Advances in Food Research, Volume 27

ADVANCES IN FOOD RESEARCH VOLUME 27

Contributors to This Volume

D. J. Casimir Ray A. Field G. W. Froning Samuel A. Goldblith Yanco Guegov J . F. Kefford Vernon L. Singleton F. B . Whitfield

ADVANCES IN FOOD RESEARCH VOLUME 27

Edited by

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

G. F. STEWART University of Califoniiu Duvis. Culifornia

E. M. MRAK University o j Califirniu Davis. Culiforniu

Editorial Bocrrcl

JOHN AYRES S. GOLDBLITH J . HAWTHORNE J . F. KEFFORD

S. LEPKOVSKY D. REYMOND EDWARD SELTZER W . M. URBAIN

1981

ACADEMIC PRESS A Suhsidiurv of Hurcourt Bruce Jovunovich, Publishers

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9 8 7 6 5 4 3 2 1

CONTENTS CONTRIBUTORST0 V O L U M E 27 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

iX

PREFACE ......................................................................

xi

Samuel Cate Prescott Samuel A . Goldblith

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

I. I1 .

............................................... 111. MIT Undergraduate Education . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Early Career . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Prescott's Work on Thermal Processing with William Lyman Underwood . . . . . . . VI . Prescott's Career during and after World War 1 ............................. VII . Prescott's Contributions to Refrigeration (and Freezing) of Foods VIII . Prescott's Contributions to the Chemistry of Coffee ......................... IX . Prescott and the Institute of Food Technologists ............................ X . Prescott 's Contemporaries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI . Prescott 's Hmors Awards. and Public Service ............................. XI1 . Prescott and MIT XI11 . In Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

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3 3 5

I 8 8 10 11

12 14 16 18 20

Mechanically Deboned Red Meat Ray A . Field

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II . Potential Yield I11 . Regulations Gov ......................... IV . Economic Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Composition of Mechanically Deboned Meat . . . . . . VI . Safety Aspects of Mechanically Deboned Meat ............................. VII . Functional Properties of Mechanically Deboned Meat ........................ VIII . Nutritional Value of Mechanically Deboned Meat ........................... IX . Palatability of Mechanically Deboned Meat ................................ X . Additional Research Needs ......... ................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

23 26 31 35 39 53 68 14 88 93 95

V

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CONTENTS

Mechanical Deboning of Poultry and Fish G . W . Froning I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................... II. Types of Mechanical Deboners . . . . . . . . . . . . . . . . . . .................... 111. Composition and Nutritive Properties . . . . . . . . . . . . . I V . Flavor Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Color Stability.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v1. Functional Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......................... VII. Utilization of Bone Residue . . . . . . . . . . . . . . . VIIl. Microbial Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........... IX. Regulations . . . . . . . . . . . . . ........... X . Research Needed. . . . . . . . . References . . . . . . . . . . . . . .

110 Ill

Ill 120 127 I29 137 138 140 142

Naturally Occurring Food Toxicants: Phenolic Substances of Plant Origin Common in Foods Vernon L. Singleton

I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... Origins and Types of Plant Phenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evolutionary Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Examples of Common Plant Phenols with Actual or Potential Significance in Animal Consumption (Toxic or Beneficial) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Mechanisms of Toxicity by Phenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusions and Assessment of Risks ..................... .. VI1. Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . ............................ 11. 111.

149 151 153 I62 210 218 22 1 22 1

Technology and Flavor Chemistry of Passion Fruit Juices and Concentrates D. 1. Casimir, J. F. Kefford, and F. B. Whitfield

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Passion Fruit Pulp and Juice . . . . . . . . . ................... 111. Concentration of Passion Fruit Juice . . . ................... IV. Chemistry of Volatile Flavoring Constitu ................... V . Needs and Applications for Research and Development . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . .............................................

