B. R. Fink, ed.), p. 24. Williams & Wilkens, Baltimore, Maryland. Collins, K. J., and Weiner, J. S. 1968. Endocrinological aspects of exposure to high environmental temperatures. Physiol. Reu. 48, 785. Cooper, C. C., Cassens, R. G., and Briskey, E. J. 1969. Capillary distribution and fiber characteristics in skeletal muscle of stress-susceptible animals. J . Food Sci. 34, 299. Cooper, C. C., Cassens, R. G., Kastenschmidt, L. L., and Briskey, E. J. 1970. Histochemical characterization of muscle differentiation. Deuelop. Biol. 23, 169. Dalrymple, R. H., and Kelly, R. F. 1966. Processing, composition and organoleptic characteristics of normal and pale, soft, exudative pork. J. Anim. Sci. 25, 883 ( abstr. ). Dalrymple, R. H., and Kelly, R. F. 1969. Incidence of PSE pork in midwestem and southern hogs. J. Anim. Sci. 29, 120 (abstr.). de Bruin, A. 1971. Fibre characteristics and lactic acid level in porcine muscle. In “The Condition and Meat Quality of Pigs” (J. C. M. Hessel-de Heer et al., eds.), Proc. 2nd Int. Symp., p. 86. Wageningen, The Netherlands. Deethardt, D., and Tuma, H. J. 1971. Effect of cooking method on various qualities of pork loin. J. Food Sci. 36, 626. DeFremery, D. 1966. Some aspects of postmorten changes in poultry muscle. In “The Physiology and Biochemistry of Muscle as a Food” ( E . J. Briskey, R. G. Cassens, and J. C . Trautman, eds.), p. 205. Univ. of Wisconsin Press, Madison. Dekker, T. P. 1969. Some data about corticosteriod levels and Na/K contents of Pietrain pig plasma. In “Recent Points of View on the Condition and Meat Quality of Pigs for Slaughter” (W. Sybesma, P. G. van der Wal, and P. Walstra, eds.), p. 117. I.V.O., Zeist, The Netherlands.

ANIMAL PHYSIOLOGY AND MEAT QUALITY

143

Dekker, T. P. 1971. Meat quality and muscle electrolytes. In “The Condition and Meat Quality of Pigs” ( J . C. M. Hessel-de Heer et al., eds.), Proc. 2nd Int. Symp., p. 43. Wageningen, The Netherlands. Denborough, M. A., Forster, J. F. A., Lovell, R. R. H., Maplestone, P. A., and Villier, J. D. 1962. Anaesthetic deaths in a family. Brit. J. Anaesth. 34, 395. Denborough, M. A., Hird, F. J. R., King, J. O., Marginson, M. A., Mitchelson, K. R., Nayler, W. G., Rex, M. A., Zapf, P., and Condron, R. J. 1973. Mitochondrial and other studies in Australian Lanrace pigs affected with malignant hyperthermia. In “International Symposium on Malignant Hyperthermia” (R. A. Gordon, B. A. Britt, and W. Kalow, eds. ), p. 229. Thomas, Springfield, Illinois. Devloo, S., Geerts, H., and Symoens, J. 1971. Effect of azaperone on mortality and meat quality after transport of pigs for slaughter. In “The Condition and Meat Quality of Pigs” (J. C. M. Hessel-de Heer et al., eds.), Proc. 2nd Int. Symp., p. 215. Wageningen, The Netherlands. Dildey, D. D., Aberle, E. D., Forrest, J. C., and Judge, M. D. 1970. Porcine muscularity and properties associated with pale, soft, exudative muscle. J. Anim. Sci. 31, 681. Dutson, T. R., Pearson, A. M., Mcrkel, R. A,, Koch, D. E., and Weatherspoon, J. B. 1971. Histochemical activity of some lysosomal enzymes in normal and in pale, soft and exudative pig muscle. J. Anim. Sci. 32, 233. Dvorak, M. 1967. 17-Hydroxycorticosteroid levels of blood plasma and the in v t r o activity of the adrenal cortex in piglets of various ages. Acta Univ. Agr. Bmo, Fac. Vet. 36,403. Dvorak, M. 1972. Adrenocortical function in foetal, neonatal and young pigs. J . Endocrinol. 54, 473. Eikelenboom, G. 1972. Stress-susceptibility in swine and its relationship with energy metabolism in skeletal musculature. P1i.D. Thesis, State University of Utrecht, Utrecht, The Netherlands. Eikelenboom, G. and Sybesma, W. 1969. Several ways of stunning and their influence on meat quality. In “Recent Points of View on the Condition and Meat Quality of Pigs for Slaughter” ( W . Sybesma, P. G . van der Wal, and P. Walstra, eds.), p. 209. I.V.O., Zeist, The Netherlands. Eikelenboom, G., and Sybesma, W. 1974. A possible mechanism for induction of porcine malignant hyperthermia. J. Anim. Sci. 38, 504. Eikelenboom, G . , and van den Bergh, S. G. 1971. Aberrant mitochondria1 energy metabolism in stress-susceptible pigs. In “The Condition and Meat Quality of Pigs” ( J . C. M. Hessell-de Heer et al., eds.), Proc. 2nd Int. Symp., p. 66. Wageningen, The Netherlands. Eikelenboom, G., and van den Bergh, S. G. 1973. Mitochondrial metabolism in stress-susceptible pigs. J. Anim. Sci. 37, 692. Eikelenboom, G . , and Verwey, M. C. 1971. A short note on the effect of halothane on muscle mitochondria. In “The Condition and Meat Quality of Pigs” (J. C. M. Hessel-de Heer et al., eds.), Proc. 2nd Int. Symp., p. 61. Wageningen, The Netherlands. Eikelenboom, G., and Weiss, G. M. 1972. Breed and exercise influence on T4 and PSE. J. Anim. Sci. 35, 1096 (abstr.). Eikelenboom, G., van der Wal, P. G., and Verwey, M. C. 1970. Plasma-transaminasen und Fleischqualitat. Fleischwirtschaft 50, 1211.

144

R. G. CASSENS ET AL.

Eikelenboom, G., Campion, D. R., Kauffman, R. G., and Cassens, R. G. 1974. Early postmortem methods of detecting ultimate porcine meat quality. 1. Anim. Sci. 39, 303. Field, R. A., Pearson, A. M., Koch, D. E., and Merkel, R. A. 1970. Thermal behavior of porcine collagen as related to post-mortem time. J. Food Sci. 35, 113. Fitts, R., Campion, D., Nagle, F., and Cassens, R. 1973. Contractile properties of skeletal muscle from trained miniature pigs. Pfliigers Arch. 343, 133. Flock, D.K. 1968. Farbhelligkeit im Musculus Longissimus Dorsi als Selektionsmerkma1 beim Schwein. Fkischwirtschuft 48, 1362. Forrest, J. C., and Briskey, E. J. 1967. Response of striated muscle to electrical stimulation. 1. Food Sci. 32,482. Forrest, J. C., Gundlach, R. F., and Briskey, E. J. 1963. A preliminary survey of the variation in certain pork ham muscle characteristics. Proc. Res. Counc. Amer. Meat Inst. Found. Uniu. Chicago, 15th, 1963 p. 81. Forrest, J. C., Judge, M. D., Sink, J. D., Hoekstra, W. G., and Briskey, E. J. 1966. Prediction of the time course of rigor mortis through response of muscle tissue to electrical stimulation. J. Food Sci. 31, 13. Forrest, J. C., Will, J. A., Schmidt, G. R., Judge, M. D., and Briskey E. J. 1968. Homeostasis in animals [Sus domesticus) during exposure to a warm environment. J. A w l . Physiol. 24, 33. Galloway, D. G., Topel, D. G., Will, J. A., Weirich, W. E., Cassens, R. G., and Briskey, E. J. 1973. Effect of environment on physiological and biochemical properties of pigs with fast and slow glycolyzing muscle. Can. J. Anim. Sci. 53, 659. Goll, D. E. 1968. The resolution of rigor mortis. Proc. FieciprocaE Meat Conf., Twenty-Farst, 1968, p. 16. Greaser, M. L., Cassens, R. G., Briskey, E. J., and Hoekstra, W. G. 1969a. Postmortem changes in subcellular fractions from normal and pale, soft, exudative porcine muscle. I. Calcium accumulation and adenosine triphosphatase activities. J. Food Sci. 34, 120. Greaser, M. L., Cassens, R. G., Briskey, E. J., and Hoekstra, W. G. 196913. Postmortem changes in subcellular fractions from normal and pale, soft, exudative porcine muscle. 2. Electron microscopy. J. Food Sci. 34, 125. Greaser, M. L. Cassens, R. G., Hoekstra, W. G., and Briskey, E. J. 1969c. The effect of pH-temperature treatments on the calcium-accumulating ability of purified sarcoplasmic reticulum. J. Food Sci. 34,633. Haase, S., and Steinhauf, D. 1971. Effects of stress on some oxygen metabolism parameters in boars. In “The Condition and Meat Quality of Pigs” (J. C. M. Hessel-de Heer d al., eds.), Proc. 2nd Int. Symp., p. 191. Wageningen, The Netherlands. Hall, J. T. 1972. Economic importance of pork. In “Proceedings of Pork Quality Symposium” (R. G. Cassens, F. Giesler, and Q. Kolb, eds.), p. IX. Univ. of Wisconsin Ext., Madison. Hall, L. W., Woolf, N., Bradley, J. W. P., and Jolly, D. W. 1966. Unusual reaction to swamethonium chloride. Brit. Med. J. 2, 1305. Hall, L. W., Trim, C. M., and Woolf, N. 1972. Further studies of porcine malignant hyperthermia. Brit. Med. J. 2, 145.

ANIMAL PHYSIOLOGY AND MEAT QUALITY

145

Hallund, O., and Bendall, J. R. 1965. The long-term effect of electrical stimulation on the post-mortem fall in pH in muscle of Landrace pigs. I . Food Sci. 30, 296. Harris, R. A., Munroe, J., Farmer, B., Kim, K. C., Jenkins, P. 1971. Action of halothane upon mitochondria1 respiration. Arch. Biochem. Biophys. 142, 435. Harrison, G. G. 1971. Anesthetic induced malignant hyperpyrexia: A suggested method of treatment. Brit. Med. J. 3, 454. Harrison, G. G., Biebuyck, J. F., Terblanche, J., Dent, D. M., Hickman, R., and Suanders, S. J. 1968. Hyperpyrexia during anesthesia. Brit. Med. J . 3, 594. Harrison, G. G., Saunders, S. J., Biebuyck, J. F., Hickman, R., Dent, D. M., Weaver, V., and Terblanche, J. 1969. Anaesthetic-induced malignant hyperpyrexia and a method for its prediction. Brit. J. Anaesth. 41, 844. Hart, P. C., and Sybesma, W. 1964. Einfluss von des Eutfernung der Speckhout auf die Fleischqualitat bei Schweinen. Proc. Eup. Meet. Meat Res. Workers, IOth,

1964. Hedrick, H. B. 1965. Influence of ante-mortem stress on meat palatability. J. Anim. S c i . 24, 255. Hedrick, H. B., Parrish, F. C., Jr., and Bailey, M. E. 1964. Effect of adrenaline stress on pork quality. J. Anim. Sci. 23, 225. Hedrick, H. B., Leavitt, R. K., and Alexander, M. A. 1968. Variation in porcine muscle quality of Duroc and Hampshire barrows. J. Anim. Sci. 27, 48. Heffron, J. J. A., and McLoughlin, J. V. 1971. The relationship between the rate of adenosine triphosphate hydrolysis and glycolysis post-mortem in skeletal muscle. In “The Condition and Meat Quality of Pigs” (J. C. M. Hessel-de Heer et al., eds.), p. 109. Wageningen, The Netherlands. Hendricks, H. B., Lafferty, D. T., Aberle, E. D., Judge, M. D., and Forrest, J. C. 1971. Relation of porcine muscle fiber type and size to postmortem shortening. J. Anim. Sci. 32, 57. Henry, M., Billion, J., and Haouza, G. 1955. La myopathie exudative dhpigmentaire du porc. Reu. Pathol. Gen. Comp. 669, 857. Henry, M., Romani, J. D., and Joubert, L. 1958. La myopathie exudative depigmentaire du porc. Maladie de l’adaptation. Essai pathogknique et consequences pratique. Reu. Pathol. Gen. Physiol. Clin. 696, 355. Herring, H. K., Haggard J. H., and Hansen, L. J. 1971. Studies on chemical and physical properties of pork in relation to quality. J. Amin. Sci. 33, 578. Hessel-de Heer, J. C. M. 1969. Serum LDH5 and muscular stress. I n “Recent Points of View on the Condition and Meat Quality of Pigs for Slaughter” (W. Sybesma, P. G. van der Wal, and P. Walstra, eds.), p. 179. I.V.O., Zeist, The Netherlands. Hessel-de Heer, J. C. M., Schmidt, G. R., Sybesma, W., and van der Wal, P. G., eds. 1971. “The Condition and Meat Quality of Pigs,” Proc. 2nd Int. Symp. Wageningen, The Netherlands. Howe, J. M., Thomas, N. W., Addis, P. B., and Judge, M. D. 1968. Temperature acclimation and its effects on porcine muscle properties in two humidity environments. J. Food Sci. 33, 235. Howe, J. M., Addis, P. B., Howard, R. D. and Judge, M. D. 1969. Environmentinduced adrenocortical lipid in “stress-susceptible” pigs. J. Anim. Sci. 28, 70. Isaacs, H., and Barlow, M. B. 1970. Malignant hyperpyrexia during anaesthesia: Possible association with subclinical rnyopathy. Brit. Med. 1. 1, 275.

146

R. G. CASSENS ET AL.

Jensen, P., Craig, H. B., and Robison, 0. W. 1967. Phenotypic and genetic associations among carcass traits of swine. J. Anim. Sci. 26, 1252. Jones, D. J., Jones, H. W., Harrington, R. B., and Judge, M. D. 1971. Effects of housing system and duration on quantitative and qualitative carcass traits in swine. J. Anim. Sci. 33, 18. Jones, E. W., Nelson, T. E., Anderson, I. L., Kern, D. O., and Burnap, T. K. 1972. Malignant hyperthermia of swine. Anaesthesiology 36, 42. Jonsson, P., Jenson, P., and Pedersen, 0. K. 1971. “Genetic Aspects of Meat Quality and Adaptation in Pigs.” Commission on Pig Production, Paris-Versailles. Jnbb, K. V. F., and Kennedy, P. C. 1970. “Pathology of Domestic Animals,” 2nd ed., Vol. 2. Academic, Press, New York. Judge, M. D. 1969. Environmental stress and meat quality. J. Anim. Sci. 28, 755. Judge, M. D. 1972. A review of possible methods to detect animal stress susceptibility and potential low quality pork. In “Proceedings of the Pork Quality Symposium” ( R . Cassens, F. Giesler, and Q. Kolb, eds.), p. 68. Univ. of Wisconsin Ext., Madison. Judge, M. D., and Marple, D. N. 1971. Adrenal insufficiency in stress-susceptible pigs. In “The Condition and Meat Quality of Pigs” (J. C . M. Hessel-de Heer et al., eds.), Proc. 2nd Int. Symp., p. 47. Wageningen, The Netherlands. Judge, M. D., and Stob, M. 1963. Stress during growth. I. Effect on physiology, carcass composition and carcass quality of lambs. J. Anim. Sci. 22, 1059. Judge, M. D., Cahill, V. R., Kunkle, L. E., and Deatherage, F. E. 1960. Pork quality. 11. Physical, chemical and organoleptic relationships in fresh pork. J. Anim. Sci. 19, 145. Judge, M. D., Briskey, E. J., and Meyer, R. K. 1966. Endocrine related post-mortem changes in porcine muscle. Nature (London) 212, 287. Judge, M. D., Cassens, R. G., and Briskey, E. J. 1967. Muscle properties of physically restrained stressor-susceptible and stressor-resistant porcine animals. 1. Food Sci. 32, 565. Judge, M. D., Briskey, E. J., Cassens, R. G . , Forrest, J. C., and Meyer, R. K. 1968. Adrenal and thyroid function in stress-susceptible pigs (Sus domesticus). Amer. J . Physiol. 214, 146. Judge, M. D., Eikelenboom, G . , Zuidam, L., and Sybesma, W. 1972. Blood acid-base status and oxygen binding during stress-induced hyperthermia in pigs. J. Anim. Sci. 35, 204 (abstr.). Kallweit, E. 1969. Effects of environmental temperature and exercise ante-mortem on blood and meat quality characteristics in pigs. In “Recent Points of View on the Condition and Meat Quality of Pigs for Slaughter” (W. Sybesma, P. G . van der Wal, and P. Walstra, eds.), p. 143. I.V.O., Zeist, The Netherlands. Kallweit, E., and Haase, S. 1971. The effect of short-term climatic stress on pigs. In “The Condition and Meat Quality of Pigs” (J. C . M. Hessel-de H e r et al., eds.), Proc. 2nd Int. Symp., p. 197. Wageningen, The Netherlands. Kalow, W., Britt, B. A,, Terreau, M. E., and Haist, C. 1970. Metabolic error of muscle metabolism after recovery from malignant hyperthermia. Lancet 2, 895. Karmas, E., and Thompson, J. E. 1964. Certain properties of canned hams as influenced by conditions of thermal processing. Food Technol. (Chicago) 18, 248. Knstenschmidt, L. L., Beecher, G. R., Forrest, J. C . , Hoekstra, W. G. and Briskey,

ANIMAL PHYSIOLOGY AND MEAT QUALITY

147

E. J. 1965. Porcine muscle properties. A. Alteration of glycolysis by artifically induced changes in ambient temperature. J. Food Sci. 30,565. ,Kastenschmidt, L. L., Hoekstra, W. G., and Briskey, E. J. 1968. Glycolytic intermediates and co-factors in fast- and slow-glycolyzing muscles in the pig. J . Food Sci. 33, 151.

KaufTman, R. G., Carpenter, 2. L., Bray, R. W., and Hoekstra, W. G. 1964. Biochemical properties of pork and their relationship to quality. I. pH of chilled, aged and cooked muscle tissue. J. Food Sci. 29, 65. Koch, D. E., Merkel, R. A., and Purchas, B. J. 1970a. The effect of postmortem niyotomy on glycolysis and ultimate qualitative charactistics of porcine longissimus muscles. J. Agr. Food Chem. 18, 1073. Koch, D. E., Merkel, R. A., and Purchas, B. J. 1970b. The effect of postmortem niyotomy on glycolysis and ultimate qualitative characteristics of porcine rectus femoris muscles. J. Agr. Food Chem. 18, 1078. Kraeling, R. R., and Gerrits, R. J. 1972. Muscle postmortem glycolysis in hypophysectomized pigs. J. Anim. Sci. 35, 1098 (abstr.). Lawrie, R. A. 1960. Post mortem glycolysis in normal and exudative longissimus dorsi muscles of the pig in relation to so-called white muscle disease. J. Comp. Pathol. Ther. 70, 273. Lawrie, R. A. 1966. “Meat Science.” Pergamon, Oxford. Leest, J. A., van Baal, J. P. W., and van Male, J. P. 1971. Effect of PSE condition of pork on stability of luncheon meat during sterilization. In “The Condition and Meat Quality of Pigs” (J. C. M. Hessel-de Heer et al., eds.), Proc. 2nd Int. Symp., p. 287. Wageningen, The Netherlands. Lendfers, L. H. H. M. 1971. Loss of pigs due to death during transport; a one-year survey at an abattoir. In “The Condition and Meat Quality of Pigs” (J.C . M. Hessel-de Heer et al., eds.), Proc. 2nd Int. Symp., p. 225. Wageningen, The Netherlands. Lewis, P. K., Jr., Brown, C. J., and Heck, M. C. 1962. Effect of stress on certain pork carcass characteristics and eating quality. J. Anim. Sci. 21, 196. Lewis, P. K., Jr., Brown, C. J., and Heck, M. C. 1967. Effect of ante-mortem stress and freezing immediately after slaughter on certain organoleptic and chemical characteristics of pork. J. Anim. Sci. 26, 1266. Lister, D. 1969. Some aspects of the physiology of pale, soft and exudative muscle. I n “Recent Points of View on the Condition and Meat Quality of Pigs for Slaughter” (W. Sybesma, P. G. van der Wal, and P. Walstra, eds.), p. 123. I.V.O., Zeist, The Netherlands. Lister, D. 1971. “Physiological Aspects of Meat Quality and Adaptation in Pigs.” Commission on Pig Production, Paris-Versailles. Lister, D. 1972. Hormonal function in relation to adaptation and stress. E.E.A.P. Pig Commission, 1972. Lister, D., and Ratcliff, P. W. 1971. The effect of preslaughter injection of magnesium sulfate on glycolysis and meat quality in the pig. In “The Condition and Meat Quality of Pig” (J. C. M. Hessel-de Heer et al., eds.), Proc. 2nd Int. Symp., p. 139. Wageningen, The Netherlands. Lister, D., Sair, R. T., Will, J. A., Schmidt, G. R., Cassens, R. G., Hoekstra, W. G., and Briskey, E. J. 1970. Metabolism of striated muscle of stress-susceptible pigs breathing oxygen or nitrogen. Amer. J. Physiol. 218, 102.

148

R. G. CASSENS ET AL.

Livestock Conservation, Incorporated. 1971. “Severe Stress: Summary of a Survey.” Omaha, Nebraska. Ludvigsen, J. 1953. “Muscular degeneration” in hogs (preliminary report). PTOC.Inst. vet. CWgT., 25th, VOl. 1, p. 602. Ludivgsen, J. 1954. Undersogelser over den sakaldte “muskeldegeneration” hos svin. p. 272. beretning fra Forsogslaboratoriet, Udginet of Statens husdy grugsudvalg, Copenhagen. Ludvigsen, J. 1957. On the hormonal regulation of vasomotor reactions during exercise with special reference to the action of adrenal cortical steroids. Acta Endocrinol. (Copenhagen) 26, 406. Ludvigsen, J. 1969a. The effect of adrenal cortical steroids in PEM-susceptible pigs. In “Recent Points of View on the Condition and Meat Quality of Pigs for Slaughter” (W. Sybesma, P. G. van der Wal, and P. Walstra, eds.) p. 109. I.V.O., Zeist, The Netherlands. Ludvigsen, J. 1969b. Some thyroid and adrenal breed characteristics and their possible relation to pale exudative muscles in pigs. In “Recent Points of View on the Condition and Meat Quality of Pigs for Slaughter” (W. Sybesma, P. G. van der Wal, and P. Walstra, eds.), p. 113. I.V.O., Zeist, The Netherlands. McClain, P. E., and Pearson, A. M. 1969. Connective tissue from normal and pale, soft, exudative (PSE) porcine muscles. 2. Physical characterization. J. Food Sci. 34, 306. McClain, P. E., Pearson, A. M., Fennell, R. A., and Merkel, R. A. 1968. Metachromasia of epimysial connective tissue from normal and from pale, soft and exudative porcine muscle. PTOC.SOC. Erp. Biol. Med. 128,624. McClain, P. E., Pearson, A. M., Brunner, J. R., and Crevasse, G. A. 1969. Connective tissue from normal and PSE porcine muscle. 1. Chemical characterization. J. Food Sci. 34, 115. McLoughlin, J. V. 1965. Studies on pig muscle. 4. pH values in the longissirnus muscle of pigs killed under commercial conditions. IT. J . Agr. Res. 4, 151. McLoughlin, J. V. 1968. Sarcoplasmic and myofibrillar protein in skeletal muscle of two breeds of pig. J. Food Sci. 33,383. McLoughlin, J. V. 1970. Muscle contraction and postmortem pH changes in pig skeletal muscle. J. Food Sci. 35,717. McLoughlin, J. V. 1971. The death reaction and metabolism post-mortem of porcine skeletal muscle. In “The Condition and Meat Quality of Pigs” (J. C. M. Hesselde Heer et d.,eds.), Proc. 2nd Int. Symp., p. 123. Wageningen, The Netherlands. McLoughlin, J. V., and Tarrant, P. J. V. 1969. Post-mortem changes in muscle taken from live pigs immediately and from pigs immediately after slaughter. In “Recent Points of View on the Condition and Meat Quality of Pigs for Slaughter” (W. Sybesma, P. G. van der Wal, and P. Walstra, eds.), p. 133. I.V.O., Zeist, The Netherlands. Marple, D. N., and Aberle, E. D. 1972. Porcine plasma growth hormone levels: Radioimmunoassay technique and its application. 1. Anim. Sci. 34, 261. Marple, D. N., and Cassens, R. G. 1972. Binding properties of porcine transcortin. J. Anim. Sci. 35,249 (abstr.). Marple, D. N., and Cassens, R. G. 1973. Increased metabolic clearance of cortisol by stress-susceptible swine. J. Anim. Sci. 36, 1139.

ANIMAL PHYSIOLOGY AND MEAT QUALITY

149

Marple, D. N., Topel, D. G., and Matsushima, C. Y. 1969. Influence of induced adrenal insufficiency and stress on porcine plasma and muscle characteristics. 3. Anim. Sci. 29, 882. Marple, D. N., Aberle, E. D., Forrest, J. C., Blake, W. H., and Judge, M. D. 1972a. Effects of humidity and temperature on porcine plasma adrenal corticoids, ACTH and growth hormone levels. J. Anim. Sci. 34,809. Marple, D. N., Aberle, E. D., Forrest, J. C., Blake, W. H. and Judge, M. D. 197213. Endocrine responses of stress-susceptible and stress-resistant swine to environmental stressors. J. Anirn. Sci. 35,576. Marple, D.N., Judge, M. D., and Aberle, E. D. 1972c. Pituitary and adrenocortical function of stress-susceptible swine. J. Anim. Sci. 35,995. Marsh, B. B. 1970. Muscle as a food. In “The Physiology and Biochemistry of Muscle as a F o o d (E. J. Briskey, R. G. Cassens, and B. B. Marsh, eds.), 2nd ed., p. 3. Univ. of Wisconsin Press, Madison. Marsh, B. B. 1972. Post-mortem muscle shortening and meat tenderness. Proc. Meat Ind. Res. Conf. p. 109. Marsh, B. B., and Leet, N. G. 1966. Studies in meat tenderness. 111. The effects of cold shortening on tenderness. J. Food Sci. 31, 450. Marsh, B. B., Woodhams, P., and Leet, N. G. 1968. Studies in meat tenderness. V. The effects on tenderness of carcass cooling and freezing before the completion of rigor mortis. J. Food Sci. 33, 12. Marsh, B. B., Cassens, R. G., Kauffman, R. G., and Briskey, E. J. 1972. Hot boning and pork tenderness. J. Food Sci. 34, 179. Merkel, R. A. 1971a. The relationship of some cardiovascular and haematological parameters to porcine muscle quality. In “The Condition and Meat Quality of Pigs” (J. C. M. Hessel-de Heer et al., eds.), Proc. 2nd Int. Symp., p. 97. Wageningen, The Netherlands. Merkel, R. A. 1971b. Processing and organoleptic properties of normal and PSE porcine muscle. In “The Condition and Meat Quality of Pigs” (J. C. M. Hesselde Heer et al., eds.), Proc. 2nd Int. Symp., p. 261. Wageningen, The Netherlands. Meyer, J. A., Briskey, E. J., Hoekstra, W. G., and Weckel, K. G. 1963. Niacin, thiamin and riboflavin in fresh and cooked pale, soft, watery versus dark, firm, dry pork muscle. Food Technol. (Chicago) 27, 485. Miller, N. M. 1972. The effects of halogenated anesthetics on mitochondria1 function. Anaesthesiology 36, 625. Miller, R. N., and Hunter, F. E., Jr. 1970. The effect of halothane on electron transport, oxidative phosphorylation and swelling in rat liver mitochondria. MoZ. Phamocol. 6, 67. Minkema, D. 1969. Possible ways of improving meat quality and stress resistance by genetic means. In “Recent Points of View on the Condition and Meat Quality of Pigs for Slaughter” ( W. Sybesma, P. G. van der Wal, and P. Walstra, eds.), p. 277. I.V.O., Zeist, The Netherlands. Moerman, P. C., and Krol, B. 1969. Technological aspects of the preparation of hams of pale watery pork. In “Recent Points of View on the Condition and Meat Quality of Pigs for Slaughter” (W. Sybesma, P. G. van der Wal, and P. Walstra, eds.), p. 237. I.V.O., Zeist, The Netherlands. Moerman, P. C., and Krol, B. 1971. Heat penetration into canned ham of different meat qualities. In “The Condition and Meat Quality of Pigs” (J. C. M. Hessel-

150

R. G. CASSENS ET AL.

de Heer et al., eds.), Proc. 2nd Int. Symp., p. 302. Wageningen, The Setherlands. Moran, T., and Smith, E. C. 1929. Post-mortem changes in animal tissues-the conditioning or ripening of beef. DSIR Food Inuest. Spec. Rep. No. 36. Morita, S., Cassens, R. G., Briskey, E. J., Kauffman, R. G., and Kastenschmidt, L. L. 1970. Localization of myoglobin in pig muscle. J. Food Sci. 35, 111. Muir, A. B. 1970. Normal and regenerating skeletal muscle fibres in Pietrain pigs. 1. Comp. Pathol. 80, 137. Muylle, E., van der Hende, C., and Oyaert, W. 1968. Stoffwechsel von Milchsaure bei Schweinen. Deut. Tieraertztl. Wochenschr. 75, 29. Nelson, T. E., Jones, E. W., Venable, J. H., and Kerr, D. D. 1972. Malignant hyperthermia of Poland Chian swine. Anuesthesiology 36, 52. Newbold, R. P. 1966. Changes associated with rigor mortis, In “The Physiology and Biochemistry of Muscle as a Food’ ( E . J. Briskey, R. G . Cassens, and J. C. Trautman, eds.), 1st ed., p. 213. Univ. of Wisconsin Press, Madison. Norman, W. 1965. Pathological conditions in muscle. Proc. Reciprocal Meat Conf., 18th, 1965, p. 177. Oldigs, B., and Unshelm, J. 1971. Influence of a stress reducing medical treatment before transport on meat quality of pigs. In “The Condition and Meat Quality of Pigs” (J. C. M. Iiessel-de Heer et al., eds.), Proc. 2nd Int. Symp., p. 205. Wageningen, The Netherlands. Omtvedt, I. T. 1968. Some heritability characteristics and their importance in selection program. In “The Pork Industry: Problems and Progress” ( D . G . Topel, ed.), p. 128. Iowa State Univ. Press, Ames. Panaretto, B. A., and Fergusson, K. A., 1969a. Pituitary adrenal interactions in shorn sheep exposed to cold wet conditions. Aust. J. Agr. Res. 20, 99. Panaretto, B. A., and Fergusson, K. A. 1969b. Comparison of the effects of several stressing agents on the adrenal glands of normal and hypophysectomized sheep. Aust. J. Agr. Res. 20, 115. Passbach, F. L., Jr., Mullins, A. M., Wipf, V. K., and Paul, B. A. 1970. Influence of aldosterone on the quality of porcine muscle. J. Anim. Sci. 30, 507. Perry, J. H., Ockerman, H. W., Parrett, N. A., and Borton, R. J. 1972. Relation of two hormone levels to postmorten porcine muscle pH and quality. 1. Anim. Sci. 35, 206. Pittman, J. A. 1972. Adrenal cortex; adrenal medulla; thyroid storm or crisis. I n “The Thyroid (S. Werner, ed.), 3rd ed., p. 644. Harper, New York. Quass, D. W., and Briskey, E. J. 1968. A study of certain properties of myosin from skeletal muscle. J. Food Sci. 33, 180. Rantsios, A. 1972. Some observations on the histology of the adrenal zona glomerulosa in Pietrain pigs. Vet. Rec. 90, 369. Reddy, M. V. V., Kastenschmidt, L. L., Cassens, R. G., and Briskey, E. J. 1971. Studies of stress-susceptibility : The relationship between serum enzyme changes and the degree of stress-susceptibility. Life Sci. 10, 1381. Romack, F. E., Turner, C. W., Lasley, J. F., and Day, B. N. 1964. Thyroid secretion rate in swine. J. Anim. Sci. 23, 1143. Sair, R. A., Lister, D., Moody, W. G . , Cassens, R. G., Hoekstra, W. G . , and Briskey, E. J. 1970. Action of curare and magnesium on striated muscle of stress-susceptible pigs. Amer. J. Physiol. 218, 108.

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Sair, R. A., Kastenschmidt, L. L., Cassens, R. G., and Briskey, E. J. 1972. Metabolism and histochemistry of skeletal muscle from stress-susceptible pigs. J. Food Sci. 37, 659. Satnick, J. H. 1968. Hyperthemia under anaesthesia with regional muscle flaccidity. Anesthesiology 30, 472. Satterlee, L. D., and Zachariah, N. Y. 1972. Porcine and ovine myoglobin: Isolation, purification, characterization and stability. J. Food Sci. 37, 909. Sayre, R. N., Briskey, E. J., and Hoekstra, W. G. 1963. Effect of excitement, fasting and sncrose feeding on porcine muscle phosphorylase and post-mortem glycolysis. J. Food Sci. 28, 472. Sayre, R. N., Kiernat, B., and Briskey, E. J. 1964. Processing characteristics of porcine muscle related to pH and temperature during rigor mortis development and to gross morphology 24 hr post-mortem. J. Food Sci. 29, 175. Scheper, J. 1971. Research to determine the limits of normal and aberrant meat quality (PSE and DFD) in pork. In “The Condition and Meat Quality of Pigs” (J. C. M. Hessel-de Heer et al., eds.), Proc. 2nd Int. Symp., p. 271. Wageningen, The Netherlands. Schmidt, G . R., Cassens, R. G . , and Briskey, E. J. 1970a. Changes in tension and certain metabolites during the development of rigor mortis in selected red and white skeletal muscle. J. Food Sci. 35, 571. Schmidt, G . R., Kastenschmidt, L. L., Cassens, R. G., and Briskey, E. J. 1970b. Serum enzyme and electrolyte levels of “stress-resistant” Chester White pigs and “stress-susceptible” Poland China pigs. J. Anim. Sci. 31, 1168. Schmidt, G. R., Cassens, R. G., and Briskey, E. J. 1970c. Relationship of calcium uptake by the sarcoplasmic reticulum to tension development and rigor mortis in striated muscle. J. Food Sci. 35, 574. Schmidt, G. R., Zuidam, L., and Sybesma, W. 1971. Biopsy technique and analysis, for predicting pork quality. In “The Condition and Meat Quality of Pigs” (J. C. M. Hessel-de Heer et ol., eds.), Proc. 2nd Int. Symp., p. 73. Wageningen, The Netherlands. Schmidt, G . R., Zuidam, L., and Sybesma, W. 1972a. Biopsy technique and analysis for predicting pork quality. J. Anim. Sci. 34, 25. Schmidt, G. R., Goldspink, G., Roberts, T., Kastenschmidt, L. L., Cassens, R. G., and Briskey, E. J. 1972b. Electromyography and resting membrane potential in longissimus muscle of stress-susceptible and stress-resistant pigs. J. Anim. Sci. 34, 379. Scopes, R. K. 1964. The influence of postmortem condition on the solubilities of muscle proteins. Biochem. J. 91,201. Searcy, D. J., Harrison, D. L., and Anderson, L. L. 1969. Palatability and selected related characteristics of three types of roasted porcine muscle. J. Food Sci. 34, 486. Sebranek, J. G . , Marple, D. N., Cassens, R. G., Briskey, E. J., and Kastenschmidt, L. L. 1973. Adrenal response to ACTH in the pig. J. Anim. Sci. 36, 41. Siers, D. G . , and Hazel, L. N. 1970. Serum growth hormone levels in swine. Growth 34, 419. Siers, D.G., and Swiger, L. A. 1971. Influence of liver weight, age and sex on circulating growth hormone levels in swine. J. Anim. Sci. 32, 1229. Sink, J. D., Bray, R. W., Hoekstra, W. G., and Briskey, E. J. 1967. Lipid composition of normal and pale, soft, exudative porcine muscle. J. Food Sci. 32, 258.

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Snodgrass, P. J., and Piras, M. M. 1966. The effect of halothane on rat liver mitochondria. Biochemistry 5, 1140. Staun, H. 1963. Various factors affecting number and size of muscle fibers in the pig. Acta Agr.. Scand. 13, 293. Staun, H., and Jensen, P. 1972. “Phenotypic and Genetic Parameters of Some Carcass Traits in Danish Landrace Pigs,” Denmark Yearbk., p. 104. Roy. Vet. and Agr. Univ., Copenhagen. Steers, A. J. W., Tallack, J. A., and Thompson, D. E. A. 1970. Fulminating hyperpyrexia during anesthesia in a member of a myopathic family. Brit. Med. J. 2, 341. Stefanovic, M. P., Bayley, H. S., and Slinger, S. J. 1970. Effect of stress on swine: Heat and cold exposure and starvation on vanilmandelic acid output in the urine. J. Anim. Sci. 30, 378. Steinhauf, D. 1969. Meat quality as a selection criterion. In “Recent Points of View on the Condition and Meat Quality of Pigs for Slaughter” (W. Sybesma, P. G. van der Wal, and P. Walstra, eds. ), p. 283. I.V.O., Zeist, The Netherlands. Swatland, H. J., and Cassens, R. G. 1972a. A brief study of muscle enlargement in the rat. 1.Anim. Sci. 34, 21. Swatland, H. J., and Cassens, R. G. 1972b. Muscle growth: The problem of muscle fibers with an intrafascicular termination. J. Anim. Sci. 35, 336. Swatland, H. J., and Cassens, R. G. 1972c. Peripheral innervation of muscle from stress-susceptible pigs. J. Comp. Pathol. 82, 229. Swatland, H. J., and Cassens, R. G. 1973a. Observations on the postmortem histochemistry of myofibers from stress-susceptible pigs. J. Anim. Sci. 37, 885. Swatland, H. J., and Cassens, R. G. 1973b. Prenatal development, histochemistry and innervation of porcine muscle. J. Anim. Sci. 36, 343. Sybesma, W., and Eikelenboom, G. 1969. Malignant hyperthermia syndrome in pigs. Neth. J. Vet. Sci. 2, 155. Sybesma, W., and Groen, W. 1970. Stunning procedures and meat quality. Proc. Eur. Meet. Meat Res. Conf.,16th, 1970. Sybesma, W., and Hessel-de Heer, J. C. M. 1967. LD& and meat quality in pigs. Proc. Eur. Meet. Meat Res. Workers, 13th, 1967, Sybesma, W., and van Logtestijn, J. G. 1966. Preslaughter temperature and its effect on post-mortem metabolism in the pig. Proc. Eur. Meet. of Meat Res. Workers, 12th, 1966. Sybesma, W., and van Logtestijn, J. G. 1967. Rigor mortis und Fleischqualitat. Fleischwirtschaft 47, 408. Sybesma, W., van der Wal, P. G., and Walstra, P., eds. 1969. “Recent Points of View on the Condition and Meat Quality of Pigs for Slaughter.” I.V.O., Zeist, The Netherlands. Sybesma, W., Minkema, D., van der Wal, P. G., and Walstra, P. 1972. Muscle biopsy analysis and meat quality. Proc. Annu. Meet. Eur. Asso. Anim. Prod. B r d . Tarrant, P. J. V., McLoughlin, J. V., and Harrington, M. G. 1972. Anaerobic glycolysis in biopsy and post-mortem porcine longissimus dorsi muscle. Proc. ROY. I T . Acad. 72, 55. Teeter, C., Carr, S. C., Tsai, R., and Briskey, E. J. 1969. A cryobiopsy technique for assessing metabolite levels in skeletal muscle. Proc. SOC. Exp. Biol., Med. 131, 5. Thomas, N. W., and Judge, M. D. 1970. Alteration of porcine skeletal muscle myoglobin by the environment. J. Agr. Sci. 74, 241.

ANIMAL PHYSIOLOGY AND MEAT QUALITY

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Thomas, N. W., Addis, P. B., Johnson, H. R., Howard, R. D., and Judge, M. D. 1966. Effects of environmental temperature and humidity during growth on muscle propertie6 of two porcine breeds. I . Food Sci. 31, 309. Topel, D. G., ed. 1968. “The Pork Industry: Problems and Progress.” Iowa State Univ. Press, Ames. Topel, D. G. 1969. Relation of plasma glucocorticoid levels to some physical and chemical properties of porcine muscle and the porcine stress syndrome. In “Recent Points of View on the Condition and Meat Quality of Pigs for Slaughter,” (W. Sybesma, P. G. van der Wal, and P. Walstra, eds.), p. 91. I.V.O., Zeist, The Netherlands. Topel, D. G. 1972. A review of animal physiology and the porcine stress syndrome in relation to meat quality. I n “Proceedings of the Pork Quality Symposium” (R. G. Cassens, F. Giesler, and Q. Kolb, eds.), p. 26. Univ. of Wisconsin Ext., Madison. Topel, D. G., and Merkel, R. A. 1966. Effect of exogenous goitrogeas upon some physical and biochemical properties of porcine muscle and adrenal gland. I . Anim. Sci. 25, 1154. Topel, D. G., Merkel, R. A,, and Wismer-Pedersen, J. 1967. Relationship of plasma 17-hydroxycorticosteroid levels to some physical and biochemical properties of porcine muscle. J. Anim. Sci. 26, 311. Topel, D. G., Bicknell, E. J., Preston, K. S., Christian, L. L., and Matsushima, C. Y. 1968. Porcine stress syndrome. Mod. Vet. Proc. 49, 40. Topel, D. G., Galloway, D. E., Will, J. A., Weirich, W. E., Grummer, R. H., Cassens, R. G., Kauffman, R. G., and Briskey, E. J. 1971. Effect of environmental temperature on physiological characteristics of pigs with fast and slow glycolyzing muscle. J. Anim. Sci. 32, 1103. Topel, D. G., Parrish, F. C., Jr., Rust, R. E., and Wilson D. G. 1972. Certain chemical and physical properties of ham muscle portions after thermal processing. J. Food Sci. 37, 907. Trey, C. H., Lipworth, L., Chalmers, T. C., Davidson, C. S., Gottlieb, L. S., Popper, H., and Saunders, S. J. 1968. Fulminant hepatitis. N . Eng. J. Med. 279, 798. Tsai, R., Goepfert, J. M., Cassens, R. G., and Briskey, E. J. 1971. A comparison of salmonella excretion by stress-susceptible and stress-resistant pigs. J. Food Sci. 36, 889. Turman, E. J., and Andrews, F. N. 1955. Some effects of purified anterior pituitary growth hormone on swine. J. Anim. Sci. 14, 7. Unshelm, J., Hohns, H., Oldigs, B., and Riihl, B. 1971. Physiological and morphological studies in pigs of different types bred and different body sizes. In “The Condition and Meat Quality of Pigs” (J. C. M. Hessel-deHeer et al., eds.), Proc. 2nd Int. Synip., p. 208. Wageningen, The Netherlands. van den Hende, C., Muylle, E., Oyaert, W., and de Roose, P. 1972. Changes in muscle characteristics in growing pigs. Histochemical and electron microscopic study. Zentralbl. Veteringer Med., 19, 102. van de Pas, J. G. C., and Walstra, P. 1971. Differences in meat quality characteristics between Dutch breeds and their crossbreeds. In “The Condition and Meat Quality of Pigs” (J. C. M. Hessel-de Heer, et al., eds.), p. 291. Wageningen, The Netherlands. van der Wal, P. G. 1969. Some data about the influence of an adrenergic blocking agent upon the ATP concentration of the musculus semimembranosus in pigs.

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In “Recent Points of View on the Condition and Meat Quality of Pigs for Slaughter” (W. Sybesma, P. G. van der Wal, and P. Walstra, eds.), p. 119. I.V.O., Zeist, The Netherlands. van der Wal, P. G. 1971. Stunning procedures for pigs and their physiological consequences. In “The Condition and Meat Quality of Pigs” (J. C. M. Hessel-de Heer et d.,eds.), Proc. 2nd Int. Symp., p. 145. Wageningen, The Netherlands. van Logtestijn, J. G. 1969. The economic significance of the stress-susceptibility of pigs. Processing characteristics of different qualities of pork. In “Recent Points of View on the Condition and Meat Quality of Pigs for Slaughter” (W. Sybesnia, P. G. van der Wal, and P. Walstra, eds.), p. 261. I.V.O., Zeist, The Netherlands. van Logtestijn, J. G., Sybesma, W., and van Gils, J. H. J. 1970. Veterinarhygienische Apekte der Stress-Empfindlichkeit von Schlachtschweinen. Arch. Lebensmittelhyg. 3, 55. van Vorstenbosch, C. J. A. H. V. 1969. Some observations concerning the ultrastructure of m. biceps femoralis. Preliminary report. In “Recent Points of View on the condition of Meat Quality of Pigs for Slaughter” (W. Sybesma, P. G. van der Wal, and P. Walstra, eds.), p. 159. I.V.O., Zeist, The Netherlands. VOS, M. P. M., and Sybesma, W. 1971. Relation between meat quantity and meat quality of market pigs. In “The Condition and Meat Quality of Pigs” (J. C. M. Hessel-de Heer et d., eds.), p. 278. Wageningen, The Netherlands. Walstra, P., Minkema, D., Sybesma, W., and van de Pas, J. G. C. 1971. “Genetic Aspects of Meat Quality and Stress-resistance in Experiments with Various Breeds and Breed Crosses,” Res. Rep. No. 1. Research Institute for Animal Husbandry, “Schoonoord” Driebergseweg 10d. Zeist, The Netherlands. Weber, A., Herz, R., and Reiss, I. 1964. Role of calcium in contraction and relaxation of muscle. Fed. Proc., Fed. Amer. SOC. Erp. Biol. 23, 896. Weiss, G. M. 1971. Cortisol and epinephrine relationships associated with stress adaptation in swine. Ph.D. Thesis, Iowa State University, Ames. Weiss, G. M., Topel, D. G., Ewan, R. C., Rust, R. E., and Christian, L. L. 1971a. Growth comparison of a muscular and fat strain of swine, I. Relationship between muscle quality and quantity, plasma lactate and 17-hydroxycorticosteroids. 3. Anim. Sci 32, 1119. Weiss, G. M., Topel, D. G., and Ewan, R. C. 1971b. Growth comparison of a muscular and fat strain of swine. 11. Protein solubility characteristics and m. longissimus and serum electrolyte. J. Anim. Sci. 32, 1124. Weiss, G. M., Topel, D. G., Siers, D. G., and Ewan, R. C. 1973. Influence of adrenergic blocking agents on stress adaptation in swine. J. Anim. Sci. 36, 1077. Wilson, R. D., Nichols, R. J., Jr., Dent, T. E., and Allen, C. R. 1966. Disturbances of the oxidative-phosphorylation mechanism as a possible etiological factor in sudden unexplained hyperthermia occurring during anesthesia. Anesthesiology 27, 231. Wilson, R. D., Dent, T. E., Traber, D. L., McCoy, N. R., and Allen, C. R. 1967. Malignant hyperpyrexia and anesthesia. 3. Amer. Med. ASS.202, 111. Wismer-Pedersen, J. 1959. Quality of pork in relation to rate of pH change postmortem. Food Res. 24,711. Wismer-Pedersen, J. 1960. Effect of cure on pork with watery structure. 11. Effect on quality of canned hams. Food Res. 25, 799. Wismer-Pedersen, J. 1968. Modem prediction practices and their influence on stress

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conditions. In “The Pork Industry: Problems and Progress” (D. G. Topel, ed), p. 163.Iowa State Univ. Press, Ames. Woolf, N., Hall, L., Thorne, C., Down, M., and Walker, R. 1970. Serum creatine phosphokinase levels in pigs reacting abnormally to halogenated anaesthetics. Brit. Med. J. 3,386. Zuidam, L., Schmidt, G. R., and Sybesma, W. 1971. Effect of fresh pork color on consumer acceptance. I n “The Condition and Meat Quality of Pigs” (J. C. M. Hessel-de Heer et al., eds.), Proc. 2nd Int. Symp., p. 282. Wageningen, The Netherlands.

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D . P. HAUGHEY. BY R . H . LOCKER.C. L . DAVEY.P. M . NOTTINGHAM. AND N . H . LAW Meat Industry Research Institute of New Zealand. Hamilton. New Zeahnd

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I Introduction ................................................... I1 Tenderness. Contraction. and Cold ................................

A. Definition of the Problem ..................................... B. Tenderness as Related to Muscle Shortening ..................... C . The Cold Shortening Effect ................................... D . Thaw Shortening ............................................ E . Aging and Contraction ....................................... I11 Application to Processing ........................................ A. From Laboratory to Factory ................................... B. Rigor Mortis and Conditioning ................................. C. Lamb Processing ............................................ D Mutton Processing ........................................... E . Beef Processing ............................................. IV Unconventional Techniques ...................................... A AlteredPosture .............................................. B. Hot Cutting and Boning ...................................... C. High-Temperature Conditioning ............................... D. Electrical Stimulation ........................................ V . Microbiology ................................................... A Factors Controlling Growth ................................... B. Application ................................................. VI . Physics of Meat Chilling ......................................... A . Cooling Phase .............................................. B. Storage Phase ............................................... VII. Research Needs ................................................ VIII Conclusion .................................................... References .....................................................

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INTRODUCTION

The relationship between meat tenderness and refrigeration technology has only begun to be understood in the last decade, although refrigeration has been with the industry for a century. Since the factors determining tenderness have been studied for many years, it is surprising that the adverse effects of cold should have been noticed so recently. It was not entirely by chance that this late recognition was made in New Zealand. Of the substantial exporters of meat, only New Zealand and Australia, by reason of remoteness from their markets, export almost all their meat in frozen form. New Zealand is the only large exporter of lamb, which still forms the major part of its meat exports. This frozen trade in small animals, involving an extreme set of cooling conditions, has led to this Institute’s developing the particular aspect of meat science concerned with cold treatment in the prerigor period and tenderness. The work has extended over the last decade, and has involved biochemists, bacteriologists, and engineers. At the present time it represents a very substantial body of results. It has taken practical form as specifications for processing lamb and beef, which allow the apparently conflicting demands of hygiene and tenderness to be met in a works’ situation. The high capacity of modern refrigerating plants, and the emphasis now given to hygiene, have led to the widely accepted belief that meat should be cooled as quickly as possible after death. The results that follow show that this belief, while having some obvious merit, disregards a very important aspect of meat quality-its tenderness. It is our experience that the temperature history of meat in the prerigor period far outweighs all other factors in determining tenderness. Too early exposure to cold can have disastrous effects on this quality. While the effects of cooling too rapidly are most evident in small animals, they are significant in all meat animals (although marginally in the pig), whether chilling or freezing is involved. The information that follows is therefore of universal relevance to meat processing. Although most of the results from this laboratory have been published, the time seems opportune to put them, together with the substanial conributions from other meat research centers, into a coherent account, to assess their significance for meat science, and to show their usefulness to meat processing. The field has not yet been the subject of an extensive review. An excellent concise review on this particular topic has appeared (Marsh, 1972), and also a short but more widely ranging one by Newbold and Harris ( 1972). Many relevant results were presented at a recent

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British symposium on meat chilling ( Meat Research Institute, Langford, 1972)

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TENDERNESS, CONTRACTION, AND COLD A. DEFINITION OF THE PROBLEM

Some attempt to outline the history of this work may be of interest for two reasons. Firstly, it illustrates how a study of a specific problem in a particular class of animals can make an impact on meat processing in general. Secondly, it is an interesting example of a fruitful, if somewhat fortuitous, interplay between basic studies on pieces of muscle from one species (ox) and practical work on carcass meat of another (lamb). It seemed neither desirable nor possible to disentangle the two, and the work is presented in roughly chronological order. If it appears to the reader in countries where sheep meat is not popular that there has been undue emphasis on ovines in this review, the justification is to be found in this history, and in the general applicability of the results. The origins of this study lie partly in an unforeseen practical problem arising from the freezing of New Zealand lamb on a large scale. As early as 1960, complaints were beginning to come back from Britain that New Zealand lamb was often tough. The quality of our frozen lamb had been a matter of national pride, and the reports were received with skepticism. When they persisted, it was felt desirable to investigate. A large experiment was set up to assess such variables as age, weight, grade, and freezing method. The results were reported eventually by Marsh et al. (1968) and will be considered in more detail in Section 111. For the moment it suffices to say that the critical customers had spoken the truth. Of the factors studied, the freezing method was found to be by far the most dominant in toughening the meat. The product of oldfashioned slow freezing after hanging was always tender, while that from blast-freezing could be anywhere between tender and very tough. It was soon evident that blast-freezing itself was not to blame, but rather freezing too soon. The postwar years had seen in New Zealand a rapid conversion from traditional freezing chambers with overhead brine pipes, to modern blast freezers. Rising lamb production had demanded much greater through-

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put. In an average works it now exceeds 10,OOO lambs per day in season ( one every 2 seconds ) , and in some 20,000. The former practice of hanging carcasses overnight on a cooling floor to dissipate excess moisture and heat before entry to the freezing chambers was thought to be no longer necessary, and carcasses went to the freezer soon after passing the scales. Most of the cooling floor space was utilized to install blast freezers. Recognition of the need for delay before freezing was therefore very unwelcome to the industry. The reasons behind this toughening during early exposure to cold were supplied by two observations on muscle made, by a happy chance, in the same laboratory at about the same time, in quite unrelated research. These, together with a third result, already known, were the foundation of the study, which has since been broadened by many new results.

B. TENDERNESS AS RELATED TO MUSCLE SHORTENING During a study on the mechanism of aging in beef, the contraction patterns of sarcomeres were observed under the microscope. It was noticed that there was a wide variation in the degree of contraction of different muscles, and of fibers within muscles, when set in rigor mortis (Locker, 1959). Could this be related to the tenderness of the muscles? To eliminate the confusing effect of connective tissue, further study was made of tenderloin (psoas), which has the least amount of such tissue. Pairs of beef psoas muscles were allowed to go into rigor mortis in the chiller, with one muscle intact on the carcass and the other partly or wholly excised. The cut muscles all shortened and were tougher than the controls. It was therefore postulated that contraction of muscles induces toughness in meat (Locker, 1960). It was suggested in this paper that the state of contraction of the muscles was related to the strains imposed upon them by hanging the carcass, and that the tenderness of these muscles might be improved by a different method of suspension. Herring et al. (1965a) confirmed this possibility by showing that if a beef side “set” while lying horizontally, with the limbs in a natural posture, marked changes occurred in sarcomere length and tenderness when compared with the other side, hung normally by the back leg. Some muscles, such as the psoas, toughened; others, such as the logissimus, improved greatly, while others were little affected. There was overall improvement and greater uniformity of tenderness. This idea has been further tested for beef and lamb by others with similar promising results (see Section 111).

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C. THECOLDSHORTENING EFFECX It had been reported by Bendall (1951) for rabbit muscle, and by Marsh (1954) for beef, that the shortening of excised muscle during onset of rigor increased with storage temperature. When an attempt was made to use this principle to shorten ox muscles to differing degrees, marked contraction was recorded on chilling to near 0°C. Further investigation of this anomaly led to the discovery of the phenomenon now known as “cold shortening” ( Locker and Hagyard, 1963). A detailed study of the shortening-temperature relationship, using beef sternomandibularis muscles, revealed a curve with a minimum (Fig. 1 ) . The shortenings of near 50% at 0°C declined to about 10% in the region of 14” to 19”C, and then rose again less steeply at temperatures above 20°C. Analysis of the time course of shortening (Fig. 2 ) showed that two distinct effects were operating-a cold shortening, which was an immediate response to chilling, and a delayed shortening at higher tem-

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FIG.1. Mean ultimate shortenings of muscles at various storage temperatures. Vertical lines represent standard deviations. From Locker and Hagyard ( 1963).

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FIG. 2 Mean shortening-time curves for muscles at various temperatures. From Locker and Hagyard ( 1963 ) .

peratures, which coincided with the onset of rigor mortis. It was this second effect that had been observed by earlier workers. Cold shortening has a rapid phase of 1 to 2 hours and is rather like a slow-motion physiological response to nervous stimulation, although it is most vigorous a t temperatures where electrical stimulation has ceased to be effective. Cold-induced contracture is stronger in beef than the rigor contracture a t higher temperatures. The ability to cold-shorten persists for about 10 hours at 25"C, declining rapidly toward the end of the period, and disappearing at about pH 6.3 when rigor is beginning. The cold contraction is at first readily reversible as the muscle returns to room temperature, but become progressively less reversible over the same period. It was found that beef psoas muscle cold-shortened on chilling, usually without delay, while longissimus began to shorten only after a period of 3 to 5 hours. Rabbit psoas and longissimus, on the other hand, did not cold-shorten. The above two results-the relation between contraction and toughness, and the induction of contraction by cold-were obviously relevant to the lamb study. This was verified by experimentation with lambs cut into sides (R. H. Locker, unpublished results, 1963). One side was subjected to rapid chilling in a blast freezer at a works, until the longissimus reached an internal temperature of 1"C, and was then allowed to go into rigor in a chiller ( 2 ° C ) . The control side was held overnight at 15°C. In each case the longissimus proved on cooking to be much tougher than the control. A similar result was obtained by immersing a side of lamb

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in ice water. It was clear that toughening could be induced by rapid chilling without freezing. Since then the practical significance of cold shortening has been confirmed in extensive studies on lamb and beef in this laboratory, including examination of a wide range of individual muscles; this will be discussed in greater detail later in the processing sections. It will be made clear that this is a practical problem affecting not only small animals but also beef carcasses when these are cooled rapidly. Under some unusual circumstances heat shortening, as represented by the right-hand side of the curve in Fig. 1, can toughen meat. This has been observed in normally hung lambs subjected to accelerated rigor at 45"C, but it can be prevented by altered posture (see Section IV,C). It also appears to be the explanation for the observation of Khan and Lentz (1973) that beef of low initial pH is tougher than that from normal animals and does not age to the same level. Their data show that in the abnormal animals the muscles had gone into rigor during dressing, while the carcasses would have been still near 37°C and the tendency for contraction therefore strong. More detailed study on the relation between degree of cold shortening and tenderness showed it to be more subtle than would have been expected from the original crude observation of Locker (1960). Marsh and Leet (1966a,b) used the cold effect to set beef neck muscles at varying lengths, and assessed the shearing force on the cooked material. The curve relating shortening to shearing force (Fig. 3 ) showed two distinct phases. Toughening increased as shortening began, but the rise was steepest between 20% and 40%. The surprising result was the 40 to 60% phase of the curve, where toughness declined equally steeply to near the original level. ( See also Fig. 9. ) A variety of treatments were used to produce shortening, such as varying delays before cooling to 2"C, different storage temperatures, and even controlled thaw rigor. The same shortening-toughness curve emerged in each case. If the muscle was restrained it did not toughen. It seemed, therefore, that the toughness was simply a function of length and was independent of the treatment used to arrive at that length. Cold shortening has been confirmed in a number of laboratories-for beef psoas and semitendinosus by Herring et al. (196513); for beef sternomandibularis by Cassens and Newbold ( 196713) ; and for lamb by Cook and Wadsworth (1966) and by Taylor et al. (1972). It has been shown by Bouton et al. (1972) that the water-holding capacity of ovine longissimus dorsi in the raw or cooked state is reduced by cold shortening.