243 247 265 269 288 290

vii

CONTENTS

Phase Transitions of Water in Some Products of Plant Origin at Low and Superlow Temperatures Yanco Guegov I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Water-The Basic Component o f Plant Tissue., . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Initial Crystallization of Water in Fruits and Vegetables . . . . . . . . . . . . . . . . . . . . . . IV. Phase Transitions at Low Temperatures (to -70°C) . . . . . . . . . V . Phase Transitions at Superlow Temperatures (-70 to - 196°C) VI. Conclusion. . .............................................

References . . . . . . . . . . . . . . . . . . . . .

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INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

297 299 307

349 352 363

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CONTRIBUTORS TO VOLUME 27 Numbers in parentheses indicate the pages on which the authors’ contributions begin.

D. J . Casimir, Division of Food Research, Commonwealth Scientific and Industrial Research Organisation, North Ryde, New South Wales, 21 13, Australia (243) Ray A. Field, Division of Animal Science, University of Wyoming, Laramie, Wyoming 82071 (23) G . W, Froning, Department of Animal Science, University of Nebraska, Lincoln, Nebraska 68583 (109)

Samuel A. Goldblith, Professor of Food Science and Vice President, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (1) Yanco Guegov, Canning Research Institute, BG 4000 Plovdiv, Bulgaria (297) J . F. Kefford, Division of Food Research, Commonwealth Scientific and Industrial Research Organisation, North Ryde, New South Wales 21 13, Australia

(243)

Vernon L. Singleton, Department of Viticulture and Enology, University of California, Davis, California 95616 (149) F. B . Whitfield, Division of Food Research, Commonwealth Scientific and Industrial Research Organisation, North Ryde, New South Wales 21 13, Australia (243)

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PREFACE In this volume of Arlvunces in Food Resetrrch, the Editors have included a short biography of Dr. Samuel Cate Prescott, one of the early leaders in the field of food science and technology, written by his student, Dr. Samuel Goldblith. The pioneering work of Prescott and Underwood and the publication of the volume Food Technology by Prescott and Proctor established the field of food science and technology in the United States. Others of the same era have made significant contributions to the growth of the field, and, since the field is still young, it will be possible to develop authentic biographical vignettes of the pioneers written by students or associates of these scientists. The Editors hope, in the forthcoming volumes, to include short biographies similar to that of Samuel Cate Prescott. It is not the Editors’ intention to include such a descriptive biography in each volume, but we hope that in the years to come, the contributions of a number of pioneers in the field will be described.

ADVANCES IN FOOD RESEARCH. VOL.

27

SAMUEL CATE PRESCOTT: -Pioneer Food Technologist -Gifted Teacher -Poet, Humanist -Mr. MIT -Friend to Many SAMUEL A . GOLDBLITH Prcdhssor of Food Science und Vice President. Mussuchusetts Institute of Technology. Cambridge, Massachusetts

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.............. 11. Early Education. . . . . . . . . . . . . . . . . 111. MIT Undergraduate Education . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Early Career . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Prescott’s Work on Thermal Processi Prescott ‘s Career during and after World War 1 ........................ , VI. VII. Prescott’s Contributions to Refrigeration (and Freezing) of Foods . . . . . . . . . VIII. Prescott’s Contributions to the Chemistry of Coffee .................... ....... IX. Prescott and the Institute of Food Technologists X. Prescott ’s Contemporaries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI. Prescott’s Honors, Awards, and Public Service ........................ ... XII. Prescott and MIT ................................. ... XIII. In Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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II

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20

INTRODUCTION

The life of Samuel Cate Prescott spans an era of time from that when the term “microbe” was first used by Sedillot in 1878 to that beyond the discovery of DNA in 1953. I Copyright @ 1981 by Academic Rcss. Inc All rights of reproduction in any form reserved. ISBN 0-12-016427-2

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S . A . GOLDBLITH

In his professional career, Dean Prescott pioneered food microbiology, transforming thermal processing of foods (canning) from an art based on experience to a technology based on science. His researches had far-reaching effects on the other basic methods of food preservation-dehydration, freezing, refrigeration, and fermentation. His work not only covered the disciplines fundamental to these basic food preservation methods but also dealt with enzymology; he and his associates pioneered studies on the chemistry of coffee; he was an active participant in the sanitary milk campaign; he was an important person in the budding

FIG. 1 . Samuel Cate Prescott (about 1904) at the blackboard. (This and subsequent photographs courtesy of Mr. Warren Seamans. Director, Historical Collections, MIT.)

SAMUEL CATE PRESCOTT

3

field of sanitary science and public health; and he made important contributions in industrial microbiology and helped to “father” the field of food technology. Prescott ’s researches remind us strongly of Pascal’s vision of knowledge: “Knowledge is like a sphere in space: the greater its volume, the larger its contact with the unknown.” This article attempts to describe the career of this remarkable man, of humble origins, who became a legend at his alma mater as well as in his own profession.

II. EARLY EDUCATION Samuel Cate Prescott was born of an old New England family in the rural village of South Hampton, New Hampshire, on April 15, 1872. After his early education in ungraded country schools (the “one-room schoolhouse”), he attended Sanborn Academy in Kingston, New Hampshire, from 1888 to 1890, and was a member of the academy’s first graduating class. A man of remarkable memory and deep feelings, with a pervasive sense of gratitude, he later served as chairman of the Board of Trustees of that institution for a number of years.

Ill. MIT UNDERGRADUATE EDUCATION

Dr. Prescott attended the Massachusetts Institute of Technology from 1890 to 1894, obtaining his Bachelor of Science degree in chemistry in 1894.’ As Prescott stated on receiving the Babcock-Hart Award of the Institute of Food Technologists in 1951 : “It is today [ 19511 somewhat difficult to visualize the segregation of sharp departmentalization which existed then in the sciences we now employ. Sixty years ago, when I planned to become a chemist, we studied chemistry, physics, and biology, largely as distinct entities rather than as a closely associated and interdependent field of knowledge. In succeeding decades, by interaction and the breaking down of limiting walls, these sciences become more coordinated and interrelated, and thus were enabled to make their great contributions to human welfare in various fields” (Prescott, 1951). During the intervening years between Prescott ’s undergraduate student days and midcareer, he quickly grasped the relationship between chemistry and biol‘Dr. Prescott served as secretary of the MIT class of 1894 from graduation until his death and was one of o d y two members of the active faculty of MIT in the entire history of the MIT Alumni Association selected to be president of the Association (the thirty-fourth president), serving a 2-year term, 1927-1928. The only other active faculty member who served as president of the MIT Alumni Association was the founder of the Association, Professor Robert H . Richards, MIT class of 1868, who was the first president, 50 years earlier than Dr. F’rescott.

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S . A. GOLDBLITH

ogy, and biochemistry became a discipline within the Department of Biology at MIT. Later, when a formal curriculum in food technology was created at the Institute, biochemistry was emphasized in the new curriculum of food technology as well as in all subfields in the department. Certain developments occurred in chemistry and physics that influenced Prescott’s subsequent career. During Prescott’s student days, Ostwald developed the ionic theory, for which he received the Nobel Prize in 1901, thereby originating the work leading to the concept of pH. In the same period that Prescott was an undergraduate student at MIT, Roentgen discovered X-rays, and Becquerel discovered radioactivity; Fischer synthesized sugar, showed caffeine and theobromine to be “purine bodies,” and made the first artificial polypeptides. It was an exciting period, opening new horizons in the sciences. Food chemistry, as Prescott noted in his Babcock Award remarks, was in its “callow youth” prior to 1890. It consisted chiefly in the search for uncommon adulterants in milk and in many food adjuncts, and in the proximate analysis of foods into fats, carbohydrates, and water, ash, and nitrogenous substances. Nutrition was just beginning as a scientific discipline, vitamins were entirely unknown, and enzymes were yet a mystery. Neither organic chemistry nor mi-

FIG. 2.

Prescott (about 1904) “hooking one.” Photogragh taken by William Lyman Underwood.