R. H. LOCKER ET AL.

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FIG. 3. Relative tenderness in relation to the shortening induced by transfer of samples from room temperature to 2°C at intervals during rigor onset. Cold shortening as percentage of initial excised length. Reprinted from B. B. Marsh & N. G . Leet, J. Food Sci. 31,450459 (1966).

Newbold (1966) showed clearly that, as had been supposed, cold shortening occurs before the chemical events leading to rigor mortis (Fig. 4). Lacourt and Charpentier ( 1971) found that isometric tension development in the psoas major of lamb follows closely the pattern of cold shortening in beef muscle. Tension generation was immediate and rapid at 2°C; it was delayed and smaller at 37°C and l6OC (Fig. 5 ) . Busch et al. (1967) showed that beef muscles generated more isometric tension at 2°C than at 37"C, and much more than at 16°C. In the case of psoas the tension rose steeply in the first 3 hours ( p H then 6.2), reached a maximum at 6 hours ( p H 6.0), and then declined slowly during the next 2 days. With semitendinosus, tension rose slowly and uniformly to a peak at 24 hours ( p H 5.6), where clearly a rigor tension was being registered. The order of tensions involved was only about 3% of that generated by tetanic contraction (Jungk et al., 1967). More recent experiments by Busch et al. (1972) again show the surprisingly slow tension development at 2°C in semitendinosus.

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FIG. 4. Chemical and physical changes in beef sternomandibularis muscle held at 1°C (average of four muscles). Mean ultimate p H 5.78. Mean total shortening, about 35% of the initial length. From R. P. Newbold, "Changes Associated with Rigor Mortis," in E. J. Briskey, R. G. Cassens, and J. C. Trautman, editors, The Physiology and Biochemistry of Muscle as a Food (Madison: The University of Wisconsin Press; @ 1966 by the Regents of the University of Wisconsin), pp. 213-224.

Hours postmortem

FIG.5. Isometric tension developed in psoas major of lamb at different tempera2°C;,).---.( 16°C; (B-B), 37°C. From Lacourt tures. Code: (A-A), and Charpentier ( 1971).

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Experiments in this laboratory (C. L. Davey and K. V. Gilbert, unpublished results, 1971) on the sternomandibularis muscle ("neck) of the ox, calf, and ewe showed a rapid generation of isometric tension, usually reaching a maximum within the first hour. Ability to lift loads varied enormously with the size (i.e., maturity) of the muscles, from 2 to 5 gm/cm2 in young calf muscles of 1 to 2 cm2 in cross section, to about 200 gm/cm2 in bull neck muscles of 30 to 40 cm2 in cross section. Strips of ox neck of average size developed approximately 80 gm/cm2 of tension in cold shock. In muscles loaded with 20 to 30 gm/cm2, the cold shortening occurred more rapidly ( M to 1 hour) than in unloaded strips (2 to 4 hours). Galloway and Go11 (1967) found pig muscle much less responsive to cold than beef muscle. They observed shortenings in longissimus dorsi strips from the pig, of 20% at 2°C and 15% at 16"C, rising to 22% at 37°C. Much greater differences were observed in isometric tension development. At 2°C tension rose steeply in the first 2 hours, then more slowly to a peak at rigor, followed by a decline to about half the maximum. At the higher temperatures, tension development was much lower and erratic in speed. Busch et al. (1972) found generation of tension in pig longissimus neither rapid nor great at 2"C, 16"C, or 25°C. It was much greater at 37°C. Hendricks et al. (1971) reported shortenings at 2°C of 13 to IS%, and 1 to 4% at 16°C. Muscles from Poland China pigs shortened much more rapidly than those from Hampshires, but to about the same length. Marsh et al. (1972) showed that some pig muscles toughened significantly on excision and exposure to 2"C, while others did not. Chicken and, to a lesser extent, turkey muscles are also capable of cold shortening (Smith et al., 1969; Wiskus et al., 1973), but the temperatur-shortening relationship is somewhat different. Chicken breast muscle shortens more at 0°C (32%) than at 14°C (23%)),but shortening at 37°C (39%) was greater than at 0°C. This result is strikingly similar to the relationship between disappearance of ATP and temperature, and between toughness and temperature, reported for the same muscle by De Fremery and Pool (1960). B. B. Chrystall (unpublished results, 1971) found that chicken breast produced tension immediately at 3"C, a rapid rise occurring in 15 minutes, with a slow further rise and decline occurring in the next few hours. Weidemann et al. (1967) have studied the histology of prerigor and postrigor ox muscles before and after cooking, and have shown the contracture bands produced on chilling at 0°C. Voyle (1969) studied the histology of beef neck muscle shortened at 2"C, using the light and electron miscroscopes. He found that less than

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half the fibers had actively shortened (mean sarcomere length 1.1 microns, electron microscope ) . The others were crimped, although these too had shortened substantially (mean 1.6 microns). A control, allowed to go into rigor at 18°C restrained only by its own suspended weight, had only straight fibers (mean 2.3 microns). Individual fibers among those actively shortened contained both contraction nodes and stretched areas, as well as breaks. Our own observations agree with this picture. Staining this muscle to differentiate red and white fibers showed comparable numbers of each. On cold shortening, both fiber types were found among the crimped as well as the strongly contracted fibers. Experiments with other ox muscles, ranging in composition from purely red fibers to predominantly white, showed little difference in ability to cold-shorten (R. H. Locker and G. J. Daines, unpublished results, 1972). An explanation of the toughening-length relationship may now be attempted in terms of the accepted sliding-filament theory of muscular contraction. Before doing so, we must ask what is actually being measured as “shear force.” The MTRINZ tenderometer ( Macfarlane and Marer, 1966) used in this laboratory has been shown by Bouton and Harris (1972) to give scores that correlate closely with those from the more widely used Warner-Bratzler machine. They showed that the latter gives shear values highly correlated with tensile strength but not with compressive strength (which is a measure of fiber adhesion due to connective tissue). In this laboratory also a good correlation has been found between the Warner-Bratzler machine and the MIRINZ tenderometer, for beef (Davey and Gilbert, unpublished results, 1972) and for lamb (W. A. Carse, unpublished results, 1974; see footnote c, Table I). The correlations for lamb and for beef longissimus are very close, but that for the tougher beef sternomandibularis is somewhat different. The blunt teeth of the MIRINZ tenderometer appear not to “feel” fine-scale connective tissue. Strips of old ewe mutton register the same score as lamb, but leave a strong bridge of uncleaved membranes (Wenham et al., 1973). The same effect is observed in beef sternomandibularis after extreme aging (18 days at 15”C), when the shear force falls from near 50 units to 15, but a bridge of tough membranes still remains spanning the bite (R. H. Locker and G. J. Daines, unpublished results, 1972). In light of the above, it may be assumed that shear force is largely a measure of the tensile strength of the myofibrils. Voyle believes that the rising phase of the toughening curve (Fig. 3 ) is related to an increasing incidence of sarcomeres, 1.5 micron or less in length, in which the thick filaments of myosin have been compressed

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into the 2-line, thus removing the I-band as a zone of weakness. This view is of a statistical population with sarcomeres below 1.5 microns as the dominant contributors to toughness. On the other hand, Marsh and Carse (1974) believe that the toughening up to 40% shortening is to be explained in terms of an increasing mechanical strength of the sarcomere as the I-band shortens. This can be tested only if a precise relation between sarcomere length and gross length can be established. This is difficult to do and may not be possible. It is our observation that at all states of stretch or contraction there is a considerable spectrum of sarcomere lengths. Problems include crimping in contraction and sliding of fibers over each other in stretch, the hazards of preparing and measuring a representative sample, and the imprecise nature of “rest length,” measured on the bench, since this may vary with individual muscles and with their handling. It will be difficult to decide which of those two theories is correct. In fact, both effects may well be contributing. It may be noted that muscle shortened to the degree that produces peak toughness in the cooked state is more tender in the raw state than is unshortened muscle (B. B. Chrystall, unpublished results, 1971). Heat coagulation is necessary to bind the filaments together. There seems little doubt about the nature of the tenderizing in the 40 to 60% phase of the curve. Marsh et al. (1972) have shown that in this range there is violent local contraction of fibers into nodes, with gross stretching and tearing between the nodes, and thus progressive weakening of the tissue. It remains to explain the reason for the massive shortening induced by cold. Since the effect was discovered, there has been great progress in understanding the control of muscular contraction (Ebashi et al., 1969). The discovery of the “relaxing factor” by Marsh (1952b) was the starting point for this advance. A key organelle in this control is the sarcoplasmic reticulum, a set of cisterns surrounding each myofibril. These cisterns have the ability to accumulate calcium, by means of an active calcium pump in the enclosing membrane. A nervous impulse arriving at a motor end plate of a muscle fiber is transmitted as a wave of depolarization over the fiber sheath, the sarcolemma. An interior network of small tubules, which have openings into the sarcolemma and are continuous with it, conducts the electrical wave through the interior of the fiber. At junctions known as the “triads,” the tubules somehow pass their message to the sarcoplasmic reticulum, perhaps as a depolarization of its membrane. The reticulum then releases calcium ions into the sarcoplasm. These diffuse to the filaments and bind to the tropomyosin-troponin complex

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located in the thin filaments. Calcium neutralizes the inhibiting effect this complex has on the interaction of actin in the thin filaments with myosin in the thick filaments. Interaction occurs, myosin ATPase is activated, and contraction ensues. Relaxation involves the reversal of this chain of reactions, beginning with active pumping of calcium back into the cisterns. It is considered likely that the ATP-driven calcium pump in the lipoprotein membrane of the sarcoplasmic reticulum may be vulnerable to cold. Horgan et al. (1971) found that at 25°C sarcoplasmic reticulum from rabbit white muscles accumulates calcium faster than that from beef sternomandibularis, and to twice the extent. At 0°C the uptake of the former was halved, while in the latter it was reduced to a very low level (see also Newbold et al., 1973). These results support pump failure as an explanation for cold shortening. The ability of the white muscle of the rabbit to maintain an active calcium pump at 0°C accounts for its failure to cold shorten. It seems possible that either a temperature-induced phase change in the lipids or a conformational change in the protein of the membrane could cause decreased efficiency or failure in the pump. It is known that the lipids of bacterial membranes adapt in composition to meet a change in the temperature of the environment, thus maintaining the same phase structure in the membrane as before (Cullen et al., 1971). Recent research has shown that chill-sensitive plants at 10°C suffer a phase change in the lipids of the mitochondria1 membrane, accompanied by lowered respiration. Chill-resistant plants suffered no such change. Warm-blooded mammals suffer a sharp phase change in the lipids of their mitochondria at 22" to %"C, but cold-blooded fish do not (Lyons and Raison, 1970). C. L. Davey and K. V. Gilbert (unpublished results, 1971) observed that myofibrils do not cold shorten, but fiber pieces (with intact reticulum) do so. Shortening is inhibited by EGTA. This is also consistent with an involvement of the calcium pump in cold shortening. Another role for calcium has been recognized in the regulation of the activity of phosphorylase, which itself is a regulator of glycolysis. It activates the kinase, which activates phosphorylase kinase, which in turn converts inactive phosphorylase b to phosphorylase a (Reimann et al., 1971). It also inhibits the phosphatase, which reverses the activation. However, the rate of ATP breakdown in resting muscle is more likely to be the factor determining the rate of glycolysis than phosphorylase activity (Scopes, 1971). Cassens and Newbold (1967a) found the rate of glycolysis in beef sternomandibularis at 1°C and 15°C to be the same for about 8 hours postmortem and lower at 5°C. This nonlinear

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R. H. LOCKER ET AL.

relationship with a minimum rate has also been found for the same muscle by Bendall (1972a) and by Scopes (1972) for 'lamb longissimus (see also Section III,B,l). This could be explained by a dominant reticular ATPase at higher temperatures, declining with temperature as adverse lipid phase changes set in. The leakage of calcium then activates fibrillar ATPase, which becomes dominant, with a net rise in ATP breakdown.

D. THAWSHORTENING At an early stage in the lamb studies it became clear that early-frozen lamb loins were more tender if thawed slowly before cooking than if put into the oven while frozen. This led to the recognition that a second effect was making a serious contribution to toughening, over and above cold shortening. This effect, known as thaw shortening, was not a new discovery, having been first observed by Moran (1930) in frog muscle. However, as a practical meat problem, it has been the subject of extensive work by Marsh and others on the whaling ship Bahenu (Sharp and Marsh, 1953), at Cambridge (Marsh, 1952a) and in this laboratory. It was found that, if a muscle is frozen prerigor and rapidly thawed, it undergoes a drastic shortening, accompanied by copious "drip." This is a special problem in processing whale meat, which is slow to go into rigor mortis. Marsh and Thompson (1958) studied thaw rigor in lamb, using excised longissimus. If lamb was frozen immediately and thawed at 16" to 20"C, there was an average shortening of 72%, with a 27% loss in weight as drip. Muscles frozen in rigor and thawed shortened by only 5%, with 3% drip. In muscle frozen pre-rigor, the drip increased with ambient thawing temperature, rising rapidly between 5°C and 10°C. Muscle thawed at -3.5"C for 4 days did not shorten or drip. The relation between shortening, drip, and pH at the time of freezing is shown in Fig. 6, and the relation between drip and thaw temperature in Fig. 7. Thaw shortening is fairly powerful, being abIe to raise loads of 500 gm/cm2 to rest length, and lift loads of 200 gm/cm2 to about half the original length. This is more powerful than cold shortening, where corresponding values of 60 gm/cmz and 20 to 40 gm/cm2 were observed (Locker and Hagyard, 1963). However, both sets of values are well below the 2 to 5 kg/sq. cm in tension which skeletal muscle can generate. A rapid rise and decline in tension during thaw rigor was observed by Jungk et al. (1967). Newbold (1966) showed that the rapid and drastic physical events of thaw rigor precede the accelerated metabolic

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FIG.7. Effect of ambient temperature on drip exudation during the thawing of muscle strips frozen before rigor onset. (O), mean of twenty results at room temperature; ( X ) , strip frozen after rigor onset. From Marsh and Thompson (1958).

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R. H. LOCKER ET AL.

run-down (Fig. 8). The pH fall and disappearance of ATP are almost complete within an hour. The thaw shortening response can persist in the aerobic surface layer of ox muscle stored under the right conditions for up to a month before freezing (Leet and Locker, 1973). The basic reason for thaw shortening appears to be the release of calcium (Kushmerick and Davies, 1968), probably by rupture of the sarcoplasmic reticulum. Thus, cold shortening and thaw rigor are related in their dependence on calcium release-in the first case by failure of the pump, and in the second by gross disruption of the fabric of the pump, with more drastic results. Rigor shortening at higher temperatures may also be due to failure of the calcium pump, in this case as the ATP which drives it runs out. Although neither carcass meat nor tenderness was studied by Marsh and Thompson (1958), they made some observations of great practical importance. It was clear from the rate of pH fall they observed in lamb muscles that blast freezers were capable of freezing these small carcasses before rigor was complete. On the other hand, they suggested that as long as the trade persisted in thawing carcasses slowly before cutting, shortening and drip would be avoided by the restraint of the skeleton and internal ice. However, they warned of the dangers involved in cutting while

FIG. 8. Chemical and physical changes in beef sternomandibularis muscle frozen about 2 hours postmortem and thawed 24 hours later at 22°C. The strip used for recording length changes was initially about 0.4 cm2 in cross section and remained under a load of 25 gm throughout. ATP was measured enzymatically. Total shortening, about 50% of the initial length. From R. P. Newbold, “Changes Associated with Rigor Mortis,” in E. J. Briskey, R. G. Cassens, and J. C . Trautman, editors, The Physiology and Biochemistry of Muscle as a Food (Madison: The University of Wisconsin Press; @ 1966 by the Regents of the University of Wisconsin), pp. 213-244.

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frozen, or in faster thawing methods. Since 1958 the expansion of supermarket trading in frozen cuts and the increasing habit of housewives of putting this meat directly into the oven or pan has produced exactly the results of which the industry was warned. This will be made clear in the process section, where it is demonstrated that cold shortening and thaw rigor are both important and additive effects in the present commercial practice of lamb freezing, and that the effects of thaw rigor ( b u t not cold shortening) may be eliminated by proper thawing of the carcass. It will also be shown that glycolysis continues in the frozen state, and a sufficient period of frozen storage will also avert thaw shortening (see Section II1,C).

E. AGINGAND CONTRACTION The well-known increase in the tenderness of meat on postmortem storage has been the subject of many studies, which cannot be reviewed here. The accumulated information from biochemical studies on the mechanism has not been conclusive. The evidence does not, on the whole, point to any substantial degradation of the proteins of the myofibril or the connective tissue (Goll et al., 1970), although it must be admitted that a quite limited breaking of covalent bonds could have a great effect on mechanical strength. Changes in the interaction of the myofibrillar proteins definitely occur and are reflected in changes in extractability. Histological studies in this laboratory have thrown some light on the process. Davey and Gilbert (1967) observed that, during aging, the Z-lines, which define the sarcomeres, disappeared, producing lines of weakness in the myofibril. The release of a complex protein material soluble at low ionic strength was observed to accompany the process (Davey and Gilbert, 1968). The fading of the Z-line occurs also in washed fiber pieces and in isolated myofibrils, where its course may be watched under the microscope ( Davey and Gilbert, 1969). The presence of EDTA inhibits the process, suggesting the need for divalent ions. It seems likely that liberation of calcium from the sarcoplasmic reticulum during onset of rigor may accelerate the process. The fading of the Z-line has also been observed by several other groups of workers (Goll et al., 1970). An important practical result emerged from a study of the effect of shortening on ability to age. Davey et al. (1967) fixed beef stemomandibularis at different lengths by stretching or by rigor shortening, and then aged part of the material for 3 days at 15°C. The relationship be-

R. H. LOCKER ET AL.

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tween shear force and tenderness is shown in Fig. 9. Muscle untoughened by shortening improves in tenderness on aging, but the effect declies rapidly over the region where shortening increases toughness. At the peak toughness produced by 40% shortening, aging has ceased to have any effect. Presumably, as more sarcomeres shorten to the point where thick filaments penetrate the Z-line and overlap their neighbors, the Z-line ceases to be significant as a weak point. In the aged meat there appears to be a distinct threshold for toughening up to about 20% shortening, which is less evident in the unaged meat. Different results were obtained by Herring et al. (1967) on beef semitendinosus from animals at two levels of maturity. They found that at all points between 48% stretch and 48% contraction aging reduced the shear force significantly, but that at the higher levels of contraction the improvement still did not produce an acceptable level of tenderness. These results at least agree with those of Davey et al. (1967) in that meat toughened by shortening cannot be restored to tenderness by subsequent aging, a most important fact for the folIowing discussion on meat processing. 4 40 O%

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FIG.9. The effect of degree of shortening on shear-force values of bovine sternoMean of five determinations, mandibularis muscles (ultimate pH values, 5.90). (O), mean of five determinations, niaximum aging ( 3 days, 15°C). unaged samples; ,).( From Davey et al. ( 1967 ) .

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When contraction is eliminated as a factor by restraining muscles during rigor, subsequent freezing and thawing result in more rapid aging at 15°C (1.4 times faster than in unfrozen) in beef sternomandibularis. ( Locker and Daines, 1973).

111.

APPLICATION TO PROCESSING A. FROMLABORATORY TO FACTORY

Although the emphasis in this review has so far been on laboratory studies, enough has been said of carcass work to show that the results are of more than academic interest to the meat processor, concerned with the practice of chilling or freezing. However, results from excised muscle in small-scale laboratory experiments may not translate directly to industrial practice. Two important questions must be asked. To what extent can cold shortening occur when most of the skeletal attachments are intact at the time of chilling? A few muscles, such as those of the neck, are severed during dressing and may cold-shorten during chilling. Other muscles, notably the longissimus, are able to shorten because of their method of attachment. Although both ends of the longissimus are attached to the skeleton, the constituent fibers lie across the muscle axis at an angle of 30 to 40 degrees. Each of these fibers has one of its ends firmly anchored to the backbone, the other having its insertion in flexible subcutaneous tissue. The fibers are therefore free to cold-shorten, and in doing so change their angle, ending up almost at right angles to the muscle axis in lambs. Even muscles that are fixed absolutely in length by firm attachments at both ends are capable of shortening in part of their length through a differential chilling rate along the muscle. This was demonstrated for beef sternomandibularis by Marsh and Leet (196613). A clamped muscle was insulated at the ends and held at 2°C. A zone of marked shortening occurred in the rapidly chilled central region, at the expense of stretching in the two ends (see the photograph in their Fig. 12). Secondly, if meat is frozen prerigor, might not thawing be so slow in bulky carcasses for shortening to be minimal or absent? Rapid thawing of meat frozen prerigor can cause very considerable toughening in lamb. This was clearly demonstrated in an experiment where one-inch chops from lambs, frozen prerigor, were sawn from the frozen legs and loins and grilled, half of each cut from the frozen state and half after an overnight thaw at 2°C (Davey and Gilbert, 1974). Slow thawing pre-

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vented thaw shortening, and the chops were acceptably tender. The shear force for the longissimus in the loin was 26, and for the gluteus medius in the leg, 23. However, thaw shortening occurred in those chops thawed rapidly on grilling, and this led to a quite undesirable level of toughening (shear force 75 and 57, respectively).

B. RIGORMORTISAND CONDITIONING If the adverse effects of exposing meat from newly killed animals to cold are to be avoided, processing must include a period to ensure that the muscles are sufficiently close to rigor mortis for shortening to be negligible during subsequent refrigeration. The term “conditioning” has been coined here to describe this stage of processing. The temperature must be high enough to ensure that no cold shortening occurs during conditioning. It should be emphasized that this is not a tenderizing process, but rather an avoidance of toughening, to enable the full tenderness potential of the meat to be realized in subsequent processing. Aging, a postrigor process, has a true tenderizing effect. Conditioning and aging are quite distinct in concept, although they may overlap in practice.

1. Temperature and Rate of Glycolysis The time-temperature relationship for the onset of rigor is therefore a matter of considerable practical importance. The most-used criterion for rigor is the attainment of ultimate pH. Although pH fall is usually close to linear over most of the range, the rate declines in the last stages, and it is not easy to give a precise time for the end point. Marsh (1954) showed that beef longissimus reached pH 5.80 in 20 hours at 7°C and in 16 hours at 17°C (ultimate p H ca. 5.6). The temperature coefficient was much smaller in this range than at higher temperatures. He considered 36 hours in a chiller adequate for completion of rigor. Cassens and Newbold (1967a) found the rate of pH fall in beef sternomandibularis during the first 8 hours to be the same at 1°C and 15”C, and slightly less at 5°C. We have also observed this relationship for the same muscle. At later times the curves diverged, and the pH was within 0.05 unit of the ultimate in 48 hours at 1°C and in 24 hours at 15°C. Cassens and Newbold considered the fall complete in 72 hours at 1°C or 5”C, and in 30 hours at 15°C. In lamb longissimus, Marsh and Thompson (1958) found a constant rate of pH fall over most of the range. Their data suggest 10 hours at 15°C to approach rigor. They found only an 11% decrease in the rate

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of fall at temperatures between 17°C and 7°C. Scopes (1972) found the rate-temperature curve for lamb muscles to have a shallow minimum at 12°C. The rate of pH fall at 0°C equaled that at 20"C, and the rate at 7°C equaled that at 15°C. Marsh and Thompson (1958) found that the rate of pH fall in ewe longissimus was only 69% of that in lamb. In recent experiments in this laboratory (Wenham et al., 1973) it was found that the p H of ewe longissimus falls to within 0.05 unit of ultimate (mean 5.77) in 13 hours (S.D. = 4) at 15"C, while biceps femoris takes 14 hours (S.D. = 3 ) . These figures are close to those obtained by Van Eerd (1972), who found that, at about 23"C, times to ultimate pH for ewe longissimus, pectoralis, biceps, and semimembranosus were 11 to 14 hours, while psoas and semitendinosus took 8 hours. It seems clear that rate of pH fall in beef or lamb is not markedly affected by temperature during the linear phase, although this may be less true for the final phase. Time appears to be more important than temperature. Data on extensibility bear this out. Cassens and Newbold (1967b) found that the time to completion of rigor in beef neck is the same at 5°C and 15°C.

2. The Eflect on Tenderness of Deluy before Freezing The data on tenderness after conditioning are more limited than those on pH fall. McCrae et al. (1971) showed clearly that, at 18"C, 24 hours was a much more desirable conditioning time than 16 hours (see Fig. 12). At 16 hours the loin and major leg muscles were 50% tougher than they were at 24 hours. There seems to be a serious discrepancy between this 24-hour period and the time to rigor in lamb, which, from pH data, appears to be nearer to 10 hours. Brunton and Gilbert (1972) have attempted to find a lower temperature limit for conditioning lamb without cold shock (see Table 1-5). They found that 16 hours in a chiller at 7°C was inadequate for loins cooked from the frozen state, but if cooked after thawing, these loins were the equal of those held 48 hours. It seems that in intact carcasses at 7°C cold shock is avoided, in contrast to experience with excised beef neck (Figs. 1 and 2 ) . Taylor et al. (1972) have shown marked toughening on chilling lambs in air at -2°C (deep loin to 7°C in 2 hours). The lower limit for safe conditioning must lie between these values and is probably close to 7°C. It is noteworthy that shear values for the lambs cooked from the frozen state after 16 hours and 24 hours at 7°C were almost identical

178

R. H. LOCKER ET AL.

with those of McCrae et al. (1971) obtained at 18°C. Conditioning for 24 hours also produces a similar result at 10°C and 18°C (see Table 1-lb, 12a). Although larger samples at 7°C and 10°C would be desirable for a firm conclusion, the rate of conditioning appears to be the same at 7"C, 1O"C, and 18°C. The reasons are discussed at the end of Section I1,C. This bears out the contention that time is more important than temperature. It is also clear that in lambs conditioned at 7°C the gain in tenderness between 16 hours and 24 hours, which occurs only on cooking from the frozen state, is due entirely to prevention of thaw shortening (see Section 111,DJ). Exposure in the freezer after 16 hours does not induce cold shortening. This is in line with results on excised beef muscle (Locker and Hagyard, 1963). The mean pH of the lambs was 5.74 at 16 hours and 5.58 at 24 hours, suggesting that thaw shortening can still operate below pH 5.8. This agrees with some other rather scattered results of our own on beef and lamb muscle. The question of how near meat must be to its ultimate pH to be immune to thaw shortening, or even whether there is a period after reaching it when the ATP level is still high enough for a response, is an important one, requiring clarification by further research. The explanation is likely to be found here for the discrepancy noted above, between adequate conditioning times for lamb, as judged by pH fall and by tenderness scores. C. LAMBPROCESSING A large amount of information has been collected on this subject from lambs cooked by a standard method and assessed on the same tenderometer. This makes many comparisons possible. The data are collected in Tables I and 11, which will be referred to as different areas are discussed. The tenderometer scores are in arbitrary units, although they correlate well with scores from our taste panel, or the more widely used Warner-Bratzler machine ( r = 0.89 for beef neck). We regard a score of 20 as tender, 20 to 30 as satisfactory, and 40 as the limit of acceptability. It should be noted that these scores are derived from roasting, which favors tenderness, since there is time during cooking for some accelerated aging, and softening of connective tissue. The roasting score should not exceed 30 if the grill score is to be below 40. In aged meat the difference in score between the cooking methods tends to disappear.

TABLE I COLLECTED TENDERNESS RESULTSFROM VARIOUS CONDITIONING, FREEZING, AND COOKINGTREATMENTS OF LAMBAND MUTTON:MEANSHEARFORCE a FOR LOINSAND LEGS8

Expt.

Treatment c

A. Normally Hung Lambs (N.H.) la Blast frozen, no delay b Conditioned 24 hours, 18°C 2 Conditioned and aged to specifications 3 Conditioned and aged to specifications 4 Conditioned and aged 72 hours, 10°C 5a Conditioned 16 hours, 7"C, one side cooked frozen, other thawed b Conditioned 24 hours. 7°C C

Conditioned 48 hours, 7°C

B. Altered Posture Lambs 6a Controls (N.H.), blast-frozen, no delay b Standing posture, blast-frozen, no delay 7a b 8a b 9a b

Controls (N.H.), 24-48 hours, l " 3 " C Standing posture, 24-28 hours, l " 3 " C Controls (N.H.), conditioned 24 hours, 15°C Standing posture, conditioned 24 hours, 15°C "Hot-box'' (N.H.), 4 hours, 45°C Standing posture, 4 hours, 45°C

Number of animals 20 20 178 6 6 2 5

5 5

Loin e

Leg e References

Cook d

LD

SM

BF

GM

ST

Fr. Fr. Fr. Fr. Fr.

73 26 22 18 18

87 28

50 24

64 20

15 15

22

28

19

14

19

15

19

12

Fr. Th. Fr. Th. Fr. Th.

39 20 25 22 22 22

Fr. Th. Fr. Th. Fr. Fr. Fr. Fr. Fr. Fr.

77 45 69 26 40 30 44 19 52 22

1 n

z

3 4

4

65

73'

62

17

28

30'

24

18

68 21

73* 23' 43 16 57' 22"

35 20 40 16 42 16

20 17 17 19 14 13

55 15 54 22

5

5 6

5 (Continued)

TABLE I (Continued)

Expt.

5

Treatment

c

10a Hung from pelvis (leg 0" to vertical) conditioned 24 hours, 15°C b leg 45" c leg 90" d leg 135" 11 Hung from pelvis, blast-chilled, 56 days, -1°C (not frozen) C. Hot-Cut Lambs 12a Control sides (N.H.), 24 hours, 10°C b Hot-cut joints, conditioned 24 hours, 10°C 13 Hot-cut, conditioned carton 24 hours, 1°C 14 Hot-cut, frozen carton, no delay

13. Normally Hung Mutton 15a Ewes, control sides frozen, no delay b Other sides conditioned 24 hours, 15°C l6a Ewes, control sides frozen, no delay b Sides conditioned 24 hours, 15°C c Sides conditioned 24 hours, 15"C, aged 48 hours, 2°C d Cf. lambs, sides frozen, no delay e Other sides conditioned 24 hours, 15°C 17a Rams, control sides frozen, no delay b Other sides conditioned 24 hours, 15°C

Number of animals

Cook d

LD

SM

BF

GM

ST

1

Fr.

38

29

19

20

19

1 1 1 6

Fr. Fr. Fr.

35 19 24 12

19 19 24

17 13 15

18 11 13

22 18 20

28 25 22

23 17 16 17

24 14 15 11

23 18 16 20

14 15 13 15

10

5 8 2 24 12 6 6 6 6

Loin e

Leg e References g

-

7

Fr. Fr. Fr. Fr. Fr.

50

Fr. Fr. Th. Th. Th.

56 29 26 22 19

62" 33" 44" 26" 33*

32 23 23 17 18

44 26 34 20 20

17 18 15 14 18

Th . Th. Fr. Fr.

32 25 28 26

32" 26" 68" 36"

22 18 23 21

40 22 43 23

14 14 19 17

-

6

8 9 9

10 10

10

E. Hot-Cut Mutton 18 Ewes, hot-cut, conditioned 24 hours, 10°C F. Electrically Stimulated Lambs 19a Controls 18OC, to freezer at 5 hours p.m. b Other sides 18"C, freezer 20 hours c Electrical stimulation: freezer at 5 hours p.m. d Other sides (stimulated) 18"C, freezer 20 hours

e

! I

12

Fr.

34

20

14

17

5

Fr. Fr. Fr. Fr.

79 34 43 31

75 29 40 27

51 21 30 18

22 32 23

5

57

17

10

11

aAll assessments on the MIRINZ tenderometer (Macfarlane and M a w , 1966), with the exception of those marked with an asterisk, which were calculated from the panel score, using a conversion graph (see Wenham et al., 1973). A linear correlation between WarnerBratzler and MIRINZ shear values applies for lamb in the range 0-70 MIRINZ units ( W. A. Carse, 1974, unpublished results.) The relation between the scores is given by the equation (WB) = 1.40 + 0.29 (MIRINZ). b The shoulder muscles were assessed in some cases, but were little affected by treatment. The mean score for three muscles-triceps brachii, infraspinatus, and supraspinatus-were as follows: Expt ( l a ) 18; ( l b ) 14; (12a) 14; (12b) 15; (13) 16; (14) 18. c In all experiments except ( 11), blast-freezing was the final treatment. d Cooking was by roasting from the frozen (Fr.) or thawed (Th.) state, as indicated, to an internal temperature of 82°C. e Muscle code: LD = longissimus dorsi; SM = semimembranosus; BF = biceps femoris; GM = gluteus medius; ST = semitendinosus. f Stimulated between neck and gambrel with 12-msec square-wave pulses (250 volts) at three per second. 0 References cited as follows: 1. McRae et al. (1971). 2. Table 11. 3. C. L. Davey and K. V. Gilbert (unpublished results, 1971). 4. Brunton and Gilbert (1972). 5. Davey and Gilbert (1973). 6 , Davey and Curson ( 1971). 7 . Gilbert et al. (1973). 8. McLeod et 02. ( 1973). 9. McLeod et al. ( 1974). 10. Wenham et al. 1973). 11. Carse (1973).

R. H. LOCKER ET AL.

182

1. Cold and Thaw Shortening in Lamb Carcasses The claim that cold and thaw shortening are matters for practical concern is completely justified in the paper of Marsh et uZ. ( 1968), where the effect on tenderness of cooling and freezing carcasses before the completion of rigor mortis was studied. In these whole-carcass experiments the tenderness of lamb loins (cooked after thawing) was greatly affected by the time-temperature pattern during the onset of rigor mortis. Such factors as carcass grade or weight and age of the animal were of minor significance. The magnitude of the effects of postmortem treatments, carried out on a large number of loins, is illustrated in Fig. 10. These histograms show that early rapid chilling and freezing has caused a substantial increase and a wider spread in shear force scores. The effect on tenderness of varying the time between the completion of dressing and freezing is summarized in Fig. 11. If freezing begins in the first 6 hours postmortem, the final toughness is considerable, but if the delay is increased beyond this point the lambs become progressively more tender, approaching their maximum at 16 hours. McCrae et aZ. (1971) extended these studies to individual muscles of the leg and shoulder (Fig. 12).

---_ I I

20 .

L

n

n

I

5

I I

I 8

I

I I

n

I

-5

I I

I

L

0 L

0"c

(0

-

3

02

' J

80

I

I

I

I

I t

4

,

I

I8

I I

I 1

I

I

I I p---->

60

40 Shearing force

20

1

0

FIG. 10. The effect of postmortem treatment on shear force. (-), early rapid freezing; ( - - - - - - ), delayed slow freezing. Forty-eight lambs in each treatment. Reprinted from B. B. Marsh et al., J. Food Sci. 33, 12-24, (1968).

NEW CONCEPTS IN MEAT PROCESSING

183

7

6

5

? 0

0 a 6 4

3

2 '2

3

6

9

42

15

24

Delay (hours)

FIG.11. The effect on panel tenderness assessment of delaying carcasses at 18" to 24°C before entry to freezer. Seven lambs per time group. Vertical lines are standard deviations. Reprinted from B. B. Marsh et al., I. Food Sci. 33, 12-24, (1968).

Differences in degree of toughening were shown to be a matter of skeletal restraint, rather than intrinsic properties, by the fact that the semitendinosus and triceps, which were always tender, trebled in shear force when cut at one end and early frozen. These results show a distinct improvement if holding is extended from 16 to 24 hours. The apparent contradiction with Fig. 11 is due to cooking from the thawed state in Fig. 12. This again demonstrates (see Section II1,B) that avoidance of thaw shortening requires longer conditioning than elimination of cold shortening. A substantial body of results accumulated in this Institute and in the industry is presented in Table 11. It can be seen that the normal commerical practice of blast-freezing with minimal delay after dressing produces lambs which, when cooked from the frozen state, have a mean toughness well above the acceptable level of 40 shear-force units. Less than a third meet the criterion. Overnight conditioning brings two-thirds within the acceptable range, while an additional aging period (see Section III,D,2) produces satisfactory tenderness in virtually all carcasses. It can be seen, however, that cooking from a thawed state gives a much better result, three-quarters of early frozen lambs and all conditioned lambs being acceptable. The difference between lambs cooked

R. H. LOCKER ET AL.

184 1

I

I

I

1

I

1

00

Mean.SS, ST, IS

FIG. 12. Effect of delay before freezing on shear force. Twenty animals in each muscle and delay group. Code: loin, LD = longissimus dorsi; leg, SM = semimembranosus, GM = gluteus medius, BF = biceps femoris, ST = semitendinosus; shoulder, TB = triceps brachii, SS = supraspinatus, IS = infraspinatus. Reprinted from S. E. McCrae et al., J. Food Sci. 36, 566372 (1971).

from the frozen and a thawed state may be taken as a measure of the effect of thaw shortening. Any residual toughness in excess of about 21 shear-force units in lamb cooked after thawing can be regarded as TABLE I1 LAMBTENDERNESS RESULTSFROM DIFFERENT PREFREEZINC TREATMENTS

Pretreatment A. Loins Roasted from Frozen State Blast-frozen at 0-6 hours Conditioned 16-24 hours Conditioned and aged B. Loins Roasted from Thawed State Blast-frozen at 0-1 hours Conditioned 24 hours, slow-frozen

Number of animals

Percent acceptable (S.F. 40)

<

Mean shear force

218 150 178

30 65 94

53 36 22

48 48

73 100

33 21

NEW CONCEPTS IN MEAT PROCESSING

185

the effect of cold shortening. Thus it can be seen in Table 11, by comparing lines 1 , 4 and 5, that both cold shortening and thaw shortening are major factors in toughening lamb under current commercial freezing procedures. The first effect is irreparable damage. The second can be avoided by the butcher or the housewife who is careful enough to thaw meat slowly before cooking. Unfortunately the industry cannot rely on such wisdom. There is some comfort in results of current work (Davey and Gilbert, 1974), which have indicated that glycolysis occurs rapidly in lamb at - 5°C and continues steadily during frozen storage at - 12°C. Within 2 to 3 weeks at -12"C, the pH had fallen below 6.0, and toughening due to thaw shortening on roasting had almost disappeared. Some toughening persisted in grilled leg steaks, but this effect declined within 2 to 3 months. This experiment is a warning that frozen storage is an important variable in experiments designed to show thaw shortening effects. From these preliminary results it appears that thaw shortening may not be a problem in frozen meat that does not reach the consumer for several months. Whether this would be true for storage at -18°C remains to be seen. However, damage due to cold shortening remains. A similar recent finding (Benke et al., 1973a,b,c) is that the rate of depletion of ATP in chicken muscle is at least as fast at -3°C as at O"C, 5"C, or 1O"C, while in beef it is faster at -3°C than at 0°C or 10°C. 2.

Conditioning and Aging of Lamb

Specifications have been developed on the basis of the previous results for producing lambs of consistently high quality (Table 111). Commercial experience over six years has shown that the apparently conflicting demands of hygiene and tenderness can be met in mass production. Bacteriological and refrigeration considerations are discussed in more detail in Sections V and VI. Tenderness results can be seen in Tables I 3 and 11. Brunton and Gilbert (1972) have since shown that conditioning and aging for 3 days at 10°C also produces lambs satisfactory in all respects (Table 1 4 ) . Although any of the routes shown produce a satisfactory product, in the light of recent results (see Section II1,B) we consider that one-temperature processes at lower levels (say 7" to 13°C) are likely to be favored in the future, since these are simpler and offer greater margins for safety. However, aging certainly has a marked temperature coefficient, and while 7°C may be the equal

R. H. LOCKER ET AL.

186

TABLE 111

NEW ZEALANDSPECIFICATION FOR CONDITIONING AND ACINGOF LAMBS Conditioning

Aging

System

1 2 3

4

Temperature ( "C)

Time ( h r )

18 16 13 13

16-24 18-27 2130 24

Temperature ( "C)

3 3 3 13

Time ( h r ) 38-96 40-96 43-96

40-48

All times are total elapsed times from completion of dressing; that is, aging times include the conditioning period. Aging is normally carried on until 38 to 48 hours postmortem , but the 96-hour maximum permits holding over a weekend. Carcasses to be unwrapped throughout. Temperature tolerance k 1°C. Air movement 50 to 150 ft/min. Relative humidity 80 to 85% during conditioning, and 85 to 90% during aging. Bacteriological standards. Aerobic plate counts at 37°C: Before conditioning, 80% of samples below 101/cm2, none above 105. After aging, 80% below 106, none above 107. At least half the samples must be taken from vulnerable areas-for example, flap and brisket. The above standard is for freezing and cutting. If lamb is to be cut or boned and rolled before freezing, read 90% instead of 80%.

of 13°C for conditioning, the higher temperature will be more suitable for rapid aging. If lamb is not to be frozen, conditioning for 16 hours is adequate protection against subsequent rapid chilling. With subsequent freezing, conditioning for 24 hours is highly desirable, although this is also a practical limit for reasons of management. Such meat is then at a very acceptable level of tenderness. The additional benefit of aging must be weighed against the economics. The advantage is most worth while in cuts for grilling. The ultimate aging attainable is shown in Table 1-11, where extreme aging for 56 days produced a mean shear force of 12 in loins. Such a level of tenderness is unnecessary.

D. MUTTONPROCESSING There is a considerable literature on lamb up to the yearling stage, but very little information on palatability of mature sheep. A recent study in this laboratory (Wenham et al., 1973) has attempted to determine the potential tenderness of the meat and the extent to which it suffers from early refrigeration. The risk of damage is real in view of experience with lambs, and also the finding that the sternomandibularis

NEW CONCEPTS I N MEAT PROCESSING

187

of ewes (42 animals) had a mean cold shortening at 2°C of 34% (S.D. = 8) (R. H. Locker and G. J. Daines, unpublished results, 1972); for the beef muscle it was 46y0 (S.D. = 6 ) (Locker and Hagyard, 1963). It was found that mutton from mature ewes or rams suffers seriously from freezing too early, although not to the same degree as lamb (Table 1-0). This is probably because the larger carcasses cool more slowly. If the carcasses are conditioned for 24 hours at 15"C,the resultant joints when roasted are not significantly tougher than lamb as assessed on small pieces, either by tenderometer or by panel. Hot-cut legs (see Section IV,A,3), from a complete series of ewes aged 3 to 9 years, gave the same low shear values as those from lambs (Table 1-18) and showed no age trend at all. However, full leg slices, which contain the definitely tougher gross connective tissue, were rated by the panel as significantly tougher when taken from ewes than from lambs, although the mutton was still very acceptable. While there was an overall preference for lamb, other palatability characteristics did not discriminate greatly against ewes or rams. It appears that the slightly greater toughness of mutton lies not in the myofibrillar material or even the microconnective tissue, but in the hardening of gross connective tissue with age. The refrigeration treatment can far outweigh these effects. It is concluded that simple improvements in processing, including hot cutting, or altered posture as described for lambs (see Section IV,A,2), could lift mutton from the category of process meat to that of an acceptable roasting meat with consequent increase in profitability.

E. BEEFPROCESSING 1. Cold Shortening in Beef Beef sides present special processing difficulties, the most important being the rapid heat removal from a massive bulk. Otherwise beef reacts in exactly the same way to cold as does lamb or mutton. Traditionally, microbial control of beef after dressing has been achieved by chilling sides rapidly enough to inhibit growth of bacteria, both internally and on external surfaces. Large-scale studies on beef have been undertaken by Davey and Gilbert (Davey, 1970) to determine whether, in practice, chilling of beef sides can be sufficiently rapid to produce cold shortening in muscles that constitute the grilling cuts. The right sides of fifty prime Angus steers

R. H. LOCKER ET AL.

188

were used as controls, and the left sides for experimental purposes. The controls were chilled at a reasonably slow rate (Fig. 13, curve 1 ) for over 48 hours to ensure that the sides were fully in rigor before boning out. Thus subsequent contraction was eliminated as a variable. The left sides were chilled at a much more rapid rate (curve 2 ) . Temperature, humidity, and air movement in the commercial chillers, and also the temperatures at various depths within the sides, were monitored throughout the chilling period. Cube roll, eye round, and strip loin cuts were chosen for quality appraisal, representing surface and deep musculature. Portions of each cut were tested after cooking, both before and after aging at 20°C for 24 hours, when tenderization was 80% of maximal. In the slowly chilled sides, the deep hind leg cooled to 17°C during the first 18 hours. This followed closely the rate achieved in the preparation of a successful experimental shipment of chilled beef in 1952 (Law and Vere-Jones, 1955). Since that time chilling rates have been increased so that the deep hind leg usually reaches 8°C in 18 hours. This was chosen as the fast chilling rate in this experiment. In both, bacterial sur35

-

30

-

25

Deep leg

-

20 -

Y

: I5 3

:

+

a':04

+$

i air

5 -

Chiller

-40 1

0

1

1

I2

1

1

l

1

24 Hours post rnortem

1

1

36

1

,

L

3

FIG. 13. Cooling curves for beef sides. The solid curves represent the deep-bone temperature of the leg under slow ( 1 ) and fast ( 2 ) chilling conditions. The dotted curves are ambient air temperatures. From Davey ( 1970).

NEW CONCEPTS IN MEAT PROCESSING

189

face counts remained low, with no sign of surface desiccation. At a relative humidity of approximately 95%, weight losses at two chilling rates were low (1.2 to 2.7%, average 2.0%). One distinct difference between the slow- and fast-chilled sides was the tendency for a sideways twist to occur in the backbone of the latter. Sometimes the neck shifted 12 inches from the vertical. The effects of the two chilling rates on the tenderness of cube rolls is illustrated in Fig. 14. Rapid chilling produced an erratic toughening, with shear-force values ranging from 10 to 120-that is, from very tender to very tough (Fig. 14a). Some 80% of cube rolls were unacceptably tough (shear force 35). As was expected from our earlier studies (see Section II,E), a large proportion of the contracted meat would not age. This is shown in Fig. 14b, where some half of the cuts were still unacceptably

75

-

50

-

25

-

Tender

Tough

Very tough

Fast- (a)

: 75 L

W

E

%

50-

W

Y Q

-

.c

c

75-

.-

2

50-

FIG.14. The distribution of tenderness and toughness in cube rolls from beef sides chilled at different rates and far different times. From Davey (1970).

R. H. LOCKER ET AL.

190

tough (shear force 35 to 110) after aging for 24 hours at 20°C. On the other hand, at the slow chilling rate a large proportion of the meat was moderately tender (Fig. 14c), and after aging it was very tender (Fig. 14d.) Results on loins and eyerounds followed this pattern. Strip loins were more sensitive to cold than cube rolls, and eye rounds rather less so. The most important conclusion is clearly that chilling of beef sides must be sufficiently slow to avoid cold shortening and also to obtain maximum dividends from aging. These results agree closely with those of Buchter (1972), who found that in young bulls, cows, and calves there was progressive toughening in the loin as chilling rate increased. Even extended aging barely brought rapidly chilled meat to the initial tenderness of slowly chilled meat. 2.

Conditioning and Aging of Beef

A specification for chilling and aging beef, based on our results, is summarized in Table IV. The whole process is carried out at one temTABLE IV NEW ZEALAND SPECIFICATION FOR CONDITIONED AND AGEDBEEF Conditions

Chiller conditioning of sides

Aging of Cuts

Packing

Bare

Temperature Time ( a )

10" -e 1°C 18-24 hours minimum in fully loaded chiller 61-72 hours (weekend)

Vacuum-pack in Cryovac S and lay horizontally in cartons; seal 10" -c 1°C 6 6 7 2 hours from end of chilling to start of freezing 0-24 hours between chilling and freezing 50 ft/min

(b) Air velocity Relative humidity Stowage

100-200 ft/min recommended (minimum 5 0 ) 90-95% 23 inches minimum clearance between sides

Cartons horizontal, with reasonable spacing top and bottom

Cuts should be handled gently to avoid increasing "drip." The minimal acceptable time is 61 hours, but this involves some loss in tenderness. Bacteriological standards. Cleanliness should be maximal throughout. In chillers, air should contain less than 10 organisms per square foot per minute, and condensation should be minimal. Aerobic plate counts at 37°C: Before chilling, 80% of samples below lO*/cm*, none above 105. After chilling, 80% below 105. After aging, 80% below 105, none above 106.

NEW CONCEPTS IN MEAT PROCESSING

191

perature ( 10°C), which is a standard boning-room temperature here. Cutting is helped by the softer fat, and chiller condensation problems are overcome. With good hygiene, vacuum-packed cuts can be aged in sealed cartons, with great economy in facilities. High-quality primal cuts can thus be produced in the brief space of 4 days and can then be frozen.

IV.

UNCONVENTIONAL TECHNIQUES A. ALTERED POSTURE

It has already been shown that tenderness is related to the final state of contraction of muscles, and that this may be much modified by alterations in the method of hanging (see Section I1,B). Recent results from unorthodox suspensions of beef and lamb carcasses show that this approach has promise for large-scale processing. This work will now be described in more detail. 1. Altered Posture in Beef

The findings of Herring et al. (1965a), already described, have been confirmed in this laboratory. K. V. Gilbert (unpublished results, 1969) chilled a side from a mature bull, likewise in the horizontal position, and found marked improvement in the longissimus, semimembranosus, and biceps, compared with the other side, hung from the hock. Other muscles were little affected. Subsequent aging increased the difference for longissimus, but decreased it for the other two muscles. A side from a mature cow suspended from the pelvis (hole in the aitch-bone) showed improvement only in the longissimus and rectus femoris over the control. In both cases the altered posture reduced variation between and within muscles. A detailed study by Hostetler et al. (1972) used sides from forty young steers, in five different postures. Their results are shown in Table V. The greatest improvement in the unconventional positions was in the longissimus, but most of the major muscles of the leg were significantly improved. Some suspensions favored one muscle, and some another. There was little to choose between the horizontal position and hanging from the pelvis. The latter is the obvious choice for practical reasons. Sarcomere lengths were generally increased in the unusual postures, with the notable exception of psoas (which was not significantly toughened). Improvement in tenderness was most marked on extension in the range 1.9

192

R. H. LOCKER ET AL. TABLE V OF NINEBEEFMUSCLES,FROM SIDESSUSPENDED TENDERNESS IN FIVE WAYS^

Mean shear force b (kg) Muscle

Vertical

Horizontal Neck-tied

Longissimus Semimembranosus Semitendinosus Biceps femoris Rectus femoris Adductor GIuteus medius Psoas major Triceps brachii

6.0 f 5.0 f 5.3 f 4.2 4.7 f 4.7 f 4.9 f 3.7 4.7 g

4.7 g 4.4 g 5.0 f d 4.3 g 4.4 f 3.8 3.9 g,’2 3.8 4.9 g

4.8 0 4.7 f,g 4.9 f,g 4.3 0 4.9 f 4.4 f,g 4.3 g 4.3

Mean of means

4.8 f

4.4 9

c

Hip-free

d

Hip-tied e

4.9 g 4.5 fd? 4.4 4.6 f,g 3.8 4.3 fag 3.6 4.4

5.4 f

4.9 g 4.3 g 4.9 f,g 5.0 f 3.8 4.0 3.7 h 4.2 4.8 g

4.7 f

4.4 g

4.4 0

5.5 f

a Reprinted from R. L. Hostetler et al., J . Food Sci. 37, 132-135 ( 1972). Copyright @ by Institute of Food Technologists. Warner-Bratzler, mean of sixteen sides. Hung from neck, fore and hind limbs tied together. d Hung from aitch-bone, limbs free. Hung from aitch-bone, limbs tied together. gj h Means on horizontal lines with different superscripts differ significantly ( P 0.05). f a

<

to 2.5 microns, but there was little effect in the range 2.5 to 3.6 microns. Overall, 40% of the tenderness variation between treatments could be attributed to sarcomere length. Bouton et at. (1973) in a similar study compared four positions: carcasses hung from the hock; hung from the hock but stretched with metal splints (after U. S. Patent No. 3,579,716); hung from the pelvis; and hung from both pelvis and hock. The splints were of little value. Again the longissimus derived the greatest benefit from pelvic suspension, but other major leg muscles also improved substantially. The two-point suspension detracted from improvement in the longissimus, but benefited other leg muscles. It also prevented toughening of the psoas. Aging for 2 to 4 weeks at 0°C narrowed the margin between pelvic suspension and control in 2 to 3-year-old steers, but much less so in mature cows. An interesting point was the decrease in compressive strength in meat from carcasses in altered postures. This implies that the strength of connective tissue had been reduced by a change in its disposition or tension. The authors concluded that hanging from the aitch-bone so improves the quality of several important muscles that their tenderness at 1 to 2 days

NEW CONCEPTS IN MEAT PROCESSING

193

is equal to that of muscles from normally hung carcasses aged for 2 to 3 weeks at 0°C.

2. Altered Posture in Lamb and Mutton The effectiveness of altered posture in improving tenderness has also been established for lamb and mutton. Davey and Gilbert (1973) found that supporting a lamb on a broomstick in a natural standing posture improved the loin (longissimus) and leg (biceps, semimembranosus, and gluteus medius). The shoulder has a standing posture regardless of hanging method and is always tender. (Table I, footnote b ) . Similar results were obtained by Davey and Curson (1971) on lambs hung from the aitch-bone by an anal hook. The leg was supported at several angles to the vertical, but the position assumed without support (at right angles to the spine) appeared best (Table 1-10). Very similar results were obtained by Quarrier et al. (1972), who found improvement in tenderness, associated with longer sarcomere lengths, in the leg and loin of lambs on pelvic suspension. Baxter et al. (1972) found the same situation in hoggets, the margins persisting after aging for 2 weeks at 0°C. As with beef, fiber adhesion was reduced as well as shear force. The effect of posture alone can be distinguished only under postmortem conditions which prevent cold shortening. In both the American and Australian work just described, all carcasses were held at 0” to 2”C, which would induce considerable toughening in the controls by contraction of the bunched muscles at the back of the leg. This does not occur in pelvic suspension, when a more uniform skeletal restraint applies. The differences are therefore magnified. In experiments here on lamb, conditioning at 18°C for 24 hours produced controls with reasonably tender leg and loin, although altered posture samples were still somewhat more tender (Table I, cf. l b and 8b). Thus, if proper conditions are employed, the effect of posture is real but small. However, the differences show dramatically in animals subjected to early blast-freezing (Table 1-6). The controls are then very tough, while legs from altered posture animals remain tender, even if cooked from the frozen state. Loins toughen, but prior thawing reduces this greatly, implicating thaw shortening. It seems that altered posture protects against the ravages of both cold and thaw shortening in the leg, but not in the loin, where skeletal restraint is less effective. The risk of thaw shortening in the loin may be overcome, however, by “tempering” in frozen storage.

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The immediate blast-freezing of lambs in altered posture now seems a real possibility. This is again discussed under hot cutting (see Section IV,B,3), which is a variant of the same principle.

3. Prospects as a Process The benefits and technical simplicity of hanging carcasses or sides from the aitch-bone provide a sound basis for the practical processing of beef and lamb. The name Tenderstretch has been applied to the process by the Texas workers. While this is a useful short title, it should be remembered that it is the avoidance of shortening, rather than actual extension, of muscles which is important. The exact degree of improvement due to posture in beef is not quite clear, since chilling too rapidly may have magnified differences. At least some protection is conferred against rapid chilling. Expensive aging may be avoided, or extra quality achieved, by the same degree of aging. With lamb it is clear that the margins are small at proper conditioning temperatures, but the greatest potential seems to lie in protection of palatability during early chilling or freezing. In the case of heavy beef carcasses there is a risk of the pelvis’ breaking under the strain. The Australian workers claim that no extra space is needed for Tenderstretch beef in hanging or transport. They overcame the awkward shape of the lamb carcass by pulling the hind shanks in with a string, and found that there was then a small reduction in stowage space over the conventional carcass. This arises from the reduction in length. We find that the same result can be achieved by a gambrel passing through the hocks and behind the shank of the pelvic hook. The leg from this “crouching” posture has been found here to be as tender as that from unmodified pelvic hanging. The conformation of cuts would be unfamiliar to the trade, although not necessarily inferior. In some cases-for example, lamb legs-it could be a selling point.

B. HOTCUTTING AND BONING Cutting the carcass immediately after slaughter offers economies in time and hanging facilities. In the case of sausage manufacture, prerigor meat has superior emulsifying properties, and hot boning of beef and pork is widely used for this purpose. Hot cutting is also established

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in the United States in the manufacture of ham and bacon. It is not yet practiced with table meat. The major hazard in such practice is the shortening of unrestrained muscles during the onset of rigor mortis. It is important that the meat be kept in the temperature range of minimal shortening. It will be shown below that in the range 10" to 15°C toughening is avoided. Earlier packaging is usually part of the process. Although this has advantages in saving space and in avoiding weight loss or contamination, it is necessary to take care that bacteria already on the meat do not multiply unduly on the warm surfaces inside the wrapping.

1 . Hot Boning of Beef Schmidt and Gilbert (1970) sought to produce acceptable beef cuts by prerigor excision. Setting in rigor for 24 hours at 15°C was followed by aging for a further 24 hours at 15°C. Microbial growth was retarded by vacuum packing in Cryovac bags. The cuts were compared with controls from the other side of the same carcass, conventionally chilled for 24 hours at 9°C (air flow 50 ft/min). At 24 hours postmortem, hot-boned samples gave shear-force values very similar to those for the controls, except for the semitendinosus, which had toughened. When the hot-boned samples were aged for a further 24 hours, shear values fell to well below those for the unaged controls (with the exception of semitendinosus, which had improved almost to the level of the control). Taste panel assessments on juiciness, texture, and general acceptability showed that these palatability characteristics had been preserved. Extensive microbial assays were carried out. Initial counts on the cuts were around 10'- to 103/cm', and these rose only slightly during 24 hours at 15°C. In the next 24 hours counts rose by less than an order of magnitude, and only in one case exceeded 104/cm2.It appears that the utilization of oxygen and the generation of carbon dioxide within the impermeable bag had effectively controlled growth. 2. Hot Boning of Young Veal

Hot boning has been extended to young calves to determine the possibility of producing wholesome and tender veal cuts, as well as processing meat (Davey and Gilbert, 1971). The pattern of rigor onset and toughening due to cold or thaw shortening was similar in these very

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R. H. LOCKER E T AL.

young animals to that in mature beasts. R. H. Locker and G. J. Daines (unpublished results, 1972) also observed that sternomandibularis of seventeen calves aged 2 to 7 days gave a mean total shortening of 27.4% (S.D. = 6.8) at 2"C, the highest value being 38y0. These values are somewhat lower than those for ox. Davey and Gilbert found cold shortening of up to 40y0 in biceps and semimembranosus, with a shear force at that level of 73; at rest length the shear force was 22. Shear values of 55 were obtained from these muscles after hot boning, freezing without delay, and cooking from the frozen state. It was recommended that the hot-boned veal be held in the carton at 7" to 10°C for 21 to 24 hours before freezing, if the meat is to be used as cuts. Patties made from early frozen meat, minced without thawing, were not distinguishable from those made with meat conditioned before freezing. Conditioning thus appears not to be necessary for young veal intended for use as process meat.

3. Hot Cutting of Lamb and Mutton Success in the hot boning of beef prompted a study on cutting lamb into primal cuts immediately after dressing ( McLeod et al., 1973). Cuts were shrink-wrapped and conditioned at 10°C for 24 hours before freezing, and were then roasted without thawing. Muscles from the loin and shoulder were the equal of those from control sides from the same animals, conditioned in the normal hanging position. The hot-cut legs were significantly more tender than legs from the controls (Table 1-12). Experiments were then extended to conditioning in a carton (24 hours in air at 1°C) before freezing (McLeod et al., 1974). The same results were obtained. Freezing without delay in a carton also produced legs and shoulders of the same tenderness, but loins toughened (Table I13, 14). The restraint of the skeleton prevents the muscles of leg and shoulder from cold shortening or thaw shortening, making conditioning unnecessary if joints are to be roasted whole. However, cutting to leg steaks permits thaw shortening and serious toughening on cooking from the frozen state. Bacterial counts were very satisfactory after conditioning of separate joints at 10°C (but not at 15°C) or cartoned cuts as described. The shrink wrap is a n essential part of the process, as it converts the initially untidy cuts into nicely rounded ones, by molding the warm fat and flesh. The leg, as a result of this effect, and of its more compact conformation, becomes a very handsome cut, resembling a ham. Long loins distorted badly in the shrink wrap and were abandoned in favor of

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double short loins and double racks, which packed well. Distortion due to packing in cartons while hot was significant only in the lower leg. If this problem can be overcome and satisfactory techniques for hot cutting developed, the method could have considerable promise for an on-line process. The shrink wrap gives early protection against contamination and eliminates weight loss. Conditioning in the carton (if necessary) would require only a tenth of the space needed for carcasses, and humidity need not be controlled. The hot-cut leg, in terms of appearance, conformation, and tenderness, could be regarded as a premium product. It would be well suited to curing as a “ham.” This would be a particularly good utilization of mutton legs. Two new possibilities could make the actual cutting much easier. It has been found that, if the carcass is blast-chilled briefly on a pelvic hook, the fat hardens to the point where cutting is as easy as for frozen meat (McLeod et al., 1974). These cuts have square edges and are barely distinguishable from those cut from a frozen carcass. Another approach is a brief electrical stimulation on the bleeding rail, which can induce stiffness at 40 minutes postmortem (see Section IV,D).

C. HIGH-TEMPERATURE CONDITIONING In practice the shortest possible conditioning time is desirable. It has already been shown (see Section II1,B) that at temperatures below 15°C the temperature coefficient of pH fall declines and changes in sign for both beef and lamb. At higher temperatures, particularly near body temperature, the coefficient rises steeply, This offers the possibility of accelerating conditioning by heating rather than cooling the carcasses during the critical prerigor phase. In earlier work at high temperatures the interest has been in rapid aging of beef. Roschen et al. (1950) patented a method in which the whole carcass was “aged for 4 hours at 37”C, before chilling to 1°C. The data presented were all from muscle excised at death, and must be suspect, but it was claimed that ultimate pH was reached in the 4 hours, and tenderness was superior to that of control muscles chilled immediately. Subsequent high-temperature work has been concerned with aging for a day or two, sometimes after prior conditioning in a chiller, and using antibiotics or irradiation to control bacteria (see Wilson et al., 1960, and references therein). Wilson et al. found cuts aged for 24 hours at 44°C or for 48 hours at 32°C to be as tender as cuts aged for 2 weeks at 2°C. It is worth noting that, after 24 hours, off-flavors were prominent

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at 49°C and noticeable at 44"C, but bacterial growth was easier to control at 44°C than at 32°C. Davey and Gilbert (1973) examined conditioning of lamb at 45"C, which is just below the point where discoloration or other deteriorative changes occur. Rigor, as judged by loss of extensibility in muscle strips, is achieved in 1% to 2 hours. In the carcass the temperature rise is slower and 3 hours are needed. As with chilling, toughening resulted in normally hung lambs ( Table I-9a ), since rigor shortening is considerable at this high temperature (cf. Fig. 1 ) . However, the opportunity again arises to use altered posture, and by this means toughening was avoided (Table I-9b). Thus with pelvic suspension it is possible to condition a lamb satisfactorily in 3 hours, whereas a minimum of 16 hours and preferably 24 hours is needed at normal conditioning temperatures. At first sight the procedure seems doubtful on microbiological grounds. However, 45°C is above the normal optimum for most organisms apart from thermophiles. Thus, growth of spoilage and pathogenic bacteria is likely to be repressed, at least in the 3 hours required for conditioning. D. ELECTRICAL STIMULATION Another approach to speeding the onset of rigor is electrical stimulation. The first to apply this to carcasses were Harsham and Deatherage ( 1951), although they were less concerned with conditioning than with accelerated aging, which they claimed, Stimulation of beef carcasses at 3000 volts produced a fall in the pH of muscles to 6.1 in 1 hour. The meat was as tender after 2 days at 1°C as that from unstimulated controls after 18 days at 1°C. De Fremery and Pool (1960) found that electrical stimulation of chicken breast muscle for 15 minutes increased the initial rates of fall in pH and ATP content. Hallund and Bendall (1965) found that, in pigs with a naturally slow glycolytic rate, 30 seconds of stimulation approximately doubled the rate of pH fall, the effect persisting throughout the fall (in contrast to a transient effect on rabbit muscle). Forrest and Briskey (1967) and McLoughlin (1970) gave similar brief stimulation to the same pig muscle, with comparable results. In experiments on lamb in this laboratory, Carse (1973) subjected carcasses, hung at 15"C, to electrical pulses for 30 minutes. Leg and loin muscles of controls took 15 hours to reach pH 6.0, while in stimulated carcasses the rate of pH fall increased steeply with voltage. At 250 volts, pH 6.0 was reached in 3 hours. Assessments of tenderness on carcasses thus stimulated (Table 1-19) showed that sides sent to the freezer

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at 5 hours postmortem were on the average just within the acceptable range, while controls were quite tough. The other sides, sent to the freezer at 20 hours, were satisfactory and indistinguishable from the controls. This last result shows that the accelerated glycolysis itself has no detrimental effect on tenderness. In the sides frozen at 5 hours, stimulation produces a great improvement, although the tenderness still leaves much to be desired. Since these lambs were cooked from the frozen state, and the pH would have been well below 6.0, it seems highly probable that the residual toughness is the result of thaw shortening (see Section II1,B) and that the treatment may be adequate to prevent cold shortening, or thaw shortening after frozen storage ( see Section III,C$). Very recent results from this laboratory (B. B. Chrystall and C. J. Hagyard, unpublished results, 1972) show that the stimulation need only be brief. When lambs were hooked up immediately after their throats were cut and pulses were applied between the hooks and a pin in the neck (3OOO volts ac, five l-msec pulses per second), violent twitching occurred for only 30 seconds and then declined to a slight tremor. The lambs skinned easily, and no damage to the viscera could be seen. At the completion of dressing (20 minutes postmortem) stiffening had begun, and it was complete in 40 minutes, when the pH of the meat was near 6.3. Subsequent freezing and assessment of the meat showed that at this point the meat is already immune to cold shortening but not to thaw shortening, and that there is no toughening due to the stimulation. The same short stimulation after dressing was much less effective in accelerating rigor. These preliminary experiments are continuing, to define optimum voltages and times and to confirm the absence of adverse effects. There seems good reason to believe that a brief stimulation on the bleeding rail could be incorporated in a production line, to give carcasses which by the end of the normal handling period would have set, and would be ready for chilling or freezing without risk of cold shortening. The carcasses would also be suitable for immediate reduction to primal cuts (see Section IV,B )

.

V.

MICROBIOLOGY

A. FACTORS CONTROLLING GROWTH Meat processing methods must satisfy international hygiene requirements. Any new process is unlikely to be accepted unless the product is

200

R. H. LOCKER ET AL.

satisfactory with rcspect to contamination by pathogenic microorganisms and has a reasonablc storage life. Microbial activity must be strictly controllcd . Although reduction of temperature is one effective means of controlling microbial growth on meat, the considerations of tenderness discussed above make it dcsirable to restrict the rate of cooling in the prerigor pcriod to a level at which cold shortening is unlikely to occur. To resolve the conflict between the requirements of hygiene and palatability, it is necessary to use other means to limit microbial proliferation during processing and chilled storage. The effects on microbial growth of other factors, such as water activity, redox potential, and carbon dioxide concentration, can be used for this purpose. These factors exert their effects under different circumstances. The water activity of surface tissues of carcasses can be reduced by drying, redox potential within muscle falls during the development of rigor mortis, and carbon dioxide concentration can be built up by suitable packaging of cuts. The effectiveness of such measures was demonstrated by the successful shipment to Britain of chilled beef from Australia and New Zealand in the 1930’s. This trade relied on good hygiene, environmental control during chilling, and the use of carbon dioxide in shipboard storage to prevent the development of spoilage during the 40 to 60 days required for processing and transportation. This process was developed after extensive studies of the effect of environmental factors on the microbial population of beef during chilling and storage. The use of lowered A,,* for microbial control in meat processing is limited by the need to avoid excessive drying, with appreciable weight loss and deterioration in appearance from loss of bloom, desiccation, and freezer bum. With beef carcasses, the surface area-to-volume ratio is such that it is possible to obtain a fair measure of control over microbial growth during the early stages of chilling (cooling phase) without these undesirable effects. The smaller lamb carcass cools more rapidly, and weight loss and appearance become more important. Thus, control of bacterial growth on lamb by surface drying is limited to shorter times than are possible with beef. Microbial contamination of carcasses from healthy animals is mainly on the surface, although small numbers of organisms may be present in the lymphatic system (Nottingham 1960). The proliferation of these organisms is determined by temperature as well as by redox potential. TO prevent deep spoilage, it is necessary to cool the deeper tissues sufficiently to inhibit growth of anaerobes by the time the redox potential has fallen to a level at which anaerobic growth will occur. The limiting

* Available moisture.

NEW CONCEPTS IN MEAT PROCESSING

201

temperature for control of bone taint has not been established precisely, but results from laboratory studies and production trials with chilled beef suggest that reduction of the deep bone temperature to about 15°C in 24 hours provides adequate protection against deep spoilage. Laboratory studies have shown that mesophilic anaerobes generally grow slowly, if at all, at temperatures below 15°C; thus, in the short time from the fall of redox potential to the end of conditioning, little growth is likely (Roberts and Hobbs, 1968). The few psychrophilic anaerobes that have been isolated show a lag before growth commences which is sufficient to prevent significant development during the conditioning and aging period (Beerens et al., 1965). The laboratory findings are supported by experience with a shipment of chilled beef involving two hundred and fifty carcasses. The deepbone temperature of four of the five sides for which temperatures were recorded did not fall below 17°C in the first 24 hours after slaughter (Law and Vere-Jones, 1955). On arrival in Britain 50 days later, this beef was subjected to a critical examination by members of the meat trade and two trained taste panels. No sign of bone taint was observed. B. APPLICATION The use of reduced water activity, low redox potentials, and carbon dioxide as an adjunct to low temperatures for the control of microbial growth has been extended to new methods of lamb and beef processing. The feasibility of controlling microbial development by these means has been demonstrated here both in laboratory experiments and in production trials.

1. Recent Laboratory

Studies

The effect of temperature and A, on the growth of mixed populations of meat microorganisms in liquid media is illustrated in Figs. 15 and 16. These results were obtained with the A, media of Scott (1953) inocu-

lated with a mixed culture of organisms obtained by swabbing the surface of lamb or beef carcasses. To determine whether the conditioning temperature would affect growth during the aging phase, simulated conditioning and aging cycles were carried out with mixed cultures in A0 media. At an A,o of 0.990 the log increase in the 25°C bacterial count after 30 hours at 13°C ranged from 0.5 to 1.0. At 18°C and A,,, 0.990 the 24-hour increase was about 2.9. The increases in the count at 37°C were similar. These results show that lower water activities are required to control growth at higher conditioning temperatures. Growth at the aging temperature, 3"C, was not affected by condi-

R. H. LOCKER ET AL.

202

90

LI

70

2 c

E 0.

5

t-

50

4

8 Days

42

FIG. 15. The effect of temperature on the time required for a hundred-fold increase in microbial count at A w (available moisture) 0.990. From P. M. Nottingham, ( unpublished results, 1971 ).

Days

FIG.16. The effect of available moisture (Aw)on the time required for a hundredfold increase in microbial count at 10°C. From P. M. Nottingham (unpublished results, 1971).

NEW CONCEPTS IN MEAT PROCESSING

203

tioning at either 18°C or 13°C (P. M. Nottingham, unpublished results, 1972). Th.e results given above were obtained with A, media incubated aerobically. Additional studies with the same A, media dispensed anaerobically under carbon dioxide in roll tubes ( Hungate, 1969) showed that under these conditions the growth rates were reduced to about half those at the corresponding A, under aerobic conditions. 2. Lamb Production

The effect of humidity and air velocity on bacterial growth on lamb carcasses during conditioning and aging is illustrated in Table VI. In this trial three carcasses were held at the same temperature, but under different conditions of humidity and airflow. The swab sampling and plating techniques described by Nottingham (1971) were used for the bacteriological examination. The results of further trials of the conditioning and aging process and production sampling from a number of meat works are summarized in Tables VII and VIII. As is shown in Table VII, bacterial counts on samples from the leg often decrease, while counts from other areas increase. This difference illustrates the effect of surface drying on bacterial growth. In conditioning rooms where the airtlow is directed across the carcasses from above, the leg area tends to dry to a greater extent than the loin or forequarter, and this drying may be sufficient to reduce the bacterial load.

3. Beef Production The results of bacteriological examination of samples taken during trials of beef chilling with a number of chillers from various meat works TABLE VI AND HUMIDITY ON BACTERIAL FLORA OF LAMBa,b EFFECTOF AIR VELOCITY

Before conditioning After aging Weight loss

0.1 m/sec 85 %

Nil

Air velocity: Relative humidity:

100% 37°C

25°C

37°C

25°C

37°C

25°C

3.90 6.72

4.04 6.53

3.88 4.08

4.15 4.11

4.11 3.20

4.28 3.38

0.6%

Nottingham ( 1971 ). Log bacterial count per square centimeter.

a From

0.25 m/sec 60 %

2.3%

4.0%

R. H. LOCKER ET AL.

204

TABLE VII EFFECTOF CONDITIONING AND

AGING ON THE

BACTERIAL FLORA OF LAhlB a , b Log bacterial count/cm

2

Conditioning and aging treatment

Number of carcasses

Time (hr )

Leg

Flap

Brisket

13"C, one temperature

32

0 48

4.25 4.09

4.56 5.58

4.66 5.45

18°C conditioning/ 3°C aging

16

0 48

3.84 3.70

4.30 5.98

4.50 5.43

a

From P. M. Nottingham (unpublished results, 1972 ). Mean log 25°C counts from lambs sampled during experimental studies. TABLE VIII LAMBCONDITIONING AND AGING IN WORKS PRODUCTION a,b Season

1968/69 1969170 1970/71 a

Number of carcasses

Mean log bacterial count/cm

2

examined

Before conditioning

After aging

253 540 656

3.92 3.83 3.85

5.30 5.65 5.18

From P. M. Nottingham ( unpublished results, 1972 ). Mean log 30°C or 37°C aerobic plate counts.

are summarized in Table IX. Apart from eighteen samples taken after chilling for 67 to 96 hours all the 847 individual counts lie within the proposed limits, even though at the beginning of the experimental program the airflow in some of the chillers was not always satisfactory. On many of the sides examined the counts dropped during the first 24 hours of chilling, but after 43 to 68 hours the numbers of bacteria were generally slightly in excess of the initial level. If chillers are operated to the specification, there should be no difficulty in meeting the bacteriological criteria. Since works trials of the new chilling procedures commenced, many thousands of carcasses have been held at the specified temperature for periods of 18 to 72 hours. In no case has any evidence of bone taint been observed during boning out (K. Garnett, unpublished results, 1971). The results indicate that chilling can be prolonged to 96 hours without excessive microbial growth, provided the humidity and air velocity are maintained at a satisfactory level.

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205

TABLE IX ON BEEFSIDESBEFORE AND AFTER CONDITIONING BACTERIA AT 8" TO 10°C ~

Number of carcasses

65 24 32 49 8

Mean log count/cm

Chilling time (hr)

0 19-23 43-48 67-68 96

2

Leg

Aitchbone

Flank

Brisket

Neck

2.39 2.20 2.29 2.60 2.11

2.54 2.17 2.36 3.41 4.18

2.94 2.75 3.20 3.91 3.02

2.80 2.57 2.85 3.56 3.20

2.65 2.98 3.14 4.75 3.11

P. M. Nottingham ( unpublished results, 1972). Mean log 30°C or 37OC aerobic plate counts.

a From

To determine the optimum conditions for holding beef cuts during aging, the effect of packaging and temperature on the microbial flora of beef was studied. Individual cuts were wrapped in polyethylene bags or vacuum-packed in an impermeable film (Cryovac S) for aerobic and anaerobic holding. As would be expected, the bacterial counts increased more rapidly on the polyethylene-wrapped cuts than on those packed in Cryovac. A satisfactory degree of tenderization, without the development of microbial spoilage, could be obtained by aging at 20"C, 15"C, or 1O"C, but the lowest of these temperatures was chosen for general use, as this gives the greatest margin of safety from the microbiological point of view. With Cryovac-wrapped cuts held at 10°C a small increase in bacterial count was generally observed (Table X), but the product showed no signs of spoilage, either visual or detectable by taste panel evaluation. In some experiments, the qualitative changes in the bacterial flora were assessed by subculturing and identifying colonies from both the 37°C and 25 "C aerobic count plates according to the methods of Cowan and Steel (1966). The initial flora before holding consisted mainly of Micrococci ( 43% of the colonies examined ) and coagulase-negative Staphylococci ( 27y0), with smaller numbers of Acinetobacter, Pseudomonas, Enterobacteriaceae ( mainly aerogenes group ), and Corynebacteria. After aging at 1O"C, the proportion of Enterobacteriaceae and Corynebacteria increased to about 32y0 and 14y0, respectively, at the expense of the Staphylococci (8%) and Micrococci (31%) (P. M. Nottingham, unpublished results, 1972).

R. H. LOCKER ET AL.

206

TABLE X

BACTERIAON BEEFCUTSBEFORE Chilling Chilling time temperature (hr) ("C)

23 20 68 34 24 a

8 10 10 18 10

Aging time

(hr) 42 44 24 66

72

Aging temperature ("C)

15 15 10 10 10

AND AFTER

AGING a,b

Bacterial count Before

After

Samples

3.22 3.45 4.85 3.00 2.57

3.55 4.28 4.89 2.92 4.31

14 14 6

21 18

From P. M. Nottingham (unpublished results, 1972). Mean log 25°C aerobic plate counts/cm 2.

VI.

PHYSICS OF MEAT CHILLING

The industrial meat processor's goal is the economic production of a high-quality meat output from a prerigor muscle input, with the quality preserved for the future benefit of the consumer, to whom the meat must be delivered in the desired form and packaging. Exploitation of the biochemical and bacteriological principles outlined earlier requires the correct physical conditions €or this transformation. The interaction of the physical and engineering aspects of a chilling process with the biological phenomena must also be considered, since the physical environment forms the link between the product and the plant. Besides having a major influence on tenderness and spoilage, as has already been made clear, the environment affects appearance, surface dryness, and weight loss ( o r shrinkage). In the present context the process is specified as a controlled physical environment of air with a given temperature, velocity, and relative humidity provided for a given time. These conditions are possible only on an industrial scale with the proper design, operation, and control of the engineering plant. This section considers the physical aspects of this type of process, which includes cooling, chilling, conditioning, aging, and postmortem holding. Depending on the specific case, the meat may be in the form of carcasses or cartons of boneless or cut meat. The physical basis of these processes is the response of a single meat module ( carcass or carton), with given initial conditions, to the boundary conditions of the surrounding air. The input of a body with a temperature differing from that of the environment results in unsteady (transient) heat transfer across the product surface and within the

NEW CONCEPTS IN MEAT PROCESSING

207

product interior. Heat transfer can be evident as the temperature/time response of sensible heat changes (cooling or heating rate), latent heat of phase changes (surface evaporation, freezing, fat hardening), and internal heat generation (heat of rigor). If the moisture contents of the air and the product surface are not in equilibrium, mass transfer across the surface interface will also occur, resulting in surface drying, weight changes due to evaporation or condensation, and internal moisture diffusion. The heat and mass transfer effects also have a controlling effect on the reaction kinetics of rigor onset after slaughter, meat aging, bacterial growth, and protein denaturation. The process variables are time, air temperature, velocity, and relative humidity, whereas the product variables are composition and geometry. The product can be a deformable solid, such as a carcass with a highly irregular shape and a complex heterogeneous structure of lean meat, fat, and bone. It is thus anisotropic in chemical composition of moisture, fat, and protein; in physical and thermal properties, such as density, enthalpy, specific heat, thermal conductivity, and thermal diffusivity; and in rheological properties. A qualitative description of the physical aspects of carcass chilling is given below to indicate the physical effects of both the process and the product variables. More detailed quantitative data and mathematical treatments of chilling physics are available (Scott and Vickery, 1939; Earle and Fleming, 1967, 1968; Fleming, 1970; Hodgson, 1964, 1966, 1970; Frazerhurst, 1971; Swenson et al., 1969; Dalgleish and Ede, 1965; Pflug et al., 1965; Rolfe, 1968; Meat Research Institute, Langford, 1972). The physical phenomena involved in chilling change in magnitude and nature during the processing time. It is thus convenient to divide the total time into two successive periods distinguished by the temperature changes undergone by the carcass; these are illustrated in Fig. 17 for mutton, and in Fig. 18 for beef. In the initial “cooling phase,” the carcass surface temperatures fall to approach the temperature level of the surrounding air. The “storage phase” then follows, when the deep meat temperatures proceed toward equilibration with the air temperature. The relative lengths of these two periods show some variations for different regions of the carcass. A. COOLING PHASE After dressing, a carcass is a hot, wet mass of prerigor muscle, fat, and bone. While the deep meat temperatures correspond closely to the body

R. H. LOCKER ET AL.

208

FIG.17. Experimental cooling curves for a ewe carcass 55 pounds in weight. From Earle and Fleming ( 1967).

(00 90

-

80

4

I

70

c 60 50

Time (hrs)

FIG. 18. Variation of beef carcass temperatures with time during chilling. Air temperature 45" to 50"F, air velocity 80 ft/min, R.H. 95%, and carcass weight 500 pounds. (---), deep leg; ( ), surface leg; (O), 2 inch in cube roll; ( X ), surface strip loin; and ( - ), air. From Frazerhurst ( 1971).

+

temperature of the living animal (38" to 40°C), the surface tissues have already cooled to around 25" to 32°C. In the early stages of the cooling phase there may be a slight rise in deep meat temperatures ( u p to ZOC), due to the heat produced from the exothermic glycolysis reactions ( the anaerobic conversion of glycogen to lactic acid).

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Initially there are relatively large differences in both temperature and vapor pressure between the carcass surface and the surrounding air. These differences are the driving forces for relatively high rates of surface heat loss by convection and evaporation (Fig. 19), with a concomitant rapid surface drying and rate of weight loss. An increased air temperature gives lower heat and mass transfer rates at this stage resulting from the effect on the temperature and vapor pressure differences. The transfer rates also increase-approximately linearly-with air velocity. Relative humidity has little effect until toward the end of the cooling phase, when a significant reduction in vapor pressure difference has been effected (Fig. 20). A lower relative humidity then increases the transfer rates. A somewhat higher ratio of evaporative heat loss to convective heat loss is given by a greater air temperature or velocity. The high transfer rates on the surface result in a rapid lowering of surface temperatures, which sets up a temperature gradient through the thickness of the carcass. As a result, heat is conducted from the internal regions to the surface, and the deep meat temperatures begin to fall. Toward the end of the cooling phase the reduction in surface transfer rates, caused by the lower surface temperatures, starts to slow down the cooling rate in the deep center regions.

-

Total - ( T O 35 " F )

0 0

0 : x

Tolal

- ( T o = 5 0 OF)

v) 0 v)

+

0

; I

Fvooorotive

0

5

1u

13

20

Time ( h r s )

FIG. 19. Variation of evaporative and convective heat loss with time during the chilling of a beef carcass. Air velocity 150 ft/min and 90% R.H. From Hodgson (1970). An estimated total heat loss curve for an air temperature of 50°F has been added.

R. H. LOCKER ET AL.

210

.a

-

V.P on carcass surface

(3

$ ._

.6

1

2 u)

2

.4

a L

a a

3

.2 V.P.-air at 90% R.H. I

0

6

12

(8

24

Time (hrs.)

FIG.20. Variation of the vapor pressure of moisture on the surface of the carcass and the air for various relative humidities. From T. Hodgson, Food Ind. S. Afr. 16, (Feb.) 41-45 and (Dec.) 47-49 (1964).

The hindquarter usually shows the slowest cooling rates, with the difference between leg and shoulder regions being more marked for beef than for lamb and mutton. The faster cooling rates shown by the neck and the thin flap and brisket regions normally result in less surface drying in these areas than in the leg region, and this tendency is accentuated by drainage down the carcass. It is therefore important to ensure adequate air movement around the lower regions of the carcass. This requires careful attention to the uniformity of air distribution; otherwise, adequate lower region drying may only be achieved at the expense of over-drying, and hence high weight loss in the leg regions. Cooling rates, and hence the duration of the cooling phase, are considerably affected by carcass size and weight (Fig. 21), with, of course, a marked difference between lamb and beef. Weight alone does not completely determine the effect of carcass geometry. Carcasses of the same weight but different shapes and/or composition can show significant differences in cooling rates (higher for long/lean carcasses than for shortlfatty ones). Although rapid cooling (low air temperatures, high air velocities) gives higher evaporation rates than slow cooling, this is generally more than compensated for by the shorter duration of the cooling phase and results in a lower overall weight loss.

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211

60

0) c

2

40

30

4 00

2 00

300

Weight of carcass side ( I b s )

FIG. 21. Variation of cooling rate with carcass weight. Indicated bone temperatures are those reached after chilling for 16 hours with an air temperature of 32°F. From T. Hodgson, Food Ind. S. Afr. 16, (Feb.) 41-45 and (Dec.) 4 7 4 9 (1964).

B. STORAGE PHASE Following the transition from the cooling phase to the storage phase, changes in some physical phenomena become apparent. Surface temperatures are now comparable with the surrounding air temperature; with lamb and mutton the surface temperatures fall below the air temperature by about half the wet-bulb depression (Fig. 17). The remaining sensible heat in the carcass is now removed at a much lower rate, with the deep meat temperatures equilibrating only slowIy with the air temperature. The surface drying rate in the storage phase is determined by the difference between the rates of surface evaporation and moisture diffusion from the underlying layers to the surface. The different types of tissue have varying diffusion rates. The diffusional resistance of muscle is relatively low, and for exposed cut surfaces appreciable moisture transfer by internal diffusion is possible. In contrast, dry connective tissue and fat, particularly, are much less permeable to moisture. Thus the presence of overlying layers of these materials adds considerably to the diffusional resistance. In fact, fat-covered regions become practically dry nonevaporating areas once the overlying connective tissue has lost its moisture. The overall percentage of wetted area

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212

70 Air velocity ( f t / m i n )

FIG. 22. The interrelationship between relative humidity and air velocity to give constant drying powers of 44 and 70 at 30°F. Air drying power: ( - ), at Ta = 30°F; ( ) at TA = 50°F.From Scott and Vickery (1939). The dotted lines have been added to show the approximate relationship at 50°F.

of the carcass surface decreases during the cooling phase, but Iater, in the storage phase, it may increase from the effect of internal diffusion. The evaporation rate throughout the storage phase is controlled by the drying power of the air and the water activity of the carcass surface. Drying power i s the rate of moisture loss of a wet surface at the same temperature as the carcass surface. The magnitude of the relative humidity, air velocity, and temperature determine the value of the drying power (Fig. 22). The air velocity across the meat surface may drop slightly during the storage phase, since natural convection can no longer make any effective contribution to airflow. Since the vapor pressure difference between the meat surface and the air is small in the storage phase, relative humidity is a significant variable for drying rate and weight loss. At high air velocities and temperatures and at low humidities, air drying power is increased. While air velocity has a significant effect at temperatures around O"C, it has less effect at 10°C or above (Fig. 2 2 ) . Control of relative humidity in the storage phase is critical in obtaining the optimum compromise between bacterial control and weight loss. Since water activity is the ratio of the vapor pressures of the carcass surface and water a t the same temperature, it is a measure of the availability of moisture at the surface. In the early stages of chilling the water activity is close to unity, but as drying proceeds, lower values obtain,

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dropping in the storage phase to about 0.95 for exposed muscle surfaces and possibly lower for connective tissue. The drop in water activity with drying contributes significantly to the decrease in vapor pressure difference (Fig. 20) and evaporation rate as chilling proceeds. The initial surface moisture content (dry weight basis) is around 300%. Adequate bacterial control requires a level of about 85% to be maintained during the storage phase. Different drying powers for the leg and shoulder regions are necessary to achieve this. The required values for beef at - 1°C were shown by Scott and Vickery (1939) to be 44 and 70 mg/cm2 per 48 hours, respectively. Compared to the steep rise in weight loss in the cooling phase, the additional loss for the storage phase is relatively small. Air temperatures of 10" to 18°C inevitably result in slightly higher total weight losses than are found with temperatures of -4" to 4°C; and weight losses are higher in lamb and mutton than in beef under the same conditions. For conditioning and aging of lamb carcasses, average weight losses at different works range from 1.8 to 3.47,; for conditioning of beef sides, the average is about 2%. At the conclusion of the storage phase the state of rigor should be achieved, and the meat will be in a firm condition suitable for further processing by cutting, boning, or freezing.

+

VII.

RESEARCH NEEDS

In this section comment will be made on some relevant matters not covered by this review, on areas requiring further study, and on some ways in which the assessment and comparison of results from different laboratories could be improved. At the basic level, the mechanism of cold shortening seems not in doubt. While research is in progress, there is still a lack of firm data in print confirming that the effect is due to impairment of the calcium pump by cold. A rather hazy area is the comparative response of red and white muscle fibers to cold. There have been unsupported suggestions that red muscles in rabbits and pigs are more responsive than white, whereas our experience with beef muscles (which are mainly "mixed" muscles) is that both kinds of fiber are active. The respective fiber types may vary in properties between species. There is good evidence, however, that the sarcoplasmic reticulum of some red fibers has an inferior capacity to bind calcium.

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In this review no attempt has been made to cover the many factors contributing to tenderness. Attention has been focused on those aspects related to muscle contractibility. Cooking causes shortening in meat, and the way this relates to cold shortening and to tenderness is not clear. The literature is not helpful, and results in this laboratory do not merit review. Another unresolved question, already mentioned, is how close to rigor a muscle must be to fail to respond to freezing and thawing. Freezing has been part of most of the experiments described, but no mention has been made of the effect of ice crystal formation on tenderness. There is a considerable literature on this subject, probably confused by cold or thaw shortening effects. This is material for another review. On the whole, recent work points to a degree of tenderization on freezing and thawing. In experiments here on beef sternomandibularis, where shortening has been eliminated as a factor, small progressive increases in tenderness were observed in three successive freeze-thaw cycles (Locker and Daines, 1973). Cooking loss rose considerably after the first cycle, but little in subsequent ones. Failure to recognize the great significance for tenderness of temperature after slaughter throws doubt on the validity of many results obtained in the past. This was excusable at the time, but papers on tenderness are still appearing which make no reference to chilling or holding conditions. All such work must be suspect. An important feature of the work described in this review has been a heavy reliance on beef sternomandibularis muscle, which was adopted early in this laboratory by B. B. Marsh as an experimental material, and is now being widely used. Although a very tough muscle, containing 10% connective tissue (Bailey, 1972), it has yielded fundamental answers on tenderness. Its length, entirely parallel structure, and easy availability at the time of slaughter without mutilation of carcass offer great practical advantages. Several identical pieces can be cut from the same pair of ox muscles, and many if the much larger bull muscles are used. Although it must be kept in mind that there are dangers in concentrating on a single experimental material, the sternomandibularis is likely to contribute more in the future, both to meat science and to muscle biochemistry. A point has been reached in meat science where consideration should be given to standardization of tenderness measurement, so that results from different schools may be readily compared, or even international tenderness standards may be developed. This would be a suitable topic for the European Conference of Meat Workers. The commendably ana-

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lytical approach of Bouton and Harris (1972) appears to differentiate rather well between tensile strength of myofibrils (shear force) and toughness of connective tissue as manifested by fiber adhesion (compressive strength). This paves the way for measurement of different parameters in standard units, preferably in standard machines, or at least in related devices which can give standard units by application of conversion factors. There seems an equally strong case for the use of standardized cooking conditions. This would be easy, at least for roasting or cooking in a water-bath. This review has dealt exclusively with tenderness. Other qualities are much less affected by the processing method in the prerigor period. Palatability factors, such as flavor and juiciness, have been assessed along with tenderness by our panels, and significant changes are sometimes observed (see, for example, Wenham et al., 1973). However, at this stage no well-defined pattern has emerged. One property mentioned in the recent literature is “drip.” Taylor (1972) found this to be two to three times as great in steaks from beef or pig carcasses cooled slowly as in those cooled quickly. The reason is not clear. Finally, the new concepts in processing described are those most relevant to the basic principles reviewed and to the authors’ experience. There have been many other advances in technology which others are better qualified to review. Many of these depend on good hygiene and new materials and methods of packaging. The emphasis has been on freezing as a means of preservation, but new developments have made possible long-term chilled storage. Although transport of chilled beef to markets on the other side of the world is not new, it can now be done more conveniently and with greater safety margins. An example is the developing container trade in vacuumpacked beef cuts from Australia to Japan or England, in which a shelflife of over 70 days is achieved at just above the freezing point of the meat, Aging occurs during transit. Similar techniques can be used with vacuum-packed lamb cuts and a storage life of 56 days at - 1°C has been achieved. (Table 1-11).

VIII.

CONCLUSION

This review has shown that the shortening of muscles by cold shock or thaw rigor, or both, is a major cause of meat toughness-one that may far outweigh other factors such as age, breed, sex, plane of nutrition,

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grade, or preslaughter stress. Increases in shear force of four times or more are not uncommon in commercial practice. In the consumer’s view such meat is inedibly tough. The adverse effects of thaw shortening may be avoided by careful subsequent handling, but damage due to cold shortening is irreversible and also inhibits aging, The final tenderness of the meat falls far short of its initial potential. These facts are powerful arguments against the early and rapid chilling or freezing of normally hung carcasses. Insistence that such refrigeration practice is necessary for bacteriological safety cannot be justified with present advanced standards of dressing and quality control. At the present time efforts are being made to define international standards for food handling in a Codex Alimentarius. Nations and groupings such as the European Economic Community impose their own requirements. In the case of meat it is important that, in protecting public health, palatability is not ignored. We have shown that the two demands of safety and tenderness are quite compatible. Meat must be seen not as an inert medium fpr the growth of microorganisms, but as a living, dynamic system, capable for many hours after slaughter of responding to external stimuli. The response determines tenderness, which is perhaps the most highly rated element in palatability. The consumer tends to take hygiene for granted as a prerequisite, and makes purchasing decisions on cost and expected pleasure in consumption. It is important for the future health of the industry that meat, an already expensive commodity, should be seen as value for money. In this age of consumer concern and rapid technical change, meat can hold its place among the products of other sophisticated food industries only if all consumer qualities are protected. In the near future it is likely that the grading of consumer packs may be based more on such qualities (as determined by processing method ) than on the traditional carcass criteria, which in many ways are becoming irrelevant. The freedom for new technology to develop is an important reason for maintaining flexibility in time-temperature tolerances for meat. Some interesting new practical possibilities, now emerging from research, have been described. Such techniques could change the whole pattern of meat processing. A critical part of some of these is a holding period at moderate temperatures to avoid toughness. The advantages in quality, speed, efficiency, and cost which such processes promise should not be frustrated by unduly restrictive regulations at national or international levels. The emphasis should be on the state of the end product, with a willingness to permit innovation in reaching a final set standard.

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REFERENCES Bailey, A. J. 1972. The basis of meat texture. J. Sci. Food Agr. 23, 995-1007. Barnes, E. M., and Ingram, M. 1955. Changes in the oxidation reduction potential of the sternocephulicus muscle of the horse after death, in relation to the development of bacteria. J. Sci. Food Agr. 6, 44-55, Baxter, R. I., Bouton, P. E., Fisher, A. L., and Harris, P. V. 1972. Evaluation of hanging methods for improving the tenderness of beef and mutton. CSIRO Meut Res. Rep. No. 2/72. Beerens, H. Sugania, S., and Tahnn-Castel, M. 1965. Psychotrophic Clostridia. J. A p p l . Bucteriol. 28, 36-48. Bendall, J. R. 1951. The shortening of rabbit muscle during rigor mortis: Its relation to the breakdown of ATP, creatine phosphate and to muscular contraction. J. Physiol. (London) 114,71-88. Bendall, J. R. 1972a. Consumption of oxygen by the muscles of beef animals and related species and its effects in the colour of meat. 1. Oxygen consumption in pre-rigor muscle. J. Sci. Food Agr. 23, 61-72. Bendall, J. R. 1972b. In “Symposium No. 2,” p. 3.5. Meat Res. Inst., Langford. Benke, J. R., Fennema, O., and Cassens, R. G . 1973a. Rates of post mortem metabolism in frozen animal tissues. J. Agr. Food Chem. 21, 5-11. Benke, J. R., Fennema, O., and Haller, R. W. 197313. Quality changes in pre-rigor poultry at -3°C. J. Food Sci. 38, 275-278. Benke, J. R., Fennema, O., and Cassens, R. C., 1973c. Quality changes in pre-rigor beef muscle at -3°C. J. Food Sci. 38, 539-541. Bouton, P. E., and Harris, P. V. 1972. A comparison of some objective methods used to assess meat tenderness. J. Food. Sci. 37, 218-221. Bouton, P. E., Harris, P. V., and Shorthose, W. R. 1972. The effects of ultimate pH on ovine muscle: Water-holding capacity. J. Food Sci. 37, 351-355. Bouton, P. E., Fisher, A. L., Harris, P. V., and Baxter, R. I. 1973. A comparison of the effects of some post-slaughter treatments on the tenderness of beef. J. Food Technol. 8, 39-49. Brunton, W. G., and Gilbert, K. V. 1972. Interim studies in lamb tenderness. Evaluation of lamb conditioned and aged at 10°C. and 7°C. Meut Ind. Res. Inst. Publ. No. 275. Buchter, L. 1972. The influence of chilling temperature on the toughness of loin muscles from young calves and bulls. In “Symposium No. 2,” pp. 4.51-4.54. Meat Res. Inst., Langford. Busch, W. A,, Parrish, F. C., and Goll, D. E. 1967. Molecular properties of post mortem muscle. 4. Effect of temperature on ATP degradation, isometric tension and shear resistance of bovine muscle. J. Food Sci. 32, 390-394. Busch, W. A., Goll, D. E., and Parrish, F. C. 1972. Molecular properties of post mortem muscle. Isometric tension development and decline in bovine, porcine and rabbit muscle. J. Food Sci. 37, 289-299. Carse, W. A. 1973. Meat quality and the acceleration of post mortem glycolysis by electrical stimulation. J. Food Technol. 8, 163-166. Cassens, R. G., and Newbold, R. P. 1967a. Temperature dependence of p H changes in ox muscles post-mortem. J. Food Sci. 32, 13-14.

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Cassens, R. G., and Newbold, R. P. 196713. Effect of temperature on the time course of rigor mortis in beef. 1. Food Sci. 32, 269-272. Cook, C. F., and Wadsworth, R. F. 1966. The effect of preslaughter environmental temperature and post mortem treatment upon some characteristics of ovine muscle. I .Shortening and pH. 11. Meat Quality. J. Food Sci. 31, 497-504 and 505-509. Cowan, S. T., and Steel, K. J. 1966. “Manual for the Identification of Medical Bacteria.” Cambridge Univ. Press, London and New York. Cullen, J., Phillips, M. C., and Shipley, G. C. 1971. The effects of temperature on the composition and physical properties of the lipids of Pseudomonas fliiorescens. Biochem. 1. 125, 733-742. Dalgleish, N., and Ede, A. J. 1965. “Charts for Determining Centre, Surface and Mean Temperatures in Regular Geometric Solids During Heating or Cooling,” Rep. No. 192. Nat. Eng. Lab., Glasgow. Davey, C. L. 1970. Beef processing and aging. Proc. Meat Ind. Res. Inst. N . Z . Conf., 12th, 1970, Publ. No. 199, pp. 73-76. Davey, C. L., and Curson, P. 1971. Interim studies in lamb tenderness. 1. Evaluation of high temperature conditioning and aging of lamb. 2. The effect of carcass posture on tenderness of lamb muscles. Meat lnd. Res. Inst. N.Z., Publ. No. 215. Davey, C. L., and Gilbert, K. V. 1967. Structural changes in meat during aging. I. Food Technol. 2,57-59. Davey, C. L., and Gilbert, K. V. 1968. Studies in meat tenderness. 6. The nature of myofibrillar proteins extracted from meat during aging. J. Food Sci. 33, 343-348. Davey, C. L., and Gilbert, K. V. 1969. Studies in meat tenderness. 7. Changes in the fine structure of meat during aging. I. Food Sci. 34, 69-74. Davey, C. L., and Gilbert, K. V. 1971. Hot boning of bobby calves. Meat lnd. Res. Inst. N.Z., Publ. No. 229. Davey, C. L., and Gilbert, K. V. 1973. The effect of carcass posture on cold, heat and thaw shortening in lamb. J. Food Technol. 8, 445-451. Davey, C. L., and Gilbert, K. V. 1974. Carcass posture and tenderness development in frozen lamb. 1. Sci. Food Agr. 25 (in press). Davey, C. L., Kuttel, H., and Gilbert, K. V. 1967. Shortening as a factor in meat aging. J. Food Technol. 2, 53-56. De Fremery, D., and Pool, M. F. 1960. Biochemistry of chicken muscle as related to rigor mortis and tenderization. Food. Res. 25, 73-87. Earle, R. L., and Fleming, A. K. (1967). Cooling and freezing of lamb and mutton carcasses. Food Technol. 21, 79-84. Earle, R. L., and Fleming, A. K. (1968). Food Technol. 22, 100-104. Ebashi, S., Endo, M., and Ohtsuki, I. 1969. Control of muscle contraction. Quart. Rev. Biophys. 2, 351-384. Fleming, A. K. 1970. Physical aspects of meat cooling. Bull. Xnt. Inst. Refrig., Annex 3, 151-160. Forrest, J. G., and Briskey, E. J. 1967. Response of striated muscle to electrical stimulation. J. Food Sci. 32, 483-488. Frazerhurst, L. F. 1971. Chiller specifications: The physical environment. Proc. Meat lnd. Res. Inst. N . Z . Conf., 13th, 1971. Publ. No. 225, pp. 17-22. Galloway, D. E., and GolI, D. F. 1967. Effect of temperature on molecular properties of post-mortem porcine muscle. J . Anim. Sci. 26, 1302-1308.

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Gilbert, K. V., Wyborn, R., and Nottingham, P. M. 1973. Storage life of chilled lamb cuts. Further studies. Meat Ind. Res. Inst. N.Z., Publ. No. 285. Goll, D. E., Arakawa, N., Stromer, M. H., Busch, W. A., and Robson, R. M. 1970. Chemistry of muscle proteins as a food. In “The Physiology and Biochemistry of Muscle as a Food” ( E . J. Briskey, R. G . Cassens and B. B. Marsh, eds.), 2nd ed., pp. 755-800. Univ. of Wisconsin Press, Madison. Hallund, O., and Bendall, J. R. 1965. The long-term effect of electrical stimulation on the post-mortem fall of pH in the muscles of Landrace pigs. J. Food Sci. 30, 296299. Harsham, A., and Deatherage, F. E. 1951. Tenderization of meat. U.S. Pat. 2,544,681. Hendricks, H. B., Lafferty, D. T., Aberle, E. D., Judge, M. D., and Forrest, J. C. 1971. Relation of porcine muscle fiber type and size to post mortem shortening. I. Anim. Sci. 32, 57-61. Herring, H. (K., Cassens, R. G . , and Briskey, E. 1965a. Further studies on bovine tenderness as influenced by carcass position, sarcomere length, and fiber diameter. 1. Food Sci. 30, 1049-1054. Herring, H. K., Cassens, R. G . , and Briskey, E. J. 196513. Sarcomere length of free and restrained bovine muscles at low temperature as related to tenderness. J. Sci. Food Agr. 16, 379-384. Herring, H. K., Cassens, R. G., Suess, G. G., Brungardt, V. H., and Briskey, E. J. 1967. Tenderness and associated characteristics of stretched and contracted bovine muscles. J. Food Sci. 32, 317423. Hodgson, T. 1964. The rapid chilling of meat-theoretical and practical considerations. Food lnd. S. Afr. 16, (Feh.) 41-45 and (Dec.) 47-49. Hodgson, T. 1966. The effect of environmental conditions on the chilling rates of meat. Bull. Int. Inst. Refrig., Annex 1, 635-646. Hodgson, T. 1970. The effect of air velocity and evaporator size on product weight losses in carcass chilling rooms. Bull. Int. Inst. Refrig., Annex 3, 161-176. Horgan, D. J., Newbold, R. P., and Tume, R. K. 1971. “Calcium-accumulating Activity of the Sarcoplasmic Reticulum” Rep. Res., p. 65, Food Div., C.S.I.R.O. Hostetler, R. L., Link, B. A., Landmann, W. A., and Fitzhugh, H. A. 1972. Effect of carcass suspension on sarcomere lengths and shear force of some major bovine muscles. J. Food Sci. 37, 132-135. Hungate, R. E. 1969. A roll tube method for cultivation of strict anaerobes. In “Methods in Microbiology” (J. R. Norris, and D. W. Ribbons, eds.), Vol. 3B, pp. 117-132. Academic Press, New York. Ingram, M. 1972. Meat chilling-the first reason why. In “Symposium No. 2,” PP. 1.1-1.3. Meat Res. Inst., Langford. Jungk, R. A., Snyder, H. E., Goll, D. E., and McConnell, K. G. 1967. Isometric tension changes and shortening in muscle strips during post mortem aging. J. Food Sci. 32, 158-161. Khan, A. W., and Lentz, C. P. 1973. Influence of ante-mortem glycolysis and dephosphorylation of high energy phosphates on beef aging and tenderness. J. Food Sci. 38, 56-58. Kushmerick, M. J., and Davies, R. D. 1968. The role of phosphate compounds in thaw contraction and the mechanism of thaw rigor. Biochim. Biophys. Acta 153, 279-287.

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Lacourt, A., and Charpentier, J. 1971. Contribution Q l'ktude de la contracture provoquke par le froid. Proc. Eur. Meet. Meat Res. Workers, 17, 1971, pp. 32-41. Law, N. H., and Vere-Jones, N. W. 1955. Shipment of chilled beef, 1952. N.Z. Dep. Sci. Ind. Res., Bull. 118. Leet, N . G., and Locker, R. H. 1973. A prolonged pre-rigor condition in aerobic OX muscle. J . Sci. Food Agr. 24, 1181-1192. Locker, R. H. 1959. Striation patterns of ox muscle in rigor mortis. J. Biophys. Biochem. Cytol. 6, 41-22. Locker, R. H. 1960. Degree of muscular contraction as a factor in the tenderness of beef. Food Res. 25, 304-307. Locker, R. H., and Daines, G. J. 1973. The effect of repeated freeze-thaw cycles on tenderness and cooking loss in beef. J . Sci. Food Agr. 124, 1273-1276. Locker, R. H., and Hagyard, C. J. 1963. A cold shortening effect in beef muscles. 1. Sci. Food Agr. 14, 787-793. Lyons, J. M., and Raison, J. K. 1970. A temperature-induced transition in mitochondria] oxidation: Contrasts between cold and warm-blooded animals. Comp. Biochem. Physiol. 37, 405-411. McCrae, S. E., Seccombe, C. G., Marsh, B. B., and Carse, W. A. 1971. Studies in meat tenderness. 9. The tenderness of various lamb muscles in relation to their skeletal restraint and delay before freezing. J . Food Sci. 36, 566-572. Macfarlane, P. G., and Marer, J. M. 1966. An apparatus for determining the tenderness of meat. Food Technol. 20, 838-839. McLeod, K., Gilbert, K. V., Wyborn, R., Wenham, L. kl., Davey, C. L., and Locker, R. H. 1973. Hot cutting of lamb and mutton. J. Food Technol. 8, 71-76. McLeod, K.. Gilbert, K. V., and Locker, R. H. 1974. Further experiments in hot cutting of lamb. J , Food Technol. 9, 179-184. McLoughlin, J. V. 1970. Muscle contraction and post niortem changes in pig skeletal muscle. 1. Food Sci. 70, 717-719. SIarsh, B. B. 1952a. Rigor mortis in whale muscle. Biochim. Biophys. Acta 9, 127132. Marsh, B. B. 1952b. The effects of ATP on the fiber volume of a muscle honiogenate. Biochim. Biophys. Acta 9, 247-260. Marsh, B. B. 1954. Rigor niortis in beef. J . Sci. Food Agr. 5, 70-75. Marsh, B. B. 1972. Post morteni muscle shortening and meat tenderness. Proc. Meat Ind. Res. Conf., (Amer. Meat Inst. Foundation, Chicago), pp. 109-124. Siarsh, B. B., and Carse, W. A. 1972. Meat tenderness and the sliding filament hypothesis. J. Food Technol. 9, 129-139. Xlarsh, B. B., and Leet, N. G. l966a. Resistance to shearing of heat denatured muscle in relation to shortening. Nature (London) 211, 635-636. hlarsh, B. B., and Leet, N. G . 1966b. Studies in meat tenderness. 111. The effects of cold shortening on toughness. J. Food Sci. 31, 450-459. Marsh, B. B., and Thompson, J. F. 1958. Rigor mortis and thaw rigor in lamb. 1. Sci. Food Agr. 9, 417-424. Marsh, B. B., Woodhams, P. R., and Leet, N. G . 1968. Studies in meat tenderness. 5. The effects on tenderness of carcass cooling and freezing before the completion of rigor mortis. J . Food Sci. 33, 12-24.

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Marsh, B. B., Cassens, R. G., Kauffman, R. G., and Briskey, E. J. 1972. Hot boning and pork tenderness. J. Food Sci. 37, 179-180. Marsh, B. B., Leet, N. G., and Dickson, M. R. 1974. The ultrastructure and tenderness of highly cold shortened muscle. J. Food Technol. 9, 141-147. Meat Research Institute, Langford. 1972. Meat chilling-why and how? “Symposium NO. 2.” MRI, Langford. Moran, T. 1930. The frozen state in mammalian muscle. Proc. Roy. Soc., Ser. B

107, 183-187. Newbold, R. P. 1966. Changes associated with rigor mortis. I n “The Physiology and Biochemistry of Muscle as a F o o d (E. J. Briskey, R. G. Cassens, and J. C. Trautman, eds.), 1st ed., pp. 213-234. Univ. of Wisconsin Press, Madison. Newbold, R. P., and Harris, P. V. 1972. Effect of pre-rigor changes on meat tenderness. A review. J. Food Sci. 37,337-340. Newbold, R. P., Tume, R. K., and Horgan, D. J. 1973.Effect of feeding a protected safflower oil supplement on the composition and properties of the sarcoplasmic reticulum and on postmortem changes in bovine skeletal muscle. J. Food Sci.

38, 821-823. Nottingham, P. M. 1960. Bone-taint in beef. 11. Bacteria in ischiatic lymph nodes. J. Sci. Food Agr. 11, 436441. Nottingham, P. M. 1971. Microbiological quality control in the meat industry. Meat Ind. Res. Inst. N.Z., Publ. No. 217. Pflug, I. J., Blaisdell, J. L., and Kopelman, I. J. 1965. Developing temperature-time curves for objects which can be approximated by a sphere, infinite plate or infinite cylinder. ASHRAE (Amer. SOC. Heat., Refrig. Air-Cond. Eng.) Trans.

71, 238. Quarrier, E. Carpenter, Z. L., and Smith, G. C. 1972.A physical method to increase tenderness in lamb carcasses. I. Food Sci. 37, 130-131. Reimann, E. M., Walsh, D. A,, and Krebs, E. F. 1971. Purification and properties of rabbit skeletal muscle adenosine 3’, 5‘-monophosphate-dependent protein kinase. J. Biol. Chem. 246, 1986-1995. Roberts, T. A., and Hobbs, G. 1968. Low temperature growth characteristics of clostridia. J. Appl. Bacteriol. 31,75-88. Rolfe, E. J. 1968. The chilling and freezing of foodstuffs. I n “Biochemical and Biological Engineering Science” ( N. Blakebrough, ed. ), Vol. 2, pp. 137-208. Academic Press, New York. Roschen, H. L., Maywood, Ortscheid, B. J., and Ramsbottom, J. M. 1950. Tenderizing meats. U.S. Pat. 2,519,931. Schmidt, G. R., and Gilbert, K. V. 1970. The effect of muscle excision before the onset of rigor mortis on the palatability of beef. J. Food Technol. 5, 331338. Scopes, R. K. 1971. Mechanisms controlling glycolysis in muscle. The biochemistry of pcst mortem glycolysis. Proc. Eur. Meet. Meat Res. Workers, 17th, 1971. pp. 14-23 Scopes, R. K. 1972.Cited by Bendall (1972b). Scott, W. J. 1953. Water relations of Staphylococcus aureus at 30°C. Aust. J. BWl. Sci. 6,549-564. Scott, W. J., and Vickery, J. R. 1939. Investigations on chilled beef. Part 11. Cooling and storage in the meat works. CSIRO Bull. No. 129. Sharp, J. G., and Marsh, B. B. 1953. Whalemeat: Production and preservation. Spec. Rep., Food Invest. Bd. London, No. 58, p. 14.