SAMUEL CATE PRESCOTT

5

crobiology gave promise at that time of syntheses of products having a direct relation to health. Prescott studied under two remarkable teachers who had a profound impact on his professional career and thereby on the development of food technology, sanitary science, and microbiology. One was Ellen Swallow Richards, the first woman graduate of MIT (1873),* instructor in sanitary chemistry, and founder of the American home economics movement. Through Mrs. Richards’s laboratory course in sanitary chemistry, Prescott learned the new and practical Babcock procedure for fat determination in milk only two years after it became available (remarkably rapid communication for those years) and eight years before Babcock was given the grand prize for his invention at the Paris Exposition of 1900. The second teacher at MIT who was to have a profound effect on Prescott’s future career was William Thompson Sedgwick, MIT’s first professor of biology, founder of the Lawrence (Massachusetts) Experiment Station, founder and first president of the Society of American Bacteriologists (later the American Society of Microbiologists), and America’s leading exponent in public health.) (Dr. Prescott became president of the Society of American Bacteriologists in 1919.)

IV.

EARLY CAREER

Immediately after graduation from MIT, Prescott became bacteriologist and assistant chemist at the sewage purification works of the City of Worcester, Massachusetts, for five months. He was selected to become private research assistant to Professor Sedgwick in 1895, and one year later was appointed an instructor in biology at MIT, where he developed the first course in industrial biology in American scientific schools. This course resulted, in part, from his researches with William Lyman Underwood, in 1895- 1899, on bacteriology and sterilization processes in the canning industry. These studies have been generally recognized as basic to the transformation of thermal processing into a rechnology under scientijic control.

Prescott’s course on industrial biology dealt with the applications of bacteriology to the wide range of food, dairy, and beverage industries. He learned to integrate biology and chemistry in seeking solutions to practical problems and was an early participant in the sanitary milk crusade, together with Sedgwick and Rosenau of Harvard. This work led to the development of a practical inspection of dairy farms, which was later required by public health authorities. Prescott ’Later married to Professor Robert H . Richards, MIT class of 1868, a distinguished mining engineer. 3ProfessorSedgwick also became the forty-third president of the American Public Health Association, elected in 1914.

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also took part in the early researches on the pasteurization of milk, when most physicians looked askance at the process. From all these areas of research and teaching, his course in industrial biology evolved into broader courses in food technology. In 1900, Dr. Prescott took five months’ leave of absence from MIT to study mycology and fermentation processes in Berlin and Copenhagen. While in Europe, he met Jean Effront and translated, from the French, Effront’s book Enzymes and Their Applications, published by John Wiley & Sons in 1902. In 1900, Prescott also taught at Simmon’s College in Boston and at the Framingham Normal School. In 1903, Prescott was promoted to Assistant Professor of Industrial Biology at MIT and prepared a manuscript of a book on Water Bacteriology with C. E. A. Winslow, published by John Wiley & Sons in 1904. In 1909 he became Associate Professor and later in 1914, Professor of Industrial Microbiology. In 1904, Prescott established the Boston Biochemical Laboratory for commercial research and consultation on milk, canning, and other food industries. This laboratory gave much attention to pasteurization and sanitary control of public milk supplies. The Boston Biochemical Laboratory remained in operation until 1921.

FIG. 3. Prescott and Underwood on annual fishing trip (circa 1910).

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7

FIG. 4. The staff of the MIT Biology Department, about 1916-1920. Dr. William Thompson Sedgwick is in the front row; Prescott is just to the right and behind the “Chief.” M. P. Honvood is fourth from the left (just his face is showing).

During the winter vacation of 191 1-1912, Dr. Prescott visited Costa Rica for the United Fruit Company to study diseases of the banana, and in 1914 he established a research laboratory in Costa Rica for the United Fruit Co., where he spent the summers of 1914-1917 in tropical food research. Dean Prescott thus initiated an association between university and industry beginning with his first researches with William Lyman Underwood, which also were among MIT’s first successful demonstrations of this powerful linkage between industry and the university. This association of Prescott’s with industry grew and developed as outlined above, and was intensified later in his career, particularly in his researches on refrigeration and coffee chemistry.