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Smith, M. C., Judge, M. D., and Stadelman, W. J. 1969. A cold shortening effect in avian muscle. J. Food Sci. 34, 4 2 4 6 . Swenson, 6. G., Grau, F. H., and Bate, H. G. 1969. Some aspects of the chilling of carcass meat. Aust. Refrig., Air Cond. Heat. 23 (11)32-36. Taylor, A. A. 1972. The influence of carcass chilling rate on drip in meat. In “Symposium No. 2,” pp. 5.14.8. Meat Res. Inst., Langford. Taylor, A. A., Chrystall, B. B., and Rhodes, D. N. 1972. Toughness in lamb induced by rapid chilling. J. Food Teclznol. 7, 251-8. Van Eerd, T. P. 1972. Emulsion stability and protein extractability of ovine muscle as a function of time post morteni. J. Food Sci. 37, 473475. Voyle, C. A. 1969. Some observations on the histology of cold-shortened muscle. 1. Food Technol. 4,275-281. Weidemann, J. F., Kaess, G., and Carruthers, L. D. 1967. The histology of pre-rigor and post-rigor ox muscle before and after cooking and its relation to tenderness. J. Food Sci. 32, 7-13. Wenham, L. M., Fairbairn, S. J., McLeod, K., Carse, W. A., Pearson, A. M., and Locker, R. H. 1973. The eating quality of mutton compared with lamb and its relationship to freezing practice. 1. Anim. Sci. 36, 1081-1087. Wilson, G. D., Brown, P. D., Chesbro, W. R., Ginger, B., and Weir, C. E. 1960. The use of antibiotics and gamma irradiation in the aging of steaks at high temperatures. Food Technol. 14, 143-147. Wiskus, K. J., Addis, P. B., and Ma, R. T-I. 1973. Postmortem changes in dark turkey muscle. J. Food Sci. 38, 313-315.

PHYSIOLOGY AND BIOCHEMISTRY OF MANGO FRUIT BY

H. SUBRAhfANYAhf, SHANTHA KRISHNAMURTHY, AND H. A. B. PARPIA* Central Food Technological Research Institute, Mysore, India

I. Introduction . ......................................... 224 A. History an ............................ . . . . . . . . . . 225 B. Principal Varieties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Botany . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 D. Production and Yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 E. Summary ......................... . . . . . . . . . . 231 11. Physiology of Fri . . . . . . . . . . 231 A. Morphology B. Compositional C. Respiration ( during Growth) .................................. 242 D. Maturity Indices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 E. Harvesting and Ripening . . . . . . . . . . . . . . ....................... 247 F. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .248 . . . . . . . . . . . . . . . . . . . 249 111. Physiology of Ripening . . . . . . . . A. Respiration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 B. Enzymes, Inhibitors, and Ethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 ............................... 257 C. Cheiiiical Constituents . . . D. Summary . . . . . . . ......................... 270 IV. Storage and Transport ......................... 271 A. Storage Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 B. Postharvest Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 C. Packaging . . . . ...................... 285 D. Transportation ...................... 286 E. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 V. Economic Aspects .................................. 288 A. World Trade . ...................... 288 ............................. 290 B. Sensory Properties . . C. Processing Quality ....................... . . . . . . . . . . . . . . . . . . . . 292 1). Nutritional Significance .......................... 292

* Present address: Food and Agriculture Industries Service, Agricultural Services Division, FAO, Via Delle Terme Di Caracalla, Rome, Italy. 223

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H. SUBRAMANYAM ET AL.

E. Waste Utilization ............................................ F. Summary ................................................... VI. Research Needs ................................................ References ....................................................

I.

294 294 294 296

INTRODUCTION

In recognition of the importance of fruits and vegetables as valuable food resources, increasing attention has been paid in recent years to the promotion and expansion of the horticultural industry. Fruits and vegetables, acclaimed universally as protective foods, are important constituents in our daily diet. With increasing urbanization and prosperity, consumer interest has shifted from starchy roots to fresh and processed fruits and vegetables which involve much more care and equipment, and the problems of marketing have become more complex. This trend has been seen especially in the last decade, owing to increased industrialization and rapid urbanization. Fruits and vegetables also represent a large and growing share of the world trade. These protective foods are highly perishable, and therefore their preservation involves maintenance of the fruit or vegetable tissue in a physiologically sound condition. The higher per capita consumption of fruits and vegetables either in fresh or in processed form in the United States, the United Kingdom, Western Europe, Oceania, and some parts of Asia and the Middle East indicates an important technological advance in methods of storage and preservation. Postharvest technological problems are more complex and numerous for fruits of tropical origin than for subtropical or temperate ones. The quality of these foods and the acceptance of newer products by people the world over largely determine international trade and future needs. The mango is the second largest tropical fruit (banana occupies the first place) in terms of production and acreage, and it is one of the important fruit crops of the world, greatly relished for its succulence, exotic flavor, and delicious taste. It enjoys the same popularity in the tropics as the apple does in temperate regions. India is the largest producer of choice table varieties of mango in the world, with an annual production of over 7.5 million tons. Although many varieties are cultivated, only a few are commercially important. In recent years, the rich qualities of this fruit have been accepted in many parts of the world; as a result, emphasis is being placed on storage and transport of the mango for long-distance shipment in either the fresh or the processed form. Therefore, preservation of this fruit in the fresh form for extended periods of time without loss of quality is an immediate necessity. This

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225

involves a sound knowledge of pre- and postharvest physiology and biochemistry, yet these subjects have received very little attention. The horticultural aspects of the mango have been dealt with admirably by Gangolly et al. ( 1957), Singh ( 1960), Valmayor ( 1962), and Mukherjee (1967). The pioneering studies of Cheema and Dani (1934), Wardlaw and Leonard (1936), and Singh et al. (1937a) on storage and transport of this fruit are noteworthy contributions. Hulme ( 1971) has reviewed this subject with primary emphasis on the biochemistry of ripening. As newer techniques become available, additional information is accumulating in the literature, and the mango is receiving greater attention in the Western and Northern hemispheres. Fruit beverages called Alegre and Nectar based on mango have become popular and are gaining importance in the Americas, Europe, and the Soviet Union. Therefore, methods of preserving this fruit in the fresh form require immediate attention, and a complete review embracing all facets of this problem will be useful to both research workers and the food industry.

A. HISTORY AND ORIGIN The mango has been known in India since very early times. It is referred to in Sanskrit literature as Amra and has been under cultivation by man for over 4000 years ( D e Canodolle, 1904). It appears, however, that Hsiian-tsang, one of the early travelers to India (632-645), was the first person to bring the mango to the notice of people outside India. This fruit occupied an important place in horticulture during the rule of the Moghul emperors in India, and Akbar the Great ( 1 5 S 1 6 0 5 ) planted an orchard of 100,OOO mango trees. The origin of most of the improved varieties in India have been traced to those days, and the encyclopedia Ain-e-Akbari (1590 A . D . ) contains a good account of the mango regarding its quality and varietal characteristics ( Mukherjee, 1967). Phytogeographical data and studies of the phylogenetic taxonomy of species of Mangifera indicate that this genus originated in the IndoBurma region. Most of the cultivated varieties have arisen from four main species-Mangifera indica, Mangifera sylvatica, Mangifera odorata, and Mangifera zeylanica. Mango cultivation is found in many countries of Southeast Asia-the Philippines, Indonesia, Java, Thailand, Burma, Malaya, and Ceylon. It has also gained popularity in Egypt, Southeast Africa, South Africa, Hawaii, the West Indies, Florida, Israel, Mexico, and Brazil. Introduction of the mango to East and West Africa and subsequently to Brazil is said to have occurred in the sixteenth century.

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Mexico acquired the mango in the nineteenth century, and it entered Florida in 1833. The cultivated mango varieties are the result of constant selection by man from the original wild plants for over 4000 years. The wild progenies are still available in India in two species, Mangifera indica and Mangifera sylvatica, which have small fruits with a big stone, thin acidic flesh, and long fibers. The knowledge of vegetative propagation gained in the sixteenth century made it possible to produce a large number of cultivars which were far superior to the wild forms. These fruits have little fiber, and are sweet in taste with more flesh. Among several hundred cultivated varieties of mango in India, only a few are polyembryonic, and these are of inferior quality. Most of the improved varieties are monoembryonic. But there are good varieties of polyembryonic nature being cultivated in Thailand, Indonesia, the Philippines, etc. (S. K. Mukherjee, 1972). Rhodes et al. (1970) examined the variability among forty cultivars of Mangifera indica L. and one each of M . odoratu Griff and M . zeylanica Hook f., using the method of cluster analysis of distance coefficients based on seventy-three characters; they found that the cultivars of M . indica were distinct from the others. Most cultivars clustered into one of the four major groups: one group with polyembryonic cultivars common to Southeast Asia, a second group of monoembryonic cultivars of India, a third group of intermediate characters from India and the West Indies, and a fourth group including several hybrids developed in Florida and Hawaii, designated the Sandersha Haden complex. Indian mangoes can be classified as seedling varieties, which are wild and monoembryonic, a few polyembryonic types, and horticultural varieties propogated exclusively by budding or grafting from monoembryonic types. Ten polyembryonic cultivars have been reported in the west coast of South India. The horticultural. varieties predominate in commerciaI cultivation, and all belong t o the single species of Mnngifera indica L. B. PRINCIPAL VARIETIES

A number of varieties of mango, estimated at over 10o0, are available in India. These varieties are mostly of choice type with little commercial value except for about a dozen cultivars. By and large, the two most outstanding varieties of India, Alphonso of the Southwest and Dashehari of the North, are very popular. Many varieties have adapted to local agroclimatic regions and cannot be cultivated in other regions. In Florida, after the introduction of Mulgoba from India, the U. S. Department of Agriculture introduced over five hundred cultivars during the

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227

years 1880 to 1937. According to Malo (1972), Florida has literally been a melting pot of mango types from countries where the mango is considered important among fruits consumed by the people. Outstanding commercial varieties have been developed out of this diversity of genetic material collected from various sources. The number of varieties in other mango-growing countries is limited. Most of these varieties are selected for fruit quality rather than for other important characters such as regularity in bearing, dwarf stature of the tree, or resistance to diseases. In Florida, varieties consistent in productivity, eye appeal, good shipping and eating qualities, and high degree of resistance to anthracnose disease claim to have been developed in recent years. It has been the endeavor of the horticulturist to develop an ideal variety with all the desirable characters of good color, flavor, texture, taste, high pulp content, and good keeping quality, combined with resistance to diseases and regular bearing habits. Constant efforts and research have been made in this direction by Singh (1972), S. K. Mukherjee (1972), and others, and several hundred hybrids have been developed in India in the last ten years. These workers have given prominence to regular bearing habits, early fruit-set, and resistance to bunchy top disease in their hybridization studies. A hybrid of recent origin, No. 65 (named Mallika), is said ta have many of the desirable characters described earlier (Singh et al., 1969). Consumer preference is the ultimate deciding factor in popularizing a variety for large-scale cultivation, but this varies considerably from country to country and among regions in the same country. A list of principal commercial varieties in different mango-growing regions is given in Table I. C. BOTANY The mango belongs to the dicotyledonous family Anacardiaceae consisting of sixty-four genera, mostly trees or shrubs. Other economic plants such as cashew and pistachio nut belong to this family. The name Mangifera is derived from the word Mangai (the Tamil name for mango) and fero (meaning to bear). The word indica means Indian and stands for the name of the species. The wild races of Mangifera indica bear fruits with scanty, slimy flesh, having a fine aroma, and almost free of fibers. Among the other species, M . oblongifolia Hook f. has small fruits which are pickled unripe in Indochina or eaten after cooking. Mangifera xeylanica Hook f. has fruits similar to M . indica but consisting of innumerable soft, short fibers. Mangifera alttissima Blanco has medium-sized fruits with flesh free from fibers. It is used by people in the Philippines

H. SUBRAMANYAM ET AL.

228

TABLE I PRINCIPALVARIETIES OF MANGOIN

THE

WORLD^

Cultivated table varieties Country Fleshy with less fiber

Juicy with soft fiber

India

Alphonso, Dashehari, Chowsa, Bombay green, Langra, Rumani, Bangalora, Swarnarekha, Banganpalli, Neelum, Pain, Mdgoba

Raspoonia, Mithwa Sundershah, Mithwa Ghazipur, Taimurya, Nauras, Rasgola, Sharbathi Begrain, Cherukurasam Peddarasam

Philippines Pakistan

Carabao, Pic0 Sindhri, Bombay, Alphonso, Banganapalli, Fazli, Chousa, Langra, Gdab-khas Boribo, Ngowe, Apple, Early Gold, Nimrod, Malindi, Kensington, Mabroka Irwin, Tommy Atkins, Smith, Kent, Palmer, Sensation, Keitt, Haden Julie, Peter

Africa

Florida

West Indies a

From Acta Horticuburae, p. 24 ( 1972); Trop. Prod. Insti. Conf. Proc. ( 1969).

for pickling. Mangifera odorata Griff has large fruits with a distinctive flavor when ripe. The flesh is sweet, with coarse fibers, and is preferred, next to that of M . indica, by the people of Malaysia. The chromosome number reported for sixty-seven cultivated Indian varieties of M . indica is n = 20 and 2n = 40. Mangifera syltmtica, M . zeylanica, and M . odorata also have n = 20 and 2n = 40. The wild forms of M . indica and M . sylvatica are found in the hill tracts of Chittagong, whereas M . zeylanica is indigenous to Ceylon, and M . odorata is found in Malaysia and the Philippines. The only variation in the chromosome number of Mangifera found until recently is in a polyembryonic seedling of the variety Vellai kolumban belonging to Ad. indica, where 2n = 80 has been reported. Thus, the varieties of M . indica and allied species show a remarkable stability in their chromosome number ( Mukherjee, 1967). The tree is evergreen, and the size differs to a great extent. Trees more than 100 years old are common in India. Growth is in periodic flushes from the terminal buds of the young branches. The flowers are borne on large panicles at or toward the ends of the branchlets, and each panicle has thousands of flowers. The mango produces both perfect

MANGO FRUIT

229

and staminate flowers, the latter outnumbering the former. Only very few flowers bear the fruit. Mango fruit is a typical drupe consisting of an outer pericarp, a fleshy mesocarp, and a stony endocarp enclosing the seed. The shape, color, and size of the fruit vary to a great extent, as described in greater detail by Gangolly et al. (1957), Singh (1960), and Mukherjee (1967) for Indian varieties, by Valmayor (1962) for the Philippine varieties, and by Ruehle and Ledin (1960) for varieties from Florida. The harvesting season for the fruit varies from country to country. In India, the harvesting season for the best table varieties extends from April to July, although some lesser-known varieties in the South are available until December. African mangoes are available all year, and in the United Arab Republic the season is from April to September. Varieties from Florida come to market from June to September, and the Philippine varieties are available from May through August. Thus, it is seen that fruits come to harvest during the summer months of May through August in most of the mango-producing countries. The peak season for harvest of fruits lasts for 4 to 6 weeks only, and this glut season differs from country to country and under different agroclimatic conditions. The peak season for harvest of mango in India is usually from May through June (Singh, 1960) and in South Africa the bulk of the harvest is gathered in February and March ( Hobson, 1969). The harvesting season is regulated in Florida by careful selection and planting of suitable varieties which mature early or late (Malo, 1972). Such an organized approach is possible in countries where this fruit has been introduced recently and acreage is small. The mango grows successfully in a wide range of environmental conditions, although it is believed that the best mango-growing regions of the world have a mean annual temperature ranging from 21.0" to 26.5"C (70" to S O O F ) , and temperature extremes are undesirable for normal growth and fruiting. D. PRODUC~ION AND YIELD The world's estimated annual production of mango is about 9.5 million metric tons, covering a total area of 3.16 million acres. In India, this fruit occupies an area of 2.2 million acres with an annual production of 7.5 million metric tons valued at 7.5 billion rupees ( Rs.) (one billion dollars) (S. K. Mukherjee, 1972). India is also the largest producer of choice table varieties of mango, with an estimated production of 2.5 million tons of grafted cultivars; the rest of the production of 5 million tons consists of

H. SUBRAMANYAM ET AL.

230

seedling varieties. Other main centers of mango production in the world are Pakistan and Bangladesh, accounting for a million tons of fruits, and all other countries put together (the Philippines, Africa, the United Arab Republic, Florida, the West Indies, Ceylon, and Southeast Asia) are believed to produce a million tons (Table 11). The annual production of mango has increased considerably in the Philippines from 57,600 tons in 1960 to 134,100 tons in 1967, valued at 8.2 million dollars (Valmayor, 1972). Current production statistics for other countries are not available, but it is likely that acreage and production have increased substantially in all the producing countries of the world, owing to improvement in horticultural practices and introduction of hybrid cultivars which are regular and heavy bearers. The number of trees per hectare varies from 100 to 175, depending on the variety. The yield of fruit per hectare ranges from 9 to 16 tons, the maximum being in South India ( Mysore State), The yield depends on the variety, the cultural practices adopted, and the environmental conditions prevailing during growth and development. The average yield per tree is about 100 kg, although some vigorous trees are reported to yield 1 ton per season in Pairi and Alphonso varieties.

TABLE I1 AND EXPORT OF FRESH MANGOES WORLD PRODUCTION

Production ( tons )

Export ( tons )

India

7,500,000

1,488

Pakistan

1,000,000

-

Country

Africa other than South Africa Philippines United Arab Republic Thailand South Africa, Brazil, Mexico, Cuba, Florida, Caribbean region, and Ceylon

154,000

-

134,000 88,000

3,243

-

2,246

624,000

9,500,000

-

3,023

10,000

Importing countries Middle East, United Kingdom, Europe

-

Hong Kong

Singapore and Malaysia

-

MANGO FRUIT

231

E. SUMMARY Geographical, phylogenetic, and taxonomic studies made by several taxonomists have clearly established the origin of the genus Mangifera in the Indo-Burma region. Species differentiation by the method of cluster analysis adopted by Rhodes et al. (1970) appears to be promising for the taxonomists. The horticulturists have begun to evolve an ideal variety of mango that is acceptable to the local populace. Dwarf stature of the tree would be most desirable for orchard management, in addition to the other desirable characters; this should be given prominence in the future hybridization programs. Information on production statistics from all the mango-growing regions of the world would be helpful in establishing a sound world market.

II.

PHYSIOLOGY OF FRUIT GROWTH AND DEVELOPMENT A. MORPHOLOGY

The morphological changes of the mango fruit during growth have been determined by various physical, chemical, and physiological characters. Singh et al. (1937a), while studying the ontogenetic drifts in the physiology and chemistry of tropical fruits, classified the life cycle of the mango fruit into four stages: (1) juvenile, ( 2 ) adolescent, (3) climacteric, and (4)senescent. The juvenile stage extends up to 21 days after fertilization and is characterized by rapid cellular growth. This stage is followed by an adolescent stage of maximum growth between 21 and 49 days and a maturation period lasting until 77 days when the respiration climacteric is reached and the fruit starts to ripen. The last phase, the senescent stage, begins after 77 days when respiration drops, the fruit becomes edible-ripe with development of characteristic aroma and taste, and finally deterioration sets in. Increase in size and changes in appearance are the main morphological characters observed during the growth of mango fruit. The growth rate is measured in the developing fruit of different varieties in terms of length, diameter, circumference, weight, volume, color, shape, and other physical attributes from the anthesis or fruit-set stage until maturation is complete. Wardlaw and Leonard (1936) made a careful study of the development of the shoulders (that part of the fruit situated around the stem end) with flesh color in Julie variety and observed that these changes are related to the development of the embryo. All observable

232

H. SUBRAMANYAM ET AL.

changes have been related to the weight of the fruit. In their opinion, maturation-is complete when the fruit reaches a weight of 350 gm, with conspicuous raised shoulders and pale orange flesh, and the endocarp is fully developed at this stage. The fruit becomes tree-ripe when the weight is about 400 gm with the stalk on mound and the flesh is orange in color. The time required for complete development and maturation of the fruit differs considerably from variety to variety, depending also on the regions where it is cultivated and the methods employed for determining the growth rate. Singh et al. ( 1937a), Leley et d.(1943), and Mukherjee (1959) observed that the mango fruit takes about 90 days for complete development, and the physical increase in size and weight stops 4 to 5 weeks before harvest maturity in Langra, Krishnabhog, Alphonso, Dashehari, and Fazli Zafrani varieties. Rolz et al. ( 1971) indicated that Mamey variety of mango takes 90 days for complete development and maturation, and a similar observation was made by Kennard (1955) for Paheri and by Kennard and Winters (1956) for Amini varieties. These observations appear to be somewhat contrary to the reports made by Teaotia et al. (1968) for Langra, by Lakshminarayana et al. (1970) for Alphonso, and by Saini et al. (1971) for Dashehari varieties, indicating that 110 to 116 days are required for full development and maturity. This difference noticed by earlier workers is likely to be due to failure to recognize the exact stage of anthesis, since several days are required to observe fruits of a mustard size. It is also reported that the ovaries in the mango fruit can grow without pollination up to the size of a pea, owing to the likely presence of cytokinin-like substances; subsequent enlargement follows as a result of pollination and fertilization (Chacko et al., 1970a). In Alphonso mango, the gain in weight of the fruit is slow for 5 weeks after fruit-set but becomes rapid thereafter. A brief lull is observed between 12 and 14 weeks, followed by a rapid rise. Similarly, the increase in length is slow between 9 and 14 weeks, and the increase in diameter is slow between 11 and 13 weeks. From these observations, it may be concluded that growth slackens between 9 and 14 weeks after fruit-set. The growth pattern in the mango fruit follows a simple sigmoid curve, and growth continues up to the stage of harvest maturity. Other observed morpological changes indicate that the stone remains soft for 9 weeks after fruit-set; subsequently it hardens, and about 14 weeks after fruit-set its development is complete. The reduced growth rate for the whole fruit occurs during this period (Lakshminarayana et al., 1970). Mango is botanically a drupe, and the growth pattern in

MANGO FRUIT

233

drupacious fruits has been broadly divided into three phases (Tukey, 1933). In the first stage, the fruit enlarges rapidly, and the embryo remains small. In the second stage, the embryo develops rapidly, but the fruit remains static. Further development of the fruit takes place in the third stage of maturity. This process has been referred to as cyclic growth (Lilleland and Brown, 1936). Similar sigmoid growth patterns for fruit and seed have been recorded in other varieties of mango (Chacko et al., 1970a,c; Saini et al., 1971) in which growth continues until harvest maturity (Fig. 1). According to earlier reports (Singh et al., 1937a; Leley et al., 1943; Mukherjee, 1959), growth of the mango fruit almost ceases 4 to 5 weeks before the fruit reaches full maturity. Rolz et al. (1971) observed in their studies on Mamey mangoes that the increase in volume of the fruit at each stage is attributable to an increase in cell size rather than to an increase in the number of cells. At this stage, it is interesting to examine the role of endogenous auxins in the development and maturation of the fruit. Fruit growth is governed by the auxins produced in the fertilized ovule following the stimulus from pollination. Developing mango seeds are a rich source of many growth substances such as auxins, gibberellin-like substances, and inhibitors. Using ether fractionation, paper chromatography, and various bioassay techniques, Chacko et al. (1970b) isolated two acidic ( A and B) and one neutral ( C ) growth substance which are similar in many characteristics to indole auxins from immature mango fruits. An attempt was made in their studies to find a correlation between the concentration of these three growth substances at different stages of fruit development and the production of auxins in various parts of the mango fruit such as the pericarp and seed. They found that the production of acidic growth-promoting substances ( A and B ) in fruits and seeds is greatest between 20 and 32 days after pollination. The neutral fraction ( C ) increased from 15 days and remained at a high level up to 45 days in both fruits and seeds (Table 111). The low concentration of growth-promoting substances present in the seeds is likely to be responsible for the slow growth rate observed during the initial period of fruit growth. The peak period of production of growth substance in the seed corresponds to the time of rapid fruit growth; thereafter, the rate of production declined gradually, although the total amount of all three growth promoters present per fruit and per seed increased consistently until the fruits reached full maturity. Hence, it is assumed that the peak period of auxin production in mango seed occurs before the full development of the embryo.

H. SUBHAhlANYAM ET AL.

234

T A B L E I11 ACIDICAND NEUTRAL GROWTH-PROMOTING SUBSTASCES PER FRUIT AND SEED IN DASHEHAR~ MANGOu

IAA equivalents ( pg ) Days after pollination

Fruit growth substances

A

0-10 15 20 25 32 38 45 50 68 90

0.00004 0.00115 0.025 0.325 1.0888 1.905 2.21 3.48 1.72 0.71

a From Chacko

Seed growth substances

B

C

0.00001 0.001 0.002 0.047 0.258 0.239 0.658 0.556 0.300 0.100

0.00001 0.0002 0.014 0.225 0.765 1.905 4.381 5.22 1.380 0.43

A 0.00065 0.0034 0.150 0.280 0.483 0.595 1.75 2.15 0.75

B 0.00006 0.0026 0.052 0.1178 0.0966 0.112 0.566 0.479 0.275

C 0.0007 0.0069 0.1071 0.330 0.483 1.86 3.922 3.594 0.316

et al. ( 1970b).

Failure to induce parthenocarpic fruits by external application of auxins (Venkataratnam, 1949; Chacko and Singh, 1969) indicated that other hormones, possibly kinins and gibberellin-like substances, are also involved in the growth and development of mango fruits (Chacko et al., 1 9 7 0 ~ )Further . investigations by the same authors revealed that extracts of mango fruits and seeds contained gibberellin-like substances. The peak production of these auxins in the fruit occurred at the beginning of endosperm development and corresponded to the time of rapid fruit growth; synthesis in the seeds decreased gradually afterward, but the total amount of active material per fruit continued to increase until the fruits attained full size, suggesting that these substances are involved in the cell enlargement phase of fruit growth in mango (Fig. 1). Histological studies of developing mango fruit are still obscure in the literature, and the precise period of cell division and enlargement during fruit growth is not clearly known. Singh (1961) found that in Dashehari mangoes the zygote rests for 8 to 10 days after its formation, and in the meantime the fruit grows beyond the mustard stage (1 to 2 mm in diameter). Sturrock ( 1968) reported that, after the initial enlargement of the ovary, the endosperm in the mango seed is in a free nuclear condition. At this stage, the embryo sac appears to be made up entirely of endosperm. As the endosperm develops, it completely consumes the nucleus and the developing embryo consumes the endosperm later.

235

MANGO FRUIT

Recent studies on meristematic activity of the pericarp in Dashehari and Chowsa mangoes have shown that, in outer and inner epidermis, cell division extends for a longer period than in other regions. This suggests that the outer epidermis keeps pace with the increase in size of the fruit by cell division rather than by cell enlargement. In the exocarp region, cell division stops 5 to 6 weeks after anthesis, and in

100

90

20

(0

Days after pollination

-

24 22 -

-

3

20 f 48u-

I6

-

Seed growth

5 44' ? !.

c

- 60 -50 - 40

12-

5- 10s

4

0

; 0

8-

- 30

6 -

-20

4-

- l o m

Of1

I

I

I

I

I

I

I

I

p

1 1 0

Days after pollination

FIG. 1. Growth pattern and quantitative changes of Gibberillic acid (GA)-like substances in Dashehari mango. ( A ) Fruits. ( B ) Seeds. 0-0, sigmoid growth; 0-0, GA-equivalents. From Chacko et al. ( 1 9 7 0 ~ ) .

236

H. SUBRAMANYAM ET AL.

the mesocarp region cell division extends up to 6 to 7 weeks after anthesis (Fig. 2 ) . The growth peak period, however, does not coincide with the maximum meristematic activity in the pericarp. From these studies, the authors (Saini et al., 1971) concluded that growth of the pericarp is affected more by cell enlargement than by cell division and also that the size of the seed contributes to the growth of the fruit. Thus, the initial period of slow growth of the fruit is due to lack of immediate initiation of seed development, the rapid growth of the fruit in the second stage is attributable to seed development, and finally the slow growth rate of the fruit is related to lack of seed growth and hardening of the endocarp region. B. COMPOSITIONAL CHANGES

Most of the studies on mango relate to postharvest changes in quality, composition, and storage. Little attention has been given to changes

FIG. 2. Meristematic activity in the exocarp and mesocarp regions in Dashehari mangoes. ( A ) Transverse section ( T . S . ) of the exocarp region enlarged, showing dividing cells. ( B ) T.S. of mesocarp region showing recently divided cell. From Saini et al. (1971).

MANGO FRUIT

237

during growth and development which ultimately determine the quality of the product. These changes differ considerably among the varieties

and the regions where the fruits are grown. The trend of chemical changes remains similar, although the actual quantity vanes to a great extent. Some of the chemical changes that take place during development and maturation in Alphonso cultivar are shown in Figs. 3 and 4.

1. Moisture Among the constituents of the fruit tissue, moisture plays a predominant role, especially in the developing fruit, in maintaining the protoplasmic movements and cell structure. The moisture content is low (70%) in the initial stages after fruit-set which coincides with the cell division phase; it reaches a maximum in the sixth week ( S S S ) , followed by a slight decline, and remains more or less steady until harvest. It is during this phase that cell enlargement takes place. Moisture content in the peel (exocarp) is lower than in the flesh (mesocarp) at all stages of fruit development.

t

I

FIG.3. Changes in sugars (1, 2 ) , titratable acidity ( 3 ) , total nitrogen (4),starch ( 5 ) , and alcohol-insoluble residue ( 6 ) (other than starch) in Alphonso mangoes during development and maturation. From Lakshminarayana et al. ( 1970).

238

H. SUBRAMANYAM ET AL.

4 8 12 Weeks after fruitset

16

FIG. 4. Phenolics in Alphonso mangoes during development and maturation. WF, whole fruit; P, peel; F, flesh. From Lakshminarayana et al. ( 1970).

2. Carbohydrates The major chemical change is the accumulation of starch throughout the period of growth and maturation. This aspect was investigated by Leley et al. (1943) in Alphonso mango, and they indicated that the starch content increases from 1 to 13% during development. A similar increase was noticed in several varieties grown in India (Mukherjee, 1959; Lakshminarayana et al., 1970), Florida ( Harkness and Cobin, 1951; Soule and Harding, 1956; Popenoe and Long, 1957), and other countries (Wahab and Khan, 1954; Rolz et al., 1971). The starch content of the peel is always higher than that in the flesh throughout the development and maturation of the fruit. Among the several varieties grown in India, it has been found that nonreducing and total sugars increase up to the stage of harvest maturity. The reducing sugars remain more or less constant throughout the period of development (Singh et al., 1937a; Wahab and Khan, 1954; Siddappa and Bhatia, 1954; Mukherjee, 1959). A similar observation has been made for Floridagrown mangoes by Soule and Harding ( 1956). While investigating the biochemical changes during growth and ripening of Alphonso mango, Leley et al. (1943) observed that the total and nonreducing sugars increase and the reducing sugars, both glucose and fructose, decrease gradually throughout the growing period of the fruit. Recent studies by Lakshminarayana et al. (1970) on the same variety indicate that sugar content declines throughout the period of growth and reducing sugars are present in higher concentration than nonreducing sugars. Sugar content is maximum by the third week after fruit-set, and this increase coincides with the respiratory maximum of the growing fruit,

MANGO F R U I T

239

which is termed the growth climacteric by the above authors. The peel registered a higher concentration of sugar than the flesh during different phases of growth, but at the time of harvest the sugar content of the peel, the flesh, and the whole fruit remained the same.

3. Acids Acidity in a developing mango fruit, expressed as citric or malic acid, shows a gradual increase in the early phase of development of the fruit and declines slowly thereafter until harvest. This pattern of acid change has been observed in many varieties grown in India (Singh et al., 1937a; Leley et al., 1943; Wahab and Khan, 1954; Mukherjee, 1959) and the West Indies (Wardlaw and Leonard, 1936). Lakshminarayana et al. (1970) have intensive investigations of the Alphonso variety regarding acid changes in the exocarp (peel), mesocarp ( flesh), and epicarp (whole fruit) in the developing fruit. The acidity reached the maximum in the seventh week (4.2%) after fruit-set and declined slowly to around 2.8y0 at the time of harvest. The acidity was always more in the mesocarp (flesh) and reached a value of 3.0% at the time of harvest.

4 . Cell Wall Constituents The cell wall is made up essentially of cellulosic and pectic substances, but this aspect has not been given due emphasis in studies of the mango fruit. However, the alcohol-insoluble residue ( AIR) other than starch gives an overall picture of the gross changes taking place in the cell wall material. This increases slightly in the early stages of growth, subsequently drops, and then remains steady until harvest maturity. In the flesh, however, AIR other than starch builds up slightly during the growth period (Lakshminarayana et al., 1970). A recent report by Rolz et al. (1971) indicates that acid-soluble pectin increases to its maximum value during the period of maximum growth of the fruit and declines later as the growth progresses. The water-soluble pectin is greatest at the time of harvest maturity, and ethylenediaminetetraacetic acid (EDTA)-soluble pectin remains constant (Fig. 5 ) .

5. Nitrogen Compounds Changes in nitrogen compounds have been studied by Singh et al. (1937a) to correlate the carb0hydrate:nitrogen ( C / N ) ratio with the

240

H. SUBRAMANYAM ET AL.

WEIGHT (grams)

FIG.5. Changes in pectic constituents in Mamey mangoes during development and water-soluble pectin; 0-0, acid-soluble pectin ( protomaturation: A-A, pectin) ; 0-0, EDTA-soluble pectates; 0-0, methyoxyl content in HClsoluble fraction; A-A, methoxyl content in water-soluble fraction; M-M, methoxyl content in EDTA-soluble fraction. From Rolz et al. ( 1971).

respiratory behavior and physiological attributes (described earlier) of the developing fruit. The nitrogen content of the fruit follows the respiratory sequence. In stage 1 there is a high rate of respiration and a low C/N ratio; in stage 2, the respiratory rate is steady but the C / N ratio increases; the respiratory rate is low and the C / N ratio is high in stage 3; in stage 4 there is an increasing rate of respiration, and an abrupt decline in sucrose with an increase in glucose, and the C / N ratio is also high. There is a confusion of climacteric in stage 3 where respiration is low, and higher respiration is noted in stage 4, the

MANGO FRUIT

241

senescent stage. Perhaps, the true respiration climacteric characteristic of fruit ripening falls between the third and fourth stages. Studies by Lakshminarayana et al. (1970) indicate that the bulk of total nitrogen during growth consists of protein nitrogen; this decreases up to the ninth week and remains steady thereafter until harvest. Total nitrogen and nonprotein nitrogen follow the same trend. The exocarp (peel) records higher nitrogen content than the mesocarp (flesh) in the developing mango fruit. 6. Pigments The development of color in terms of pigments during growth of the fruit has not been studied except at the final stage when the fruit is ready for harvest. Visual changes in the surface color are clearly seen during growth and development of the fruit, changing from dark-green to olive-green shades; sometimes reddish or yellowish hues appear, depending on the variety. The flesh changes in color from white to cream and then to yellow and orange shades as the fruit approaches maturity. Wardlaw and Leonard (1936) correlated the changes in flesh color of Julie mangoes with other chemical and physiological attributes of the developing fruit. Similar observations have been made on many varieties grown in India, but the actual amount of pigments (chlorophyll, xanthophyll, carotenoids ) responsible for characteristic color changes have not been examined. Preliminary observation made by Laksminarayana (1972) in Alphonso variety indicates that carotenoids (total and @-carotene) remain at very low concentrations in the mesocarp (flesh) during the first two stages of development and increase gradually as the fruit approaches maturity. Rolz et al. (1971) found similar trends for total carotenoids in the exocarp (peel) of Mamey mangoes.

7. Vitamins Although the ripe mango is an excellent source of many vitamins, information is lacking for the developing fruit except for vitamin C (ascorbic acid), Siddappa and Bhatia ( 1954) reported that the vitamin C content is maximum (300 mg per 100 gm) in the Pain variety in early stages of growth. Vitamin C determinations made by Spencer et al. (1956) in the developing fruits of mango varieties Mulgoa, Pico, Amini, and Turpentine showed a downward trend from 88 mg% to 22 mg% within 5 to 10 weeks after fruit-set. The final maturation period of 4 to 6 weeks was accompanied by little change. Studies on developing

242

H. SUBRAMANYAM ET A L .

Dashehari and Fazli mangoes indicate that the vitamin C content declines during the first 7 weeks after fruit-set and remains fairly constant until harvest maturity (Singh and Chada, 1961). The apparent ascorbic acid content of the whole fruit (Alphonso) increases soon after fruit-set and reaches its peak value (240 mgyo) in the fifth week, declines thereafter up to the eighth week (130 mgyo), and remains more or less steady until harvest maturity (Lakshminarayana et al., 1970). 8. Astringents

The astringency of the fruit during the early part of the growth is due to its high phenolic content. There is a sharp decline in the polyphenol content of the developing Alphonso mango fruit up to about the eighth week, after which the values remain constant. The total phenols are higher in the exocarp (peel) at all stages of development (Fig. 4 ) ( Lakshminarayana et al., 1970).

9. Growth Substances Biennial bearing in mango is a major problem encountered in many commercial cultivars. The hormonal status of the shoots is supposed to control this alternate bearing habit. While studying this problem, Chacko et al. (1970a) examined the endogenous growth factors which control fruit development in Dashehari mangoes. They isolated three growth-promoting substances besides gibberellin-like substances during development of the fruit. Changes in the level of these endogenous auxins are involved in the growth, development, and maturation of the fruit which were discussed earlier. However, no attempt has yet been made to correlate the hormonal balance of the tree with alternate bearing habits.

C. RESPIRATION(DURING GROWTH) The first reference to the occurrence of an early climacteric between 7 and 21 days after fruit-set in mango (varieties Krishnabhog and Langra ) , besides the one occurring during normal ripening and senescence, was made by Singh et al. (1937a). They correlated the respiratory drifts during growth, development, and maturation with physical, physiological, and biochemical attributes of the developing mango fruit. This study, which is one of the noteworthy early contributions in this field, has often

MANGO FRUIT

243

been cited by subsequent workers. Later, Leley et al. (1943) and Mukherjee ( 1959), while studying the biochemical and physiological aspects of developing mango fruit, did not observe a respiratory peak in other varieties grown in India. Their failure to recognize an early respiratory peak in a developing mango fruit is likely to be due to their missing the early stages of the fruit when their experiments were started. The respiratory drifts as observed by Lakshminarayana et al. (1970) during growth and development of Alphonso mango are shown in Fig. 6. The respiration rate was high soon after fruit-set and reached a peak value in the fourth week. Then it gradually declined u p to the eighth week and remained steady thereafter until harvesting time. Similar patterns were recorded when tissue discs were used in a Warburg respirometer. The appearance of early climacteric in CO, production during the fourth week after fruit-set is accounted for by extreme cellular activity of the fruit at the initial stages; this is termed the growth climacteric, and it is different from the climateric phase noticed in postharvest stages which is associated with normal ripening of the fruit. The respiratory behavior of the developing fruit shows different trends when expressed per fruit or per unit weight basis. In the former case, the respiration rate gradually increases until the fruit attains harvesting maturity, closely following the sigmoid growth curve; in the latter, it declines initially and remains steady until fruit development is complete.

D. MATURITYINDICES The quality of the ripe fruit is influenced by many factors of which the stage of maturity at harvest is an important aspect. Several parameters have been suggested for determining maturity of fruits on the basis of their external appearance and chemical constituents at the time of harvest (Table IV) . But these indices vary considerably from variety to variety; hence one cannot generalize for all the varieties. The degree of maturity in mango has been correlated with physical appearance and surface color by Cheema and Dani (1934). They defined four different stages of maturity, termed A, B, C, and D, based on shoulder growth, size, and surface color of the fruit for Alphonso variety. Wardlaw and Leonard (1936) described three stages of maturity for Julie variety of West Indian mango; they agreed, in principle, that the parameters recommended by Cheema and Dani (1934) are applicable to West Indian mangoes. However, the Julie variety of West Indies shows little skin coloration, but other varieties from India show a marked color change

H. SUBRAMANYAM ET AL.

244

-

A

900 -

700 L

2 r 0"

-

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500-

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4

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8

16

Weeks after fruit-set

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MANGO F R U I T

245

TABLE IV

MATURITYSTANDARDS FOR HARVEST: ALPHONSO AND PAIFU MANGOESa Physical and chemical factors Weight ( gm ) Specific gravity Total soluble solids ( % ) Acidity (% as inalic acid)

Group A

> 320 > 1.02 > 10 < 3.2 > 800 > 12.5

Group B

Group C

300 f 20 1.01-1.02 8*1 3.5 c 0.2

250 2 20 1.0-1.01 7Ll 3.9 ? 0.2 400-800

Group D

< 225 < 1.0 <

6

> 4.1 <

Total carotenoids (fig% ) 600-800 400 Alcohol-insoluble residue ( % ) 11.5-12.5 10.5-11.5 10.5 Maturity Over mature Physiologically Physiologically Physiologically mature immature immature

<

a From Bhatnagar and Subramanyam ( 1971 ).

on ripening. Harkness and Cobin (1951) classified Florida-grown mangoes on the basis of the specific gravity of the fruit. In their opinion, Haden mangoes having a specific gravity between 1.01 and 1.02 and a sucrose content of more than l.OY0 at the time of harvest are suitable for picking. Popenoe et al. (1958) suggested that starch content (5%) a t the time of harvest could be a reliable index of maturity for some varieties grown in Florida. Mukherjee ( 1960) examined several physical, morphological, and chemical indices in Indian varieties of mango for defining maturity for harvest and suggested that (water sinkers : >LO specific gravity) specific gravity grading is the most reliable method. Suryaprakasha Rao and Srinath (1967) observed that harvesting time can be roughly predicted, depending on the heat units available after flowering. Teaotia et al. ( 1968) suggested that the starch-to-acid ratio (4or more) could be used as an index for determining maturity in Langra variety of mango. More recently Shantha Krishnamurthyl and Subramanyam ( 1970a) studied various physical, physiological, and chemical attributes for fixing the optimum stage of maturity for Pairi mangoes. Fruits were classified into three groups on the basis of weight, appearance (shoulder growth and pit formation at the stalk end), and surface color. The physical parameters were compared with chemical indices such as sugars, soluble solids, and color of the flesh pulp at harvest. These attributes were correlated with fruit quality at ripe stage and respiratory behavior of the harvested fruits. On the basis of these studies, it was 1

For references, see under Krishnaniurthy, Shantha.

246

H. SUBRAMANYAM ET AL.

observed that optimum maturity for Pairi mangoes could be indicated by a weight of 260 +- 20 gm, an olive-green surface color, and outgrown shoulders. In addition, p H and color of the pulp in terms of the chromaticity coordinate ( x ) were useful indices, but other parameters were inconsistent. The respiration climacteric maximum was delayed in immature fruits (group I ) and advanced in overmature fruits (group 111) (Fig. 7 ) . For fruits of optimal maturity (group 11) the climacteric maximum was recorded on the ninth day at ambient storage. Ripening characters, spoilage, and consumer preference indicated that the fruits belonging to group I are physiologically immature and those of groups I1 and I11 are at the optimum stage of maturity. However, fruits in group I11 are available only to the limited extent of the total harvest; therefore, group I1 is preferred for all practical purposes. Hulme (1971), in attempting to draw an analogy for Indian and Florida-grown mangoes, suggests that Florida mangoes contain more sugar a t the unripe stage than Indian mangoes at a comparable stage of maturity; hence this chemical index is not useful for other varieties.

\ \

250

\

\ \

,

\ \

! !

200 L

Jz

50

L

I

4

8

t2

I

(6

I

20

Days after harvest

FIG. 7 . Respiratory pattern of Pairi mangoes at different stages of maturity: - - 0, immature; 0 -.-.-. 0, optimum maturity; .@---0, advanced ma-

0-

turity. From Shantha Krishnamurthy and Subramanyam ( 1970a).

MANGO FRUIT

247

Kennard and Winters (1956), while studying the effect of 2,4,5trichlorophenoxypropionic acid ( 2,4,5-TP) at various concentrations on maturity of Amini mangoes, observed that the fruit size was reduced and maturity was hastened 1 to 2 weeks by early sprays of the growth regulator. Late sprays as the fruit was nearing maturity had little effect on growth. Subramanyam et al. ( 1972d) examined several growth regulators to determine whether they delay or hasten maturity in Alphonso mango, with the object of spreading the peak harvest season over an extended period of time. They observed that P-naphthoxyacetic acid (P-NOA) at 25 ppm, as a foliar spray at monthly intervals from fruit-set, hastened maturity by 2 weeks, and maleic hydrazide ( M H ) at 750 ppm delayed harvest maturity by 2 weeks. Fruits treated with p-NOA ripened earlier after harvest, developed attractive skin color, and recorded higher carotene content in the flesh of the ripe fruit. Maleic hydrazide, on the other hand, increased the fruit size, delayed the ripening process, interfered with carotene formation, and increased the susceptibility of fruits to fungal infection. Gibberillic acid, 2,4,5-T, and 2,4,5-TP had no consistent effect on maturity or on quality of the ripe fruit.

E. HARVESTING AND RIPENING Green and mature fruits are harvested individually by manual labor with the help of a bamboo pole and net attached to it at the end. The fruits are lowered to the ground in a basket by means of a rope. In some areas, fruits are clipped, leaving a stalk end of 1 cm to avoid injury. Mechanical harvesting is not possible in view of the large spreading habit of the tree. Fruits are graded on the basis of weight and surface color. Specific gravity grading by water and brine flotation appears to be useful and more reliable than the other methods in vogue. Fruits having a specific gravity of more than 1.02 ripen faster and have a reduced storage life, but they are suitable for consumption in the fresh state. Fruits with a specific gravity of 1.0 to 1.02 require a longer period for ripening, have a longer storage life, and are superior for dessert purposes as well as for processinz. Fruits having a specific gravity lower than 1.0 take a longer time to ripen and have a longer storage life with an increased susceptibility to infection. The quality is often poor in either fresh or processed form (Bhatnagar and Subramanyam, 1971) . However, these grade standards vary considerably from variety to variety and cannot be generalized for all the varieties of mango grown in different regions.

248

H. SUBRAMANYAM ET AL.

Mangoes take about 1 to 2 weeks to attain an edible-ripe condition at ambient storage of 30°C k 5”, 60 to 85% R.H. However, the storage life depends on the stage of maturity at harvest, the variety, and the storage temperature. Fruits of lower maturity ripen in 3 weeks, and those of higher maturity take about a week to attain an edible-ripe stage. Although refrigerated storage conditions have been recommended from time to time for several table varieties, recent investigations on an intensive scale indicate that fruits stored at a temperaure below 25°C do not ripen satisfactorily even though the critical temperature for development of chilling injury is below 10°C (Lakshminarayana and Subramanyam, 1970, 1971). Several factors responsible for low-temperature injury in tropical fruits have been critically reviewed by Fidler and Coursey (1969); some of these aspects are discussed in greater detail in the next section. In commerce, dry straw or paper cuttings are used for ripening mangoes. These cushioning materials conserve heat, and contaminating molds produce ethylene in sufficiently large amounts to stimulate ripening. Several chemical methods are in vogue to stimulate ripening and the development of surface color in mangoes. Acetylene produced from calcium carbide also degreens the immature fruits effectively, but the edible quality remains poor ( Mirchandani, 1965; Srivastava, 1967). Ethylene gas or compounds that release ethylene in situ, on the other hand, are likely to increase the susceptibility of fruits to microbial infection, more so in fruits of lower maturity; therefore, artificial ripening should be employed with caution ( Hobson, 1969; Subramanyam, 1973). Heat treatment, using hot water as a momentary dip, accelerates ripening and is safer ( Subramanyam et al., 1972c; Shantha Krishnamurthy and Subramanyam 1970b).

F. SUMMARY The term “climacteric” used by Singh et al. (1937a) with reference to the development and maturation of mango fruit has led to serious confusion in view of the exhaustive literature that has accumulated on postharvest respiratory climacteric in edible fruits. Two distinct respiratory maxima have been observed-one during development of the fruit, and the other during ripening-and it would be more logical to designate the early respiratory maximum as the “growth climacteric” and the one occurring after harvest as the “ripening climacteric.” Recent literature indicates that the growth pattern follows a simple sigmoid curve, and the growth continues until harvest maturity, although the rate of physical

MANGO FRUIT

249

expansion slows down at the final stages. The relationship between physical expansion, flesh color, and morphological characters of the developing fruit as shown by Wardlaw and Leonard (1936) does not appear to be correct, since soil moisture can greatly influence the physical expansion of the fruit with little effect on maturation, and it would be reasonable to correlate fruit development with number of days after anthesis. The scattered information available on chemical constituents of the fruit during development and maturation contributes very little to our understanding of the developmental physiology. A recent attempt to correlate the endogenous growth-promoting substances with the development and maturation of the fruit is interesting, since these auxins or cytokinins are known to regulate fruit maturation. Little attempt has been made to evolve maturity indices that have practical significance, and existing practices depend essentially on morphological characters of the fruit which vary from cultivar to cultivar. Development of objective methods, especially nondestructive ones, based on textural properties and internal color of the flesh determined by pressure meters, magnetic resonance, and light transmittance techniques as suggested for temperate fruits appears to be the choice of the future. Regulation of the maturity of the fruit by application of synthetic plant growth regulators is promising, since this would alleviate several problems encountered in peak harvesting seasons. Mechanical harvesting is a far-fetched idea with the existing cultivars and current orchard practices. Postharvest treatments using hot water coupled with chemical regulators are helpful in regulating uniform ripening and color development.

111.

PHYSIOLOGY OF RIPENING

Mangoes are usually harvested at a proper stage of maturity, based essentially on physical attributes as described in the previous section. They are then allowed to ripen at ambient conditions (30°C, 85% R.H.) of storage. Fruits are not allowed to ripen on the tree ( 1 ) because of the economic aspects of the problem-the majority of fruits drop from the tree before they are ripe enough for consumption; and ( 2 ) because tree-ripe fruits are inferior in taste and aroma to fruits that ripen after harvest, and their keeping quality is reduced. The physiology of ripening involves numerous metabolic activities resulting in fruits of acceptable quality. Of these physiological activities, changes in carbohydrates and acids to give the desired sugar-to-acid ratio, development of color and

250

H. SUBRAMANYAM ET AL.

flavor characteristic of the variety for consumer appeal, and softening of the texture for acceptable quality are of prime importance. All these biochemical changes take place within a short-period of 10 to 14 days at ambient temperature, depending on the variety and stage of maturity at harvest.

A.

RESPIRATION

The most significant postharvest change is in the rate of respiration. Since the work of Kidd and West (1922), the study of fruit respiration has gained importance, as it provides a rational basis for the storage of fleshy fruits. Banerjee et al. (1934) observed that the respiratory rate of Neelum mangoes is closely correlated with ripening. Singh et al. (1937a), while studying the ontogenetic drifts in the physiology and chemistry of tropical fruits, observed a climacteric maximum in respiration during ripening in Krishnabhog and Langra varieties of mango. Karmarkar and Joshi (1941) studied the respiratory behavior of Alphonso mango under different storage conditions. They observed that the respiratory activity of the green, hard fruit of B-stage maturity of Cheema and Dani (1934) increased during the first 4 or 5 days and then declined as the fruit ripened. This decrease was attributed to depletion of acids present in the fruit. Leley et al. (1943), working with Alphonso mango, noted a climacteric rise in respiration after harvest with the commencement of ripening. The maximum value for the rate of respiration occurred when the fruits were still hard and green ( 2 to 5 days) or were just beginning to change color. The fruits were fully ripe after 9 days. During this latter period of ripening, the rate of respiration decreased. The study of respiratory drifts in different varieties of mango such as Pairi, Alphonso and Neelum during ripening have clearly indicated that the sequence follows a regular pattern and shows a definite climacteric peak in respiration after harvest (Fig. 8 ) (Shantha, 1969; Shantha Krishnamurthy and Subramanyam, 1973). This peak is very different from the one noticed during growth by Singh et al. (1937a) and Lakshminarayana et al. (1970). The pattern of respiration was classified into four distinct phases based on observable changes: (1) a preclimacteric phase lasting for 3 days when the fruits are green and firm and CO, release is at a low rate; ( 2 ) a climacteric rise extending up to 6 days when a sudden spurt in CO, production is observed and the fruits remain green and firm;and (3) a climacteric peak occurring between 6 and 10 days after harvest marked by a peak in CO, release, The fruits at this phase tend to break color, become soft, and develop an odor characteristic of the variety.

MANGO FRUIT

250

t

2.51

3

200

0

E

100

50

4

0

42

46

Days after harvest

FIG.8. Respiratory pattern of Pairi mangoes at 28°C when 0-0, same fruits and 0 - - - 0, fruits at random were used. ( 1 ) Preclimacteric; ( 2 ) climacteric rise; ( 3 ) climacteric peak; ( 4 ) senescent. From Shantha Krishnamurthy and Subramanyam ( 1970a). This is followed by stage 4, a postclimacteric phase, lasting from 10 to 14 days, when CO, release shows a sudden decreasing trend; the fruit develops an attractive color and odor and is soft and edible-ripe. After this stage, senescence of the fruit sets in, and the fruit is susceptible to infection due to microorganisms, resulting in decay and death of the fruit material. A similar respiratory pattern in terms of 0, consumption as well as CO, release was noticed in the peel and flesh tissue slices, and the respiratory quotient (R.Q.) was always above unity, reaching a maximum value of 3.5 in the peel as the fruit approached the climacteric peak. Respiratory patterns and rates are influenced to a great extent by the stage of maturity at harvest. Preliminary studies on the effect of different stages of maturity on the respiratory drifts of Alphonso mango are discussed by Lakshminarayana (1973). Fruits were picked for this purpose at different stages of development from 1 to 16 weeks after fruit-set. The respiratory pattern of these fruits during storage for 10 to 20 days indicated that the climacteric peak in CO, production occurs at all stages of development. The intensity of the peak varies with different develop-

252

H. SUBRAMANYAM ET AL.

mental stage. Fruits harvested in the first 6 weeks after fruit-set showed the respiratory peak in CO, release within 2 to 5 days of harvest, whereas fruits of 7 to 9 weeks’ maturity recorded the climacteric around the eighth day of harvest. Fruits harvested at later stages of development, from 9 to 14 weeks, exhibited the peak in CO, release within 5 to 7 days of harvest. Lakshminarayana noticed a correlation between the maturity of the fruit and extension of the preclimacteric stage which lasted for a longer time when stone hardening was taking place. At other stages of fruit development, the preclimacteric stage was of a shorter duration. The climacteric peak noticed in immature fruits is of little signscance, since it is not associated with normal fruit ripening, although the preclimacteric phase extends over a longer period. In these studies, no correlation was seen with physical appearance of the fruit at harvest, age of the fruit, or respiratory behavior after harvest. However, Shantha Krishnamurthy and Subramanyam ( 1970a) clearly observed in Pairi mangoes a close correlation between physical appearance of the fruit at harvest, and onset of respiratory climacteric, characteristic of different stages of maturity (Fig. 7). The respiratory climacteric during ripening of the fruit has a great impact on storage life, as this process releases a sufficiently large amount of energy. For the purpose of storage at low temperature, the amount of energy released during ripening is calculated from the respiration rate of the fruit. Physiological losses in weight indicate the total moisture lost during storage and ripening, which results in desiccation and a shriveled appearance of the fruit. The marketable quality of the fruit also depends on this factor to a great extent. Loss of moisture is greater in immature fruits than in the fruits of optimum maturity, and, in general, mangoes lose 10 to 12% of their weight during storage and ripening at ambient conditions. This physiological loss is essentially due to transpiration and respiration ( Shantha Krishnamurthy and Subramanyam, 1973). The effect of various external factors on the respiratory drifts of mango fruit will be discussed in the subsequent section under postharvest treatments. Studies on respiration were extended to the subcellular level (mitochondrial level). Patwardhan ( 1965) isolated active protoplasmic particles from seven varieties of mango using a sucrose phosphate buffer. These preparations oxidized succinate and, less actively, citrate. Several of the mitochondria1 dehydrogenases were shown to be present, but they required the addition of cytochrome c for maximum activity. They were, therefore, not entirely intact; since mangoes contain phenolics in SUBciently large amounts in the unripe stage, and since no precautions were

MANGO FRUIT

253

taken to prevent the inhibitory effect of phenolics, apart from the presence of cysteine in the extracting medium, the preparations made by Patwardhan ( 1965) were fairly crude. Mattoo et al. (1968) isolated mitochondria using trismaleate buffer containing 0.4 M sucrose, but, again, no precautions were taken to prevent interference by phenolics. In the mitochondria of ripening mango, both phosphorylation and oxidation increase, but the P/O ratio is not affected. This increase may be due to the increased demand of ATP by the enhanced rate of various metabolic activities during ripening.

B. ENZYMES, INHIBITORS, AND ETHYLENE

1 . Enzymes and Znhibitors Brill ( 1919), while studying the enzymes of some tropical plants, found that mango contains a proteolytic enzyme which has properties similar to those of bromelin. Banerjee and Kar (1941) studied catalase and peroxidase activities of developing mango from fruit-set to ripening (13 to 115 days). Catalase activity was shown in five distinct well-marked phases: (1) an early phase of very low activity, ( 2 ) a phase of rapid and steady increase, ( 3 ) a period of high activity, with ( 4 ) a steep rise to maximum activity, and (5) a rapid decline to a minimum. A positive correlation of these enzyme activities with hemin Fe content of the tissue was shown at each stage. Catalase activity was enhanced in fruits ripened under artificial doses of ethylene at 28" to 32"C, and reduced activity was seen in cold-stored fruits. Aganvala et al. (1960) found that in apical parts of diseased mango tissue (black tip) of Safeda and other varieties these enzymes were found to be more active. Recently, Mattoo et al. (1968) have shown that, during ripening of Alphonso mango, phosphatase activity increased twofold and that the enzyme activity could be doubled by the addition of ,&carotene but not vitamin A. They concluded that the increased carotene levels found during ripening might regulate carotenogenesis by promoting phosphatase activity. They further demonstrated that there is a four- to fivefold increase in the activities of peroxidase and catalase enzymes during ripening of Alphonso mango. They attributed this increased activity to disappearance of a heat-labile and nondialyzable inhibitor in unripe fruits. They also (Mattoo and Modi, 1969a) showed by means of in vitro studies that ethylene synthesized in the fruit before the onset of climacteric in mango activates the enzymes and inactivates the inhibitor (Fig. 9 ) . Further, these inhibitors, proteinic in nature, were isolated and purified. Peroxidase enzyme inhib-

254

H. SUBRAMANYAM ET AL.

o-o-

0

15

30

45

70

Hours a f t e r treatment

FIG.9. Effect of ethylene on disappearance of catalase inhibitor from Alphonso mango slices incubated at 25°C for 70 hours. 0 = controls; A = 10 ppm ethylene; 0 = 50 ppm ethylene. From Mattoo and Modi ( 1969a j.

itor was purified 300-fold and amylase inhibitor 100-fold from unripe mangoes (Mattoo and Modi, 1970a). According to Mattoo and Modi, there are individual proteinic inhibitors of various enzymes in the unripe mango, which inhibit by occupying either part of the substrate site or some active site of the enzyme. The mechanism of action of the inhibitors remains to be investigated. They have further suggested that a complex is formed between the enzyme and its inhibitor. Enzyme activity is reduced linearly by increasing amounts of inhibitor, indicating that the effect is on the free enzyme. These inhibitors have also been found to bc effective on enzymes isolated from banana. Modi and Reddy (1967) observed an increase in the activity of NADP-dependent malic enzyme, glucose-6-phosphate dehydrogenase, and 6-phosphogluconic dehydrogenase during ripening of mango. Cegarra (1966) made a study of the relative abundance of peroxidase and phenolase enzymes in eleven grafted varieties. Mattoo and Modi (1969b) observed that activity of pectinesterase in chill-injured mango tissue was one and a half times as great as that in the healthy tissue. Amylase activity decreased two- to threefold, while invertase activity increased by more than twofold in the injured tissue (Table V ) , accounting for lower sugar content. Mattoo and Modi ( 1970b) found an increasc in ATP:citrate OAA-lyase (citrate-

255

MANGO FRUIT TABLE V PECTINESTERASE, INVERTASE, AND AMYLASE IN CHILL-INJURED AND HEALTHYTISSUESOF MANGOES b Pectin-esteraseactivity

Condition Chill-injured Healthy ‘I

b

Invertase activity

Unripe fruit

Ripe fruit

Unripe fruit

1.8-2.2 0.8-1.0

3.5-4.5 2.0-2.6

0.85-1.30 0.36-0.85

Ripe fruit

2.1-4.5 1.6-2.0

Amylase activity Unripe fruit

Ripe fruit

0.8-0.90 1.15-2.40

1.2-3.5 3.6-5.0

Units per milligram of protein. From Mattoo and Modi ( 1969b).

cleaving enzyme ) activity during the ripening of mangoes and suggested that the acetyl coenzyme A (CoA) and oxaloacetic acid (OAA) formed in thc reaction of thc rnzyme on citrate may contribute to synthetic processes taking placc during the ripening period. A crude fatty acid preparation from mango pulp when added to the enzyme preparations doubled their activity at the optimum conditions. From this, they suggest th:it the natural lipid breakdown products might regulate the activity of the enzyme in vivo. Shantha Krishnammthy et al. (1971) have shown that, durins the onset of climacteric rise, there is the development of malate decarboxylating system. Malic enzyme ( M E ) activity has been shown to increase manyfold during the first 8 days of storage. The enzyme activity developed gradually in the early ripening period, reached its maximum slightly ahead of the climacteric peak in respiration, and then dropped down. This enzyme was further isolated and purified from mango pulp 200-fold, and its properties were studied (Shantha Krishnamurthy and Patwardhan, 1971) . Besides this, the activities of glutamic decarboxylase ( GDC ) and aspartate oxoglutarate amino transferase (AGT) increased during the ripening period. The peak activity of ACT occurred just before the climacteric peak, while that of GDC began to increase at this point and rose rapidly at the climacteric peak and beyond (Fig. 10). While examining the causes for low-temperature breakdown in mangoes, Subramanyam and Patwardham ( 1968) isolated the enzyme and substrates responsible for discoloration in the necrotic lesions of the peel. Enzyme substrate specificity was determined, and the gallic acid extracted from the peel of the fruit reacted with the polyphenol oxidase isolated from the peel portion, resulting in discoloration. The pH optimum for this enzyme was between 5.0 and 5.5. The activity of polyphenol oxidase increased during ripening, with a corresponding

H. SUBRAMANYAM ET AL.

256

0

I

I

I

I

4

8

42

Days after harvest

FIG. 10. Changes in enzyme activity during ripening in Pairi mangoes at 28°C: malic enzyme; 0- . - - 0, aspartate glutamate transaminase; 0- - - 0, glutamate decarboxylase. From Shantha Krishnammthy et al. ( 1971).

0-0,

increase in total tannins and gallic acid in the peel of the fruit (Subramanyam et al., 1972a). The enzyme was purified fiftyfold by Venkaiah (1970), and its properties were studied. It was shown that copper chelates inhibited the enzyme activity and ascorbic acid inhibited the enzyme reaction by binding the product formed. Although increased activity of various enzymes has been shown during the ripening process, synthesis of the actual enzyme protein as shown in pears has not been demonstrated. This appears to be the sum total of our knowledge of the enzymes of the mango fruit, illustrating that serious attempts are being made to investigate the complex biochemical changes taking place in the mango fruit, especially during the ripening period. 2. Ethylene

Ethylene is a fruit-ripening hormone. Its evolution accompanies the process of maturation and aging. The real nature of ethylene reactions in inducing fruit ripening is still not clearly understood. In mangoes, not much is known about the exact role of ethylene action. Kar and Banerjee

MANGO FRUIT

257

(1939) studied the effect of ethylene on the ripening behavior of mangoes. Mango was once thought to be a nonethylene-producing fruit according to Biale et al. (1954) and Biale (1960). Burg and Burg (1962) have shown a normal pattern of ethylene evolution coinciding with the respiratory peak in Haden and Kent mangoes (Fig. 11).They (1965) have reported that Kent mango contains 0.08 ppm of ethylene at the onset of respiratory rise and at the time of preclimacteric respiratory minimum, sufficient to influence the metabolic activity in the fruit. Mango fruit is said to contain approximately 1.6 ppm of ethylene on the tree and occasionally 11 ppm. Yet, this concentration fails to stimulate the ripening process when the fruit is on the tree (Burg and Burg, 1967a). Hence they suggested the possibility of the presence of a natural inhibitor of fruit ripening which prevents the action of ethylene. Burg and Burg (1967b), while examining the inhibition of polar auxin transport by ethylene in etiolated pea stem sections, observed certain responses in which the gas produces symptoms of auxin deficiency. It is therefore likely that the ethylene present in the mango fruit tissue might inhibit the auxin transport from the endocarp, resulting in an inducement of ripening. Mattoo et al. (1968) showed that mangoes (Alphonso) produce ethylene during ripening in the range of 0.02 to 0.18 ppm. A threefold increase (from 18 pl/hr/gm to 50 pl/hr/gm) in ethylene production was observed in the ripening fruit slices (Mattoo and Modi, 1967) in the presence of methionene ( 3 to 7 pmoles ), suggesting that methionene may be a precursor of ethylene in mangoes.

C. CHEMICAL CONSTITUENTS Respiration rates during ripening are accompanied by many chemical changes resulting in fruits of edible quality. General compositional changes in mangoes after harvest have been studied by several workers in a number of varieties of local importance. Srikantia and Kantiengar (1942) examined the chemical composition in Raspuri (syn. Pairi) and Badami (syn. alphonso) mangoes in unripe and ripe fruits. Basu et al. ( 1947) investigated the chemical composition of several varieties of mangoes cultivated in India in the unripe and ripe stages to examine the relationship between nutritive value and chemical constituents of the fruit. They found that the unripe fruits contain more vitamin C and ripe mangoes are a richer source of sugar and carotene, the precursor of vitamin A. However, no definite relationship between vitamin C , acidity, sugars, and carotene was established. Nandi (1958) reported the chemical composition of ripe mangoes in several varieties cultivated in India.

110 110

- 100 0

100

0

90

IT \

5 80

\

..-.

8

90 L

x

x CT,

>a. -2

,” 80

Internal CH2 CH2

-E, Cl

70

0

I

N

60

0

N

V

-

50

CH,

0

2 40

CH,.

\

Production CH,

Productioi J

3

CT,

3c

N

0 V

2c 1C

Days

FIG. 11. Relationship between ethylene production rate, internal content of ethylene, and rate of respiration during the ripening of ( A ) Kent mangoes and ( B )Haden mangoes at 24°C. From Burg and Burg (1962).

MANGO FRUIT

259

Bakshi and Bajwa (1959) examined the chemical constituents of sixty varieties of mangoes grown in the Punjab and classified twenty-four varieties among them as high quality mangoes based on high total soluble solids (T.S.S.). Agnihotri et al. (1963), while examining the effect of several treatments after harvest, followed the changes in sugars, acidity, color, and texture in Dashehari mangoes during ripening at ambient conditions. Bruno and Goldberg (1963) made a survey in Northern Nigeria and selected sixteen local and seven introduced varieties of mangoes based on morphological characters and time of harvest. They concluded that local cultivars are rich in vitamin C, carotene, and protein when compared with the introduced varieties, although these are larger in size. De and Debnath (1966) followed the changes in acidity and sugars during maturation and ripening in varieties grown in East Pakistan. A gradual decline in acidity and an increase in sugar content were noted, which are common features during ripening. However, in the local variety, Kanchamitha, the acidity remained more or less constant and reducing sugars were predominant in the ripe stage. Biale ( 1960), while reviewing the postharvest biochemistry of tropical and subtropical fruits, discussed various physiological and biochemical changes that take place after harvest in the mango fruit. Johnson and Raymond (1965) reviewed the literature on chemical constituents of mangoes cultivated in different countries. The composition of peel (skin), flesh (pulp), and stone of several cultivars of mangoes has been critically examined by those authors.

1 . Sugars Among the chemical changes during ripening, a drastic increase in sugars is noticed. It forms a high proportion of the soluble solids in ripe mango fruit. The unripe mango consists mainly of reducing sugars, and the ripe fruit consists more of nonreducing sugars (sucrose). The chemistry of sugars in the fruit during the process of ripening was studied by Patwardhan (1927). The nature and concentration of sugars in different varieties during ripening have been studied by various workers (Soule and Harding, 1956; Krishnamurthy et al., 1960). Sarkar (1963) noticed free xylose in the ripening pulp and stone of the unripe fruit. Xylose and arabinose were found in the acid hydrolyzate of the skin, pulp, and stone. Ghosh (1965) has shown the formation of a new oligosaccharide when the fruit was inoculated with Colletotrichum gloeosporoides. The concentration of this reached a maximum value 4 to 6 days after inoculation. Wali and Hassan (1965) noticed arabinose

H. SUBRAMANYAM ET AL.

260

in two varieties of mango. Mostly glucose and fructose are the reducing sugars ( 3 to 4oj,), and sucrose (12%) forms the major portion among total sugars (Fig. 12). 2.

Organic Acids

Usually total acidity is measured and expressed in terms of citric or malic acid. The acid content vanes from 4 to 5% in the green fruit to 0.5 to 0.1% in the ripe fruit. Qualitative changes of total acids have been found in a few varieties. Ishii (1933) made a preliminary study of organic acids in the mango. Cheema et al. (1954) observed a correlation between the acid content of green fruit and the length of the storage life, the latter being short in fruits with low acidity and long in fruits with high acidity. Jain et nl. (1959) made a qualitative study of organic acids in pickling mangoes and reported the malic and citric acids are predominant. According to Fang (1965), the organic acid contents of Kent and Hsaing-Ien mango include malic, citric, tartaric, oxalic, and glycolic acids and an unidentified acid (Table V I ) . Shantha Krishnamurthy et al. (1971) made a detailed study of the 0x0 acids of Pairi mangoes during the climacteric rise. They identified pyruvic, oxaloacetic, and a-ketoglutaric acids in the pulp portion. The study of concentration of these acids during the climacteric rise revealed that a-ketogluturate and pyruvate followed the respiration rate of the whole fruits (Fig. 13). The peak of these acids was observed 2 or 3 days ahead of the climacteric peak. Fair amounts of oxaloacetic acid were noticed in r~arlystages of ripening, hut they were reduced to traces afterwards. TABLE VI ORGANICACIDS OF MANGOFHUITS a,b Variety

Acid

Kent

Hsaing-Ien

Glycolic Oxalic Malic Citric Tartaric

0.061 0.036 0.074 0.327 0.081

0.026 0.008 0.045 0.194 0.051

Fang ( 1965). Values expressed as percent of fresh weight.

a From

MANGO FRUIT

261

Days a f t e r harvest

FIG.12. Changes in sugars, total soluble solids (T.S.S. ), alcohol-insoluble residue (AIR), and titratable acidity in Alphonso mango during ripening at 28°C. ( A ) 1, glucose; 2, fructose; 3, sucrose. ( B ) 1, T.S.S.; 2, AIR; 3, acidity. From Subramanyam et al. ( 1972a).

Studies of the effect of Krebs cycle acids such as succinate, malate, and fumarate on mango tissue respiration showed that succinate was toxic a t 0.05 M concentration at the senescent stage, - whereas malate and fumarate were not toxic even a t concentrations of 0.1 M (Subramanyam et al., 1972a).

262

H. SUBRAMANYAM E?' AL.

-a3 a

0

0

3.6

P

; c 0 c al 0

0

0.4 L

0

al c

>

0.2

g

a

F 4

8

(2

Days a f t e r harvest

FIG.13. Changes in 0x0 acids during ripening of Pairi mango at 28°C: 0-0, oxaloacetic acid; 0- - - 0, a-oxoglutaric acid; 0- . - * - . - 0, pyruvic acid. From Shantha Krishnamurthy et al. ( 1971).

3. Proteiiw and Amino Acids 'The free amino acid content of four varieties of mangoes by partition chromatography indicated the presence of glutamate, aspartate, alanine, glycine, serine, and y-aininobutyric acid ( Govindarajan and Sreenivasayya, 1950). Mango, like other fruits, is not a rich source of protein. Maximum protein in mango was 1.57 to 5.427" in the Peruvian variety La Molina (Jain, 1961). Generally in a majority of cultivars it varies from 0.5 to l.O<%. Although the enzyme activity has been shown to increase during the ripening process, simultaneously there was no net increase in protein content ( Shantha Krishnamurthy and Subramanyam, 1970a). This is perhaps due to the possibility that the existing protein may become active or the increase in enzyme protein may not be very significant. However, this subject is a controversial one, as there are evidences for and against protein synthesis in banana fruit by Brady et al. (1970) and Sacher (1966). Frenkel et al. (1968) have shown directed synthesis of malic enzyme required for ripening in pears. Shantha Krishnamurthy et al. (1971) identified twelve amino acids in Pairi cultivar by paper chromatography, only six of which are present

MANGO FRUIT

263

in high concentrations. During their studies on amino transferase, concentrations of aspartic, glutamic, and y-aminobutyric acids were determined during the ripening period. Aspartate and glutamate concentrations were high in the early part of storage period but showed a sudden fall at the climacteric peak (Fig. 14). y-Aminobutyrate increased throughout the ripening period. Further examination of Alphonso mango by automatic amino acid analyser revealed the presence of seventeen amino acids, six of which are present in large amounts (Shantha Krishnamurthy and Subramanyam, 1973) (Table VII). The relationship among the 0x0 acids, amino acids, and enzymes in mango is schematically represented as shown. Aspartate

+ a-Oxoglutarate

-

GDC

I

Malate

+ Pyruvate

Glutamate

+ con

J

y-Aminobntyrate

0

+-

Oxaloacetate

AGT

+

COn

I

I

I

4

6

12

Days a f t e r harvest

FIG. 14. Changes in free amino acids during ripening of Pairi mango at 28°C: aspartic acid; 0- . - - 0, glutamic acid; 0- - - - 0, y-aminobutyric acid. From Shantha Krishnamurthy et al. ( 1971).

0-0,

-

H. SUBRAMANYAM ET AL.

264

TABLE VII FREE AMINOACIDSOF MANGOFRUITa Micromoles/100 gm of pulp wAmino acids

Aspartic Threonine Serine GIutamic Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine y-Amino-n-butyric acid a

Unripe (at harvest)

Edibleripe

134.42 11.20 23.21 68.04 9.69 2.74 51.10

3.52 7.53 14.76 39.90 12.43 27.01 126.00 12.11 Trace 5.86 8.74 2.81 6.25 Trace Trace Trace 139.54

-

2.74 2.30

-

17.15 5.45

-

63.75 79.61

From Shantha Krishnaniarthy and Sitbramanyam ( 1973).

4. Pigments The emphasis on factors of quality has led to studies on pigments because of their obvious effect on eye appeal. The color of mango fruit is essentially due to carotenoids and chlorophyll. Yamamoto et al. (1932) isolated carotenes from mango fruit for the first time and concluded that they are a mixture of a- and p-carotenes. Ramasarma and Banerjee (1940) followed the changes in carotene content during ripening, and later Ramasarma et al. ( 1946) crystallized p-carotene from Badami (syn. Alphonso) mango fruit. The effect of ripening and of differences among the varieties on carotene content has been studied by Sadana and Ahmad (1949) and by Chowdhury (1950). According to Chowdhury (1950), the total carotenoid and the individual pigments rise rapidly to a maximum and then fall. At temperatures above 36"C, these changes are accelerated without affecting the maximum value for carotene. Exposure to ultraviolet light increases the carotenoid content during the ripening period.

MANGO FRUIT

265

Jungalwala and Cama (1963) characterized the carotenoids of mango and studied their quantitative distribution. According to them, mango fruit pulp contains sixteen different carotenoids (Table VIII) . Luteoxanthin and violaxanthin, which are rarely found in fruits, are present in significant amounts in mango. About 60% of the total carotenoids consists of ,&carotene. Modi and Reddy (1967) studied the carotenogenic pathway in Alphonso mango. The utilization of geraniol and farnesol for carotene formation in the cell-free extracts and of geraniol as an intermediate in carotenogenesis in mangoes has been demonstrated. Further studies by Mattoo et al. (1968) indicated an increased concentration of mevalonic acid (0.5 to 3.0 pmoles) and geraniol (0.0 to 0.5 pmole) in the preclimacteric fruit and 5 to 10 pmoles and 1 to 5 pmoles, respectively, in the ripe fruit. This is also accompanied by increased activity (twofold) of the enzyme phosphatase. They concluded that carotenogenesis in the ripe fruit is regulated by phosphatase which seems to dephosphorylate the intermediates of carotenogenic pathway. Jacob et al. (1970) noticed a tremendous increase in carotenoid content during ripening. Fifteen different carotenoids were present in unripe TABLE VIII

RELATIVE AMOUNTSOF INDIVIDUAL CAROTENOIUS IN FULLYRIPE ALPHONSO MANGOPULPa Carotenoid Carotene hydrocarbons Phytoene Phytofluene cis-p-Carotene p-Carotene y-Carotene Oxycarotenoids 5,6-Monoepoxy-/3-carotene

Mutatochrorne Crypotoxanthin Violaxanthin cis-Violaxanthin Antheraxanthin cis-Antheraxanthin Zeaxanthin Luteoxanthin Mutatoxanthin Auroxanthin a

From Jungalwala and Cama ( 1963).

THE

Percent of total carotenoids present

3.70 6.89 0.36

59.50 0.01 0.85 1.52 0.66 1.25 9.02 1.01 0.50 0.01 11.25 0.76 2.71

266

IT. SUBRAMANYAM ET AL.

mangoes, fourteen in partly ripe fruit, and seventeen in fully ripe fruit. Carotene constituted the major carotenoid pigment in unripe (37% ) and fully ripe ( 5 0 % ) mangoes. Total carotenoids showed a twentyfold increase and p-carotene a tenfold increase during ripening of Alphonso and Pairi mangoes (Fig. 15). The ratio of these two varied widely, depending on the variety, soil condition, maturity, and date of harvest in the same orchard (Subramanyam et al., 1972a). A single anthocyanin pigment has so far been isolated from the skin of the fruit; it has been identified as peroxidin 3-galactoside which is known to be present only in red cranberry ( Proctor and Creasy, 1969). 5. Cell Wall Constituents

Although the change in the texture of the mango fruit during ripening is a drastic one, little attempt has been made to study the pectic con-

stituents which are mainly responsible for the texture. Other cell wall components are also not known. Generally, there is a decrease in the molecular size and esterification of pectin during ripening of fruits. Dennison and Ahmed (1967) studied the changes in the pectic constituents of Kent mango in relation to storage by irradiation. Irradiated fruit con-

FIG.15. Development of carotenoids in Alphonso mango during ripening at 28°C: 1, total cartenoids; 2, p-carotene. From Subramanyam et al. ( 1972a).

MANGO FRUIT

267

tained higher water-soluble and lower versene-soluble pectic fractions than did untreated fruit. Rolz et al. (1971) studied the texture of the Mamey mango in terms of firmness. Overall changes in cell wall material are determined by the alcohol-insoluble residue which showed a gradual decline during the ripening process. The uronic and carbonyl contents of the water-soluble fraction of pectin increased to a maximum when the fruit was mature (ripe), then decreased as overmaturation ( overripening ) started at ambient storage conditions. These values have been compared with those for fruits stored at low temperature (between 8" and 10°C) for 27 days and for fruits removed from cold storage after 17 and 20 days to ambient conditions, in which case the ripening process is reported to be totally abnormal. The authors (Rolz et nl., 1972) added an element of confusion by using the terms maturc imd overmature to refer to the ripe and overripe condition of the fruit, since it i s helieved that the maturation process is complete while the fruit is on the tree and the ripening syndrome set in when the mature fruit is removed from the tree. Recent investigations of cell wall constituents in Alphonso and Pairi cultivars by Mizuta and Subramanyam ( 1973) indicate that water-soluble pectin (high methoxyl ) increased; the hexametaphosphate fraction ( calgon, low methoxyl) increased up to 6 days and declined later; and the hydrochloric acid-soluble fraction ( protopectin) decreased during ripening at ambient storage. The total pectin content increased gradually with the onset of ripening in both cultivars, and all the fractions were found in higher concentration in Alphonso than in Pairi. The inherent viscosity of the first two fractions decreased with ripening, whereas the viscosity of the acid-soluble fraction at 85°C increased. The holocellulose and the cellulose content did not change appreciably during ripening in both cultivars. The intrinsic viscosity of cellulose was low at harvest, increased slightly with the onset of ripening, and remained steady until the ripening process was completed. The Pairi cultivar had higher intrinsic viscosity of the cellulose than did the Alphonso cultivar. In the above studies, the carbazole assay has been employed for quantitative determination of fractionated pectins, and it is observed that the water-soluble fraction of pectin increases with softening of texture and the onset of the ripening process. An increase in the water-soluble fraction of pectin, a decrease in the calgon-soluble fraction and protopectin, followed by a decrease in the inherent viscosity of the soluble-fractions during ripening, suggest that the bonding strength of protopectin diminishes, owing to decomposition, de-esterification, and depolymerization. However, an increase in the viscosity of the protopectin fraction at 85°C:

268

H. SUBRAMANYAM ET AL.

suggests release of low-molecular-size substances in the earlier stages of ripening; high-molecular-size substances are released with the onset of ripening, and the converse is true in the soluble fractions. Cellulose and hemicellulose appear to have an insignificant role in textural changes during ripening of this tropical fruit. 6. Tannins Das Gupta et al. (1955) made a preliminary study of the deposits in the necrotic tissue of mango. They suggested that the deposits belong to a group of tannins, phlobatannins, or phlobaphenes or their derivatives. Soule and Harding (1956) made a quantitative study of total tannins in Florida mangoes. They varied from 31 to 76 mg in green fruit and from 31 to 75 ins in ripe fruit. Jain (1961) found 160 mg in pulp and 105 mg in the skin. Recent studies on the nature and chemistry of tannins in mango have revealed the presence of gallotannin, a toxic substance, in significant amounts in the unripe fruit ( E l Ansari et al., 1971). Further analysis of these gallotannins was made by El Sissi et al. ( 1971) in Pairi and Rumani mangoes.

7. Vitamins The vitaminic vaIue of mango fruit lies mainly in its vitamin C (ascorbic acid), vitamin A (carotene), and small amounts of vitamin B group. The vitamin B, (thiamine) content of two varieties of Florida cultivars has been reported by Stahl (1935) as 35 and 60 pg per 100 gm of fresh weight, and the vitamin B, (riboflavin) content of three varieties as between 45 and 55 pg per 100 gm. Quinones et al. (1944) have given 0.057 to 0.060 m,o per 100 gm as the thiamine content and 0.037 to 0.073 mg per 100 gm as the riboflavin content of four varieties of Philippine mangoes. Ghosh (1960) found 36 pg of folic acid in 100 gm of green mangoes. Floch (1958) has shown that Guiana fruits are rich in carotene-8000 I.U. per 100 gm of pulp. According to Chavez and Jaffee (1965), Venezuelan mango contains vitamin C in amounts varying from 5 to 70 mg per 100 gm of pulp, depending on the variety. Among thirty varieties of Puerto Rico mangoes studied by Iguina de George et al. (1969), the vitamin A content varied from 5000 to 7900 I.U. per 100 gm of pulp. Julie and Francisque mangoes had the highest vitamin C content, ranging from 6 to 63 mg per 100 gm in the fully grown fruit, providing the minimum daily requirement with 200 gm of pulp. The vitamin

MANGO FRUIT

269

C concentration was higher in the peel than in the edible portion (Spencer et al., 1956). Miller et al. (1956) studied the changes in ascorbic acid content during ripening in mangoes while comparing the vitaminic values of foods used in Hawaii. According to a comparative study made by Vilar (1962), mango is the richest source of vitamin C among various fruits and vegetables sold in the Angolan market. 8. Minerals

Minerals are important for the various metabolic activities of the living tissue and even more so far the fruit, which exhibits tremendous activity during the ripening process. Singh (1960) has tabulated the mineral content of mango pulp as percentage of ash. Singh (1962) estimated the mineral content of thc flesh, peel, stone, kernel, seed coat, and the whole mango fniit. The flesh contained 507:,of the total mineral content with the exception of calcium. The main minerals were sodium, phosphorus, potassium, and magnesium. Mattoo and Modi (1969b) estimated the mineral content (sodium, potassium, and calcium ) in the chill-injured mango tissue in both the free and total forms. An increase in concentration of all three minerals was noticed in the injured tissue. This is due to accumulation of minerals either by release from the cell membranes or by translocation from other parts. Minerals were estimated in the fruit tissue of Alphonso mango in order to determine causes for physiological breakdown. There was no significant difference in the concentrations of sodium and potassium, but calcium content was lower and phosphorus was higher in the diseased tissuc than in normal tissue (Subramanyam et al., 1971). 9. Fatty Acids Cholap et al. ( 1971) analyzed the oil from ripe Alphonso mango pulp. According to this study, it contains a triglyceride consisting of myristic acid, palmitic acid, palmitoleic acid, stearic aid, oleic acid, and linoleic and linolenic acids. 10. Flavor Constituents According to Soule and Harding (1957) peak flavor development is noticed on the sixth day of ripening. The actual measurement of flavor constituents has not been attempted so far. Pattabhiraman et al. (1968, 1969) extracted odor constituents from Alphonso mangoes using chloro-

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form and ether. For this extraction, mango pulp was made into a slurry with water and passed through a cyclone separator. The odor constituents were condensed by a steam stripping technique in a trap kept in chilled water. The chloroform extract of this condensate possessed the characteristic mango flavor. The odor constituents were susceptible to heat and were separated by column, thin-layer, and gas-liquid chromatography. These workers found that carotene modified the overall aroma. Spectroscopic and chromatographic analyses of the extract indicated that odor ingredients are esters and carbonyl compounds, but no definite identifications of individual compounds were made.

D. SUMMARY Changes in respiratory drifts after harvest have been followed in some cultivars of manso, and an attempt has been made to establish a relation between respiration rate and the major chemical constituents of the fruit. The magnitude in respiration differs considerably among cultivars, in addition to depending on the physiological age of the fruit. However, the data available give a disjointed account of changes in chemical constitutents and the related enzymes that are required for ripening, except for a recent report from the authors’ laboratory on the development of a malate decarboxylating system and of transaminases during ripening. Information on the changes in the Krebs cycle intermediates and amino acids which are essential building blocks for synthesis of specific proteins during ripening in mango fruit has not been elucidated. The new concept of protein synthesis or protein turnover in relation to enzyme requirements during ripening has evoked considerable interest in recent years, and such a possibility has been demonstrated in pomes (Hulme et al., 1971; Frenkel et d., 1968), avocado (Richmond and Biale, 1966), and banana (Brady et al., 1970). Studies on energy metabolism and cell membrane permeability associated with fruit ripening would contribute to an understanding of the mechanism of this respiratory climacteric. The precise function of ethylene in fruit ripening needs to be investigated in greater depth, since this gas at very low concentrations is known to initiate the sequence of biochemical reactions and to regulate ripening. The reports on biosynthesis of pigments and odorous constituents during ripening of mango fruit are of a very preliminary nature. It is anticipated that information on these complex biochemical changes will be available in the near future, since many groups are actively associated with the physiology of this tropical fruit.

MANGO FRUIT

IV.

27 1

STORAGE AND TRANSPORT A. STORAGEDISEASES

Fruits, being perishable, undergo heavy losses during handling, transport, and storage. Mango fruit is susceptible to various diseases caused by fungi, bacteria, and physiological factors in the fruit tissue. Among these, fungi constitute the major group of pathogens responsible for fruit rot diseases. Although a number of fungi have been reported to bc associated with different parts of thc mango plant, only a few arc pathogenic to mango fruits and caiisc considerable damagr to them in storage and market (Table I X ) .

1 . Antlwmxose One of the most serious diseases is caused by the fungus Colletotrichum gloeosporioides Penz. It is widely prevalent in all the mango-growing regions of the world. Various manifestations of the disease on the mango include blossom blight, leaf spot, fniit nisseting or staining, and fruit rot. The severity of the injury depends mostly on the humidity of the TABLE IX STORAGEDISEASES OF MANGOES ~~

Type of spoilage

Decay percentage

Stem-end rot

15-20

Anthracnose Lateral rot

10-15 3-5

Tip rot Sooty mold

1-2 50-60 in coastal areas 3-5

Soft rot Black spot Spongy tissue Black tip Soft nose

3-5 3555 ( in Alphonso ) 10-15

10-15

Organisms or causes responsible for decay Gloeosporium mangiferae P. Henn Botryodiplodia theobromae Pat Diplodia natalensis Pole-Evans Cohtotrichiim gloeosporioides Pen2 Aspergillus niger van Teigh Phomopsis sp. A ~ p e ~ g i l l usp. s Meliola mangiferae Earle Bacillus carotoyonus Patel Pseudomonas mangiferae indicae Patel Physiological; causes not known

Physiological, brick kiln contamination Calcium deficiency

272

H. SUBRAMANYAM ET AL.

atmosphere. Most of the infection takes place during the blossoming period and remains in the developing fruit as latent infection. These latent infections become active and serve as centers of decay when the fruit approaches maturity. During ripening these spots develop rapidly, causing considerable loss in transit and storage. Symptoms of the disease are circular or irregular large spots, dark brown in color, which gradually enlarge until they cover the whole fruit.

2. Aspergillus Rot This rot is caused by Aspergillus niger van Tiegh and is seen as stalkend rot and lateral rot. The extent of the lesions depends on the area of injury of the fruits. The disease usually appears a t the stalk end of the fruit. At first, it appears as a light-brown circular patch. The lesion grows in a regular manner and forms a large, circular spot encircling the stalk. After 3 to 4 days, the infection reaches an advanced stage and the affected region becomes sunken and crumpled. 3. Stem-End Rot Stem-end rot of mango fruit is a common postharvest disease in many mango-growing countries. The condition is caused by the organism Diplodia natalensis ( Srivatsava, 1972). The disease manifests itself only in the ripe mango. In the initial stage, the epicarp darkens around the base of the pedicel. Later, the affected area enlarges to form a circular, brownish-black patch which extends rapidly within 2 to 3 days in a humid atmosphere. Fruits without pedicels are more susceptible to disease than those with pedicels. Srivastava et al. (1965) have studied in detail the fungal disease of mango caused by Aspergillus, Botryodiplodia, and Colletotrichum sp. The percentage losses due to these three pathogens in seventeen varieties of mango from various localities have been recorded by them. The variations in percentage loss in different varieities as well as the differences in the same variety at different places arc attributed to a number of factors, especially environmental ones. 4. Bacterial Rot Bacterial rot of stored mangoes has been reported in various countries. Pate1 et al. (1948) reported the occurrence of a bacterial disease, the symptoms of which are similar to those of black spot disease caused by Bacillus mangiferae in South Africa. The pathogen was named Pseudo-

MANGO FRUIT

273

m o m mangiferae indicae sp. nov. Infected fruit shows deep, longitudinal cracks with a heavy, gummy exudation. Pate1 and Padhye (1948) observed another bacterial soft rot disease caused by Bacterium caratovorum. Sundararaj et al. (1972) reported bacterial rot in stored Bangalora and Neelum mangoes. The infected fruits develop light-brown, water-soaked areas initially. Later, these areas enlarge, covering the whole fruit in 2 to 3 days. The area is soft to the touch, depressed, and light-brown in color. In advanced stages it exudes a gummy fluid and the epidermis of the fruit comes off as a thin papery covering. Polyethylene film covers which were reported to delay ripening and extend the market life were seen to promote rapid development of bacterial rot. Treatment with antibiotics ( tetracycline, oxytetracycline, and chloramphenicol) were not effective, although the incidence of the disease was lessened.

5. Black Tip Among the physiological diseases, black-tip necrosis of mango is another common condition prevalent in India and Florida. Orchards near a brick kiln suffer heavy losses every year. The first symptom of the disease is the appearance of a small etiolated area at the distal end of the fruit after 3 to 4 days of fruit-setting. This area gradually increases in size, and the tip becomes necrotic, often exposing the stone of the fruit as a result of disintegration of outer tissues. Affected fruits do not mature properly, and the tip becomes hard and black. This is common in many varieties of mango. The disease has been given various names, such as necrosis of mango fruit (Das Gupta et al., 1955), black-tip disease and tip pulp of mango (Das Gupta, 1958). Prasad and Singh (1965) reported that this disease can be controlled by regular sprays of boron after the flowering stage. Nauriyal et al. (1972) suggest that spraying mango trees with aqueous solutions of sodium hydroxide and sodium carbonate minimizes losses due to black tip. They claim that these alkaline solutions neutralize the acidic reaction of the toxic brick-kiln fumes. 6. lnternal Breakdown

This physiological disease is thought to be due to internal factors of the fruit. Young (1960) has reported “soft nose,” a physiological disorder of Florida mangoes. The symptoms are the breakdown of the flesh on the ventral side and toward the apex in the fruit while it is still on the tree.

274

H. SUBRAMANYAM ET AL.

Investigations during several seasons have fairly well established the disorder to be physiological. In Haden mango there is a yellowing of the green skin at the apex, and the area becomes soft. The tissue will be overripe when compared with the surrounding tissue, and it is bitter in taste. In advanced cases the tissue becomes a spongy, greyish black mass. The nature and mechanism of breakdown are not known. Fertilizer trials with nitrogen, phosphorus, potassium and calcium have been made to control this disorder (Young and Miner, 1961). In India, another physiological disorder is noticed in Alphonso, which is an important commercial variety (Subramanyam et al., 1971). It is called internal breakdown, spongy tissue, or soft center. So far it has been observed only in this cultivar. Externally, the fruit appears to be sound, and the disorder is noticed only when the fruit is cut into halves. It is observed only in semiripe and ripe fruits. The breakdown tissue is characterized by its pale yellow color; the tissue is soft or spongy, with or without an off-flavor. The condition starts with the tissue adhering to the stone portion and gradually spreads to the periphery. In extreme cases, the whole fleshy portion becomes too soft, resembling bacterial rot. (Fig. 16). The causative factors for the onset of this breakdown and its control are not clear. A recent survey (Subramanyam et al., 1971) regarding the prevalance of this disorder indicated that abount 25 to 30% of the mangoes (Alphonso) from different areas are subject to this disease, and remedial measures have still to be investigated. Chatpar et al. (1972) have observed that spongy tissue is associated with spore-forming bacillus, since they could cause spongy tissue to develop artificially by inoculation of the spore suspension into fresh and healthy mangoes. The basis of selection of healthy mangoes for their inoculation studies is not clearly known, since the breakdown appears inside the semiripe or ripe fruits. Moreover, low pH, high acidity, and phenolics in the flesh tissue at harvest appear to be highly unfavorable for the growth of bacillus. However, it is likely that the physiological abnormality in the tissue predisposes the fruit to secondary bacterial infection at the semiripe or ripe stage.

B. POSTHARVEST TREATMENTS Various methods have been adopted to extend the storage life, reduce the losses, and improve the quality of mango fruit. These can be classified as physical and chemical methods, which include storage of fruits at low temperature, controlled atmosphere storage, irradiation, heat treatment, and other chemical treatments.

MANGO FRUIT

275

FIG.16. Internal breakdown, a physiological ripening disorder in Alphonso mango. From Subramanyam et al. ( 1971).

1. Low-Temperature Storage Extension of storage life by maintaining a cool temperature is based on the fact that the respiration rate will be lowered, which in t u n reduces the rate of metabolic activity. Thus, the ripening rate is slowed down, resulting in extension of storage life. The keeping quality of the fruit is an important factor to be considered in selecting the varieties for storage. Storage life also depends on other factors such as the stage of maturity, the method of packing and the packing materials used, and the duration of storage. Ram Ayyar and Joshi (1929) kept ripe fruits for 3 weeks a t 10°C (50°F) and partially ripe ones for 6 weeks. When unripe ( green) mangoes were stored at this temperature, they ripened unevenly, owing to

276

H. SUBRAMANYAM ET AL.

the fact that the ripening enzymes are apparently inactive after 3 weeks. Work done in this connection at the low-temperature research station at Trinidad (Wardlaw and Leonard, 1936) revealed that most of the commercial varieties were susceptible to low temperature and developed chilling injury. Cheema et al. (1939) studied the behavior of several (twenty-eight) Indian varieties and found that the Alphonso variety of Bombay kept best. The determination of the temperature and the duration of storage are the most important factors governing cold storage. Mathur et al. (1953) studied the storage temperatures for Seedling, Peter, and Alphonso mangoes. They observed that Seedling and Peter mangoes can bc stored for 42 days in a fresh condition at 5.5" to 7.2"C (42" to 45"F), while Alphonso can be stored for a maximum of 28 days at 8.3" to 10°C (47" to 50°F). They have also shown that ripe Alphonso mangoes can be stored at 5.5" to 7.2"C (42" to 45°F) for 28 days. Similar studies by Singh et al. (1954) for Totapuri mangoes revealed that at a temperature of 5.5" to 7.2"C (42" to 45°F) and R.H. 85 to 90% the fniit could be stored for 7 weeks. Gandhi (1955) found a temperature of 7.2" to 9°C (45" to 48°F) to be suitable for storage of mature Alphonso mangoes for 7 weeks. At a lower temperature the fruit is injured and it fails to ripen properly when shifted to room temperature. Mukherjee (19%) suggests a safe storage temperature of 9°C (48°F) for Langra, Dashehari, and Chowsa mangoes grown in North India, since the critical temperature for development of chilling injury is 7.2"C (45°F) or lower. Krishnamurthy et al. (1963) recommend a temperature range of 7.2" to 9°C (45" to 48°F) and R.H. of 85 to 90% for all varieties of mango. Ripe Haden mangoes can be stored at a temperature range of 2" to 13°C (35.6" to 5 4 ° F ) . Temperatures not exceeding 7°C (44.6"F) are recommended because higher temperatures reduce the storage life from 4 weeks to 1 week (Akamine, 1963). Vickers (1964) has also suggested low-temperature storage for mangoes grown on the Kenya coast. According to Hatton et al. (1965), Florida mangoes (unripe) stored at 1.6"C (35"F), 4.4"C (40"F), and 7.2"C (45°F) developed chilling injury, and hence storage at these temperatures was not successful. The optimum storage temperature suggested was 12.7"C (55"F),at which fruits could be kept for 2 to 3 weeks. With longer storage periods, excessive decay and softening during storage were observed. Mattoo and Modi (1969b) reported that the optimum storage temperature for Alphonso mangoes was 5" to 10°C (41" to 50°F). Thompson (1971b) examined optimum refrigerated storage conditions for several varieties of mangoes grown in the West Indies. An interaction between minimum temperature tolerance

MANGO FRUIT

277

and stage of physiological maturity was noticed. Low-temperature injury occurred on immature fruits, at temperatures below 10"C, but it did not become obvious until temperatures were below 5°C. Wrapping the fruits in polyethylene bags delayed ripening of fruits at higher temperatures, but had less effect at lower temperatures. Anthracnose disease was related to place of harvest and storage temperatures. Julie and Ceylon varieties harvested at stage B maturity (Wardlaw and Leonard, 1936) could be held at 7°C for 3 to 4 weeks, and the flavor of the ripened fruit was reported to be consistently good. Valmayor (1972) recommends a temperature of 10°C (50°F) for Carabao and Pic0 mangoes. Ripe fruits could be stored for 18 to 21 days, and freshly picked mature green fruits for 23 to 26 days. These studies on the storage of mangoes from different countries revealed that they are very susceptible to low-tempcrature injuries, collectively known as chilling injuries. Such injuries manifest themselves as definite skin blemishes, failure to develop normal color on ripening, failure to ripen on removal from cold storage, and a marked decline in resistance to the attack of pathogens. Physiological and biochemical changes during cold storage of mangoes have been studied in detail by Singh et al. (1954). According to them, acidity was high, total soluble solids were less, and ascorbic acid content was greater in the coldstored fruit. They observed that the rate of respiration was very low and the climacteric peak was reached earlier at higher storage temperatures. Loss in weight was less, and the percentage of wastage during different weeks is also indicated. However, studies on respiration by Singh et al. (1954) are not clear, since measurements of respiration were made at a higher temperature than that found in storage conditions. Mattoo and Modi (1969b) studied in detail the changes in sugars, enzymes, and minerals in the chill-injured mango tissue. The development of chilling injury in mango peel, as in the pulp, was marked by a significant decrease in the soluble sugar content, no significant change in the total hexose content, and less starch breakdown. Ivertase activity increased, whereas that of amylase decreased. Mango invertase showed two temperature optima-one a t 0°C and the other at 37°C (Chatpar et al., 1971). Rolz et al. (1971) observed that the best storage temperature for Mamey mango is 13°C (55°F) with a storage life of 2 to 3 weeks. At temperatures of 8" to 12°C there was no chilling injury, but such fruits did not ripen normally when removed to an ambient condition of storage. They further observed that the amount of insoluble pectic material remained higher than the amount of soluble material, resulting in a hard texture of the fruit.

278

H. SUBRAMANYAM ET AL.

Pilot studies on storage of Alphonso mango at 13°C (55°F) under a forced-air circulation system to remove the accumulated volatiles and excessive CO, in the storage atmosphere have also not been successful in improving poststorage ripening quality, although there was no evidence of chilling injury (Lakshminarayana and Subramanyam, 1971) . Intensive studies on biochemical constituents have been followed by Shantha Krishnamurthy and Subramanyam ( 1973). Changes in carotenoids and other chemical constituents during cold storage are indicated in Fig. 17. A typical respiratory pattern obtained for fruits stored at low temperature is shown in Fig. 18.

2. Controlled Atmosphere Storage Controlled atmosphere storage, either alone or coupled with cold storage, has been recommended for various fruits and vegetables. Singh et al. (193713) examined the response of the respiratory system in mango to alterations in the concentration of oxygen and nitrogen with a view to suggesting controlled atmosphere storage for this fruit. Date and Mathur (1958) indicated that the life of mangoes is 6 to 10 weeks at 45" to 47°F; with gas storage, the life can be extended to 10 to 16 weeks. They also examined the optimum maturity of the fruit for gas storage. They recommend that Alphonso mangoes with a 7.7-cm diameter and Pairi with a 6.9-cm diameter are most suitable for gas storage. Kapur et al. (1962) have shown that Alphonso mangoes can be stored for 35 days at 8.3" to 10°C (47" to 50°F) and Pairi mangoes for 49 days at 5.5" to 7.2"C (42" to 45°F) within 10% wastage level under refrigerated gas storage. Burg and Burg (1966) found that the storage life of several tropical fruits including mango can be prolonged under subatmospheric air pressure, since this procedure accelerates the escape of ethylene from the fruit tissue and also reduces the oxygen tension in the atmosphere, thereby lowering the sensitivity of the fruit tissue to ethylene action. Hatton and Reeder (1967) noted that mango fruits stored in an atmosphere of increased CO, and reduced O2 for 20 days at 13°C (55°F) required more time to soften than did normal fruits when ripened at 21°C ( 7 0 ° F ) . A report by Lakshminarayana and Subramanyam ( 1970) on storage of mango fruits in static atmospheres of 5%, 1070, and 15% CO, concentration has shown the occurrence of fermentative decarboxylation with accumulation of acetaldehyde and alcohol in the tissue. Accumulation of these toxic products was greater at higher CO, concentration in the storage atmosphere. However, this study has to be confirmed under a continuous gas flow system. Valmayor (1972) has stated

0

30

42 18 24 Days after harvest

6

Days after harvest

2,500

a

C

2,000

FIG. 17. Changes in sugars, total soluble solids, alcohol-insoluble residue, titrable acidity, and carotenoids in Alphonso mango during storage at 10°C. ( A ) 1, glucose; 2, fructose; 3, sucrose. ( B ) 1, T.S.S.; 2, acidity; 3, AIR. ( C ) 1, total carotenoids; 2, p-carotene. From Subramanyam et d.( 1972a).

0

P

\

rn 1,500

?

0 .0

2 4,000

?

V

500

0

6

12

48

24

Days after h a r v e s t

30

H. SUBRAMANYAM ET AL.

280

140 019p I \

120 L

loo-

0;

‘\O

I I

“8

r m

>

I

80-

0

E

h’.o 2

I

0

I

60 40 -

I

I I

I I

that Philippine mangoes can be stored for 40 to 45 days in a storage atmosphere of 5% CO, and 5% 0, at 10°C. 3 . 1wadiation

Storage and preservation of mangoes by means of radiation has been attempted. Mathur and Lewis ( 1961) studied the effect of y-rays (12 X lo3 rads) on Alphonso mangoes using cobalt 60 at a dose rate of 120 rads/min. Retention of moisture was greater in irradiated fruit, and nonreducing sugars and ascorbic acid content were less than in the control. Treated mangoes were stored for 24 days in sound condition, as compared with 16 days at 23” to 39°C for control fruits. Hatton et al. (1961) studied the effects of 7-radiation on the market life of Florida mangoes, the rate of ripening, and the control of anthracnose decay. Radiation doses ranged from 10,OOO to 20,000 rads. Most radiation doses retarded ripening, and radiation seems to influence the anthracnose decay indirectly through its effect on rate of ripening. In Hawaii, Haden and Pairi mangoes were exposed to 7-radiation in an attempt to destroy the seed weevil (Upadhya and Brewbaker, 1966). This treatment extended the shelf-life by 100% at room temperature from 2 to 4 days to 4 to 8 days, and from 4 to 7 days to 10 to 14 days at 9°C. The treatment also reduced weight loss during storage. Off-flavors developed only at a dose rate of 150 krads or more.

MANGO FRUIT

281

According to Dharkar et al. (1966a), the extension of the storage life of unripe, mature Alphonso mangoes can be achieved at a dose of 25 krads when the fruit is irradiated under air or N, or CO,. A nitrogen atmosphere was more advantageous with respect to good quality and less spoilage of the fruit. Ripening of irradiated fruits was delayed by 6 days. These workers also found that irradiation combined with skin coating had a more beneficial effect on extension of the storage life of mangoes than did irradiation alone ( 1966b). The effect of irradiation on ripening of Kent mangoes was studied by Dennison and Ahmed (1967). The effect on pectic constituents is dealt with in detail. Ali Farooqi and Muhammed (1968) have shown that ripening of hard, green mangoes can be delayed by irradiation from a cobalt 60 source at a dose of 30 krads. Upadhya (1969) studied the biochemical changes induced by the low disinfestation dosages of y-irradiation and discussed the biochemical lesions produced by the irradiation. Dharkar and Sreenivasan ( 1972) tried different dosages of y-radiation, ranging from 12 to 200 krads. They observed a maximum delay in ripening with minimum damage at 25 krads. Storage life was extended by 10 days at 25" to 32°C. These preliminary trials on extension of the storage life of the mango were limited to a few fruits. The effect of radiation on large-scale storage has yet to be determined. This would also involve the economics of the problem which is an important factor in commercial storage. According to Maxie et al. ( 1971), irradiation holds little promise for perishable commodities in terms of economics. The effect of irradiation on the consumer's health should also be looked into.

4. Heat Treatment During recent years, the use of postharvest heat treatments has gained recognition as a promising method of reducing decay in various fruits and vegetables. In mango this method has been used to reduce anthracnose decay (Pennock and Maldonaldo, 1962). A hot-water dip treatment was given for 15 minutes at 124" to 125°F before storage and ripening. According to Smoot and Segall (1963), a hot-water dip of mangoes at 130" to 132.5" 4 for 5 minutes was most effective in controlling anthracnose decay in Florida mangoes. Hatton and Reeder ( 1964) confirmed this observation by trials on a commercial scale. According to them, anthracnose was effectively controlled by a 5-minute immersion in water at 131°F. This was true for eight of nine varieties selected for treatment. Treated fruits developed slightly less skin color than untreated fruits, but the grade and market-

282

H. SUBRAMANYAM ET AL.

ability of the fruit were not affected. These authors suggested that the heat tolerance of a given variety should be determined before commrcial treatment under specific conditions of time and temperature. Tandon and Singh (1968b) have shown that anthracnose disease can be effectively controlled in Langra and Dashehari by treating the fruits in hot water a t 50°C for 15 minutes. After treatment, they stored the fruits for 5 weeks a t 9" to 10°C without affecting the quality of the fruit. Shantha Krishnamurthy and Subramanyam ( 1970b) studied the effect of heat treatment of Pairi mangoes with and without growth regulators. Heat treatment at 52°C k 1" for 5 minutes resulted in uniform and accelerated ripening. Treated fruits were ripe within 10 to 12 days, while the untreated group took 14 to 16 days to ripen. Incorporation of growth regulators such as maleic hydrazide and 2,4,5-TP separately in the hotwater bath counteracted the accelerated ripening without affecting the quality of the ripe fniit. Hot-water treatment has also been reported to enhance surface color and the carotene content of the fruit (Subramanyam and Sebastian, 1970). This effect was more pronounced in fruits of lower maturity than in those of higher maturity (Subramanyam, 1973). There was an overall reduction (from 25% to 5y0) in fungal spoilage in heat-treated fruits. Perhaps the development of fungal infection is delayed if not eradicated completely.

5. Chemical Treatments Various chemicals have been used to control ripening, to improve the color, and to reduce losses during storage and ripening (Table X ) . Major losses due to anthracnose occur. Dharam Vir et al. (1967) have shown that the postharvest dip treatment of mangoes with aureofungin (500 ppm-an antibiotic developed at Hindustan Antibiotics Ltd., Pimpri, India) prolonged their life by 18 to 20 days. This was also used to treat the padding material utilized for packing fruits and to check the development and spread of infections in packed boxes. This treatment inhibited the growth of Diplodia natalensis for a period of 20 to 25 days. Tandon and Singh ( 1968a) used various fungicides including 507::;,copper oxychloride ( Blitox), SOY0 cuprous oxide, zineb, ferbam, captan, ziram, and bordeaux mixture. They found that mango anthracnose can be controlled effectively by spraying the trees with zineb or bordeaux mixture from flowering time to the time of harvesting, and dip treatments had no effect. Exposing the fruits to gases such as NH3, SOr, or CO, or to hot air had little effect in controlling the disease. Subramanyam et al. (1969) studied the effect of various fumigants on ripening and spoilage.

MANGO FRUIT

283

TABLE X POSTHARVEST

TREATMENTS WITH FUNGICIDES AND ANTIBIOTICS TO REDUCE DECAY I N MANGOES( ALPHONSO,P A I R I ) ~

Fimgicides/antibiotics

Benzimidazole ( thiabendazole ) ( 2- ( 4-thiazolyl ) benzamidazole) Benomyl (benlate) (methly-l( butylcarbamoyl) -2-benzimidazole carbamate ) Captan ( N-trichloromethylniercapto4-cycloliexene-l,2-dicarboxi1nide ) Thirani ( tetramethylthiurani disulfide ) Zineb (zinc ethylenebisdithiocarbamate ) Allisan ( 2,5-dichloro-4-1iitroaniline ) Aureofungin (heptaene antibiotic) Streptocycliae Untreated a

Effective concentration (%)

Decay after 16 days of ambient storage

(%)

0.05-0.10

3-5

0.05-0.10

35

0.25-0.50 0.25-0.50 0.25-0.50 0.25-0.50 0.05-0.10 0.05-0.10

7-10 5-7 7-10 8-12 5-10 5-10

-

550

From Bhatnagar and Subramanyam (1971).

Ethylene oxide inhibited ripening, delayed fungal spoilage, and prolonged the storage life. Methyl formate and methyl bromide were effective only under ambient temperature storage conditions. Chloropicrin caused severe injury to fruits. Zineb (0.375%) in hot water as a postharvest dip reduced the spoliage due to fungi and delayed the ripening process in Alphonso and Pairi mangoes. Delayed treatment with sodium diethyldithiocarbamate ( 0.4%) was also effective in reducing spoliage of Pairi mangoes ( Subramanyam and Moorthy, 1973). Subramanyam et al. (197213) studied the effect of fungicides such as captan ( 0.5% ) , a Bordeaux mixture (44-50), and copper oxychloride (cuprovit 8-50) on Alphonso mango as preharvest sprays to reduce spoliage and improve keepins quality. They observed that treatment with captan fungicide was beneficial in controlling spoliage when it was given as a drench spray at the inflorescence stage. Spoilage was reduced from 27% to 9% with the captan spray. In addition, captan-treated fruits were superior with regard to color, odor, flavor, and taste on ripening. Sundararaj et al. (1972) noted that the use of the antibiotics, tetracycline, oxytetracycline, and chloramphenicol did not reduce the bacterial rot of mangoes.

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Growth regulators were used either alone or in combination with skin coating or in hot water to regulate ripening and to improve the quality of the fruit. Date and Mathur (1960) suggested the use of 2,4,5-T (lo00 ppm) as a postharvest dip treatment in aqueous solutions prior to storage at 18" to 29.5"C. The treatment delayed the ripening process, and the quality of ripe fruits at the end of storage was acceptable. Subramanyam et nl. (1962) used 2,4-D1 (20 ppm) and M H (250 ppm) and MH (250 ppm) in combination with fungicidal wax as a coating for Alphonso mangoes to extend their storage life under ambient conditions. Storage life was extended from 6 to 12 days as a result of the treatment, and the spoilage was also minimum in ripe fruits at the end of storage. The respiration rate was reduced from 130 mg to 80 to 85 mg/kg/hr after treatment with the growth regulator. Shantha Krishnamurthy and Subramanyam ( 1970b) examined the effect of 2,4,5-TP and MH as a postharvest dip treatment in hot water on Pairi mangoes in ambient storage. Hot water alone accelerated the ripening process and reduced fungal spoilage. This accelerated ripening was counteracted by incorporation of MH in the dip water, thereby extending the storage life with minimum spoilage. 2,4,5-TP also delayed the ripening process, but did not improve the skin color. These compounds did not alter the chemical composition or quality of the fruit. Further experiments with synthetic growth regulators such as SADH,' ethephon, and chloromequat have shown that fruit quality in terms of carotenoids was improved when these chemicals were incorporated in dip water at 53°C and the fruit was given a momentary dip after harvest (Shantha Krishnamurthy and Subramanyam, 1913). The total and &carotene content in Alphonso mango increased from 20 to 40%:,over that in untreated controls. Skin coatings of various types with an without an added fungicide have increased the storace life of mangoes in nonrefrigerated storage to a considerable degree. Bose and Basu (1954a,b) used molten paraffin wax as a coating in an attempt to increase the storage life of Fazli mangoes. Mathur and Srivastava (1955) used water wax emulsion and a refined mineral oil on three varieties of mangoes to extend their keeping quality. Wax coating as an aqueous emulsion increased storage life by SO:, and reduced respiration and transpiration rates. Partial coating of thc fruit top (stalk-end) with mineral oil had a similar effect, but coating of the entire surface with mineral oil resulted in skin injury and breakdown of the fruit. Mathur and Subramanyam (1956) examined the use of fungicidal wax emulsion as a skin coating on Alphonso mangoes; 2,4-D: 2,4-dichlorophenoxyaceticacid. SADH: succinic acid 2,2-dimethylhydrazide; ethephon: 2-chloroethylphosphonic acid; chloromequat: 2-chloroethyltrimethylammonium chloride. 2

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the treatment containing 2.7% solids increased the storage life from 12 to 19 days in nonrefrigerated storage, reduced the respiration rate, and delayed the climacteric peak by 4 days. The treated fruits were sound and acceptable at the end of storage. In subsequent years, several wax formulations were developed based on sugar-cane wax, carnauba wax, and microcrystalline paraan wax, with and without fungicides, and these were tested on a number of varieties of mango grown in India. The treatment delayed the ripening process and extended storage life by 50 to 7597, in nonrefrigerated conditions. However, development of skin color was delayed, which was not advantageous for immediate marketing. Perhaps the treatment interferes with breakdown of chlorophyll and development of carotenoids in the peel portion. However, Srivastava et al. (1962) claimed that skin coating of mangoes has brought about drastic changes causing a serious biochemical imbalance within the fruit, which, it appears, is highly improbable. Mattoo et al. (1967) examined several postharvest treatments to regulate ripening and to improve the keeping quality of mangoes in ambient and refrigerated storage. Subramanyam et al. ( 1972c) recommended optimum conditions for harvesting, handling, transport, and storage of Alphonso and Pairi mangoes under ambient conditions based on commercial trials. They tried to improve the ripening qualities of mangoes stored at low temperature (12"C, 90 to 95% R.H.) by a brief shock treatment in hot water as a momentary dip. However, the ripe fruits were organoleptically inferior. Several postharvest chemical dip treatments to control low-temperature breakdown were examined, but none were found to be effective in controlling this physiological ripening disorder.