V.

PRESCOTT’S WORK ON THERMAL PROCESSING WITH WILLIAM LYMAN UNDERWOOD

So much has been written elsewhere of the great achievements of these two doughty pioneers, who, under primitive conditions, did so much for canning technology in particular, and food technology in general, that little space will be devoted here to this, perhaps Prescott’s most important and seminal r e ~ e a r c h . ~ 4 F ~ more r details on Underwood and Prescott’s work on thermal processing, see Bitting (1937) and Goldblith (1971, 1972).

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S . A. GOLDBLITH

More important to Prescott, it stirred in him the idea to devote most of his professional career to food science and technology, polarized initially around sanitary science and bacteriology. Instead of focusing narrowly and deeply on canning, Prescott broadened his researches into dehydration, refrigeration, freezing, sanitation of food and water, chemistry of coffee, miscellaneous chemicals as sanitizers, and other food additives.

VI.

PRESCOTTS CAREER DURING AND AFTER WORLD WAR I

In 1917, prior to America’s entry into World War I, Prescott entered the military service as a major in the Sanitary Corps of the United States Army, where he was in charge of research and dehydration of food and was inspector of quartermaster stores in large training camps. His interests and researches in dehydration continued through World War I1 (Prescott, 1919, 1920, 1943; Prescott et al., 1922). He remained in the Officers’ Reserve Corps until he became 65, being made a lieutenant colonel in 1920 and a full colonel in 1935. During World War 11, at the age of 70 plus, he served as special consultant to the Secretary of War on dehydration of foods and prepared, for the Office of Scientific Research and Development, the most comprehensive report on troop feeding programs covering the history of army feeding from 1775 to 1940 (Prescott, 1944). Thus, Prescott’s interest in dehydration continued from early in his life to almost the end of his active research career. His major (but by no means sole) contribution was his delineation of the tremendous advantages of this method of preservation during World War I. Needed research still remained incomplete even during World War 11, and we have yet to realize the full potential of this means of preservation.

VII.

PRESCOTTS CONTRIBUTIONS TO REFRIGERATION (AND FREEZING) OF FOODS

Dean Prescott’s work on refrigeration as a method of food preservation is indicative of the catholicity of his interests as a scientist and technologist. During the 1920s and 1930s, Prescott and his students and colleagues studied the technology of refrigeration originally with particular reference to the public health aspects. His work with Sedgwick and Rosenau in the first decade of the twentieth century, on milk and its sanitation, demonstrated the importance not only of pasteurization but also of refrigeration in preserving both the sanitary and the nutritional quality of milk. His work with Proctor (Prescott and Proctor, 1936); Geer (Prescott and Geer, 1936), and Bates and Highlands (Prescott et ul., 1932) demonstrated the impor-

SAMUEL CATE PRESCOTT

FIG. 5 .

9

Major Samuel Cate Prescott, U . S . Army, Sanitary Corps (Res.) (circa 1918)

tance of temperature control to prevent growth of Salmonella and germination of botulinum spores. In his work with Bates and Highlands (Prescott et al., 1932), they pointed out that “the general decreases in numbers of bacteria noted make it seem still more unlikely that microorganisms are the sole or even the principal cause of these changes” (referring to physical and chemical changes), “which probably are enzymic.” Thus, he had the concept of the need of heat inactivation of enzymes, or blanching, which was later demonstrated quantitatively by Sizer and Josephson (1942; Sizer, 1943) in their seminal work.5 ‘Sizer, who obtained his doctorate at Rutgers working with the eminent protein chemist Dr.James Allison, was a young colleague of Dr. Prescott’s, beginning as an instructor at MIT in 1935. He later

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Dr. Prescott was an active member of the Scientific Advisory Committee of The Refrigeration Research Foundation (TRRF) and served as its chairman and later honorary chairman from its founding in 1943 until his death. He also served for several years, prior to World War 11, as president of Commission I11 of the Institut International du Froid. Thus, although not pioneering the technological basis of freezing or refrigeration as methods of food preservation, Dean Prescott and his colleagues made significant and lasting contributions to these techniques. Because of his knowledge, experience, and prestige, he became a dominant figure in TRRF, thus making this organization an important factor in later researches, which established the scientific basis of the freezing preservation of foods.