C. PACKAGING Packing of fruits is essential for their safety during transit. In India, baskets made of bamboo with paddy straw as cushioning material are preferred because of their low cost (Naik, 1949). This type of packaging fruits is often found to be unsatisfactory owing to delay in transit and rough handling ( Contractor, 1951) . In view of this, ventilated wooden boxes of different sizes have been recommended for packing mangoes of different varieties ( Cheema et al., 1954; Gandhi, 1955). Ventilated lugs, fiber board boxes, and corrugated cardboard cartons of different dimensions, depending on the variety to be packed, have been used in India, Trinidad, the Philippines, Florida, and Jamaica. Wood wool or excelsior is often used at the bottom and top as a cushioning material (Jain, 1961;

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Ruehle and Ledin, 1960; Srivastava, 1967). Stahl (1951) examined different wrapping materials for mangoes and suggested the use of Vinylite and Pliofihn wrappers for room-temperature storage. Wrapping of individual fruits in tissue paper treated with biphenyl is useful in reducing decay and damage during transit and storage of mangoes. Tissue paper or craft paper lining between layers has also been suggested when fruits are packed in ventilated wooden boxes (Srivastava, 1967; Jain, 1961). Teaotia et al. (1964) tested various packing media for short-term storage and ripening of three varieties of mangoes. Weight losses were found to be minimum, and ripening was complete and satisfactory at the end of the week at room-temperature storage. Lakshminarayana et al. ( 1971) examined bamboo containers and wooden boxes for packing mangoes for inland transport by rail as a commercial shipment (18 metric tons). Paper cuttings, paddy straw, and newsprint were used as cushioning materials. Ventilated wooden boxes with paper cuttings in alternate layers as cushioning materials were found to be the best packing materials for rail shipment of mangoes. Wrapping the fruit in tissue paper or in polyethylene and packing it in ventilated cardboard cartons are suggested for export purposes (Hobson, 1969; Thompson, 1971a). P. K. Mukherjee ( 1972) examined different packages and cushioning material, fruit wrappers, and skin coating for transportation of mangoes by rail and road. Wrapping of fruits individually in tissue paper was found to be superior, since it improved the external appearance and the fruit developed an attractive skin color. Physical and chemical changes that take place in the fruit during packaging, storage, and transport have been examined by the above investigators to correlate the fruit quality with consumer acceptance. Proper packaging of fruits has generally been found to be beneficial, since the moisture loss is reduced, damage due to mechanica1 injury and bruises is minimized, decay due to microorganisms is prevented, and the ripening process is delayed. Some of the flexible packages are semipermeable to gases and moisture. A simplified controlled atmosphere is thus created around the fruit, and the ripening process is delayed.

D. TRANSPORTATION Mangoes are usually transported by road or rail for internal trade. Road transport is preferred over rail in view of the reduction in time for shipment over longer distances and more efficient distribution of the produce. Modes and methods of transport of mangoes in India have been discussed in detail by Mirchandani ( 1963, 1965) and Srivastava (1967).

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Singh (1960) reports the historical development of long-distance transport of mangoes and emphasizes that the first successful shipment of mangoes from India to England was in 1896. During the same period, fruits were shipped in cool chambers from Queensland to England and from Trinidad to England in 1907. New Zealand exported fruits in cold storage at 45°F to England in 1915. Simultaneously, trade in mangoes was attempted with success, and fruits were shipped with great care under refrigeration from the Windward Islands to New York (Anonymous, 1904). Similarly, Carabao and Pic0 varieties from the Philippines were shipped in bulk quantities to the China Coast, Japan, Singapore, and Java (Wester, 1920). These early investigations have led to the development of trial shipments of mangoes on a larger scale from India to England by Cheema and Dani (1934), from the West Indies to England by Wardlaw and Leonard (1936), and from Israel to England and the Netherlands ( Oppenheimer 1947). Air-cooled ventilated wagons have been suggested for long-distance rail shipment of mangoes (Lakshminarayana et al., 1971). The bulk of the raw material in the fresh form is exported from India to the Middle East countries in cargoes at ambient temperature, and a small quantity is airlifted to the United Kingdom and European countries (Indian Institute of Foreign Trade, 1968). Recent investigations indicate that trial shipments of mangoes grown in the Caribbean countries to Britain and Canada by air and refrigerated cargoes show promise (Thompson, 1971a). Refrigerated transport and cool-temperature storage are not recommended at present for Alphonso and Pairi varieties cultivated in India because of the development of low-temperature breakdown in mangoes ( Lakshminarayana and Subramanyam, 1970, 1971). The problems and prospects related to air freighting of tropical fruits have been reviewed (Kay Daisy, 1969) . Emilsson ( 1969) discussed in great detail the problems that arise in long-range transport of tropical fruits in the fresh form and the future scope for large-scale expansion of the world trade for these exotic fruits. From these studies it is concluded that not all varieties are suitable for long-distance shipment, and fruits invariably develop low-temperature breakdown when stored at temperatures below 10°C during transportation. Therefore, refrigerated storage and transport of mangoes should be employed with caution.

E. SUMMARY The principal cause for decay in the mango fruit after harvest and during ripening is fungal infection, and stem-end rot caused by Gloeo-

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sporium mangiferae is predominant in all mango-growing regions of thc world. Other types of decay are localized and appear in some cultivars. Bacterial diseases are endemic in nature. Physiological disorders in commercial cultivars are due either to environmental pollution or to mineral deficiencies in the soil and fruit trees. Epidemiology has to be established and effective prophylactic measures should be adopted to control or reduce the decay in this fruit. With the information now available it is clear that mature, green, firm, unripe mango fruit develops chilling injury or low-temperature breakdown (L.T.B.) in refrigerated storage, Ripe fruits also develop surface discoloration and breakdown when stored at low temperatures. It is desirable to examine the causes for L.T.B. and to work out alternative methods for long-term storage. Studies on preservation by irradiation are too preliminary to permit any conclusion to be drawn on its commercial value, but it seems to offer little promise for the future. The use of paper liners or wrapping individual fruits and packing them in cardboard cartons or polyvinyl crates rather than wooden lugs, which are used at present, appear to be ideal techniques for long-distance transport and export. Air-cooled ventilated wagons or trucks and fast shipping facilities are essential if this highly perishable fruit is to reach the consumer in perfect condition. To achieve this, a concerted effort is necessary.

V.

ECONOMIC ASPECTS A. WORLDTRADE

The mango is relished throughout the world for its succulence, taste, and exotic flavor. Although India is the largest producer, international trade in this fruit is relatively small in view of the several problems encountered during the postharvest stages. Increased emphasis in recent years on overcoming these problems will boost the development and expansion of world trade in this tropical fruit and its products. Data on world trade in mangoes are difficult to obtain because they are combined with other tropical fruits in the foreign trade statistics of most countries. The Philippines and Thailand are the largest exporters of fresh mangoes, and India takes the next place (Table 11). Exports of the Philippines are directed almost exchisively to Hong Kong; the only

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outlets for Thailand are Singapore and Malaysia. About 80% of India’s exports are absorbed by Kuwait, Bahrain, and Trucial Oman States. The United Kingdom and France are the leading consumers of fresh mangoes in Europe. The export of fresh mango from India, which was barely 636 tons in 1965 (valued at Rs. 0.85 million), increased to 1488 tons in 1969-1970 (valued at Rs. 3 million). The target set for 1975-1976 is 10,000 tons ( Bhatnagar and Subramanyam, 1971). Trade of fresh mango in Europe is about 300 tons for EEC (European Economic Community) ; for EFTA (European Free Trade Association), the total is estimated at about 500 tons. For other countries of Western Europe except Spain, which is a producing country, the total imported is not more than 50 tons (Cadillat, 1970). Data for other countries are not available. However, exports of fresh mango constitute only a very small fraction of the production, and concerted efforts must be made if the goals are to be met. It is particularly important to take precautions to attain the high standards of quality and appeal needed for sophisticated export markets. Mangoes have not yet become popular in the European countries; in fact, they are hardly known in most European markets, with the exception of the United Kingdom and France. There are indications, however, that the potential is immense. According to a recent survey of the European Economic Commission, the demand is likely to rise to the level of 50,000 tons in the next few years. The production base in African countries is limited; therefore countries that have the advantage of geographical proximity to the main consuming markets have the greatest scope for expansion of world trade of this tropical fruit. The world production of processed mango products is estimated at about 20,000 tons, of which India’s share during 1969-1970 was 16,000 tons, valued at Rs. 37.5 million. International trade in processed mango products is not likely to exceed 15,000 tons and is dominated by India. Total Indian exports of mango products during 1969-1970 were about 11,000 tons, valued at Rs. 24.5 million. Other exporting countries are the Philippines and the United Arab Republic, accounting for about 1700 tons in 1966. This includes 700 tons of mango slices in syrup and 1000 tons of mango nectar. The export of processed mango products from India increased considerably during 1970-1971, and about 6OOO tons of mango juice alone, valued at Rs. 11.25 million, was exported to the U.S.S.R. in 1971 ( Subramanyam and Bhatnagar, 1972). The total foreign exchange earned from exports of fresh mango and its processed products has increased fourfold in the last five years, owing to the popularity of this fruit in

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E?' .4L

the Gulf countries, the United Kingdom, the U.S.S.R., and some parts of Europe. Mango-based beverages have become popular with Spanishspeaking Latin ethnic populations in the United States. A recent test survey conducted by the Foods Division of the Coca Cola Company in Southern United States clearly indicates a substantial market for mangobased beverages such as Alegre and fruit drink, and this has opened a new outlet for the Indian mango. Sizable quantities of mango solids from the best varieties valued at Rs. 2.5 million were exported for this purpose in 1972, and the future potential appears to be great ( Coca-Cola Company, 1972).

B. SENSORY PROPERTIES Sensory analysis is a branch of analytical science which measures and evaluates the properties of products by one or more of the several human senses. It is an interdisciplinary science involving basic physiology, psychophysics, mathematics, and biochemistry, applied quality evaluation methodology, and very complex correlation studies producing instrumental and chemical results (Tilgner, 1971 ) . The consumer is attracted to a fruit mainly by its physical attributes such as appearance, freshness, color, flavor, aroma, and texture. These physical attributes are influenced by the chemical constituents but properties such as nutritive value are generally of a complex nature and difficult to measure. Very little effort has been made to decide the physical attributes that determine the sensory properties of tropical fruits. The quality of the fruit is judged mainly by its chemical constituents rather than its sensory properties. An instrument to measure certain physical attributes in arbitrary units has yet to be designed. The importance of color measurement in the food industry is well known. The product's appearance is of paramount importance, and color becomes one of the most critical factors, especially in tropical fruits like the mango, which has an attractive yellow to orange shade with a red blush. Fruits are assessed on their visual color, which is a subjective determination; hence, objectivized measurements by different colorimeters or color matching standards and reflectometers are preferred. The light transmittance technique is one of the recent objectivized nondestructive methods being introduced in the U.S. Department of Agriculture for assessing quality in fresh fruits. This method is claimed to be useful for grading fresh fruits and predicting the quality and internal defects in apples, peaches, tomatoes, and potatoes (Birth and Norris, 1965).

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Attributes like flavor and aroma are of utmost importance in determining the quality of food, either fresh or processed, since these properties have an esthetic appeal to the consumer. Flavor analysis is a complex subject, and basic research is needed on the nature of volatile and nonvolatile constituents and their interactions. Flavor also depends on the quality and intensity of the compounds present, It involves psychophysiology of the senses and also human beliefs, fads, and prejudices. In recent years, flavor in foods is assuming greater importance, and it should not be too long before measurements can be made for all the food material consumed by man. Studies on flavor and aroma are very limited in fresh fruits, and especially so in tropical fruits. Specialized odor measurements can be made by means of instruments that complement the human senses and permit necessary correlations to be obtained. An attempt was made by Pattabhiraman et nI. (1968, 1969) to characterize the odorous constituents in fresh and processed mango fruit. Much more work is necessary to correlate these constituents with those that determine flavor and aroma. Textural properties of fruit are measured in terms of hardness, cohesiveness, chewiness, and juiciness, which give the texture profile. These properties are determined to a great extent by chemical constituents such as starch, pectin, fiber, and other cell wall constituents present in the fruit. Analytical methods are available for quantitative measurements of these chemical constituents. Texture of the fruit is often judged by feel or touch, rather than by an objective method of analysis. The texturometer, the tenderometer, the penetrometer, the instron unit, and instruments that measure sonic vibration response are used to determine the texture profile in fresh and processed fruits. ( Finney, 1971a,b; Sobotka et nl., 1972). A correlation between physical and chemical measurements would be more useful in deciding the textural properties of fruits. Such studies have been confined only to temperate fruits; more basic research on the mango is required. Taste is determined by several heterogeneous physical and chemical characters. It is the critical and most important sensory property of foods and the one that determines market quality and consumer preference. Objectivized and instrumental methods have not been developed yet to assess the taste of food, since an instrument cannot replace human senses. The food industry has made phenomenal progress in this direction, and people have been trained to evaluate the taste of specific foods. This is a subjectivized method of analysis with a considerable range of error, but it is the only reliable method to date for assessing taste of foods.

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C. PROCESSING QUALITY The processing quality of the mango depends mainly on the nature of the product to be prepared. As was mentioned in the first section, mangoes are classified essentially into two groups-the fleshy and the juicy types. Fruits for dessert purposes have firm flesh, are free of fiber, and have good color and flavor. Such fruits are preferred for packing in slices. The juicy types are fibrous with a rich color and flavor and are used essentially for nectar or fruit beverages. De Leon and De Lima (1966) examined four varieties and three color stages of Philippine mangoes for canned juice. On the basis of acceptability for canned juicc and the physical and chemical properties of the fresh fruit, they suggest that pH value, total soluble solids, and total titratable acidity of the fresh fruit are better guides for processing. Generally, fruits preferred for processing are ripe and firm,rich in color and flavor, with a high pulp and a high sugar contcnt, low acidity or with a proper blend of acid and sugar. No clear-cut index of fruit quality in terms of chemical constituents has yet been defined for the requirements of the processing industry, since this is in its infancy. Much of the flavor is lost during thermal processing, and no way has yet been found to supplement the lost flavor. Color in mangoes is fairly stable during thermal processing. For pickles and chutneys, which are prepared from raw and unripe material, fruits with less tannins (astringents) and high acidity are preferred, depending on whether the finished product is to be sweet or sour. Czyhrinciw ( 1969), while discussing the technology of tropical fruits, mentioned certain qualities to be considered in the processing of mango fruit, such as juiciness, edible part, specific gravity and porosity of the edible flesh, texture, color, and flavor. However, these qualities differ considerably, depending on the variety, the stage of maturity at harvest, and the degree of ripeness of the fruit. More intensive research is essential in this field.

D. NUTRITIONAL SIGNWICANCE The mango has many uses at all stages of growth and development, whether green, half-ripe, or fully ripe. A number of processed products are prepared from ripe mango, such as slices in syrup, nectar, juice, pulp, jam, jelly, powder, fruit bars, and flakes. Slices in brine and pickles and chutney are prepared from unripe mango. The principal use of the ripe mango is as a dessert fruit. In tropical countries, no other fruit contributes as much toward an adequate diet as mango. The flesh of

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the ripe mango constitutes 70 to 80% of the total weight, and the stone (seed) 7 to 20% of the total weight, depending on the variety. Seedling varieties contain a soft pulp of juicy consistency with a large proportion as stone. Table varieties are firm,compact, and less fibrous, with a low percentage of stone, The main constituents of the fruits are water, carbohydrates, protein, fat, minerals, pigments, tannins, vitamins, and ethereal substances which impart the rich flavor to the fruit. These fruits are classified under the category of protective foods; they owe their nutritional importance chiefly to the presence of minerals and vitamins. Carbohydrates form the major portion (10 to 14%), including sugars, starch, cellulose, and pectic substances. The caloric value of the fruit lies mainly in its sugars, which provide sources of readily available energy ( Gopalan et d.,1971). The average composition of ripe mango and seed kernal is given in Table XI. Mangoes are a rich source of vitamin C. Immature, unripe mangoes have more vitamin C than the mature and ripe ones. Ripe mango is probably the richest source of carotene, the precursor of vitamin A. A small mango is adequate to provide the required amount of vitamins A and C for a balanced diet. The carotene content (vitamin A ) of Indian varieties of mango varies from lo00 to 12,000 pg and the vitamin C content from 13 mg to 80 mg per 100 gm. of the edible portion of the fruit, depending on its ripe or unripe condition. Ripe mangoes are also fair sources of thiamine and niacin, but only a small amount of riboflavin is present. The fruit also contains a fair amount of calcium, TABLE XI NUTRITIVE VALUEOF MANGOFRUIT Percent of fresh weight Proximate composition Edible portion Moisture Protein Fat Minerals Fiber Carbohydrates Energy (kcal)

Milligrams percent of fresh weight

Seed ‘1 kernel

Ripe prilp

54.0 55.0

74.0 81.0 0.6

2.6 4.2 1.4 0.9 35.9 192.0

0.4 0.4 0.7 16.9 74.0

Minerals and vitamins Calcium Phosphorus Iron Carotene ( F g ) Thiamine Riboflavin Niacin Vitamin C

a From Bhatnagar and Subramanyam ( 1971) .

From Gopalan et al. ( 1971).

Seed ‘L kernel

Ripe h pulp

40.0

14.0 16.0 1.3

110.0 0.7 20.00 0.21 0.19

9.0

2743.0 0.08 0.09 0.9 16.0

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phosphorus, and iron but is a poor source of other minerals. Flour from the seed kernel is rich in starch, protein, and fat.

E. WASTEUTILIZATION Seeds (stones) and peels are the important wastes, constituting 25 to 55% of ripe as well as unripe mangoes. Mango kernel flour is potentially a rich source of good-quality starch and also contains proteins of high biological value. The flour can be used for human consumption as well as for animal feed. The seed kernels are comparable to most of the cereals, particularly in respect to carbohydrates, fat, protein, minerals, calcium, and phosphorus, and defatted kernel can be utilized in the preparation of chapaties, etc., by replacing wheat flour to the extent of 10%. It has not yet been a commercial success in India, however, because of the problems incurred in collection of the raw material (Jain, 1961). The possibility of utilizing the peels needs to be explored.

World trade in fresh mango is insignificant, considering the total production. This is obviously because of the numerous problems encountered during development, maturation, storage, transport, and ripening of the mango, in addition to lack of an organized marketing system and insufficient knowledge of the versatility of this fruit among the peoples of the Western and European hemispheres. Convenience foods including processed products and fruit beverages based on the mango have gained importance in recent years, the world over. To make this fruit and its products popular throughout the world, basic research is essential in the field of sensory properties and processing qualities. The fruit is a rich source of vitamins and minerals besides carbohydrates. The peel and mango kernel form a major portion of the waste products of this fruit, the utilization of which could ultimately determine the cost of the primary product.

VI.

RESEARCH NEEDS

There is a need to collect comprehensive information on the economic characteristics of all the important cultivars of the mango. Good varieties should be developed, possessing desirable characters from different cultivars by means of an intensive hybridization program. This program may

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also involve exchange of germ plasm between different mango-growing countries, which would possibly help in overcoming the biennial bearing habit of many commercial cultivars. There is also a need to develop suitable varieties intended for trade in the fresh form to meet export requirements. Similarly, varieties have to be screened among the existing cultivars, particularly for canning and freezing. The desired variety should possess qualities such as high flesh yield, a small and thin stone, and good color, and it should also be nonfibrous, with a compact and firm texture. Research on the physiology of flowering and the role of hormones in controlling flower-bud initiation is necessary for a regular mango crop. The developmental physiology during growth and maturation of all the commercially important cultivars intended for trade in the fresh or processed form should be given due attention, as this determines the quality of the ripe fruit. Development of methods, preferably nondestructive techniques for objective determination of picking maturity in the field, is essential, and methods of grading the fruit after harvest should be investigated on all important varieties for inland and export trade. Very little is known about the postharvest physiology and biochemistry of the mango fruit, especially regarding its flavor development and carotene biogenesis; intensive research is essential in this field. Biosynthesis of ethylene which initiates the sequence of chemical reactions in ripening fruits has been little studied. Conditions for packing, transit, storage, and ripening need to be standardized for all the important cultivars meant for local trade as well as export. Economic and effective methods of postharvest treatment need to be developed to reduce heavy spoilage, extend life under ambient conditions, and improve the quality of raw materials, keeping in view the consumer’s exact requirements. Physiological disorders during low-temperature storage and ripening disorders such as soft center in the choicest varieties should be examined carefully. The possibility of controlled atmosphere storage is indicated, and this method should be extended considerably. Long-distance transport by air and fast shipping facilities for the fresh fruit are indispensable if export trade is to be built on a sound basis. Recent products developed from this fruit have opened new avenues for economic utilization of pulp, a by-product in the canning industry, as well as surplus fruits available in the fresh market. Quality evaluation based on sensory properties has to be given a serious thought. The success of international trade is essentially dependent on marketing research to fulfill the consumer’s exact requirements, and to this effect an organized, integrated approach is essential.

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H. SUBRAMANYAM ET AL. ACKNOWLEDGMENTS

We are thankful to Mrs. N. V. Subhadra for valuable assistance during the preparation of this review and to the copyright owners for permission to reproduce some of the figures and tables.

REFERENCES Agarwala, S. C., Shanna, C. P., and Kumar, A. 1960. The effect of the “Black-tip” disease on the catalase and peroxidase activity of the mango fruit. Curr. Sci. 29, 195. Agnihotri, B. N., Kapoor, ,K. L., and Srivastava, J. C. 1963. Physico-chemical changes in Dashehari mango during storage. Puniub Hort. J. 3, 286. Akamine, E. K. 1963. Haden mango storage. Hawaii Farm Sci. 12, 6. Ali Farooqi, W. A., and Muhammed, A. 1968. Preservation of mangoes (Mangifera indica L . ) by gamma radiation. Food lrradiat. 9, 8. Anonymous. 1904. Grenada mangoes in New York. Agr. News W. lndies, 3, 264. Bakshi, J. C., and Bajwa, B. S. 1959. Studies on varietal differences in fruit quality of the mango varieties grown in the Punjab. Indian J . Hort. 16, 216. Banerjee, B. N., Karmarkar, D. V., and Row, G. R. 1934. Storage of mangoes. Agr. Liue-Stock India 4, 36. Banerjee, H. K., and Kar, B. K. 1941. Studies in the physiology of some Indian fruits. 11. Catalase activity in Mangifera indica. Curr. Sci. 10, 289. Basu, N. M., Ray, G. K., and De, N. K. 1947. On the possible relationship between carotene, vitamin C, total acidity pH and sugar content of different varieties of mangoes during their green and ripe conditions. J. Indian Chem. Soc. 24, 355. Bhatnagar, H. C., and Subranianyam, H. 1971. Some aspects of preservation, processing and export of inango and its products. Expert Group Meet., UNIDO ID/WG, 88/15. Biale, J. B. 1960. Post-harvest biochemistry of tropical and sub-tropical fruits. Aduan. Food Res. 10, 293. Biale, J. B., Young, R. E., and Olmstead, A. 1954. Fruit respiration and ethylene production. Plant Physiol. 29, 168. Birth, G . S., and Norris, K. H. 1965. The difference meter for measuring interior quality of foods and pigments in biological tissues. U . S . Dep. Agr., Tech. Bull. 1341, 4. Bose, A. N., and Basu, G. 1954a. Extension of storage life of Fazli mango by coating with paraffin. Sci. Cult. 19, 263. Bose, A. N., and Basu, G. 1954b. Studies on the use of coating for extension of storage life of fresh Fazli mango. Food Res. 9, 424. Brady, C. J., Palmer, J. K., O’Connell, P. B. H., and Smillie, R. M. 1970. An increase in protein synthesis during ripening of the banana fruit. Phytochemistry 9, 1037. Brill, H. C. 1919. Enzymes of mango. Agr. J. India 14, 662. Bruno, A., and Goldberg, P. H. 1963. The morphology and the chemical colnposition of Nigerian mangoes (Mangifera indica L ) . Trop. Agr. (Trinidad) 40, 143. Burg, S. P., and Burg, E. A. 1962. Role of ethylene in fruit ripening. Phnt Physiol. 37, 179.

MANGO FRUIT

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Burg, S. P., and Burg, E. A. 1965. Ethylene action and the ripening of fruits. Science 148, 1190. Burg, S. P., and Burg, E. A. 1966. Fruit storage at subatmospheric pressures. Science 153, 314. Burg, S. P., and,Burg, E . A. 1967a. Evidence for a natural occurring inhibitor of fruit ripening. Plant Physiol. 39, Suppl. X. Burg, S. P., and Burg, E. A. 1967b. Inhibition of polar auxin transport by ethylene. Plant Physiol. 42, 1224. Cadillat, R. M. 1970. Trade in tropical fruit in Europe. Trop. Sci. 12, 113. Cegarra, B. J. R. 1966. A comparative study of some chemical and physical indices which are important from the industrial aspect in grafted mango varieties. Mem. 6an J . Agron. (Maracaibo) 1, 21; Hort. Abstr., 37, 7896 (1967). Chacko, E. K., and Singh, R. N. 1969. Growth regulator induced parthenocarpy in mango (Mungifera indica L). Curr. Sci. 38, 249. Chacko, E. K., Singh, R. N., and Kachru, R. B. 1970a. Physiology of flowering and fruit growth in inango. Characterization of naturally occurring auxins and inhibitors in immature fruits. Indian J. Exp. Biol. 8, 135. Chacko, E. X., Kachru, R. B., and Singh, R. N. 1970b. Changes in the level of acidic and neutral growth promotors during fruit development in Dashehari mango (Mangiferu indica L.) J . Hort. Sci. 45, 341. Chacko, E. K., Singh, R. N., and Kachro, R. B. 1970c. Gibberellin-like substances in developing frnits of the mango (Mangiferu indica L). J . Hod. Sci. 45, 371. Chatpar, H. S., Matoo, A. K., and Modi, V. V. 1971. Biochemical changes on chilling injury in mangoes. Phytochemistry 10, 1007. Chatpar, H. S., Geetha, G., Mattoo, A. K., and Modi, V. V. 1972. Some problems pertaining to storage and ripening in mango fruit. Acta Hort. 24, 243. Chavez, J. F., and Jaffee, W. G. 1965. The nutritional importance of fruits in the Venezaeliui diet. Proc. Carib. Reg. Anzer. SOC. Hort. Sci. 8, 254. Cheenia, G . S., and Dani, P. G. 1934. Report on export of inango to Europe in 1932-3. Bombay Dep. Agr. Bull. 170. Cheenia, G . S., Karinurkar, D. V., and Joshi, B. M. 1939. Investigations on the cold storage of mangoes. Imp. Counc. Agr. Res. New Delhi, Misc. Bull. 21. Cheenia, G. S., Bhat, S., and Naik, K. C. 1954. “Commercial Fruits of India.” Macmillan, New York. Cholap, A. S., Bandyopadhyay, C., and Sreenivasan, A. 1971. Studies on the triglyceride component of mango (Mangifera indica). Indian J . Technol. 9, 309. Chowdhury, M. T. 1950. Carotenoid pigments of different varieties of mangoes, changes during ripening. J . Sci. Food Agr. 1, 173. Coca Cola Company. 1972. “Operation mango.” Zndian Food Pucker 26, 11. Contractor, J. 1951. The choicest fruit of Hindustan. Florida Mango, Forum, Mango studies. 127, 000. Czyhrinciw, N. 1969. Tropical fruit technology. Advan. Food Res. 17, 153. Das Gnpta, S. N. 1958. On the prevention of mango necrosis (Black tip). Ct~rr.Sci. 27, 446. Das Gupta, S. N., .4sthana, S. N., and Bhatt, R. S. 1955. Studies on the diseases of Mangifera indica L. VII. Occurrence of deposits in necrotic mangoes. Indian J . Agr. Sci. 25, 237. Date, W. B., and Mathur, P. B. 1958. Preliminary studies on the refrigerated gas storage of mangoes. Food Sci. 7, 283.

298

H. SUBRAMANYAM ET AL.

Date, W. B., and Mathur, P. B. 1960. Effect of post harvest treatment with growth regulators on the ripening of mangoes. Food Sci. 9,248. De, H. N., and Debnath, J. C. 1966. Biochemical and nutritional studies on East Pakistan fruits. Part 11. Differential mechanism of ripening of ordinary varieties and Kanchamitha (unripe green sweet) varieties of mangoes (Mangifera indica). Pak. J . Sci. Ind. Res. 9,57. De Candolle, A. 1901. “Origin of Cultivated Plants.” Kegan, Paul, Trench, London. De Leon, S. Y., and De Lima, L. 1966. Acceptability of canned mango juice from four varieties and three color stages of maturity. Philipp. J. Sci. 95, 401. Dennison, R. A., and Ahmed, E. M. 1967. Irradiation effects on the ripening of Kent mangoes. J. Food Sci. 32, 702. Dharam Vir, Roychoudhuri, S. P., and Thirumalachar, M. J. 1967. Aureofungin as fruit dip and wrap treatment for the control of Diplodia rot of mango and alternaria rot of tomato fruits during transit. Indian Phytopath. 20, 301. Dharkar, S. D., and Sreenivasan, A. 1972. Irradiation as a method for improved storage and transportation of mangoes. Acta Hort. 24,259. Dharkar, S. D., Savagaon, K. A., Srirangarajan, A. N., and Sreenivasan, A. 1966a. Irradiation of mangoes. I. Radiation induced delay in ripening of Alphonso mangoes. J. Food Sci. 31, 863. Dharkar, S. D., Savagaon, K. A., Srirangarajan, A. N., and Sreenivasan, A. 1966b. Irradiation of mangoes. 11. Radiation effects on skin coated Alphonso mangoes. J. Food Sci. 31, 870. El Ansari, M. A,, Reddy, K. K., Sastry, K. N. S., and Nayndaninia, Y. 1971. Polyphenols of Mangifera indica. Phytochemistry 10,2239. El Sissi, H., Ishak, M. S., Wahid, M. S., and El Ansari, M. A. 1971. The gallo tannins of Rhus and Mangifera indica. Planta Med. 19, 342; Hort. Abst. 42, 2537. 1972. Eniilsson B. 1970. Problems in long range transport of fresh avocados, mangoes and pineapples. Proc. Trop. Prod. Inst. Conf. 1969. p. 65. Fang, T. T. 1965. Chromatographic fractionation of non-nitrogenous organic acids of mango and guava fruits by silica gel column. Mem. CoZZ. Agr. Nut. Taiwan Univ. 8, 236. Fidler, J. C., and Coursey, D. G. 1970. Low temperature injnry in tropical fruit. Proc. Trop. Prod. Inst. Conf. 1969. p. 103. Finney, E. E. 197la. Random vibration techniqnes for nondestructive evaluation of peach firmness. J . Agr. Eng. Res. 16, 81. Finney, E. E. 1971b. Dynamic elastic properties and sensory quality of apple fruit. J . Text. Stud. 2, 62. Floch, H. 1958. Guiana fruits rich in carotene. Qiral. Plant Muter. Veg. 3 / 4 , 327, Hort. Abst. 29, 958 (1959). Frenkel, C., ,Klein, I., and Dilley, D. R. 1968. Protein synthesis in relation to ripening of pome fruit. Plant Physiol. 43, 1146. Candhi, S. R. 1955. “The Mango in India,” Farm Bull. No. 6. Indian Connc. Agr. Res., New Delhi. Gangolly, S. R., Singh, R., Katyal. S. L., and Singh, D. 1957. “The Mango.” Indian Connc. Agr. Res., New Delhi. Ghosh, A. K. 1965. Formation of a new oligosaccharide in mango fruits under pathogenesis. Ctrrr. Sci. 34, 465. Ghosh, S. 1960. The content of folic acid and its conjugates in some common Indian fruits. Sci. Cult. 26. 287.

MANGO FRUIT

299

Gopalan, C., Ramasastri, B. V., and Balasubramanian, S. C. .1971. “Nutritive Value of Indian foods.” Nat. Inst. Nutr., Indian Counc. Med. Res., Hyderabad, India. Govindarajan, V. S., and Sreenivasayya, M. 1950. A papyrographic study of the nonprotein nitrogen of mangoes. Curr. Sci. 19, 234. Harkness, R. W., and Cobin, M. 1951. Haden mango: Maturity observations during 1950. Flu. Mango Forum p. 141. Hatton, T. T., Jr., and Reeder, W. F. 1964. Hot water as a commercial control of mango anthrachose. PTOC.Carrib. Reg. Amer. SOC. Hort. Sci. 8, 76. Hatton, T. T., Jr., and Reeder, W. F. 1967. Controlled atmosphere storage of Keitt mangoes. Proc. Carrib. Reg. Amer. SOC. Hort. Sci. 10, 114. Hatton, T. T., Jr., Beraha, L., and Wright, W. R. 1961. Preliminary trials of gamma radiation on mature green Irwin and Sensation mangoes. Fk. Mango Forum p. 15. Hatton, T. T., Jr,, Reeder, W. F., and Campbell, C. W. 1965. Ripening and storage of Florida mangoes. U.S., Dep. Agr., Res. Rep. 725. Hobson, L. 1970. Mango growing. Proc. Trop. Prod. Inst. Conf., 1969. p. 211. Hulme, A. C. 1961. Some metabolic changes in fruits during senescence. Advan. H07t. Sci. 1, 78. Hulme, A. C. 1971. The mango. In “The Biochemistry of Fruits and their Products” ( A . C. Hulme, ed.), Vol. 2, p. 233. Academic Press, New York. Hulme, A. C., Rhodes. M. J. C., and Wooltorton, L. S. C. 1971. The relationship between ethylene and the synthesis of RNA and protein in ripening apples. Phytochemistry 10, 749. Iguina de George, L. M., Collazo de Rivera, A. L., Benaro, J. R., and Pennock, W. 1969. Provitamin A and Vitamin C contents of several varieties of mango (Mangifera indica L.) grown in Puerto Rico. J. Agr. Uniu. P . R. 53, 100; Hort. Abst. 40, 2456 (1970). Indian Institute of Foreign Trade. 1968. “Survey of India’s Export Potential of Fresh and Processed Fruits and Vegetables,” Vol. 1B. Min. Commerce, Govt. of India, New Delhi. Ishii, M. 1933. Studies on the sugars and organic acid of Mangifera indica L. Biol. Abstr. 7 , 12888 (1933). Jacob, J., Subbarayan, C., and Cama, H. R. 1970. Carotenoids in 3 stages of ripening of mango. J. Food Sci. 35, 262. Jain, N. L. 1961. Chemistry and technology of mango. Rev. Food Technol. 3, 131. Jain, N. L., Krishnamurthy, G . V., and Lal, G. 1959. Non-volatile organic acids in unripe pickling mangoes and salted mango slices by paper chromatography. Food Sci. (India) 8, 115. Johnson, R. M., and Raymond, W. D. 1965. The chemical composition of some tropical food plants. V. Mango Trop. Sci. 7 , 156. Jungalwala, F. B., and Cama, H. R. 1963. Carotenoids in mango (Mangifera indica) fruit. lndian J. Chem. 1, 36. Kapur, N. S., Sarveshwara Rao, K., and Srivastava, H. C. 1962. Refrigerated gas storage of mangoes. Food Sci. 11, 228. Kar, B. K., and Banerjee, H. K. 1939. Effect of ethylene on Mangifera indica. Nature (London) 144, 597. Karmarkar, D. V., and Joshi, B. M. 1941. Respiration studies of the Alphonso mango. Indian J. Agr. Sci. 11, 993.

300

H. SUBRAMANYAM ET AL.

Kay Daisy, E. 1970. The air freighting of fruits, with particular reference to tropical countries. Proc. Trop. Prod. lnst. Conf. 1969. p. 81. Kennard, W. C. 1955. Development of the fruit, seed, and embryo of the Paheri mango. Bot. Gaz. (Chicago) 117, 28. Kennard, W. C., and Winters, H. F. 1956. The effect of 2,4,5-TP application on the size, matnration and quality of Amini mangoes (Mangifera indica L.). Proc. Amer. Soc. Hort. Sci. 67, 290. Kidd, F., and West, C. 1922. Rep. Food Invest. R d . London, p. 17 (quoted by Hulme, A. C., 1961). Krishnamurthy, G . V., Jain, N. L., and Bhatia, B. S. 1960. Changes in physico chemical composition of mangoes during ripening after picking. Food Sci. 9, 277. Krishnamnrthy, Shantha and Patwardhan, M. V. 1971. Purification and properties of malic enzyme (decarboxylating) from pnlp of mango (Mangifera indica L ) . Phytochemistry 10, 1811. Krishnamiirthy, Shantha and Snbramanyam, H. l97Oa. Respiratory climacteric and chemical changes in the mango fruit (Mangifera indica L ) . J. Amer. Soc. H o e . sci. 95, 333. Krishnamiirthy, Shantha and Siibramanyam, II. 1970h. Effect of maleic hydrazide and 2,4,5-trichlorophenoxypropionicacid on ripening and qiiality of mango friiit. Pestic. Sci. 1, 63. Krishnamnrthy, Shantha and S~rbram;inyani, H. 1973. PIP- and post-harvest physiology of the mango fruit. (Mangifera indica L.). Trop. Sci. 15, 167. Krishnamnrthy, Shantha, Patwardhan, M. V., and Subramanyam, H. 1971. Biochemical changes dnring ripening of mango. Phytochemistry 10, 2577. Krishnamnrthy, S., Sham Singh, and Katyal, S. L. 1963. “Frnit Cnltnre in India.” Indian C o m e . Agr. Re.r., New Delhi. Lakshminarayana, S. 1973. Respiration and ripening patterns in the life cycle of the mango frnit. J. Hort. Sci. 48, 227. Lakshminarayana, S., and Subramanyam, H. 1970. Carbondioxide injury and fermentative decarboxylation in mango frnit at low temperatnre storage. J. Food Sci. Technol. 7, 148. Lakshminarayana, S., and Subramanyam, H. 1971. Control of microclimate in cold storages for tropical fruits with reference to mango. Climate Cont. 4, 37. Lakshminarayana, S., Subhadra, N. V., and Subramanyam, H. 1970. Some aspects of developmental physiology of the inango fruit. J. Hort. Sci. 45, 133. Lakshminarayana, S., Vijayendra Rao, A. R., Moorthy, N. V. N., Anandaswaniy, B., Dalal, V. B., Narasimham, P., and Subranianyam, H. 1971. Studies on rail shipment of mango. J. Food Sci. Technol. 8, 121. Leley, V. K., Narayana, N., and Daji, J. A. 1943. Biochemical studies in the growth and ripening of the Alphonso mango. Indian J. Agr. Sci. 13, 291. Lilleland, O., and Brown, J. G. 1936. Growth study of the apricot frnit. 111. The effect of girdling. Proc. Amer. Soc. Hort. Sci. 34, 264. Malo, S. E . 1972. Mango ciiltnre in Florida. Acta Hort. 24, 149. Mathur, P. B., and Lewis, N. F. 1961. Storage behavionr of a-irradiated mangoes. Znt. I. A p p l . Radiat. Zsotop. 11, 435. Mathur, P. B., and Srivastava, H. C. 1955. Effect of skin coatings on the storage behaviour of mangoes. Food Res. 20, 559. Mathur, P. B., and Subramanyam, H. 1956. Effect of a fungicidal wax coating on the storage behaviour of mangoes. J. Sci. Food Agr. 7, 673.

MANGO FRUIT

301

Mathru, P. B., Singh, K. K., and Kapur, N. S. 1953. Cold storage of mangoes. Indian J. Agr. Sci; 23, 65. Mattoo, A. K., and Modi, V. V. 1967. Methionine utilization in ripening mangoes. Indian J. Exp. Biol. 5 , 126. Mattoo, A. K., and Modi, \’. V. 196Ya. Ethylene and ripening of mangoes. Plant Physiol. 44,308. Mattoo, A. K., and Modi, V. V. IYB9b. Biocheniical aspects of ripening and chilling injury in mango fruit. Proc. Trop. Prod. Inst. Conf., 1969, p. 111. Mattoo, ‘4.K., and Modi, V. V. 1970a. Partial purification and properties of enzyme inhibitors from unripe mangoes. Enzymologia 37, 237. Mattoo, A. K., and Modi, V. V. 1970b. Citrate cleavage enzyme in niango fruit. Biochem. Biophys. Res. Coninirin. 39, 895. Mattoo, A. K., Modi, V. V., and Reddy, V. V. R. 1967. Studies on ripening of mangoes, Proc. Int. Symp. Troll. Subtrop. Hort. Mattoo, A. K., Modi, V. V., and Reddy, V. V. R. 1968. Oxidation and carotenogenesis regulating factors in mangoes. Indian J. Biochem. 5, 111. , Maxie, E. C., Sonimer, N. F., and Mitchell, F. G. 1971. Infeasibility of irradiating fresh fruits and vegetables. HortScience 6, 202. Miller, C. D. et al. 1956. Vitamin values of foods used in Hawaii. Hawaii Agr. E x p . Sta. Tech. Bull. 30, 94; Hort. Abstr. 27, 845 (1957). Mirchandani, R. T. 1963. “Containers Used for Fruits and Vegetables in India,” 135-B. Directorate of Marketing and Inspection, Nagpur. Mirchandani, R. T. 1965. “Marketing of Mangoes in India,” Mkt. Ser. 153. Directorate of Marketing and Inspection, Nagpur. Miznta, T., and Subramanyam, H. 1973. Changes in pectic and cellulosic constitnents in Alphonso and Pairi mangoes during post-harvest ripening. J. Jap. SOC. Hort. Sci. (submitted for publication). Modi, V. V., and Reddy, V. V. R. 1967. Carotenogenesis in ripening mangoes. Indian J . EX/,. Biol. 5, 233. Mukherjee, P. K. 1958. Cold storage of n ~ a n g o Hort. . Aduun. 2, 14. Mukherjee, P. K. 1959. Biochemical and physiological studies during developnient of mango fruit. Hort. Aduun. 3, 95. Mukherjee, P. K. 1960. Studies on right stage of maturity of mango for storage. Anntr. Rep. Hort. Res. Inst. 78. Mnkherjee, P. K. 1972. Harvesting, storage and transport of mango. Acta Hoit. 24, 251. Mukherjee, S. K. 1967. History, origin and botany In “The Mango: A Hand Book,” p. 1. Indian Counc. Agr. Res., New Delhi. Mukherjee, S. K. 1972. Symposium on mango and mango culture. Acta Hort. 24, 9. Naik, K. C. 1949. “South Indian Fruits and their Culture” Varadachary and Co., Madras, India. Nandi, N. L. 1958. Chemical constituents of different varieties of mangoes (Mangifera indica). Sci. Cult. 23, 618. Nauriyal, J. P., Chadha, K. L., and Rajpoot, M. S. 1972. Investigations on the control of black tip disorder of mango. Acta Hort. 24, 215. Oppenheimer, C. 1947. The acclamatization of new tropical and subtropical fruit trees in Palestine. Bull. Agr. E x p . Sta., Rehouot p. 44. Patel, M. K., and Padhye, Y. A. 1948. Bacterial soft rot of mango in Bombay. Indian Phytopathol. 1, 127.

302

H. SUBRAMANYAM ET A L .

Patel, M. K., Moriz, L., and Kulkarni, Y. S. 1948. A new bacterial disease of Mangifera indica. Curr. Sci. 17, 189. Pattabhiraman, T. R., Rao, Pramila, and Sastry, L. V. L. 1968. Preliminary Studies on the preparation of odor concentrates and identification of odorous ingredients in mango and guava. Perfum. Essent. Oil Rec. 59, 733. Pattabhiraman, T. R., Sastry, L. V. L., and Abraham, K. 0. 1969. Preparation of odor concentrates and identification of odorous ingredients in mango and guava. Part 11. Perfum. Essent. Oil Rec. 60,233. Patwardhan, M. V. 1965. Preparation of an active mitochondria1 fraction from the fruit of Mangifera indica. Nature (London) 207, 983. Patwardhan, V. G. 1927. Studies in the chemistry of sugars in the fruits, especially mango during the process of ripening. Poona Agr. Coll. Mag. 19, 32. Pennock, W., and Maldonaldo, G. 1962. Hot water treatment of mango fruits to reduce anthracnose decay. Piierto Rico Agr. J . 46, 272. Popenoe, J., and Long, W. G . 1957. Evaluation of starch content and specific gravity as measures of maturity of Florida mangoes. Proc. Flu. Sta. Hort. SOC. 70, 272. Popenoe, J., Hatton, T. T., Jr., and Harding, P. L. 1958. Determination of maturity of hard green Haden .and Zill mangoes. Proc. Amcr. Soc. Hort. Sci. 71, 326. Prasad, A., and Singh, M. P. 1965. A short note on the control of mango necrosis. Sci. Cult. 31, 251. Proctor, J. T. A., and Creasy, I,. L. 1969. The anthocyanin of the mango fruit. Phytochemistry 8, 2108. Quinones, V. L., Guernant, N. B., and Dutcher, R. A. 1944. Vitamin content of some tropical fruits, their juices and nectars. Food Res. 9, 415. Ramasarma, G. B., and Banerjee, B. N. 1940. Changes in the carotene and ascorbic acid content of mangoes during ripening. J. Indian Inst. Sci., Sect. A 23, 1. Ramasama, G. B., Rao, S. D., and Hakim, D. N. 1946. Carotenoid pigments of badami mango fruit. Biochem. J. 40, 657. Ram Ayyar, C. S., and Joshi, N. V. 19.29. Preservation of mangoes by cold storage. Agr. J. India 24, 124. Rhodes, L. A. M., Campbell, C., Malo, S. E., and Camier, S. G. 1970. A numerical taxonomic study of the mango (Mangifera indica L ) . J. Amer. Soc. Hort. Sci. 95, 252. Richmond, A., and Biale, J. B. 1966. Protein synthesis in avocado friiit tissue. Arch. Biochem. Biophys. 115, 211. Rolz, C., Flores, M. C., De Ariola, M. C., Mayorga, H., and Menchu, J. F. 1971. Development and cold storage of mango “Mamey.” A Guatemalan local variety. Expert Group Meet., UNlDO ID/WG, 88/15. Rolz, C., Deshpande, S., Paiz, L., Ortiz, J., Flores, M. C., Sanchez, M., and De Ortega, M. 1972. Chemical changes and fruit quality during the ripening of tropical fruits. Turrialba 22, 65. Ruehle, G . D., and Ledin, R. B. 1960. Mango growing in Florida. Flu. Unia., Agr. Ext. Sera. Bull. 174. Sacher, J. A. 1966. Permeability characteristics and amino acid incorporation during senescence (ripening) of banana tissue. Plant Physiol. 41, 701. Sadana, J. C., and Ahmad, B. 1949. Metabolism of the caretenoid pigments of the mango during the development of the fruit. Indian J. Med. Res. 37, 193. Saini, S. S., Singh, R. N., and Paliwal, G. S. 1971. Growth and Development of

MANGO FRUIT

303

mango (Mangifera indica L . ) Fruit. 1. Morphology and cell division. Indian 1. Hort. 28, 1. Sarkar, K. P. 1963. Pentose sugar in mangoes ( Mangifera indica L). Sci. Cult. 29,51. Shantha, H. S. 1969. Respiration of mango fruit (Mangifera i d i c a ) with special reference to climacteric rise. Ph.D. Thesis, University of Mysore, India. Shantha Krishnamurthy. See Krishnaniurthy, Shantha. Siddappa, G. S., and Bhatia, B. S. 1954. Tender green mangoes as a source of Vitamin C . Indian 1. Hort. 11, 104. Singh, B. N., Seshagiri, P. V. V., and Gupta, S. S. 1937a. Ontogenetic drifts in the physiology and chemistry of tropical fruits under orchard conditions. Indian J. Agr. Sci. 7, 176. Singh, B. N., Seshagiri, P. V. V., and Gupta, S. S. 1937b. Response of the respiratory system in mango and guava to alteration in the concentration of oxygen and nitrogen. Ann. Bot. (London) [N.S.] 1, 11. Singh, K. K., and Chada, K. L. 1961. Factors affecting the Vitamin C content of mango. Punjab Hort. J. 1, 171. Singh, K. K., Kapur, N. S., and Mathur, P. B. 1954. Further studies on the cold storage of mangoes. Indian J. Agr. Sci. 24, 137. Singh, L. B. 1960. “The Mango.” Leonard Hill, London (Second Impression, 1968). Singh, M. P. 1962. Studies in the macronutrient depletion of mango through crop removal in var: Dashehari. Indian J. Hort. 19, 103. Singh, R. N. 1972. An assessment of the existing and also potential commercial cultivars of mango in India. Acta Hort. 24, 24. Singh, R. N., Majunidar, P. K., and Shanna, D. K. 1969. New mango hybrids of greater export potential. Indian Hort. 13, No. 4, 3. Singh, U. R. 1961. Studies on fruit drop in mango. IV. Enibryo development, its degeneration and stiidies on frriit pedicel and abscission zone. Hort. Aduan. 5, 218. Smoot, J. J., and Segdll, H. €1. 1963. Hot water :is :I post harvest control of mango anthiacnose. Platit. Uis. Rep. 47, 739. Sobotka, F. E., Watada, A. E., and Diener, H. C;. 1972. Effectiveness of tlie pressure load-meter in measuring firmness of tomato fruit. HortScience 7, 34. Soule, M. J., Jr., and Harding, P. C. 1956. Effects of size and date of sampling on starch, sugars, solitble solids and phenolic compounds in mangoes. Proc. Fla. Mango Fortint 16, 13. Sorile, M. J., Jr., and Harding, P. C. 1957. Changes in physical characters and chemical constituents of Haden mangoes during ripening at 80°F. Proc. Fla. State Hort. Soc. 69, 282, Biol. Abstr. 31, 39400 ( 1958). Spencer, J. L., Morris, M. P., and Kennard, W. C. 1956. Vitamin C concentration in developing and mature friiits of mango (Mangifera indica L ) . Plant. Physiol. 31, 79. Srikantia, C . , and Kantiengar, N. L. 1942. Analysis of Raspuri and Badanii varieties of mango grown in Mysore. Proc.. Indian Acad. Sci., Sect B 15, 280. Srivastava, D. N. 1972. Epidemiology and prevention of diplodia stem-end rot of ripe mangoes. Acta Hort. 24, 235. Srivastava, H. C. 1967. Grading, storage and marketing. In “The Mango. A Handbook,” Chapter 5, p. 99. Indian Counc. Agr. Res., New Delhi. Srivatsava, M. P., Tandon, R. N., Bhargava, S. N., and Ghosh, A. K. 1965. Studies on fungal diseases of some tropical fruits. 111. Some post harvest diseases of mango (Mangifera indica L ) . Proc. Nat. Acad. Sci., India, Sect. B . 35, 69.

304

H. SUBRAMANYAM ET AL.

Srivastava, H. C., Narasimham, P., Kapur, N. S., Sreenivasan, A., and Subramanyam, V. 1969. Role of respiration on development of carotenoids during ripening of mangoes and tomatoes. Food Sci. Technol., Proc. lnt. Cong., lst, 1962 Vol. 1, p. 529. Stahl, A. L. 1935. Composition of miscellaneous tropical and sub-tropical fruits of Florida. Flu. Agr. E x p . Sta. Bull., 283. Stahl, A. L. 1951. Tropical fruit products research. Flu. Mango Forum, Mango Stud. 108. Sturrock, T. T. 1968. Nacellar embroyos of the mango. Proc. Flu. State Hort. SOC. 80, 350. Subramanyam, H. 1973. Effect of 2-chloroethyl phosphonic acid ( Ethephon) on carotene development and storage behaviour of mangoes. J. Sci. Food Agr. (to be published). Subramanyam, H., and Bhatnagar, H. C. 1972. Problems and prospects in export trade of mango and its products. lnt. Symp. Subtrop. Trap. Hort; Hort. Soc., 3rd, 1972. p. 98. Subramanyam, H., and Moorthy, N. V. N. 1973. Control of spoilage and ripening in inango fruit by zineb and sodinni diethyl dithiocarbanate. Pestic. Sci. 4, 25. Subramanyam, H., and Patwardhan, M. V. 1968. Enzymatic browning reaction in the mango fruit (Mangifera indica L). PTOC. l n t . Congr. Biochem. 7th, 1967. Subramanyam, H., and Sebastian, K. 1970. Effect of succinic acid 2,2-dimethylhydrazide on carotene development in Alphonso mangoes. HortScience 5 , 160. Subramanyam, H., Moorthy, N. V. N., Dalal, V. B., and Srivastava, H. C. 1962. Effect of a fungicidal wax coating with or without regulators on the storage behaviour of mangoes. Food Sci. 11, 236. Subramanyam, H., Moorthy, N. V. N., Snbhadra, N. V. and Mntliu, M. 1969. Control of spoilage and inhibition o f ripening in Alphonso mangoes by fumigation. Trop. Sci. 11, 120. Subramanyam, H., Krishnaninrthy, Shantha, Subhadra, N. V., Dalal, V. B., Randhawa, G . s., and Chacko Elias, K. 1971. Studies on internal breakdown: A physiological ripening disorder ill Alplionso mangoes ( Mangifercr indica L. ) Trap. Sci 13, 203. Sitbramanyam, H. et al. 1972a. Studies on low temperature breakdown in mangoes. Annu. Re),., Cent. Food. Tech. Res. Inst.. 1967-1972. Subramanyam, H., Narayana Moorthy, $1. V., Lakshminarayana, S., and Dalal, V. B. 1972b. Control of fungal spoilage in Alphonso mangoes by pre-harvest application of fungicides. Acta Hort. 24, 224. Subramanyam, H., Narayana Moorthy, N. V., Lakshminarayana, S., and Krishnamurthy, Shantha, 1972c. Studies on harvesting, transport and storage of mango. Acta Hort. 24, 260. Subramanyam, H., Nafiiyana Moorthy, N. V., Snbhadra, N. V., and Lakshminarayana, S. 1972d. Improvement of quality and control of maturity in Alphonso mangoes by pre-harvest application of growth regulators. Annu. Rep., Cent. Food Tech. Res. Inst., 1967-1971. Sundararaj, J. S., Muthuswamy, S., and Palaniswainy, A. 1972. Bacterial rot of stored mangoes. Acta Hort. 24, 217. Suryaprakasha Rao, P. V., and Srinath, M. K., 1967. Heat unit requirements for the maturation of mango variety, Baneshan (Syn: Banganapalle). Indian J. Hort. 24, 156.

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Tandon, I. N., and Singh, B. B. 1968a. Control of mango anthracnose by fungicides. Indian Phytopathol. 21, 212. Tandon, I. N., and Singh, B. B. 1968b. Control of mango anthracnose by hot water treatment. Indian Phytopathol., 21, 331. Teaotia, S. S., Singh, L. P., Maurya, V. N., and Agnihotri, B. N. 1964. Study of the changes in physical characters and chemical constituents of mango varieties during artificial ripening in different ripening media. Indian J. Hod. 21, 136. Teaotia, S. S., Singh, R. D. , and Maurya, V. N. 1968. Studies on maturity standards for Mangifera indica L. Var. “Langra.” Indian J. Hort. 25, 24. Thompson, A. ,K. 1971a. Transport of West Indian mango fruits. Trop. Agr. (Trinidad) 48, 71. Thompson, A. K. 1971b. The storage of mango fruits. Trop. Agr. (Trinidad) 48, 63. Tilgner, D. J. 1971. A retrospective view of sensory analysis and some considerations for the future. Advun. Food Res. 19, 216. Tukey, H. B. 1933. Growth of the peach embryo in relation to growth of fruit and season of ripening. Proc. Amer. SOC. Hort. Sci. 30, 209. Upadhya, M. D. 1969. Effect of low dose gamma radiation on the thiobarbituricacid-reacting substances (TBRS), SH content and certain enzymes of pirie mangoes. Indian J. Plant Physiol. 12, 179. Upadhya, hl. D., and Brewbaker, J. L. 1966. Irradiation of mangoes for control of the mango seed weevil. Hawaii Farm Sci. 15, 6. Valmayor, R. V. 1962. “The Mango, its Botany and Production.” University of Philippines, College of Agriculture, Lagnna. Valmayor, R. V. 1972. The Philippine mango indiistry-its problenis and progress. ilcta Hort. 24, 19. Venkaiah, B. 1970. Effect of plant growth regulators on properties of rat liver mitochondria. Chapter 6. Polyphenol oxidase from peel of the mango fruit. Pl1.D. Thesis, Aligarh Muslim University, India. Venkataratnam, L. 1949. Hormone indnced set and parthenocarpy in mango. Cuff. sci. 18, 409. Vickers, M. E. H. 1964. “An experiment on the cold storage of mangoes of the Kenya coast.” East. Afr. Agr. Forest. J . 30, 46. Vilar, H. S . D. 1962. Sitbsidio para-o-estiido da riqneza em, vitamina C nos fnitose ehortalicas vendaveis no inercado do lrianda. ( Contribution to the study of Vit. C content of the fruits and vegetables sold on the market of Luanda.) Agron. Angolana 14, 35; Trop. Abstr. 18, k1855 (1963). Wahab, A,, and Khan, A. J. 1954. Changes in the chemical composition of mangoes during ripening. Pnk. J. Sci. Res. 6, 121. Wali, Y. S., and Hassan, Y. M. 1965. Qualitative chromatographic survey of the sugars prevailing in some horticnltural crops. Proc. Amer. Soc. H o d . Sci. 87, 265. Wardlaw, C. W., and Leonard, R. E. 1936. The storage of West Indian mangoes. Low Temp. Res. Stntion ~Mem.3, 1. Wester, P. J. 1920. Tlie mango. Brill. Bur. Agr. Philipp. Is. 18. Yamamoto, R., Osima, Y., and Goma, T. 1932. Carotene in mango fruit (Mungifera indica L ) . Sci. Pap. In.st. Phys. Chem. Res., Tokyo 19, 122; Chem. Abstr. 27, 349 ( 1933). Yonng, 3. W. 1960. Soft nose: A physiological disorder in mango fruit. Punjab Fruit J. 23, 259. Young, J. W., and Miner, J. T. 1961. Relationship of nitrogen and calcium to “soft nose” disorder in mango fruit. Proc. Aner. Soc. Hort. Sci. 78, 201.

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FORMATION AND CONTROL OF CHLOROPHYLL AND GLYCOALKALOIDS IN TUBERS OF Solanurn tuberosurn L. AND EVALUATION OF GLYCOALKALOID TOXICITY *

BY S. J. JADHAV Department of Food Science. University of Alberta. Edmonton. Alberta. Canada

AND

D. K . SALUNKHE Department of Nutrition and Food Science. Utah State Unioersity. Logan. Utah

I. I1. I11. IV .

V.

Introduction . . . . . . . . . . . . . . . . . ....... .............. Distribution of Chlorophyll and Glycoalkaloids ...................... Biosynthesis of Chlorophyll and Glycoalkaloids ..................... Factors Affecting Chlorophyll and Glycoalkaloid Formation . . . . . . . . . . . A . Cultivar .................................................... B . Location, Climate. and Environment ........................... C . Maturity and Specific Gravity ................................. D . Storage and Temperature ..................................... E . Relative Humidity . . . . . .................................. F . Light Intensity and Quality ................................... G . Duration of Light Exposure ................................... H . Mechanical Injury and Preprocessing Treatments .............. Control of Chlorophyll and Glycoalkaloid Formation .............. A Genetics ................................................... B . Packaging .................................................. C . Colored Lights and Colored-Film Filters ........................ D . Wax, Oil, Soap, and Surfactant Treatments ...................... E . Chemicals . . . . . . . . . . . . . . . . . . . . . . . . .......................

.

* Contribution

308 310 312 316 316 317 319 319 324 324 329 330 331 332 332 332 333 337

No . 1777 from Utah Agricultural Experiment Station. 307

308

S. J. JADHAV AND D. K. SALUNKHE

F. Controlled Atmosphere Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Hypobaric Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Ionizing Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Pharmacology and Toxicology of Glycoalkaloids ..................... VII. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ....................................................

I.

340 340 340 342 348 349

INTRODUCTION

The potato (Solanurn tuberosum L.), with an annual production of nearly 300 million metric tons, is one of the major world food crops ( FAO, 1966). Because they yield heavily, are relatively inexpensive, and can be grown in a wide variety of soils and climates, potatoes are the mainstay in the diets of people in many parts of the world. The USSR, Poland, Germany, France, and the United States are the leading producers of this crop. A total production of over 16 million metric tons in 1970 (USDA, 1972) reflected a general upward trend since 1920 in the United States (Wadleigh and Dyal, 1972). Potatoes are an excellent source of carbohydrates and have a significant content of phosphorus, potassium, calcium, and vitamins, especially vitamin C. Their protein content of over 10% on a dry-weight basis brings them relatively close to the 11% protein iii wheat flour. Because of the high nutritive value and lysine content of potato protein, it is a valuable supplement to cereal proteins. Considerable losses of potatoes occur between the field and the marketing place owing to physiological and mechanical damage. When potatoes are exposed to light during postharvest handling and marketing, a green pigmentation develops at the surface. This condition, known as “greening,” indicates the formation of chlorophyll ( Hilton, 1951) . Although chlorophyll is harmless and tasteless, green potatoes are highly unattractive and are considered unfit for human consumption. Thus, additional losses of potatoes can occur in supermarkets and food stores where high-intensity lights are used to attract customers. According to United States standards for potatoes issued on July 15, 1958, greening is defined as “damage” if more than 5% of the total weight of the potato must be removed to eliminate the greened tissue, and as “serious damage” if the loss is over 10%. Surveys conducted by Motts (1937) and DeLoach and Sitton (1941) indicated that severity of potato greening varied from 14 to 27%. According to the observations of Patil (1972) all stores in Logan, Utah, suffered from losses of potatoes due to greening. Gull and

CHLOROPHYLL/GLYCOALKALOIDS OF SOLANUM TUBEROSUM L.

309

Isenberg (1958) conducted a survey of eighty-five stores in various localities in the State of New York. They observed that the potatoes were exposed to artificial light of up to 350 foot-candles ( f c ) in intensity, and the malady of potato greening was noticed in all stores. Although recent literature does not reveal reports on losses due to greening, present-day merchandising practices do not eliminate the incidence of potato greening. Certain environmental conditions stimulate a synthesis of solanine, a bitter-tasting glycoalkaloid possessing poisonous characteristics and normally present in all tubers in very small amounts. Green potatoes are usually associated with an increased level of this component. However, the processes of chlorophyll and solanine formation in potatoes are independent of each other (Conner, 1937). Currently, solanine is known as a mixture of a-solanine and a-chaconine, the predominant glycoalkaloids of potato (Fig. 1).

CHI

CHIOH

@

HO

I

HO

d-SOLANINE

FIG.1. Chemical structures of a-solanine and a-chaconine.

310

S. J. JADHAV AND D. K. SALUNKHE

The importance of the problem of potato greening and glycoalkaloid production is twofold-economic considerations and food poisoning hazards. It is significant that glycoalkaloids of potato are not destroyed by cooking, baking, or frying. Therefore, the only effective way to control these alkaloids is to inhibit their synthesis in the tubers. The United States Department of Agriculture and the Canadian Department of Agriculture in a joint action have withdrawn from commerce the potato cultivar Lenape because of its high glycoalkaloid content. This regulatory action has renewed the interest in the pharmacological and toxicological aspects of potato glycoalkaloids (Zitnak, 1970). Hardenburg (1964) reviewed earlier work on certain aspects of the greening problem. Emphasis is given here to the formation, distribution, and control of chlorophyll and glycoalkaloids in potato tubers and to the evaluation of glycoalkaloid toxicity.

II. DISTRIBUTION OF CHLOROPHYLL AND GLYCOALKALOIDS A normal whole potato contains very little chlorophyll; however, light will induce synthesis in its peripheral (periderm and outer parenchyma) zone. According to Larsen (1949), the synthesis is confined mainly to the first 3 mm of tissue, with the highest concentration in the first, where it seldom exceeds 1 mg per 100 cm’ of surface area. The formation of chlorophyll is especially vigorous in areas of high metabolic activity such as the apical end, eyes, and meristematic region. Patil (1972) showed that slightly more chlorophyll a than b developed when White Rose potatoes were exposed to light (100 fc) for a period of 1 to 15 days. The amounts of other forms of chlorophyll are negligible in potato tubers. The peripheral cells of potato tubers contain large amyloplasts (leucoplasts ), which are formed from proplastids. During the process of greening, these are converted into chloroplasts. Electron micrographs indicate large vesicles at the edges of amyloplasts during the early stages of chlorophyll formation. As chloroplast formation proceeds, these vesicles are replaced by vacuolated grana. The chloroplasts that are formed still have large starch granules. These chloroplasts, the cell organelles, are the photosynthetic apparatus and synthesize chlorophyll pigments which are confined to the lamellae.

CHLOROPHYLL/GLYCOALKALOIDS OF SOLANUM TUBEROSUM L.

311

The chemistry of alkaloids present in solanaceous plants was reviewed extensively by Schreiber (1968). Until 1954, it had been considered that the cultivated form of potato contains only one glycoalkaloid, solanine, discovered nearly 150 years ago. Kuhn and LQw (1954) reported the discovery of another glycoalkaloid, a-chaconine, in the leaves and shoots of cultivated potato and in the leaves of the wild potato Solanum chacoense, from which it was named. a-Solanine and a-chaconine have the same aglycone-solanidine-but differ with respect to the sugar chain (Fig. 1). The sugar portion of a-solanine is composed of -galactoseglucose-rhamnose, and that of a-chaconine is composed of -glucoserhamnose-rhamnose. Besides these main alkaloids representing up to 95% of the total alkaloids, p and y forms of both solanine and chaconine possessing a shortened chain (Kuhn and Low, 1955a,b) were found in leaves of Solanum tuberosum and Solanum chacoense. The occurrence of leptinines and leptines (hydroxy and acetoxy derivatives of solanines and chaconines) have been reported only in wild potato, Solanum c h coense ( Schreiber, 1968). However, the several alkaloids existing in potatoes may have arisen through hybridization of such species with the cultivated potato plant. Zitnak (1961) detected free solanidine in concentrations up to 33% of the total glycoalkaloid level in bitter Netted Gem potatoes. Additionally, other alkaloids containing different aglycones such as tomatidenol, demissidine, and 5p-solanidan-3a-01have been identified in Solanum tuberosum L. (Schreiber, 1968). Zitnak ( 1968) indicated some unknown alkaloids that could be obtained from flowers of potato plants by different extraction procedures. It is interesting to note that Kennebec potatoes, when sliced and exposed to air at room temperature for 48 hours, synthesized two new alkaloids not previously present, identified as a- and p-solamarine (Shih, 1972). In recent literature, glycoalkaloid content or TGA (total glycoalkaloids ) content has been used in place of the earlier term solanine content, referring to the predominant mixture of a-solanine and a-chaconine. Analytical methods based on color development by the sulfuric acid-formaldehyde (Pfankuch, 1937; Dabbs and Hilton, 1953; Baker et al., 1955; Gull and Isenberg, 1960) or antimony trichloride ( Wierzchowski and Wierzchowska, 1961; Sanford and Sinden, 1972; Sinden and Webb, 1972) reagent method detect total alkaloids of potato tissue obtained in the extraction procedure and are more reliable than the old gravimetric method (BOmer and Mattis, 1923) of potato alkaloid determination. In the potato plant, most of the tissues, including leaves, shoots, stems, blossoms, tubers, tuber eyes, peels, and sprouts, contain the major glycoalkaloids. The glycoalkaloid concentration is high in the tip shoots, and

312

S. J. JAUHAV A N D D. K. SALUNKHE

the flowers are particularly rich in glycoalkaloids (Lampitt et al., 1943). As was mentioned by Wolf and Duggar (1940), glycoalkaloid content is high in the meristematic regions such as leaf buds and young leaves down to about the eighth node, beyond which there is a marked decrease. It is known that the maximum amount of the alkaloids is found in sprouts. According to Guseva et al. (1960) a-solanine represents about 40% of the total glycoalkaloids of sprouts, and a-chaconine represents about 60%. Results on the glycoalkaloid contents of different potato cultivars obtained by Wolf and Duggar (1946) revealed that glycoalkaloids accumulated continuously in the tubers of all cultivars studied. The glycoalkaloids are formed in the parenchyma cells of the periderm and cortex of the tubers and in areas of high metabolic activity such tis eye regions (Wolf and Duggar, 1940; Hilton, 1951; Reeve et al., 1969); the concentration is arranged in a descending gradient from the outside inward. Little or none is found in the pith, and only small amounts are present in the intermediate region ( Lampitt et al., 1943). Bomer and Mattis ( 1924) stated that potato tubers with more than 20 mg of solanine (glycoalkaloids) per 100 gm of fresh weight were beyond the upper safety limit for food purposes. Studies on the distribution and concentration of solanine (glycoalkaloids) and its aglycone, solanidine, in peels and peeled potatoes (Netted Gem) of high glycoalkaloid content by Zitnak (1961) revealed that potato peels contained solanidine in amounts equal to those in the peeled tuber, although the peels represented only one-seventh of the whole tuber weight. According to Zitnak and Johnston (1970), the glycoalkaloids diffuse through the entire tubers on reaching a high concentration. The anatomical distribution of chlorophyll, a-solanine, and a-chaconine is shown in Fig. 2.

111.

BIOSYNTHESIS OF CHLOROPHYLL AND GLYCOALKALOIDS

A biosynthetic pathway to chlorophyll via glycine, succinyl-CoA, S-aminolevulinic acid, and porphyrins has been reviewed by Bogorad (1965, 1966) and Ellsworth (1972). Biosynthetically, all steroidal compounds such as sterols, certain sapogenins, terpenes, hormones, and alkaloids are interrelated, and pathways leading to a synthesis of a structurally similar compound could be postulated on the basis of known ones. Thus, the regular pathway starting

Apical end

ENLARGEMENT OF SECTION "A"

,-Chlorophyll Bud

Solonine Choconine

.ion

'la" Pith branch

Basal end LONGITUDINAL SECTION Periderm

\

,-Pith P i t h branch

Perirnedullal' Y zone

Bud

zone CROSS SECTION

FIG.2. Anatomical distribution of solanine and chaconine glycoalkaloids and chlorophyll in the peridem, upper cortex, and vascular region in a potato tuber. From Jeppsen et al. (1973).

S. J. JADHAV AND D. K. SALUNKHE

314

from acetate via mevalonate, isopentyl pyrophosphate, farnesyl pyrophosphate, squalene, and cholesterol is applicable to steroidal alkaloids (Fig. 3). Reviews of biochemistry and possible biogenetic relationships of steroidal alkaloids of the Solanurn group have been conducted by several authors ( Heftmann and Mosettig, 1960; Heftmann, 1963; Clayton, 1965; Willuhn, 1965; Schreiber, 1966a,b, 1968). The most significant findings in relation to the glycosidic steroidal alkaloids of potato are summarized here. The first tracer work on the biogenesis of potato alkaloids was initiated by Guseva and Paseshnichenko (1958). They demonstrated the uptake and utilization of radioactive acetate by potato sprouts. The glycoalkaloids isolated from such sprouts grown under conditions of normal illumination had the labeled carbon chiefly in the aglycone, and from sprouts grown in the dark it was in the sugar 'portions of the glycoalkaloids. Radioactivity in the glycoalkaloids reached maximum when labeled acetate was fed for two days. In the later experiment, Guseva et d.(1960) found that a-chaconine contained nearly twice as much specific activity as a-solanine. Mevalonate was more effectively utilized in the biosynthe-

2

CHPCOOH Acetic Acid (Cz]

COOH

HOHzC

Mevalonic acid lCsl CHz-OPP

6-

Faraesyl pyrophosphate

[Cisl

hCHz-Ot'P

4sopentenyl pyrophnsphata [CsJ

1 R

@ & : Slycnalkaloidr [Cts)

Solanidine [ C n )

(R=Sugarsl

FIG.3. Outline of biogenesis of steroidal glycoalkaloids of potato.

CHLOROPHYLL/GLYCOALKALOIDS OF SOLANUM TUBEROSUM L.

315

sis of glycoalkaloids of potato seedlings than was acetate (Guseva et al., 1961). Recently, Jadhav et al. (1973b) reported the incorporation of labeled carbon from P-hydroxy-P-methylglutaric acid ( HMG ) , L-leucine, L-alanine, and D-glucose into the glycosidic steroidal alkaloids of potato sprouts. The higher amount of radioactivity in the glycoside moiety, compared with that in the aglycone part of the glycoalkaloids, indicated predominant glycosylation when labeled D-glucose was administered to the potato sprouts. On the basis of incorporation rates of the label (Jadhav et al., 1973), the role of various precursors in the biogenesis of potato glycoalkaloids is outIined in Fig. 4. Subsequently, glucosylation of solanidine catalyzed by the crude enzyme preparations from potato sprouts or tubers was demonstrated (Jadhav and Salunkhe, 1973). These results and the occurrence of 7,P,and forms of solanine and chaconine (one, two, and three sugars in the glycosidic part, respectively) in potato tissue supported a hypothesis on a stepwise synthesis of a-solanine and a-chaconine from solanidine. Cholesterol has been shown to be metabolized to solanidine when applied to leaf surfaces of potato plants ( Tschesche and Hulpke, 1967). The hypothesis that labeled carbon atoms are distributed in all the steroidal rings of solasodine, synthesized from radioactive acetate or mevalonate by S. aviculnre, agrees with that expected on the basis of Alanine

MUA

I

Cholesterol

Acetoocelvl- CoA

__

---

HMG-CoA

-

Solonidine I

Leucine

Glucose I

- -I

Solonine

t Choconine

FIG.4. A proposed role of various precursors in the biogenesis of potato glycoalkaloids. From S. J. Jadhav et al. ( 197313). Reprinted from J . Food Sci. 38, 453-455. Copyright @ by the Institute of Food Technologists.

S. J. JADHAV A N D D. K. SALUNKHE

316

the known biosynthetic and cyclization scheme of squalene (Guseva and Paseshnichenko, 1962) and may be applied to a-solanine and a-chaconine because of their structural similarity to solasodine. The origin of nitrogen atom in potato alkaloids remains an unsolved biosynthetic problem. However, according to the hypothesis of Heftmann (1967), cholesterol may be undergoing cyclization in the side chain subsequent to the formation of 27-hydroxycholestero1 followed by a direct replacement of the hydroxyl group by an amino function.

IV.

FACTORS AFFECTING CHLOROPHYLL AND GLYCOALKALOID FORMATION A. CULTIYAF~

Potato cultivars differ markedly in their rate of greening on exposure to light. The rough russeting skin of some cultivars and the presence of pigment in others can mask greening, which could be revealed by scratching the skin. In cultivars such as White Rose and Kennebec, the high rate of greening is due to a smooth white skin, and consumers want tubers as light in color and smooth-skinned as possible. Several cultivars have been known for their glycoalkaloid contents. Wintgen (1906) reported a normal range of 2 to 10 mg per 100 gm of fresh material of eleven German cultivars. Von Morgenstern (1907) found a much wider range, from 4.6 to 35 mg per 100 gm of fresh tissue in thirty-nine cultivars. The expected glycoalkaloid values extended over a wider range in nine cultivars tested by Bomer and Mattis (1923). Wolf and Duggar (1946) analyzed thirty-two cultivars and found their glycoalkaloid contents to be within the range of 1.8 to 13 mg per 100 gm of a fresh tuber. Most of the results up to this stage by the above authors were based on the gravimetric method of determination of glycoalkaloid contents. Gull and Isenbcrg (1958) exposed potatoes to light (100 f c ) for 2.5 days and observed that Kennebec developed chlorophyll (0.82 mg per 100 gm of fresh peel) more readily than did Cherokee and Katahdin (0.75 and 0.45 mg per 100 gm of fresh peel). They (1960) determined the glycoalkaloid content of peel colorimetrically and noted that Katahdin, although greening the least, contained the maximum amount of glycoalkaloids (88 mg per 100 gm of fresh peel), while Cherokee, the cultivar that greened most rapidly, increased only moderately in glycoalkaloid content (52 my; per 100 gm of fresh peel). The highest and lowest mean glycoalkaloid concentrations of fifteen cultivars studied by

CHLOROPHYLL/GLYCOALKALOIDS OF SOLANUM TUBEROSUM L.

317

Zitnak ( 1955) in Canada were in Early Dewey (7.6 mg per 100 gm of fresh tuber) and in cultivar 177 (1.9 mg per 100 gm of fresh tuber). Investigations conducted by Bomer and Mattis ( 1924), Lepper (1949), Zitnak (1970), Sanford and Sinden (1972), and Sinden and Webb (1972) revealed that cultivars with high mean glycoalkaloid contents are more likely to produce excessive glycoalkaloid contents than are cultivars with low mean glycoalkaloid contents when subjected to less than ideal environmental conditions or to improper handling. Among the five cultivars and thirteen seedlings tested by Akeley et al. ( 1962), tubers of four seedlings ( B-3556-13, B-4523-8, B-922-3, and B-922-6) developed either little or no greening, while the phenomenon was pronounced in Katahdin and Kennebec (0.075 mg and 0.153 mg of chlorophyll per square centimeter). Forsyth and Eaves ( 1968) found that Cherokee, Hunter, Sebago, and Kennebec were unmarketable because of greening after 48 hours of high-intensity fluorescent light (210 fc) exposure. Green Mountain potatoes showed less greening and were still marketable. Average total glycoalkaloid contents of five cultivars grown in 1970 at thirtynine locations in the United States were as follows: Kennebec, 9.7 mg per 100 gm fresh weight of tuber; Russet Burbank, 7.9 mg per 100 gm; Katahdin, 7.9 mg per 100 gm; Irish Cobbler, 6.2 mg per 100 gm; and Red Pontiac, 4.3 mg per 100 gm (Sinden and Webb, 1972). B-5141-6 (Lenape) showed the highest glycoalkaloid content (about 29 mg per 100 gm of fresh tuber) of all cultivars studied by Zitnak and Johnston ( 1970) and Sinden and Webb ( 1972). Patil et al. (1971a) reported that various cultivars differed significantly in chlorophyll and glycoalkaloid formation (Table I ) ; their results were in conformity with those of Wolf and Duggar (1946), Larsen (1949), Lepper (1949), Zitnak (1955), Gull and Isenberg (1958), Akeley et al. (1962), and Sinden and Webb (1972). It appears, therefore, that greening potential and glycoalkaloid content are genetically controlled characteristics which vary with the cultivar. B. LOCATION, CLIMATE, AND ENVIRONMENT Sinden and Webb (1972) reported significant differences in tuber glycoalkaloid contents of five commercial cultivars and of Lenape when all were grown at thirty-nine locations in the United States. Besides genetic differences, the effects of location were significant. They concluded that deviations of glycoalkaloid contents above the average to excessive levels (above 20 mg per 100 gm) in the five commercial cultivars at certain locations were rare and usually could be explained by abnormal growing conditions ( environment) or improper handling. On the con-

318

S. J. JAUHAV AND D. K. SALUNKHE TABLE I

CHLOROPHYLL AND GLYCOALKALOID CONTENTS OF ELEVENCULTIVARS EXPOSED TO WHITEFLUORESCENT LIGHT( 100 Fc) FOR A PERIODOF 5 DAYSa Cultivar b LaChipper Platte Cascade LaRouge Sioux Norchip Red LaSoda Shurchip Russet Burbank F Kennebec Bounty

Chlorophyll c (mg/100 gm fresh peel)

Glycoalkaloids d (mg/100 gm fresh peel)

0.691 0.720 0.966 1.175 1.247 1.395 1.566 1.808 2.083 2.228 2.431

44.81 55.86 65.69 73.06 58.93 79.20 69.99 44.50 69.07 96.40 70.30

From Patil et al. ( 1971a). Reprinted from J. Food Sci. 36, 474-476. Copyright @ by the Institute of Food Technologists. b Initial glycoalkaloid contents were not reported. Initial chlorophyll is negligible. Determined by the AOAC method ( 1965) . d Determined by the method of Gull and Isenberg ( 1960). e Twenty-four milligrams of initial glycoalkaloid content per 100 gm of fresh peel as determined by Wu and Salunkhe (1972b).

trary, Zitnak (1955) observed that the influence of locations was of an insignificant nature. The possibility of slightly higher levels of glycoalkaloids in potatoes being associated with certain locations was considered a result of conditions during transportation of the tubers. The variability in glycoalkaloid concentrations in certain cultivars was attributed to natural (environmental) variations rather than to the influence of soil conditions in different locations. Investigations of Hutchinson and Hilton (1955) showed that very bitter tuber samples from several locations in Alberta (Canada) contained toxic levels of glycoalkaloids, but showed no greening. They noted that one or more unfavorable climatic conditions had probably prevailed, such as nutritional unbalance and frost or hail damage to the tops of the plants before tuber maturity. An unusually cool growing season accompanied by an abnormally high number of overcast days can also result in the excessive glycoalkaloid content of a potato crop (Bomer and Mattis, 1924; Sinden and Webb, 1972). In spite of the obvious relationships between planting date, tuber size, and tuber maturity, Braun (1948) reported no correlation between glycoalkaloid content and planting date. Arutyunyan (1940) claimed that the glycoalkaloid content of

CHLOROPHYLL/GLYCOALKALOIDS OF SOLANUM TUBEROSUM L.

319

potatoes grown in mountainous sections is always less than that of those grown in hot climates. The observations of Yamaguchi et ul. (1960b) led them to conclude that White Rose potatoes harvested in winter are more susceptible to greening than are those harvested during the summer. Reports of the effects of the major elements, moisture, and organic content of the soil on the glycoalkaloid content of potatoes are conflicting (von Morgenstern, 1907; Pallman and Schindler, 1942), some claiming no effect (Bomer and Mattis, 1924; Braun, 1948; Lepper, 1943). It is probable that the nature of physiological stress under different conditions of location, climate, and environment may lead to variations in the total glycoalkaloid contents.

C. MATURITYAND SPECIFICGRAVITY Environmental factors that tend to retard the maturation process are associated with higher than normal levels of glycoalkaloids (Sinden and Webb, 1972). Such potatoes are more likely to turn green than are mature ones. Since thickness of the periderm depends on tuber development, greening can be easily detected in smooth- and thin-skinned and newly harvested young potatoes. According to Bomer and Mattis (1924), immature and small potatoes show more increases in glycoalkaloids on exposure to light than do old and large ones. Wolf and Duggar (1946) noticed an inverse relationship between the tuber size (31.3, 135, 210, and 270 gm average weight of White Rural) and glycoalkaloid concentration (18, 14.8, 8.9, and 7.3 mg per 100 gm fresh weight). The results of Buck and Akeley (1967) indicated (Table 11) less greening in tubers harvested at an early date than in those harvested at a later date. Specific gravity is another factor in relation to the maturity of potato tubers (Salunkhe et al., 1954; Talburt and Smith, 1959). Patil et al. (1971a) reported the formation of chlorophyll and glycoalkaloids in Kennebec potatoes of three different specific gravities-1.06 to 1.08, 1.08 to 1.10, and 1.10 and above. Chlorophyll development was inversely related (2.6, 2.1, and 1.46 mg per 10 gm of fresh peel) to specific gravity; however, glycoalkaloid synthesis (98, 98, and 85 mg per 100 gm of fresh peel) was independent of specific gravity. D. STORAGE AND TEMPERATURE Gull and Isenberg (1960) observed that potato tubers were less susceptible to greening after storage for 8 months at 4.4"C than they

S. J. JADHAV AND

320

1). K. SALUNKHE

TABLE I1

EFFECTOF MATURITY DATEON GREETING OF POTATO TUBERS FOLLOWISG EXPOSURE TO FLUORESCENT LIGHT(35 Fc) FOR 100 HOURS AT 21.1"Cn Maturity date

Cnltivar August 20 B4523-8 Blanca B4846-14 Russet Burbank Katahdin Kennebec

638

57 51 47 41 42

September 16

October 20

50 40 36 34 22 12

65 58 51 43 38 31

From Buck and Akeley ( 1967). b Optical density values (inversely proportional to greening) as measured by Ratiospect, mean of four replications.

were after 3 months. On the other hand, the glycoalkaloid content was higher and was less affected by light at the end of the longer storage period, although sprouts were removed. In contrast, the storage of tubers at 4" to 5°C for 6 to 7 months after harvest did not appreciably affect the glycoalkaloid content of the cultivars tested by Wolf and Duggar (1946). Hilton (1951), on the basis of his results (Table HI), generalized that low-temperature storage maintained or caused more bitterness of the tubers than did storage temperatures above 10°C. Subsequently, Zitnak ( 1953) established the fact that high glycoalkaloid concentrations could develop at low temperatures (4" to 8°C) during postharvest storage of Netted Gem tubers whether continuously illuminated or kept in darkness (Table IV). Yamaguchi et al. (1960b) reported the ef€ect of temperature during light exposure on chlorophyll accumulation by White Rose potatoes. The results are illustrated in Table V. Freshly harvested potatoes greened slowly at 5"C, at an intermediate rate at 1O"C, and at a quite high rate at 15"C, 20"C, and 25°C. The potatoes stored for 18 days in the dark at various temperatures were similarly exposed to light at the corresponding storage temperatures. These tubers developed chlorophyll s l o ~ l yat 5°C (Table V ). However, the rate of greening was relatively high compared with that of freshly harvested tubers. T~ibersstored at 10°C contained less chlorophyll after nearly SO hours of light exposure than did tubers held at 15°C or 20°C. At temperatures above lO"C, chlorophyll accumulation was less compared with results for freshly harvested tubers. Similar

CHLOROPHYLL/GLYCOALKALOIDS OF SOLANUM TUBEROSUM L.

321

observations-that chlorophyll development is slower at low temperatures (Larsen, 1949), and faster at room temperature (Folsom, 1947) than at low temperatures-agreed with the results of Yamaguchi et al. (196Ob). Buck and Akeley (1967) conducted long-term storage at temperatures of 4.4"C, 12.8"C, and 21.1"C and found more greening of TABLE I11 EFFECTOF STORAGE TEMPERATURE ON TUBER BITTERNESS a IN NETTEDGEM POTATOES AFTER COOKING b Taste score Storage temperature ("C) 0-5 5-10 10-15 15-20

Tubers originally not bitter 2.3 3.0 2.1 1.9

( 9) (18) (18) ( 9)

Tubers originally bitter

Mean

( 9) (18) (18) ( 9)

2.60 2.75 2.15 1.80

2.9 2.5 2.2 1.7

a Abnormally high glycoalkaloid content, evaluated by organoleptic tests based on taste score such as 0 = not discernible, 1 = trace, 2 = slight, 3 = moderate, 4 = marked, 5 = very marked. Values in parentheses are number of readings. b From Hilton (1951). Reprinted from Sci. Agr. 31, 61-70. c Stored for 5 months at the indicated temperatures.

TABLE IV STORAGE CONDITIONS ON GLYCOALKALOID CONTENT EFFECTOF VARIOUS OF NETTEDGEM POTATOES b Storage time in weeks 1 2 3 4 5 6 Average

Cold, humid storage at 4"-8"C

Dry, warm storage at 12"-15"C

Dark

Light

Dark

Light c

7.93 5.63 11.30 13.75 11.09 15.42 10.86

19.83 19.01 18.51 17.38 16.34 23.50 __ 19.09

7.50 7.96 3.52 5.78 4.01 8.72 6.26

7.20 5.04 3.20 7.26 3.65 6.98 5.86

a Determined colorimetrically using sulfuric acid-formaldehyde reagent and expressed in mg% of untreated tissue; 5.9 mg% average value of normal Netted Gem tubers (Zitnak, 1955) . ZI From Zitnak ( 1953 ). c A weak Mazda light ( 15 watts) at a distance of 30 inches from tubers.

322

S. J. JADHAV AND D. K. SALUNKHE

tubers stored at 4.4"C than of those stored at 12.8"C or 21.1"C. Tubers greened less after 4 month's storage than did those stored for 2 months (Table VI). Yamaguchi et al. (196Ob) also studied the effect of storage temperature on the rate of subsequent greening at room temperature (Table VII). Tubers stored at 5°C were visibly green in 24 hours, and after 36 hours were intensely green. After storage at higher temperatures, TABLE V DURINGLIGHTEXPOSURE (100 F c ) EFFECTOF TEMPERATURE GREENINGOF WHITE ROSEPOTATOES a Chlorophyll Exposure temperature ("C)

(&lo0

6

Freshly harvested

5

5

10

44 79 95 103

15 20 25

cm2 tuber surface)

80 hr

40 hr

18 days storage

Freshly harvested

12 37 68

5 87

81 66

-d

ON

120 hr

18 days storage

Freshly harvested

18 days c storage

66

13 168

210

158

-d

26 142 -a

-a

142 134

-d

-d

-d

-d

18

From Yainaguchi et al. ( 1960b). Determined by the AOAC ( 1950) method C Storage temperature is same as exposore temperature. d Values not reported. a

b

TABLE V I EFFECTOF LENGTH OF TIMEIS STORAGE AND STORAGE TEMPEHATURE ON THE GREENING OF POTATO T U B E R S FOLLOWING EXPOSURE TO LIGHT(35 F C ) FWR 100 HOURS AT 21.1"c a Tinie in storage Cultivar Blanca B4523-8 B4846-14 Russet Burbank Katahdin Kennebec

2 months

4 months

68 b 64 63 52 51 36

69 68 64

54 59 46

Storage temperature 4.4"C

60 54 50 43 42 32

12.8"C

21.1"C

72 70 69

74 74 72 58 64 50

58 60 42

From Buck and Akeley ( 1967 ) . Optical density values (inversely proportional to greening) as measured by Ratiospect, mean of follT replications. a

b

CHLOROPHYLL/GLYCOALKALOIDS O F SOLANUM TUBEROSUM L.

323

chlorophyll accumulation was at slower and slower rates. These authors further noticed that reconditioning at 20°C for a longer time decreased the rate of chlorophyll formation of stored potato tubers exposed to light at room temperature (Table VIII). TABLE VII TEMPERATURE ON GREENING OF STORED a WHITEROSE EFFECTOF STORAGE POTATOES DUHNGLIGHTEXPOSURE (90 Fc) AT ROOMTEMPERATURE b Storage temperature ("C)

Chlorophyll

5 10

15 20 25 30

c

( pg/cm2 tuber surface)

24 hr

48 hr

72 hr

68 36 28 20 12 12

216 121 85 63 44 32

279 224 158 121 111

55

aTubers were stored in the dark for 18 days at the temperatures indicated. b From Yamaguchi et al. ( 1960b). Determined by the AOAC ( 1950) method.

TABLE VIII EFFECTOF RECONDITIONING OF STORED a WHITEROSEPOTATOES ON GREENING DURINGEXPOSURE OF LIGHT(90 Fc) AT ROOMTEMPERATURE b Duration of reconditioning

0 day

Storage temperature ("C)

10 days

17 days

Duration of light exposure

36 hr

5 10 15 20 25 30

c

125 d 100 58 36 35 25

72 hr

36 hr

72 hr

36 hr

72 hr

237 239 175 137 106 75

97 75 58 36 25 25

219 225 163 102 66 58

23 18 18 12 18 13

119 102 86 50 47 50

Tubers were stored in the dark for 14 days at the temperatures indicated. From Yamaguchi et al. ( 1960b) c Reconditioning was conducted at 20°C. dValues represent micrograms of chlorophyll per 100 cmp of tuber surface as determined by the AOAC ( 1950) method. a

b

.

324

S. J. JAVHAV AND D. K. SALUNKHE

E. RELATIVEHUMIDITY According to Larsen (1949), variation in the humidity did not affect the development of greening. Nothing is known about possible effects of relative humidity on the glycoalkaloid content of tubers.

F. LIGHTINTENSITY AND QUALITY The probability that tubers will be exposed to a certain quantity and duration of light, singly or in certain combinations including daylight, sunshine, and ultraviolet, fluorescent, or incandescent light, varies with environmental factors and marketing conditions. Several workers ( Larsen, 1949; Gull and Isenberg, 1958, 1960; Isenberg and Gull, 1959; Liljemark and Widoff, 1960; Yamaguchi et al., 1960a; Patil et al., 1971a) have studied the effect of light on greening and glycoalkaloid contents of tubers. Light intensity as low as 5 fc produces chlorophyll which increases with increased light intensity but not proportionately. Yamaguchi et nl. (1960a) tested various light intensities (12, 22, 52, 62, 90, and 135 fc) at 21.1"C on White Rose potatoes, and found increasing quantities of chlorophyll (73, 78, 108, 122, 134, and 153 pg per 100 cm2 of tuber surface) as light intensity increased. The efficiency of light in causing greening was lower at the higher light intensities, which was attributed to the lower rate of protochlorophyll synthesis or to a light-filtering effect from the chlorophyll already present. Liljemark and Widoff (1960) studied the effect on chlorophyll development of increasing light intensities from 2.3 to 334 fc (Table IX). They observed an increase in chlorophyll content with an increase in light intensity and further noted that the chlorophyll content appeared to rise in proportion to the logarithm of light intensity values in lux. Glycoalkaloid contents in this series almost paralleled chlorophyll development, despite the fact that the glycoalkaloid contents had already been high at the onset of the illumination. The findings of Patil et nl (1971a) indicated synthesis of chlorophyll with increased light intensity up to 100 fc, slow and gradual degradation up to 150 ft, and rapid degradation at 200 fc during 5 days of light exposure (Fig. 5 ) . These authors predicted that the insignificant differences in the high glycoalkaloid contents after exposure to four light intensities (50, 100, 150, and 200 fc) could be a result of storage at low temperatures for a long period of time. In contrast, Gull and Isenberg (1958) found no significant increase in the amount of chlorophyll at light intensity above 50 fc when fresh peels of Kennebec (0.7 mg per 100 gm, 25 fc; 0.74 mg per 100 gm, 50 fc), Cherokee (0.42 mg per 100 gm, 25 fc; 0.56

CHLOROPHYLL/GLYCOALKALOIDS O F SOLANUM TUBEROSUM L.

325

TABLE IX EFFECTOF LIGHTINTENSITIES ON GREENING AND GLYCOALKALOID COXTENTOF MAJESTICPOTATOES a,b Light intensity (fc) 2.3 14 56 334

Chlorophyll

0 day 1.2(387) 1.2(387) 1.2(387) 1.2(387)

c

3 days

7 days

1.7(437) 2.2(450) 3.0(532) (--d

-d

and ( glycoalkaloid) content

)

3.2(537) 5.2(625) 5.6(675) 8.0(720)

11 days 4(475) 8.2(562) 9.5(600) 21.6(1057)

17 days 4.5(550) 6.9(662) 11.9(750) 17.5(1200)

From Liljemark and Widoff ( 1960). Tubers exposed to light (daylight lamp) at room temperature. c Expressed in milligrams per 100 gm of dry weight of surface discs ( 3 mm thick) as determined by the AOAC (1950) method. Values in parentheses represent glycoalkaloid content as determined colorirnetrically by a modified Baker et al. (1955) method. d Values not reported. a

b

mg per 100 gm, 50 fc ) , and Katahdin ( 0.32 mg per 100 gm, 25 fc; 0.37 mg per 100 gm, 50 fc) were analyzed after 60 hours of illumination at room temperature. Tubers of Netted Gem (2.8 mg% TGA) and Katahdin (2.5 mgo/b TGA) developed excessive amounts of TGA when illuminated with a Mazda lamp (100 watts, 30 inches distance) for eight days. Netted Gem developed 17 m3yo at 6.7" to 8.9"C and 11.8 mg% at 12.2" to 30

120

FIG. 5. Chlorophyll and glycoalkaloid contents of Kennebec potato tubers exposed to four light intensities for 5 days at 21.1"C and 90% R.H. Data expressed as fresh weight of peels. Chlorophyll and glycoalkaloids were determined by the AOAC ( 1965) and Gull and Isenberg ( 1960) methods. From Patil et al. ( 1971a). Reprinted from J. Food Sci. 36, 474-476. Copyright @ by the Institute of Food Technologists.

326

S. J. JADHAV A N D D. K. SALUNKHE

16.6"C; and Katahdin developed 7 mg% at 6.7" to 8.9"C and 12.6 mgyo at 12.2" to 16.6OC (Zitnak, 1955). Freshly harvested tubers may remain exposed to solar radiation prior to their transfer to postharvest storage rooms. Zitnak (1953) exposed tubers of Netted Gem (1.69 mgyo original TGA) and Katahdin (1.46 mg% original TGA) to sunlight during clear sunny days (October 14-22, 1952) for 6, 12, and 36 hours and found exceptionally rapid synthesis of glycoalkaloids (12 and 4.95 mg% TGA; 13.18 and 7.10 "8% TGA; 20.38 and 16.28 mg% TGA). These treatments were conducted for 6 hours a day followed by storage (10"to 15°C) in the dark until the next suitable day. Traces of greening after a 12-hour exposure and intense greening after a 36-hour exposure were observed in the tubers of both cultivars. Conner (1937) exposed tubers to different wavelengths of light. The blue end of the spectrum encouraged glycoalkaloid formation the most, while the yellow-red end of the spectrum was most efficient for chlorophyll but did not increase glycoalkaloids. According to the investigations of Zitnak (1953), dormant tubers of Netted Gem cultivar responded by a rapid and significant increase of the glycoalkaloid concentration from 5.72 mg% to 11.00, 18.88, 22.42, 21.54, and 23.17 mg% when exposed to the efficient (13,000-A) infrared light (18" to 22°C) source for 4, 6, 8, 10, and 16 days. Such tubers developed further increases in glycoalkaloid concentrations after 2 months of storage at 4" to 8°C. Greening was noted in all samples, with an intense color after only 4 days of exposure. Ineffectiveness of infrared light on glycoalkaloid synthesis as reported by Conner (1937) resulted from defective treatments. In a subsequent experiment (Zitnak, 1953), tubers that were stored for 3.5 months and then irradiated with ultraviolet light (14" to 18°C) for 4, 6, 8, or 10 days showed a gradual increase in glycoalkaloids over control tubers by 47y0, 14101,, 190%, and 222%. At temperatures of 7" to 10°C, however, tubers showed a decrease in glycoalkaloid content when treated for more than 4 days. The effect noted in this treatment was not attributed solely to the most efficient wavelength at 3654 A because of other wavelengths emitted by the ultraviolet source in the visible spectrum of light. No greening was observed in the tubers irradiated with ultraviolet light. Pink, blue, and daylight fluorescent lights caused more greening, while green, gold, and warm fluorescent lights reduced greening (Isenberg and Gull, 1959; Yamaguchi et al., 1960a; Liljemark and Widoff, 1960). The results of Yamaguchi et al. ( 196Oa) and Liljcmark and Widoff (1960) are reproduced in Tables X and XI. Glycoalkaloid determinations using the outer layer of the tubers gave irregular results (Liljcmark and Widoff, 1960).

CHLOROPHYLL/GLYCOALKALOIDS OF SOLANUM TUBEROSUM L.

327

TABLE X EFFECTOF COLORED FLUORESCENT LIGHTON CHLOROPHYLL FORMATION IN WHITE ROSEPOTATOES a Chlorophyll content ( % of control)

Fluorescent lamp

Experiment I c Control (cool white standard) Cool white deluxe Warm white standard Warm white deluxe Daylight Blue Green Gold Pink Red Frosted incandescent (75 watts)

b

Experiment I1 d

100 100 85 80 164 188 23 33 121 33 e 46 g

100 104 79 82 108 149 56 37 84 13 f 62h

From Yamaguchi et al. ( 1960a). Determined by the AOAC method ( 1950). c Experiment I: 75 fc and 72 hours of illumination. d Experiment 11: 75 fc and 96 hours of illumination. e 32 fc light intensity. f 60 fc light intensity. 80 fc light intensity. h 75 fc light intensity. a

b

TABLE XI EFFECTOF COLORED FLUORESCENT LIGHTON CHLOROPHYLL FORMATION IN KING EDWARD VII POTATOES a Chlorophyll content c ( % of control) Duration of exposure

Fluorescent lamp b

Control (daylight lamp) Red Blue Green a From Liljemark and Widoff ( 1960). b A constant light intensity ( 1 4 fc) at

10 days

20 days

100 81 70 33

100 127 80 46

the surface of tubers throughout the experiment. c Determined by the AOAC method ( 1950).

328

S. J. JADHAV AND D. K. SALUNKHE

Gull and Isenberg (1958) compared uncovered tubers with tubers in red, blue, yellow, and colorless polyethylene bags subsequent to the light exposure (75 fc, 60 hours). They observed no significant greening variations. Pigmented bags did not affect greening because the actual amount of light reaching the tubers through the bags was sufficient to induce the formation of chlorophyll. Yamaguchi et al. (1960a) tested several colored polyethylene films (Table XII) and concluded that the reduction of greening under many of the polyethylene films probably was partially caused by the reduction in light intensity. In practically all films tested, light absorption was low in the region where chlorophyll formation takes place. Lutz et al. (1951) evaluated several types of consumer bags for Triumph and Pontiac potatoes and reported that the amount of greening was in proportion to the visibility the package permitted. According to Yamaguchi et al. (1960a), the spectrum of light incident on the potatoes from the cool white fluorescent light was changed by colored cellophane filters. The chlorophyll contents of tubers under TABLE XI1 EFFECTOF VARIOUSCOLORED POLYETHYLENE FILMS ON CHLOROPHYLL DEVELOPMENT IS WH~TE ROSE POTATOES Colored polyethylene Control ( n o film)' Yellow 1 Blue Brown Green 1 Red 1 Amber 1 Yellow 2 Red 2 Amber 2 Amber 3 Aluminium Green 2 Amber 3 Turquoise White Red 3 Black

Intensity under film (fc)

75 67 58 54 53 48 46 11 13 30 11

37 36

35 35 35 13 1

Chlorophyll content ( % of control) 100 100 86 81 94 78 93 31 88 71 73 54 46 60

70 80 62 12

From Yamaguchi et d.( 1960a). Determined b y the AOAC (1950) niethod. c Tubers exposed tinder cool white standard flirorescent light (75 f c ) for 3 days.

a

b

CHLOROPHYLL/GLYCOALKLOIDS OF SOLANUM TUBEROSUM L.

329

yellow (36 pg per 100 em2), tango (30 pg per 100 cm2),green (25 pg per 100 em2), and blue (41 pg per 100 cm2) were significantly less than that of the control (70pg per 100 cm') after 115 hours of light (90 fc) exposure. Light intensities under yellow, tango, green, and blue cellophane were 74, 60, 20, and 39 fc, respectively. A similar study conducted by Forsyth and Eaves (1968) using green, blue, and red cellophane indicated that green and blue were somewhat better than red in retarding greening. Jeppsen et al. (1973) reported that orange and yellow cellophane reduced greening by 20% and 30% of clear ( control ) , respectively, while glycoalkaloid formation under green cellophane was 27% less than that under clear (control) cellophane (Fig. 6 ) .

G. DURATION OF LIGHTEXPOSURE The exposure time is an important factor in greening because of its cumuhtive effect. A mechanical device interposed between the source of light and the tubers can reduce the light intensity and subsequently affect the rate of greening (Hardenburg, 1954). According to the results of Gull and Isenberg (1958), the rate of greening of Kennebec tubers under 75 fc light intensity is almost directly proportional to the duration

6

-

ynY 5

c

z4 e 0

7

I = z n -r

3

c

E ' 0

Clear

Rod

Oranre

Vellsr

61001

Blue

Violet

Colored Cellophane

FIG. 6. Effect of fluorescent light passing through colored cellophane filters on chlorophyll and glycoalkaloid formation in Russet Burbank tubers after exposure to 50 fc light intensity through each filter for 10 days at 16°C and 60% R.H. Data expressed on fresh weight of peels. Chlorophyll and glycoalkaloid contents as determined by the AOAC ( 1965) and Gull and Isenberg ( 1960) methods. From Jeppsen et al. ( 1973).

330

S. J. JADHAV AND D. K. SALUNKHE

of exposure (0.4/1, l.l6/2, 1.6/3, and 1.91/4 mg of chlorophyll per 100 gm of fresh peel/number of days exposed at room temperature) to either continuous or alternating 12-hour periods of light and dark. The length of exposure required to cause greening and an increase in glycoalkaloid formation varies in the literature reports. When the tubers of White Rose cultivar were exposed to 9 fc intensity at 6.loC, Larsen (1949) found no visible greening for up to 5 days. Gull and Isenberg ( 1960) observed that glycoalkaloid contents of fresh peelings increased with the time of exposure for up to 4 days in Cherokee, Katahdin, and Kennebec, but not in Red Pontiac. The amounts of alkaloids in the tubers of all cultivars had declined considerably from the peak concentrations reached at 4 days (Table XIII). Patil et al. (1971b) reported that the glycoalkaloids and chlorophyll contents increased linearly up to the sixth and tenth days, respectively (Fig. 7). There was no difference in the amounts of chlorophyll at the end of the tenth and fifteenth days. The glycoalkaloid contents were relatively constant from the sixth to the tenth day but had declined slightly at the end of the fifteenth day. It was postulated that the continued formation of glycoalkaloids and chlorophyll beyond 4 days may be a cultivar characteristic. Yamaguchi et al. (1960a) observed a lag period of 12 to 24 hours before greening was visible in the tubers of the White Rose cultivar. Greening was visible after about 24 hours of light exposure (Gull and Isenberg, 1958; Howard et al., 1957).

INJURY H. MECHANICAL

AND

PREPROCESSING TREATMENTS

The production of steroid-glycoalkaloids in wounded potatoes was reported by McKee (1955). Injury of tubers caused by either bruising EFFECTOF

TABLE XI11 DURATION OF EXPOSURE OF LIGHT(75 Fc) CONTENTOF POTATOTUBERS a Glycoalkaloid content

b

ON

GLYCOALKALOID

(mg/100 g m fresh peel)

Cultivar Red Pontiac Cherokee Kennebec Katahdin

0 day

2 days

4 days

6 days

28.8 35.0 47.7 52.7

31.6 48.5 71.1 85.5

37.2 65.5 93.3 100.0

22.7 67.7 91.6 86.6

From Gull and Isenberg ( 1960). Determined by a colorimetric ( sulfuric acid-fomlaldehyde reagent ) method originally reported by Pfankuch ( 1937) and subsequently improved by Dabbs and Hilton ( 1953), Baker et al. ( 1955), and Gull and Isenberg ( 1960). a

b

CHLOROPHYLL/GLYCOALKALOIDS O F SOLANUM TUBEROSUM L. 5 ,

.

,

,

,

,

,

,

I

I

,

, ,

, ,

,100

0

1

2

3

k

5

6

7

331

20

I 9 1 0 1 1 1 2 1 3 1 4 1 5

Days

FIG.7. Chlorophyll and glycoalkaloid contents of White Rose potato tubers exposed to light (100 fc) up to 15 days at 21.1% and 90% R.H. Data expressed on fresh weight of peels. Chlorophyll and glycoalkaloids were determined by the AOAC (1965) and Gull and Isenberg (1960) methods. From Patil et al. (1971b).

or mechanical grading after harvesting induced glycoalkaloid synthesis (Sinden, 1972). KuE ( 1964) reported that in fresh potato slices the concentration of glycoalkaloids increased from an undetectable amount to 20 mg per 100 gm after storage at room temperature in the dark for 3 days. A similar study conducted by Salunkhe et al. (1972) indicated that potato slices (0.3 mg of glycoalkaloids per 100 gm) when held in the dark at relatively high temperatures (15°C or 24°C) for 2 days synthesized the glycoalkaloids (1.3 and 2.05 per 100 gm). The rate of alkaloid formation increased (4.94 and 7.4 mg per 100 gm) when the slices were stored under high-intensity light (200fc). In many instances, in potato-processing plants, slices, cubes, mash, strings, strips, and shreds are stored at relatively high light intensity and temperature for some time before cooking or processing. This may cause synthesis and subsequent accumulation of the glycoalkaloids-an effect that Salunkhe et al. (1972) attributed to a physiological defense mechanism.

V.

CONTROL OF CHLOROPHYLL AND GLYCOALKALOID FORMATION

Several physicochemical methods for the control of greening and of formation of glycoalkaloids have been studied. Practical use of these methods has certain limitations because of marketing trends and priority of health problems.

332

S. J. JADHAV AND D. K. SALUNKHE

A. GENETICS Gortner (1949) reported that the alkaloid content of plants may be increased or decreased by appropriate selection of strains, by plant breeding, and by fertilization. The breeding of cultivars resistant to greening may be possible because genetic differences controlling this biochemical change are apparent among existing strains (Akeley et al., 1962). None of the commercially acceptable cultivars of potatoes now on the market are immune to greening and glycoalkaloid formation. Consumer protection, therefore, requires the development and growing of cultivars with low glycoalkaloid contents.

B. PACKAGING One approach to the problem of greening is to protect tubers from light. Using mesh or transparent bags (polyethylene and Pliofilm), Lutz d al. (1951) found that greening was not serious until after 3 days of exposure to moderate light-25 fc for 9 hours a day. No significant differences in the greening were observed between tubers packed in polyethylene and those packed in mesh bags. Solid kraft paper bags gave better protection against greening. A similar study by Hardenburg (1954) indicated that the extent of greening was highest in polyethylene bags, intermediate in h a f t bags with mesh windows, and least in solid kraft paper bags. Howard et al. (1957) observed that tubers under burlap were less green than those exposed to light, Those under 10-02. burlap showed less greening than those under 7Yz-o~.burlap. Liljemark and Widoff (1960) referred to the use of amber-colored polyethylene to protect tubers against light without appreciably diminishing their visibility so that they could be displayed for 6 to 10 days in a retail store. Larsen (1949) suggested covering the windows in potato bags with blue or green cellophane to reduce greening. Newman (1966) recommended dark-colored polyethylene bags. LIGHTS AND COLORED-FILM FILTERS C. COLORED Although placing tubers under green light or green cellophane filters reduces greening, the appearance does not attract the consumer (Larsen, 1949; Liljemark and Widoff, 1960). Yamaguchi et al. (1960a) suggested tango (amber) cellophane as a satisfactory and inexpensive canopy filter between the light source and the tubers. According to their views, tango (amber) seemed the most promising to give normal

CHLOROPHYLL/GLYCOALKALOIDS OF SOLANUM TUBEROSUM L.

333

appearance to the tubers. Present knowledge does not show any convincing evidence that packaging, colored lights, and colored-film filters protect tubers from glycoalkaloid development.

D. WAX,OIL, SOAP,AND SURFACTANT TREATMENTS Waxing the tubers had no effect on chlorophyll formation (Lutz et al., 1951; Howard et al., 1957; Yamaguchi et d.,1960a). Lutz et al. (1951) observed the expected masking effect of red-colored wax on greening. Of the two wax formations, only one considerably retarded greening (Yamaguchi et al., 1960a). The waxing of potatoes was once a fairly widespread marketing practice because it attracted consumer attention, but it appears to be rapidly declining in importance in the United States. However, the finding of Wu and Salunkhe (197%) that hot paraffin wax effectively controls the formation of chlorophyll and glycoalkaloids in potato tubers has created a new interest in this area (Fig. 8). These authors treated Russet Burbank potatoes with paraffin wax at 60"C, 8O"C, lOO"C, 120"C, 140"C, and 160°C for yZ second and exposed them to fluorescent light (200 fc) for 10 days at 16°C and 607,R.H. The results indicated no inhibition of chlorophyll and glycoalkaloid synthesis at 60°C and 80"C, signscant inhibition at 100°C and 12O"C, and almost complete inhibition at 140°C and 160°C. Heating the

FIG.8. Effect of waxing and heating at different temperatures on chlorophyll and glycoalkaloid formation in peels (fresh) of Russet Burbank tubers after exposure to 200 fc light intensity for 10 days at 16°C and 60% R.H. ( A ) Original (zero-time) sample; ( B ) control (nonwaxed); ( C ) - ( H ) waxing at 60"C, 80"C, lOO"C, 120°C, 140°C, and 160°C; ( I ) heating with air at 160°C for 3 minutes; ( J ) heating with air at 160°C for 5 minutes. Chlorophyll and glycoalkaloids as determined by the AOAC (1965) and Gull and Isenberg (1960) methods. From Wu and Salunkhe ( 1972a). Reprinted from J. Food Sci. 37, 629-630. Copyright @ by the Institute of Food Technologists.

334

S. J. JADHAV AND D. K. SALUNKHE

tubers at 160°C in air for 3 to 5 minutes and subsequent exposure to light did not prevent chlorophyll and glycoalkaloid formation. It was the combined treatment of waxing and heating that retarded the chlorophyll and glycoalkaloid formation. They further concluded that this treatment is especially useful because paraffin wax does not create problems like most physicochemical treatments and coated wax can be easily removed by peeling the tubers before processing or cooking. In a later study, corn oil dips were given to potato tubers at 22"C, 60"C, lOO"C, and 160°C for l/z second, and excess oil was removed with tissue paper (Wu and Salunkhe, 1972b). Oiling at 22°C reduced chlorophyll by 93 to 1 0 0 ~ oand glycoalkaloid formation by 92 to 97%. A t elevated temperatures such as W'C, 1OO"C, and 160°C, the treatment completely inhibited both chlorophlyy and glycoalkaloid synthesis ( Fig. 9). Wu and Salunkhe (1972c) further reported that treatments with corn oil, peanut oil, olive oil, vegetable oil, or mineral oil at 22°C were equally effective, but the tubers appeared oily. Moreover, oxidative rancidity of the oils and fat might develop in the course of time. To decrease the amount of oil used, corn oil was diluted with acetone. Treatment with 1/2, 1/4, and l/s oil significantly and effectively inhibited the formation of chlorophyll and glycoalkaloids (Fig. 10). Treatments with 1/*,and l/s4 oil had 95%, 72y0, and 22% inhibitory effect on

x6,

Red Pontiac

Russet Burbank

A

B

C

D

t

F

A

B

C

O

t

F

A

O

t

F

FIG.9. Effect of oil dipping on chlorophyll and glycoalkaloid formation in peels (fresh) of potato tubers after exposure to 200 fc light intensity for 10 days at 16°C and 60% R.H. ( A ) Original (zero-time) sample; ( B ) control (nontreated); (C)( F ) oiling at 22"C, 60°C, 100°C, and 160°C. Chlorophyll and glycoalkaloids as determined by the AOAC (1965) and Gull and Isenberg (1960) methods. From Wu and Salunkhe ( 1972b).

CHLOROPHYLL/GLYCOALKALOIDS OF SOLANUM TUBEROSUM L.

H

l

J

335

K

FIG. 10. Effect of different dilutions of corn oil with acetone on chlorophyll and glycoalkaloid formation in peels (fresh) of Russet Burbank tubers after exposure to 200 fc light intensity for 10 days at 16°C and 60% R.H. ( A ) Original (zero-time) 1/4-3/4, 1/8-7/8, sample; ( B ) control (non-treated); (C)-(J) 1-0, 1/2-1/2, 1116-15/16, 1/32-31/32, 1/64-63/64, and 11128-127/128, oil-acetone; ( K ) acetone. Chlorophyll and glycoalkaloids as determined by the AOAC (1965) and Gull and Isenberg (1960) methods. From Wu and Salunkhe ( 1 9 7 2 ~ ) .

chlorophyll, and 827", 49%, and 28% inhibitory effect and glycoalkaloids. Tubers treated with acetone alone or with 1/128 oil behaved in the same way as check tubers. A concentration of l/S corn oil and 78 acetone was the minimum effective dilution. According to their estimate, 1 gm of oil in 50 gm of acetone is enough to treat 8000 gm of potato tubers. The acetone treatment had no apparent harmful effect on the tubers. For practical application, acetone can be recovered by passing the treated tubers through a warm-air chamber and condensing the acetone in the warm air by a cooling coil system. Jadhav and Salunkhe (1974) reported the effectiveness of mineral oil at different concentrations (Fig. 11). The efficiency of treatment increased with increasing concentration up to 10% (w/v in petroleum ether) and then remained almost constant and maximum up to 100%. Tubers treated with mineral oil up to 10% concentration showed an attractive appearance compared with that of untreated and excessively treated ones. At a concentration of loc); mineral oil, tubers did not turn green after exposure to light for 4 weeks, while glycoalkaloid inhibitions at the end of the first, second, third, and fourth weeks were 93%, 67%, 49%, and 65% of control compared with initial values. In general, oil treatment is important because it is a simple, effective, and inexpensive method of controlling greening and glycoalkaloid formation in potato tubers. Sinden (1971) immersed tubers in a 2% or 376 detergent solution at 21.1"C for 10 to 40 minutes and then rinsed them under tap water. On

336

S. J. JADHAV AND D. K. SALUNKHE

0

20

GIycorlkJoids ng/100 gn 40 60 80 100

120

Initial Control P. Ether

1

-=.

1

5

z

-

E

10

15

%

I

20 100

0

2

4 6 Chlorophyll mp/lOO gm

FIG.11. Effect of mineral oil at different concentrations (w/v in petroleum ether) on chlorophyll and glycoalkaloid formation in peels (fresh) of Norgold Russet potatoes after exposure to 200 fc light intensity for 7 days at 16°C and 60% R.H. The values in parentheses represent percent reduction of the difference between control and initial values. Chlorophyll and glycoalkaloids as determined by the AOAC ( 1965) and Gull and Isenberg ( 1960) methods. From Jadhav and Salunkhe ( 1974).

exposure to light (120 fc) for 10 days, greening and light-induced glycoalkaloid synthesis were inhibited. Inhibition of chlorophyll in Russet Burbank tubers was 92y0 for the first 2 days when treatment was given with 3% detergent solution for 30 minutes. The inhibitory effect decreased with extended exposure, but inhibition was still more than 50% after 10 days. With Kennebec and Sebago cultivars, both of which green rapidly under routine light conditions, the treatment with 2y0 detergent (Joy) for u) minutes resulted in 47% and 33% inhibition. The glycoalkaloid content of the fresh peels of Kennebec was 61% less than in the control. With the cultivar Green Mountain, which greens less rapidly, inhibition was only 14%. Poapst and Forsyth (1973) reported that photoinduced greening of potatoes can be prevented by simply washing or spray rinsing the tubers with an aqueous solution of an edible surfactant known commercially under the brand name of Tween 85. An application of 0.04y0of the tuber weight prevented chlorophyll for a period of 15 days or more while a spray containing 4 4 % Tween 85 formed an effective film on the surface of the most susceptible cultivars.

CHLOROPHYLL/GLYCOALKALOIDS OF SOLANUM TUBEROSUM L.

337

E. CHEMICALS Numerous chemicals are known to prevent greening and/ or glycoalkaloid synthesis in potato tubers. Gull and Isenberg (1958) applied several chelating compounds as preharvest foliar sprays in concentrations of 4OOO pprn and 8000 ppm at the rate of 100 gallons per acre of potato crop. Four compounds ( Chelate 600 NaFe, Sequestrene Na,, Sequesterene Na,Cu, and copper gluconate) effectively reduced the greening of the tubers when they were exposed to fluorescent lights (75 fc) for 60 hours. CheIate 330 Fe was effective only at the 8000-ppm concentration. Preharvest foliar spraying with Ethrel and N6BA reduced glycoalkaloid synthesis in Shurchip potatoes exposed to light (200 fc, 7 days) by 32.5% and 25% (Jeppsen et al., 1973). Chlorophyll increased slightly with Ethrel and decreased by 30.5% with N6BA. Schwimmer and Weston (1958) found that potatoes dipped for 30 minutes in a 0.0084 M solution of 3-amino-1,2,4-triazole (ATA) developed 29% chlorophyll of control under 600 fc light after 6 days. Tubers tested with nicotinic acid ( 4 x M ) by a vacuum injection method had a 73% depressed glycoalkaloid level during a 4-day illumination (100 fc) at 10°C (Parups and Hoffman, 1967). Inhibition of chlorophyll and glycoalkaloids by Ethrel and Alar was first reported by Patil et al. (1971a) when these chemicals (10,OOO ppm) were applied by the technique of Parups and Hoffman (1967). After 5 days under continuous white fluorescent light (100 fc), inhibition of chlorophyll by Ethrel was 75% of control value, and that of glycoalkaloids was 27%; for Alar the values were 55% for chlorophyll and 32% for glycoalkaloids. Recently, investigations by Jadhav and Salunkhe (1974) revealed that postharvest applications of Phosfon, Phosfon-S, Amchem 72A42, Amchem 70-334, Nemagon, and Telone (250, SOO, and lo00 ppm) significantly inhibited glycoalkaloids and chlorophyll formation (Figs. 12 and 13). Amchem 72-A42 was most effective in preventing the synthesis of both glycoalkaloids and chlorophyll in potato tubers. Inhibition by Nemagon and Telone was low at lower concentrations. In general, the higher the concentration, the greater was the inhibition regardless of the specific chemical used. Chlorophyll and glycoalkaloid syntheses were inhibited by 25% and 32%, 53% and 57%, and 86% and 69% when tubers of Norgold Russet were subjected to glycerine treatments at concentrations of lo%, 20%, and 30% (w/v in water) followed by light exposure (Jadhav and Salunkhe, 1974). The various sources of effective chemicals are presented in Table XIV.

338

S. J. JADHAV AND D. K. SALUNKHE 0

20

60

40

80

Initial Control Phosfon Phosfon.S Inchem. 12442 Amchem70.334 Nenagon Telonc

21

I

I

0

20 40 60 Glycoalkaloids mg/lOO g m

80

FIG. 12. Effect of chemicals on glycoallcaloid formation in peels (fresh) of Russet Burbank potatoes after exposure to 200 fc light intensity for 6 days at 16°C and 60% R.H. The values in parentheses represent percent reduction of the difference between control and initial values. Glycoalkaloid content as determined by the Gull and Isenberg ( 1960 ) method. From Jadhav and Salunkhe ( 1974 ) .

0

1

0

1

2

3

Initial

Control

Phosfon Phosfon-S Amchen. 72442 Anchem70.334

Nenagon Telone 2 3 Chlorophyll mg/lOO gm

4

4

FIG. 13. Effect of chemicals on chlorophyll formation in peels (fresh) of Russet Burbank potatoes after exposure to 200 fc light intensity for 6 days at 16°C and 60% R.H. The values in parentheses represent percent reduction of the difference between control and initial values. Chlorophyll content as determined by the AOAC ( 1965) method. From Jadhav and Salunkhe ( 1974).

CHLOROPHYLL/GLYCOALKALOIDS O F SOLANUM TUBEROSUM L.

339

TABLE XIV SOURCEOF EFFECTIVE CHEMICALS Trade Name

Chemical Name

Source _ _ _ _ _ _ _ _ _

Amchem 68-240 2-Chloroethylphosphonic (Ethrel or Ethepon) b acid 2-(p-Chlorophenylthio)triAmchem 72-A42 ethylamine Amchem 70-334 or 2-(pChlorophenylthio)triGSN-7057 (CPTA) ethylamine hydrochloride Alar (SADH) h Succinic acid-2,e-dimethylhydrazide

5. Phosfon 6. Phosfon-S

7. Nemagon 8. SD49O-Code 4-1-9-11 (N6BA) b 9. Telone

10. (ATA) b

11.

12. Chelate 330 Fe

13. Chelate 600 NaFe

14. Sequesterene Na4

15. Sequesterene NazCu 16. Copper gluconate a Effective b

Amchem Products, Ambler, Pennsylvania Amchem Products, Ambler, Pennsylvania Amchem Products, Ambler, Pennsylvania UniRoyal Chemical, Division of UniRoyal, Inc. Bethany, Connecticut Tributyl2,4-dichlorobenzyl- Mobil Chemical Co., phosphonium chloride New York Tributyl2,4-dichlorobenzyl- Mobil Chemical Co., ammonium chloride New York 1,2-Dibromo-3-chloropro- Shell Chemical Co., pane Modesto, California NG-Benzyladenine Shell Chemical Co., Modesto, California 1,3-Dichloropropene and Dow Chemical Co., related chlorinated hydroMidland, Michigan carbons 3-Amino-l,2,4-triazole Aldrich Chemical Co., Cedar ,Knolls, New Jersey Nicotinic acid Aldrich Chemical Co., Cedar Knolls, New Jersey

Pentasodium diethylene triamine pentaacetate with Fe 1,2-Diaminocyclohexane tetraacetic acid with NaFe Tetrasodium ethylenediamine tetraacetate dihydrate Disodium cupric ethylenetrih ydrate

Geigy Chemical Co., Ardsley, New York Geigy Chemical Co., Ardsley, New York Geigy Chemical Co., Ardsley, New York Geigy Chemical Co., Ardsley, New York

Copper salt of gluconic acid Pfizer Inc., Chemical Division, New York

in controlling chlorophyll and glycoalkaloid formation. Abbreviation.

340

S. J. JADHAV AND D. K. SALUNKHE

The following chemicals have been reported as ineffective in controlling greening and glycoalkaloid synthesis: sulphurous acid and ammonium thiocyanate ( Larsen, 1949); 0-methyl threonine and chloroisopropyl phenyl carbamate (Schwimmer and Weston, 1958); EDTA, tramcinnamic acid, and dibenzoylresorcinol ( Yamaguchi et al., 1960a); Cycocel and maleic hydrazide (Patil et al., 1971a); maleic hydrazide and gibberellic acid (Parups and Hoffman, 1967) as dip treatments; maleic hydrazide (Schwimmer and Weston, 1958; Parups and Hoffman, 1967) as a foliar spray; and So% ethylene gas (Larsen, 1949) as a 24-hour exposure. F. CONTROLLED ATMOSPHERESTORAGE

Controlled atmosphere storage of tubers was explored by Forsyth and Eaves (1968). They found no immediate greening in an atmosphere of 100% N., or 75% COz, and the storage time could be extended to 1week without noticeable development of greening. It appeared to them from organoleptic tests of these potatoes after baking that potatoes stored under 1 0 0 ~ Nz, o but not those stored under 75y0 COz,developed off-flavor due to the formation of glycoalkaloids. After further studies using different levels of COz, they concluded that 15% or higher concentrations of CO, will prevent greening without affecting palatability (Table XV) . Patil et al. (1971b) found no significant effect of 157, COm2 on the formation of potato glycoalkaloids, while chlorophyll contents were decreased by 33% of control values. STORAGE G. HYPOBARIC Application of subatmospheric pressure (hypobaric ) is a recent approach to increasing the storage life of horticultural produce. Tubers stored at 126 mm Hg pressure did not turn green, while treatments at 253, 380, 507, and 633 mm Hg pressure were ineffective in controlling chlorophyll formation (Jadhav et al., 1973a). There were no differences in glycoalkaloid levels among tubers subjected to any of the storage pressure treatments and the control (Table XVI). H. IONIZING RADIATION

Several workers studied ionizing radiation in relation to the formation of chlorophyll in illuminated potato tubers (Schwimmer and Weston, 1958; Gull and Isenberg, 1958; Ziegler et al., 1968; Patil et al., 1971b).

CHLOROPHYLL/GLYCOALKALOIDS OF SOLANUM TUBEROSUM L.

341

TABLE XV OF SEBAGO POTATOES EFFECTOF COZ ON GREENING IN CONTROLLED ATMOSPHERE STORAGE a

Percent COz Mean

Range

0.03 7.5 12.0 14.9 20.3 29.3

4-10 7-15 9-22 12-26 18-38

Greening 6

Market grade c

Severe Medium Slight Very slight None None

Below grade Below grade Below grade 33.3%below grade Canada #1 Canada #1

a From Forsyth and Eaves ( 1968). Reprinted from Food Technol. 22, 4850.Copyright @ by the Institute of Food Technologists. b Scores based on eye observations after 48 hours of illumination (210 fc). c Graded b y Canada Department of Agriculture Inspector 168 hours after commencement of experiment. Rejections are due to a n excess of green coloration.

TABLE XVI EFFECTOF STORAGE AT SUBATMOSPHERIC PRESSURETREATMENTS ON CHLOROPHYLL AND GLYCOALMLOID CONTENTS OF RUSSETBURBANK POTATO TUBERS TO 210 Fc LIGHT INTENSITY FOR 15 DAYS a EXPOSED Treatment pressure (mm H g ) Control 633

507 380 253 126

Chlorophyll IJ

Glycoalkaloids c

(mg/100 gm fresh peel)

(mg/100 gm fresh peel)

9.43 7.52 10.89 9.79 9.83 0.26

46.34 42.66 40.82 51.87 35.82 52.94

a From

Patil ( 1972). Determined by the AOAC ( 1965) method. c Determined by Gull and Isenberg ( 1960) method. 6

Increasing doses ( 5 , 15, 50, and 250 krads) inhibited (62%, 67%, 75Yob, and SOY0 of control compared to initial) but did not completely suppress chlorophyll formation in Russet Burbank tubers illuminated (600 fc) for 3 days after irradiation (Schwimmer and Weston, 1958). Gull and Isenberg (195s) observed a nearly 50% reduction in the chlorophyll contents of tubers subjected to a 40-krad dose followed by illumination (75 fc, 60 hours) after 2 months of storage at 4.4"C. However, the radiation

342

S. J. JADHAV AND D. K. SALUNKHE

treatments seriously injured the surface tissues of the tubers. A 10-krad dose, alone or in combination with 15% CO,, reduced chlorophyll from 4.3 mg to 0.88 mg and 0.68 mg per 100 gm of fresh peels in tubers of Russet Burbank held under light (100 fc) for 5 days (Patil et al., 1971b). These treatments did not affect glycoalkaloid synthesis. Ziegler et al. (1968) exposed tubers to 280 fc of continuous illumination, postirradiation, for 12 days and found increasing inhibition of greening with increasing doses of irradiation from 0 to 400 krads (Table XVII). The results of Ziegler et al. (1968) also showed two apparent trends: decrease of greening with irradiation, irrespective of CO, treatment, after 4 days of illumination; and decrease of greening with increasing CO, in the atmosphere, irrespective of irradiation, after 12 days of illumination.

VI.

PHARMACOLOGY AND TOXICOLOGY OF G LYCOALKALO I DS

Accidental consumption of potatoes containing high amounts of glycoalkaloids has caused severe illness and, on some occasions, death. Damon (1928) briefly narrated cases of potato poisoning reported by several investigators. The largest number of such cases occurred on the European continent, mostly among soldiers. Although the poisoning was attibuted to the eating of potatoes, no analysis was made on the content of glycoalkaloids. Harris and Cockburn ( 1918) investigated an epidemic among the civilian population of Glasgow, Scotland. They examined TABLE XVII IRRADIATIONON THE CHLOROPHYLL FORMATION IN KENNEBEC EFFECTOF GAMMA POTATOES DURING 12 DAYSOF ILLUMINATION (280 F c ) ~ Chlorophyll content b ( % of control)

Irradiation dose ( krads )

4 days

8 days

12 days

0 50

100

100

100

50

100 200

29 24 14

59 70

400

24 24

80 65 35 26

a From Ziegler et al. ( 1968). Reprinted from J. Food Sci. 33, 533-535. Copyright @ by the Institute of Food Technologists. b Determined by the Ziegler and Egle ( 1965) method.

CHLOROPHYLL/GLYCOALKALOIDS OF SOLANUM TUBEROSUM L.

343

sixty-one cases, one of which was fatal-that of a boy five years of age. The analysis of potatoes that caused the poisoning revealed 410 mg of glycoalkaloids per kilogram of potatoes. Rothe (1918) reported an outbreak in Leipzig, Germany, affecting forty-one persons. The circumstances clearly implicated the potato, and the chemical examination made in this case disclosed the glycoalkaloid contents of the potatoes to be as high as 430 mgl kg. Since then, several cases of glycoalkaloid poisoning from eating potatoes have been described by Bomer and Mattis (1923), Griebel ( 1923), and Willimott ( 1933). In the United States, Hansen (1925) and Damon (1928) reported cases in Illinois and Missouri, respectively. The case reported by Hansen is reproduced here. About October 15, 1924, James B. Matheney, of Vandalia, Illinois, gathered about one and one-half bushels of tubers from the patch of strawed potatoes. The tubers were green. . . . On October 18, the family started to use the greened potatoes and two days later began to show symptoms of poisoning. All the members of the family, consisting of the father, mother, two daughters, and five sons were ill with the exception of the father, who did not partake of the tubers, and a child of 18 months, who lived on milk almost exclusively. The mother, age 45, died on October 25, a daughter, Cynthia, age 16, died two days later. The other five members of the family recovered. (Hansen, 1925, p. 341.)

In Canada, several complaints from potato growers and consumers concerning unpalatable and bitter-tasting potatoes have been noted and traced back to high glycoalkaloid contents ( Zitnak, 1961). Losses of livestock and poultry caused by ingestion of potato vines, sprouted potatoes, cull potatoes, and potato peels containing glycoalkaloids have been reported (Hansen, 1925; Willimott, 1933). These products had been exposed to light when discarded by the processing plants or left in the field by farmers. The literature on outbreaks of poisoning due to glycoalkaloids in potatoes has been summarized by Wilson (1959), who also reported a recent occurrence in Great Britain. According to him such outbreaks are extremely uncommon and apparently occur only when a batch of potatoes containing an unusually high glycoalkaloid concentration happens to have been eaten. The symptoms are generally those of an acute gastrointestinal upset with abdominal pain, vomiting, and diarrhea. While referring to 1 out of 61 cases in Glasgow in 1918, Wilson (1959) pointed out that apparently it is not usually fatal. The first attempt to establish the toxicity of glycoalkaloids by using experimental animals was made by Meyer in 1895. He could not isolate either a-solanine or solanidine from the urine of a dog that had been fed a-solanine. Willimott ( 1933) made unsuccessful experiments to study the effects of feeding potato plants to adult rats, Golubeva (1966) exam-

344

S. J. JADHAV AND D. K. SALUNKHE

ined thirty-five patients with food allergy mainly referring to nightshade. He treated these patients with a-solanine and observed curative effects. Satoh (1967) observed that the pretreatment of rats with adrenergic blockers or reserpine prevened the glycemic effects of solanine. Nishie et al. (1971) reported that a-solanine prolonged sleeping times induced by pentobarbital, reduced spontaneous motor activity in mice, and caused contraction of guinea pig ileum strips with certain minor exceptions. The metabolic fate and distribution of tritiated a-solanine in rats recorded by these authors indicated (1)partial as well as complete gastrointestinal hydrolysis, ( 2 ) poor absorption from the gastrointestinal tract, ( 3 ) rapid urinary and fecal excretion of metabolites, and (4)buildup of high but descending order of concentrations in various tissues such as spleen, kidney, liver, lung, fat, heart, brain, and blood. The inhibitory effect of a-solanine on cholinesterase was established through the results of Orgell et d.(1958), Pokrovskii ( 1956), Harris and Whittaker ( 1959, 1962), and Orgell (1963).Orgell et al. ( 1958) demonstrated the presence of cholinesterase inhibitor in the aqueous extracts of potato tissue (tuber, sprouts, leaves, flowers, and stem). The tuber peel contained ten to forty times the concentration of the inhibitor present in the innermost flesh, while extracts of tuber sprouts were as active as the extracts of tuber peel. Subsequently, Harris and Whittaker (1959) showed that this naturally occurring inhibitor differentially inhibited the three serum cholinesterase phenotypes in much the same way as dibucaine. At that time, however, it was not realized that the glycoalkaloid a-solanine (known to be present in appreciable amounts in potatoes and other solanaceae) inhibited serum cholinesterase. On the basis of the possible presence of a-solanine (or solanidine in its numerous forms) in potato-plant extract and the inhibition pattern of various parts of the potato plant or tuber coinciding remarkably with that of glycoalkaloid distribution, Zitnak ( 1960) interpreted potato alkaloid as the cholinesterase inhibitor in the experiments described by Orgell et al. (1958). Zitnak (1960) further predicted the probable presence of free alkaloid solanidine in the extract as a result of enzymatic hydrolysis of glycoalkaloids. Pokrovskii (1956) proved that the inhibitor described by Orgell et al. (1958) and indicated by Harris and Whittaker (1959) was asolanine. He (1956) further proposed that the antiesterase property of a-solanine can be considered the basis for the development of methods to identify biologically harmful concentrations of glycoalkaloids in potatoes. The anticholinesterase action of a-solanine was further supported by the experiments of Harris and Whittaker (1962), who showed that

CHLOROPHYLL/GLYCOALKALOIDSOF SOLANUM TUBEROSUM L.

345

a-solanine and solanidine differentially inhibited the serum cholinesterase of individual persons of “usual,” ‘‘intermediate,’’ and “atypical” phenotypes. This effect was similar to that obtained with dilute aqueous extracts of potatoes. They concluded that, if the inhibition of serum cholinesterase is to be attributed to the toxic effects of a-solanine, then presumably persons with the “atypical” enzyme will be to some extent less susceptible than persons with the “usual” enzyme. Patil et al. (1972) studied the pattern of plasma and erythrocytic cholinesterase inhibition of asolanine in rabbits. They deemed it a weak to moderate inhibitor of both specific and nonspecific cholinesterase. Less inhibition of erythrocytic rather than plasma cholinesterase was considered to be caused by: ( 1 ) the different distribution of a-solanine at these two sites, (2) differences in the mechanism of inhibition of the enzyme as it was produced by plasma and erythrocytes, or ( 3 ) dilution of red blood cells, if the inhibition was reversible. After small multiple doses of a-solanine, a quick inhibition was followed by rapid recovery of serum cholinesterase without the inhibition of red cell cholinesterase. They therefore speculated that, while small doses of a-solanine may cause discomfort on ingestion, repeated doses will have little noticeable effect resulting from acetylcholinesterase inhibition. Several animals have been tested for differences in sensitivity to a total potato alkaloid extract as well as to a-solanine. The data are presented in Table XVIII. Apparently, the high activity of a-solanine injected into the bloodstream may be due to cholinesterase inhibition, while the lesser effects of orally administered a-solanine could result from its poor absorption from the gastrointestinal tract ( Nishie et al., 1971; Patil et al., 1972). The postmortem examination results observed in the experiments of Patil et al. (1972) and also reported by Gull et al. (1970) did not reveal any well-defined symptoms directly attributable to the toxic effects of asolanine. Information on the pharmacology and toxicity of a-chaconine, although it represents nearly 60% of total potato alkaloids, is meager. A mode of action similar to that of a-solanine seems possible because of its structural resemblance to a-solanine. Nishie et al. (1971) reported that the free alkaloid solanidine was less toxic than a-solanine. Except for the inhibitory action of leptine I (Orgell, 1963), pharmacological and toxicological properties of trace alkaloids of potato (leptines and leptinines) are unknown. In an attempt to find a quick, simple, and inexpensive method to determine a-solanine toxicity, Patil et al. (1972) noted a depression in the growth of the fungus Trichoderma virde. The daily rate of growth was the same on the control medium (PDA) without a-solanine and on the

TABLE XVIII OF (Y-SOLANINE TOXICITY EVALUATION W A

Dose of a-solanine a Experiment

Administration

Amount

a,

Effect

Reference

~

Man b

Pregnant rat

Oral Oral Oral Oral Intravenous Intravenous Oral

Rat

Gastric intubation

Sheep

Mice

Chick embryo ( 4 days old) Rabbit

Intraperitoneal Oral Intraperitoneal Intraperitoneal Intraperitoneal Intraperitoneal Injection into yolk sac

2 2 . 8 mg/kg c 20-25 mg C 225 mg/kg 500 mg/kg 17 mg/kg 50 mg/kg 10% of sprout diet 590 mg/kg 75 mg/kg 1000 mg/kg 42 -C 1.8mg/kg 10 mg/kg 32.3 mg/kg 2 50 mg/kg 18.8 zk l m g / k g

Intraperitoneal

20 mg/kg

Intraperitoneal

30 mg/kg

Intravenous

10 mg/kg

Toxic d Toxic Toxic Lethal Toxic Lethal Death of all pups before weaning age 50% death within 24 hours 50% death in a few hours Nontoxic 50%death in 7 days Toxic 50% death Lethal 50% mortality in 18 days Overnight death Death in 2.5-24 hours; recovery if survived for at least 24 hours Death in 6.25 hours Death in 50 minutes Death in 2 minutes

Source of a-solanine: ( 1 ) Aldrich Chemicals; ( 2 ) K & K Laboratories Inc.; ( 3 ) Sigma Chemical Co. In a case of potato poisoning (total alkaloid). C Determined from potatoes consumed (total alkaloid). d General symptoms of food poisoning. a b

Riihl ( 1951) Wilson (1959) Konig ( 1953) Konig ( 1953) Konig (1953) Konig ( 1953) Kline et al. ( 1961 ) Gull et al. ( 1970) Gull et al. ( 1970) Nishie et al. ( 1971 ) Nishie et al. ( 1971 ) Patil et al. ( 1972) Patil et al. ( 1972) Patil et al. ( 1972) Nishie et al. ( 1971 ) Nishie et al. ( 1971 ) Patil et al. ( 1972)

Nishie d al. ( 1971 ) Patil et al. ( 1972) Nishie et al. ( 1971)

CHLOROPHYLL/GLYCOALKALOIDS OF SOLANUhl TUBEROSUM L.

347

medium containing 10 mg of a-solanine per 100 ml. The increasing concentration of a-solanine in the medium progressively inhibited growth. The lethal concentration (LC,,) was 102.2 mg per 100 mI of the medium. Many workers have attributed the disease-resistance character of potato cultivars to the presence of glycoalkaloid in the tuber and plant. Thus, Allen and KuC (1968) mentioned that a-solanine and a-chaconine were highly fungitoxic to Helminthosporium carbonurn and accounted for at least 90% of the fungitoxicity of potato peel extracts to this fungus at p H 5.6. Inhibition of the growth of Alternaria solani on potato dextrose agar by potato alkaloids ( a-solanine, a-chaconine, and solanidine ) has been reported by Sinden et al. (1973). Solanidine was most inhibitory, followed by a-chaconine and a-solanine. At a concentration of 250 ppm, a-solanine caused 33% inhibition of growth after 96 hours of incubation at 24°C. Zimmer et al. (1967) developed a bioassay technique for the quantitative evaluation of saponin content in alfalfa using Trichodermu. Similarly, the fungitoxic effect of a-solanine on Trichoderma may be further developed to formulate a bioassay technique for rapid estimation for a-solanine. To counteract a-solanine toxicity, Patil et nl. (1972) explored the possible therapeutic uses of atropine sulfate ( 2 mg/ kg ), pargyline hydrochloride ( 5 mg/kg), and amphetamine sulfate ( 5 m g / g ) . A prior dose of atropine sulfate lowered the mortality associated with i p injection into mice of 40 mg of a-solanine per kilogram from 9/10 to 5/10. Similar applications of pargyline hydrochloride and amphetamine sulfate resulted in 8 / 9 and 10/10 mortality. The mice injected with amphetamine sulfate were very active and stimulated before the administration of a-solanine. In comparison with the effect of other drugs, this period of stimulation persisted for some time after the a-solanine injection. Thus, amphetamine and pargyline had no important effects, while the atropine influence appeared antagonistic to and able to counteract the a-solanine toxicity. Renwick (1972) of the London School of Hygiene and Tropical Medicine put forth a theory suggesting that two severe birth defects, spina bifida cystica and anencephaly, could result from a woman eating blighted potatoes during her first month of pregnancy. However, the nature of substances present in potatoes stressed by pathogens or certain environmental agents which can cause such birth defects is not known. Subsequently, a study was undertaken by Poswillo et al. (1972) to establish a causal relationship between blighted potatoes and anencephaly. In spite of gross abnormalities in four of eleven fetuses in six pregnant animals and severe teratogenic defects in those conceived after a prolonged period of consumption of the “blighted potato concentrate, no cases of

348

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ASB (anencephaly or spina bifida) were reported by Poswillo et (11. ( 1972). Moreover, the experimental procedure of Poswillo et ul. ( 1972) did not show that Phytophthora infesturn was responsible for an increase in the glycoalkaloid content of potatoes. There have been no previous reports of a suspected teratogen in stored potatoes. Ansell (1972), referring to the occurrence of ASB in countries not consuming potatoes and the low level of ASB in areas where potato consumption and the level of blight are high, claimed that Renwick's hypothesis is invalid. Further investigations are needed to prove or disprove the Renwick hypothesis.

VII.

SUMMARY

Potato tubers turn green on a cumulative exposure to light as a result of the formation of chlorophyll. Such tubers are usually associated with an increased level of glycoalkaloids. Abnormally high amounts of glycoalkaloids (>20 mg per 100 gm fresh weight) in tubers produce a bitter taste and off-flavor, and cause health hazards. The quality of potatoes and potato products, especially in respect to appearance and taste, are adversely affected. Although chlorophyll and glycoalkaloids develop in the peripheral zone of the tuber and glycoalkaloids in the cortex, the biosynthetic processes are independent of each other. Biosynthesis of chlorophyll is based on porphyrin, whereas glycoalkaloids follow a pathway similar to that of isoprenoids. Several factors influence the rates of greening and glycoalkaloid formation. The greening potential and glycoalkaloid development are inherent cultivar characteristics. Abnormal growing and environmental conditions and improper handling lead to differences in glycoalkaloid concentrations. Immature and small potatoes show more tendency toward greening and glycoalkaloid production than do mature and large ones. In general, intense light, high temperatures during light exposure, and low storage temperatures enhance the rate of greening. The longer the duration of light exposure, the greater is the accumulation of chlorophyll and potato glycoalkaloids. The problem of greening and the formation of glycoalkaloids could be approached by at least five routes: (1)breeding of resistant cultivars, ( 2 ) protection from light, ( 3 ) special storage conditions, ( 4 ) application of nonresidual chemicals such as wax and oils, and ( 5 ) careful handling and sorting procedures. Outbreaks of glycoalkaloid poisoning, the toxicity and pharmacology of glycoalkaloids, and current views relating to potential health problems are discussed.

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REFERENCES Akeley, R. V., Houghland, G. V. C., and Schark, A. E. 1962. Genetic differences in potato-tuber greening. Amer. Potato J. 39, 409-417. Allen, E. H., and Kuk, J. 1968. a-Solanine and a-chaconine as fungitoxic compounds in extracts of Irish potato tubers. Phytopathology 58, 776-781. Ansell, J. 1972. Renwick‘s hypothesis. New Sci. 56 (820), 419-420. AOAC. 1950. “Official Methods of Analysis.” Association of Official Agricultural Chemists, Washington, D.C. AOAC. 1965. “Official Methods of Analysis.” Association of Official Agricnltural Chemists, Washington, D.C. Arutyunyan, L. A. 1940. The solanine content of potatoes. Vop. Pitan. 9, 30-36. Baker, L. C., Lampitt, L. H., and Meredith, 0. B. 1955. Solanine, glycoside of the potato. 111. An improved method of extraction and determination. J. Sci. Food Agr. 6, 197-202. Bogorad, L. 1965. Porphyrins and bile pigments. In “Plant Biochemistry” (J. Bonner and J. E. Varner, eds.), pp. 717-760. Academic Press, New York. Bogorad, L. 1966. The biosynthesis of chlorophylls. In “The Chlorophylls” ( L . P. Vernon and G. R. Seely, eds.), pp. 481-510. Academic Press, New York. Bomer, A., and Mattis, H. 1923. High solanine content of potatoes. Z. Unters. Nahr.Genussm. Gebrauchsgegenstaende 45, 288-291. Bomer, A., and Mattis, H. 1924. The solanine content of potatoes. Z. Unters. Nahr.Genussm. Gebrauchsgegenstaende 47, 97-127. Braun, H. 1948. Wber Solanin-Anreicherungen in Kartoffelknollen. Beitr. Agrarwiss. 2, 61-75. Buck, R. W., Jr., and Akeley, R. V. 1967. Effect of maturity, storage temperature, and storage time on greening of potato tubers. Amer. Potato I. 44, 56-58. Clayton, R. B. 1965. Biosynthesis of sterols, steroids, and terpenoids. Part 11. Phytosterols, terpenes, and the physiologically active steroids. Quatt. Reu., Chem. SOC. 19, 201-230. Conner, H. W. 1937. Effect of light on solanine synthesis in the potato tuber. Plant Physiol. 12, 79-98. Dabbs, D. H., and Hilton, R. J. 1953. Methods of analysis for solanine in tubers of Solanum tuberosum. Can. J . Technol. 31, 213-220. Damon, S. R. 1928. “Food Infections and Food Intoxications.” Williams & Wilkins, Baltimore, Maryland. DeLoach, D. B., and Sitton, G. R., 1941. Marketing central Oregon and Klamath Basin late-crop potatoes. Oreg., Agr. Exp. Sta., Bull. 400, 1-36. Ellsworth, R. K. 1972. Chlorophyll biosynthesis. In “Advances in the Chemistry of Plant Pigments” ( C . 0. Chichester, ed.), pp. 85-102. Academic Press, New York. FAO. 1966. “Production Yearbook,” Vol. 20. United Nations, Rome. Folsom, D. 1947 Permanence of greening of potato tubers. Amer. Potato I. 24, 336340. Forsyth, F. R., and Eaves, C. A. 1968. Greening of potatoes: CA cure. Food Technol. 22, 48-50. Golubeva, S. N. 1966. Experiences in the diagnosis of food poisoning and treatment with solanine. Vestn. Otorinolaringol. 28, 23-27.

350

S. J. JADHAV AND D. K. SALUNKHE

Gortner, R. A. 1949. Nitrogen bases. In “Outlines of Biochemistry,” (R. A. Gortner, Jr., and W. A. Gortner, eds. ), pp. 488-513. Wiley, New York. Griebel, C. 1923. Injurious potatoes rich in solanine. 2. Unters. Nuhr.-Genussm. Gebrauchsgegenstaende 45, 175-183. Gull, D. D., and Isenberg, F. M. 1958. Lightburn and off-flavor development in potato tubers exposed to fluorescent lights. Proc. Amer. SOC. Hoe. Sci. 71, 446454. Gull, D. D., and Isenberg, F. M. 1960. Chlorophyll and solanine content and distribution in four varieties of potato tubers. Proc. Amer. SOC. Hort. Sci. 75, 545556. Gull, D. D., Isenberg, F. M., and Bryan, H. H. 1970. Alkaloid toxicology of Solanum tuberosum. HortScience 5, 316. Guseva, A. R., and Paseshnichenko, V. A. 1958. A study of the biogenesis of potato glycoalkaloids by the method of labeled atoms. Biokhimiya 23, 385-388. Guseva, A. R., and Paseshnichenko, V. A. 1962. A study of solasodine biosynthesis by the method of oxidative breakdown. Biokhimiya 27, 721-725. Guseva, A. R., Borikhina, M. G., and Paseshnichenko, V. A. 1960. Utilization of acetate for the biosynthesis of chaconine and solanine in potato sprouts. Biokhimiya 25, 213-214. Guseva, A. R., Paseshnichenko, V. A., and Borikhina, M. G. 1961. Synthesis of radioactive mevalonic acid and its use in the study of the biosynthesis of steroid glycoalkaloids from Solanum. Biokhimiya 26, 631-635. Hansen, A. A. 1925. Two fatal cases of potato poisoning. Science 61, 340441. Hardenburg, R. E. 1954. Comparison of polyethylene with various other 10-pound consumer bags for Sabago, Katahdin, and Green Mountain potatoes. Amer. Potato 1. 31, 29-39. Hardenburg, R. E. 1964. Greening of potatoes during marketing-a review. Amer. Potato 1. 41, 215-220. Harris, F. W., and Cockburn, T. 1918. Alleged poisoning by potatoes. Analyst 43, 133-137. Harris, H., and Whittaker, M. 1959. Differential response of human serum cholinesterase types to an inhibitor in potato. Nature (London) 183, 1808-1809. Harris, H., and Whittaker, M. 1962. Differential inhibition of the serum cholinesterase phenotypes by solanine and solanidine. Ann. Hum. Genet. 26, 73-76. Heftmann, E. 1963. Biochemistry of plant steroids. Annu. Reu. Plant Physiol. 14, 225-248. Heftmann, E. 1967. Biochemistry of steroidal saponins and glycoalkaloids. Lloydia 30, 209-230. Heftmann, E., and Mosettig, E. 1960. “Biochemistry of Steroids.” Van NostrandReinhold, Princeton, New Jersey. Hilton, R. J. 1951. Factors in relation to tuber quality in potatoes. 11. Preliminary trials on bitterness in Netted Gem potatoes. Sci. Agr. 31, 61-70. Howard, F. D., Yamaguchi, M., and Timm, H. 1957. Effect of illumination and waxing on the chlorophyll development in scrubbed White Rose potato tubers. Amer. Potato I. 34, 324-329. Hutchinson, A., and Hilton, R. J. 1955. The influence of certain cultural practices on solanine content and tuber yields in Netted Gem potatoes. Can. J. Agr. Sci. 35, 485-491.

CHLOROPHYLL/GLYCOALKALOIDS OF SOLANUM TUBEROSUM L.

351

Isenberg, F. M., and Gull, D. D. 1959. Potato greening under artificial light. N . Y., Agr. E x p . Sta., Ithaca, Bull. 1033, 1-8. Jadhav, S. J., and Salunkhe, D. K. 1973. Enzymatic glucosylation of solanidine. J. Food Sci. 38, 1099-1100. Jadhav, S. J., and Salunkhe, D. K. 1974. Effects of certain chemicals on photoinduction of chlorophyll and glycoalkaloid synthesis and on sprouting of potato tubers. Can. Inst. Food Sci. Technol. J . 7, 178-182. Jadhav, S. J.. Patil, B. C., and Salunkhe, D. K. 1973a. Control of potato greening under hypobaric storage. Food Eng. 45, 111-112. Jadhav, S. J., Salunkhe, D. K., Wyse, R. E., and Dalvi, R. R. 1973b. Solanum alkaloids: Biosynthesis and inhibition by chemicals. J. Food Sci. 38, 453-455. Jeppsen, R. B., Salunkhe, D. K., and Jadhav, S. 1973. Formation and anatomical distribution of chlorophyll and solanine in potato tubers and their control by chemical and physical treatments. 33rd Annu. Int. Food Technol. Meet., 1973 p. 153. Kline, B. E., von Elbe, H., Dahle, N. A., and Kupchan, S. M. 1961. Toxic effects of potato sprouts and of solanine fed to pregnant rats. Proc. SOC. E x p . Biol. Med. 107, 807-809. Konig, H. 1953. Investigations concerning the action of solanine in cattle and sheep in connection with feeding the potato foliage. Schweiz. Arch. Tierheilk. 95, 97-118. KuC, J. 1964. In “Phenolics in Normal and Diseased Fruits and Vegetables” (V. C. Runeckles, ed.), p. 63. Plant Phenolics Group of North America, Norwood, Massachusetts. Kuhn, R., and Low, I. 1954. Die Konstitution des Solanins. Angew. Chem. 66, 639640. Kuhn, R., and Low, I. 1955a. Die alkaloidglykoside de bliitter von Solanum auiculare. Chem. Bet-. 88, 289-294. Kuhn, R., and Low, I. 195513. Chaconine. Ann. Acad. Sci. Fenn., Ser. A2. 60, 488495. Lampitt, L. H., Bushill, J. H., Rooke, H. S., and Jackson E. M. 1943. Solanine, glycoside of the potato. 11. Distribution in the potato plant. J. Soc. Chem. Ind., (London) 62, 48-51. Larsen, E. C. 1949. Investigations on cause and prevention of greening in potato tubers. Idaho, Agr. E r p . Sta., Res. B d l . 16, 1-32. Lepper, W. 1943. Beitrag zur Solaninfrage. Belichtung und Solaningehalt der Kartoffeln. Z. Lebensm.-Unters. -Forsch. 86, 247-250. Lepper, W. 1949. Solaningehalt von 58 Kartoffelsorten. Z. Lebensm.-Unters. -Forsch. 89, 264-273. Liljemark, A., and Widoff, E. 1960. Greening and solanine development of white potato in fluorescent light. Amer. Potato I. 37, 379-389. Lutz, J. M., Findlen, H., and Ramsey, G. B. 1951. Quality of Red River Valley potatoes in various types of consumer packages. Amer. Potato J. 28, 589-602. McKee, R. K. 1955. Host-parasite relationships in the dry-rot disease of potatoes. Ann. Appl. Biol. 43,147-148. Meyer, G. 1895. Ueber Vergiftungen durch Kartoffeln. I. Ueber den Gehalt der Kartoffeln an solanin und iiber die Bildnng desselben wahrend der Keimung. Arch. E x p . Pathol. Pharmakol. 36, 361-372.

352

S. J. JADHAV AND D. K. SALUNKHE:

Motts, G. N. 1937. Marketing potatoes in Michigan. Mieh., Agr. Exp. Sta., Spec. Bull. 288, 1-68. Newman, L. 1966.Trends in merchandising quality potatoes. Prcrc. Potato Znd. Conf., 8th, 1966, pp. 1 5 . Nishie, K., Gumbmann, M. R., and Keyl, A. C. 1971. Pharmacology of solanine. Tmicol. Appl. Phurmmol. 19,81-92. Orgell, W.H. 1963. Inhibition of human plasma aholinesteeeee in uitro by alkaloids, glycosides, and other natural substances. L b & u 26, 36-43. Orgell, W. H., Vaidya, K. A., and Dahm, P. A. 1958. krhibition of human plasma cholinesterase in uitro by extracts of solanaceous plants. Scisnce 198, 1136-1137. Pallman, M., and Schindler, K. 1942. Beeinflusst die Duengung den Solaningehalt der Kartoffeln? Schweiz. Landwirt. Monatsh. 20,21-27. Parups, E. V., and Hoffman, I. 1967. Induced alkaloid levels in p o t a b tubers. Amer. Potato 1. 44,277-280, Patil, B. C. 1972. Formation and control of chlorophyll a d solanine in tubers of Solanum tuberosum L. and evaluation of solanine toxicity. Ph.D. Thesis, Utah State University, Logan. Patil, B. C., Salunkhe, D. K., and Singh, B. 1971a. Metabalism of solanine and chlorophyll in potato tubers as affected by light and specific chemicals. J. Food Sci. 36, 474-476. Patil, B. C., Singh, B., and Salunkhe, D. K. 1971b. Formatien of chlorophyll and solanine in Irish potato ( Solanum tuberosum L. ) tubers and their control by gamma radiation and C02 enriched packaging. Lebensm.-Wiss. u. Technol. 4, 123-125. Patil, B. C., Sharma, R. P., Salunkhe, D. K., and Salunkhe, K. 1972. Evaluation of solanine toxicity. Food Cosmet. Tmicol. 10,395-398. Pfankuch, E. 1937. Photometric determination of solanine. Biochem. Z. 29.5, 44-47. Poapst, P. A., and Forsyth, F. R. 1973. Control of greening in cold stored potatoes. Res. Sta. Kentuille, Nova Scotia Annu. Report 1973, Res. Sta. Can. Dept. Agr., 73-7, 99. Pokrovskii, A. A. 1956. The effect of the alkaloids of the sprouting potato on cholinesterase. Biokhimiya 21,705-710. Poswillo, D. E., Sopher, D., and Mitchell, S. 1972. Experimental induction of foetal malformation with “blighted” potato: A preliminary report. Nature (London) 239, 462464. Reeve, R. M., Hautala, E., and Weaver, M. L. 1969. Anatomy and compositional variation within potatoes. 11. Phenolics, enzymes, and other minor components. Amer. Potato J. 46,374-386. Renwick, J. H. 1972. Hypothesis: Anencephaly and spina bifida are usually preventable by avoidance of a specific but unidentified substance present in certain potato tubers. Brit. 1. Preu. SOC. Med. 26,67-88. Rothe, J. C. 1918. Illness following the eating of potatoes containing solanine. Z.H y g . 88, 1-12. R h l , R. 1951. Beitrag zur pathologie und Toxikologie des Solanins. Arch. Pharm. (Weinheim) 284, 67-74. Salunkhe, D. K., Wheeler, E. J., and Dexter, S. T. 1954. The effect of various environmental factors on the suitability of potatoes for chip-making. Agron. J. 46, 195-199.

CHLOROPHYLL/GLYCOALKALOIDS OF SOLANL’M TUBEROSUM L.

353

Salunkhe, D. K., Wu, M. T., and Jadhav, S. J. 1972. Effects of light and temperature on the formation of solanine in potato slices. J . Food Sci. 37, 969-970. Sanford, L. L., and Sinden, S. L. 1972. Inheritance of potato glycoalkaloids. Amer. Potato 1. 44,209-217. Satoh, T. 1967. Glycemic effects of solanine in rats. Jap. J . Pharmucol. 17, 652-658. Schreiber, K. 1966a. Int. Symp. Biochem. Physiol. Alkaloid, 3rd, 1965 p. 54. Schreiber, K. 196613. Abh. Deut. Akad. Wiss. Berlin, K1. Chem., Geol. Biol. No. 3, p. 65. Schreiber, K. 1968. Steroid alkaloids: Solunum group. In “The Alkaloids” (R. H. F. Manske, ed.), Vol. 10, pp. 1-192. Academic Press, New York. Schwimmer, S., and Weston, W. J. 1958. Chlorophyll formation in potato tubers as influenced by gamma irradiation and by chemicals. Amer. Potato J. 35, 534542. Shih, M. 1972. The accumulation of isoprenoids and phenols and its control as related to the interaction of potato ( Solunum tuberosum L.) with Phytophtohora infestam. Ph.D. Thesis, Purdue University, Lafayette, Indiana. Sinden, S. L. 1971. Control of potato greening with household detergents. Amer. Potato I. 48, 53-56. Sinden, S. L. 1972. Effect of light and mechanical injury on the glycoalkaloid content of greening-resistant potato tubers. Amer. Potato J . 49, 368. Sinden, S. L., and Webb, R. E. 1972. Effect of variety and location on the glycoalkaloid content of potatoes. Amer. Potato l. 49, 334-338. Sinden, S. L., Goth, R. W., and O’Brien, M. J. 1973. Effect of potato alkaloids on the growth of Alternuria solani and their possible role as resistance factors in potatoes. Phytopathology 63,303-307. Talburt, W. F., and Smith, 0. 1959. “Potato Processing.” Avi, Westport, Connecticut. Tschesche, R., and Hulpke, H. 1967. Zur Biosynthese von Steroid-Derivaten im Pflanzenreich. VIII. Biogenese von Solanidin aus Cholesterin. Z. Naturforsch. B 22, 791. USDA. 1972. “Agricultural Statistics.” USDA, Washington, D.C. von Morgenstern, F. 1907. Wber den Solaningehalt der speise- und Futterkartoffeln und iiber den Eiduss der Bodenkultur auf die Bildung von solanin in der Kartoffelpflanze. Landwirt. Vers.-Sta. 65, 301-338. Wadleigh, C. H., and Dyal, R. S. 1972. The best kept secret. HortScience 7, 369372. Wierzchowski, P., and Wierzchowska, Z. 1961. Colorimetric determination of solanine and solanidine with antimony trichloride. Chem. Anal. (Warsaw) 6 , 579-

585. Willimott, S. G. 1933. An investigation of solanine poisoning. Analyst 58, 431-439. Willuhn, G. 1965. Biogenesis of pharmaceutically important plant steroids. pharm. Ztg. 110, 96-106. Wilson, G. S. 1959. A small outbreak of solanine poisoning. Mon. Bull. Min. Health Pub. Health Lab. Sera 18, 207-210. Wintgen, M. 1906. Wber den Solaningehalt der Kartoffeln. Z. Unters. Nahr.-Genussm. Gebrauchsgegenstaende 12, 113-123. Wolf, M. J., and Duggar, B. M. 1940. Solanine in the potato and the effects of some factors on its synthesis and distribution. Amer. J . Bot. 27, Suppl. 20s. Wolf, M. J., and Duggar, B. M. 1946. Estimation and physiological role of solanine in the potato. 1. Agr. Res. 73, 1-32.

354

S. J. JADHAV AND D. K. SALUNKHE

Wu, M. T., and Salunkhe, D. K. 1972a. Control of chlorophyll and solanine syntheses and sprouting of potato tubers by hot paraffin wax. J. Food Sci. 37, 629-630. Wu, M. T., and Salunkhe, D. K. 1972b. Inhibition of chlorophyll and solanine formation, and sprouting of potato tubers by oil dipping. J. Amer. SOC. Hort. Sci. 97, 614-616. Wu, M. T., and Salunkhe, D. K. 1972c. Control of chlorophyll and solanine formation in potato tubers by oil and diluted oil treatments. HortSceince 7, 466-467. Yamaguchi, M., Hughes, D. L., and Howard, F. D. 1960a. Effect of color and intensity of fluorescent lights and application of chemicals and waxes on chlorophyll development of White Rose potatoes. Amer. Potato J. 37, 229-236. Yamaguchi, M., Hughes, D. L., and Howard, F. D. 1960b. Effect of season, storage temperature, and temperature during light exposure on chlorophyll accumulation of White Rose Eotatoes. Proc. Amer. SOC. Hort. Sci. 75, 529-536. Ziegler, R., and Egle, K. 1965. Determination of chloroplast pigments. I. Critical examination of the spectrophotometric determination of chlorophyll. Beitr. Biol. Pj7unz. 41, 11-37. Ziegler, R., Schanderl, S. H., and Markakis, P. 1968. Gamma irradiation and enriched COz atmosphere storage effects on the light-induced greening of potatoes. J . Food Sci. 33, 5334535. Zimmer, D. E., Pedersen, M. W., and McQuire, C. F. 1967. A bioassay for alfalfa saponins using the fungus, Trichodenna uiride. Pers. ex Fr. Crop Sci. 7, 223224. Zitnak, A. 1953. The influence of certain treatments upon solanine synthesis in potatoes. M. S. Thesis, University of Alberta, Edmonton, Alberta, Canada. Zitnak, A. 1955. Factors influencing the initiation and rate of solanine synthesis in tubers of Solanum tuberosum L. Ph. D. Thesis, University of Alberta, Edmonton, Alberta, Canada. Zitnak, A. 1960. Cholinesterase inhibitors. Science 131, 66-68. Zitnak, A. 1961. The occurrence and distribution of free alkaloid solanidine in Netted Gem potatoes. Can. J. Biochem. Physwl. 39, 1257-1265. Zitnak, A. 1968. Separation of glycoalkaloids from Solanum tuberosum L. by thinlayer chromatography. Proc. Can. SOC. Hort. Sci. 7,75437. Zitnak, A. 1970. “Occurrence of Bitter Potatoes in Ontario,” Inform. Bull. No. 257/ 81, pp. 1-2. University of Guelph, Ontario, Canada. Zitnak, A. and Johnston, G. R. 1970. Glycoalkaloid content of B5141-6 potatoes. Amer. Potato J. 47, 256-260.

Subject Index A Adipose tissue, food regulation, 25-34 Adrenal hormones, and meat quality relationship, 89-96 Adrenocorticotropin, 89-90, 93 Appetite, causes of, 9-14 cessation of, 15-17 factors affecting, 10-17 hormonal relationships, 11-12, 14, 2324, 27-34 sensory stimulus, 35-43

B Beef processing, 187-193, 195,203-206 Body weight, set point, 25-34

C Carcass cooling, 137-138, 158-159 Chewing, role in nutrition, 17-18 Chlorophyll, control of formation in potato, 331-342 distribution and biosynthesis in potato, 310-316 factors affecting formation in potato, 316-331 Controlled atmosphere storage, mango fruits, 278-280 potato, 340 Cooling treatments, meat quality relationship, 137-138 Corticoid hormones, and meat quality, 90-95 Cryobiopsy, 108

D Diet, life span relationship, 53-55

E Ethylene, use in mango ripening, 253254, 256-258

F Flavors, affect on food intake, 3 7 4 8 food processing, 46-51 meat processing, 46, 47 Food intake, adipose tissue, role of, 25-34 gastrointestinal role, 17-24 obesity impairment of, 51-53 regulation of, 1-69 sensory stimulus, 35-43 Food processing, flavors, 46-51 pork, normal, PSE, and DFD, 124-128 Food, sensoring stimulus, 35-43 Fruit ripening, physiology of mango, 249-270

G Glycoalkaloid, control of formation in potato, 331-342 formation in Solanun tuberosum, 312331 pharmacology and toxicology, 342-348

355

356

SUBJECT INDEX

Growth hormone, and meat quality, 8889

I Ionizing radiation, see y-radiation

H Halothane, effect on muscle metabolism, 84-87 Heat treatment, use in storage of mango fruits, 281-282 Hypobaric storage, 340 1

L

Lamb processing, 178-186, 193-194, 196, 203, 204 Light, glycoalkaloid formation in potato, 324-330 Low-temperature storage, mango fruits, 255, 275-278

M Magnesium, effect on meat quality, 102, 110, 136 Malignant hyperthermia, 82-87 Mango fruit, physiology and biochemistry, 223306 storage and transport of, 271-288 Meat chilling, cooling phase, 206-211 storage phase, 211-213 Meat, flavor due to processing, 46, 47 Meat quality, animal physiology and, 71-155 beef, 187-193, 195, 203-206 conditioning, 173-178, 185-186, 1%)191, 197-198 control methods to maintain, 134-138 cooling and quality relationship, 137138 detection methods, 131-134 electricaI stimulation of rigor, 198-199 high-temperature conditioning, 197198

hormonal affects, 87-98 hot cutting and boning, 194-197 lamb, 178-186, 193-194, 196,203, 204 mutton, 186-187, 193-194 new concepts, 157-222 preslaughter environment, 74-79 rigor mortis relationship, 80-81 Meat tenderness, cold shortening effect, 137, 159-170, 187-190 thaw shortening, 170-173, 182-185 Microbiology,, meat processing, 195-197, 199-206 MIRINZ tenderometer, 167 Monotony, flagging appetite, 16-17 Muscle biochemistry, role in meat quality, 9%113 Muscle morphology, as related to meat quality, 113-121 Muscle, pale, soft, exudative, biochemistry of, 106-1@, 111-113 endocrine interrelationship, 87-98 occurrence and processing of, 123-128 rigor mortis, 80-81 thyroid role, 9 6 9 8 Mutton processing, 186-187, 193-194 Myotomy, effect on meat quality, 101102

0 Obesity, impairment of food intake regulation, 51-53

P Packaging, mango fruits for shipment, 285-286 role in chlorophyll formation in potato, 328, 332 Polyethylene film, bacterial rot of mango, 273 chlorophyll development in potato, 328-329 Pork, animal physiology and meat quality, 71-155 Potato, chlorophyll formation and glycoalkaloid toxicity, 307-354

357

SUBJECT INDEX

T

Processed foods, effect on nitrogen metabolism, 24

R y-Radiation, mango fruit storage and preservation,

280-281 potato storage, 340-342

Temperature, cold shortening effect in meat, 161-

170, 182-191 effect on mango, 255 glycoalkaloid formation in potato, 319-

323 high-temperature conditioning of meat,

197-198 meat quality and animal stress, 74-79,

S Sarcoplasmic reticulum, 86-87, 105-106,

169 Sensory stimulus, food, 3 9 4 3 Solanine, 309 distribution in potato, 310-312 toxicology of, 342-348 Storage, diseases of mango fruits, 271-274 methods for mango fruits, 274-285 hypobaric, 340 potatoes and glycoalkaloid formation,

319-323 Sugars, mango fruits, 238-239, 246, 259-

260

A

5

€36 c 7 0 8 E 9 F O

G 1 H 2 1 3 J 4

132 microbical growth during meat conditioning, 199-206 physics of meat chilling, 206-213 processing hams, 124-128 thaw shortening in meat, 170-173,

182-185

V Vitamins, mango fruit content, 268-269,

293

w

Warner-Bratzler machine, 167 Wisconsin system, meat quality judging,

73