VIII.

PRESCOlT’S CONTRIBUTIONS TO THE CHEMISTRY OF COFFEE

The exact date of Dean Prescott’s researches on the chemistry (macro) of coffee is not too difficult to establish. Ukers ( I 935) mentions, on p. 408 of his magnum opus on this subject, that in 1921 “the NCRA (National Coffee Roasters’ Association) received a preliminary report from Prof. Samuel C. Prescott, Massachusetts Institute of Technology, on a scientific research into coffee he was conducting for the Association.” We know further from Ukers’ book that in 1920 “twenty two thousand five hundred dollars of the American Fund (of the Joint Coffee Trade Campaign) was appropriated for a scientific research which was conducted by Prof. S. C. Prescott at Massachusetts Institute of Technology whose final report, made and widely published in 1923, was that coffee is a wholesome, helpful, satisfying drink for the great majority of people. Prescott’s studies on the chemistry (macro) of coffee and the organoleptic properties of the coffee brew as affected by degree of roast, variety, temperature of extraction, etc., were carried on over the period beginning in 1920 and lasted until the late 1930s. His co-workers in this mammoth undertaking were R. L. Emerson, L. V. Peakes, R . Heggie (then a graduate student at MIT, later executive vice president, American Chicle Co.), and R. B. Woodward (then a graduate student at MIT, later professor of organic chemistry at Harvard and Nobel Laureate in Chemistry in 1965) (see Prescott et af., 1937a,b). The work of the MIT “coffee lab” not only covered the macrochemistry of coffee, especially the effect of roasting on the chemistry of the coffee bean, but also resulted in the development of vacuum-packed coffee (work sponsored by the American Can Co.), the determination of optimal brew times and tempera”

became head of the Department of Biology in 1955 and Dean of the MIT Graduate School in 1967. Josephson was a graduate student of Sizer’s.

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FIG. 6. Prescott and Underwood at work (circa 1920-1925).

tures, and studies of glass versus metal for brewing coffee, as well as the development of the vacuum-brewing Silex type of coffee maker. Prescott’s researches with the above-mentioned workers on the chemistry of coffee have withstood the test of time. He contributed more to our knowledge of the macrochemistry of coffee than any other individual or group of workers until the advent of the gas chromatograph, the mass spectrometer, and ultraviolet and infrared identification equipment, two decades later. Here, as with his other studies, Prescott established industrial linkages-in this case, not only with the coffee growers, roasters, and blenders, but also with a can manufacturer and equipment company. Prescott’s coffee work has another aspect, insofar as MIT is concerned, which will be dealt with later.

IX. PRESCOTT AND THE INSTITUTE OF FOOD TECHNOLOGISTS Prescott more than any other individual was probably responsible for the first two Food Technology Conferences at MIT in 1937 and 1940, which were the basis for the foundation of the Institute of Food Technologists (IFT). He served as its first president in 1940 and received its two highest awards-the Nicholas

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Appert Medal in 1942, and the Babcock Award (later renamed the Babcock-Hart Award) in 1951. He remained active both locally and nationally in the IFT until his death on March 19, 1962.

X.

PRESCOTTS CONTEMPORARIES

Prescott’s list of friends and contemporaries were as cosmopolitan as his interests in science and in life itself. His (and later his wife’s) friends included all senior officers of MIT and many of the diverse faculty. The Institute was relatively small in his day. The faculty members were less spread out in terms of their residences, and this made it easier to be close to his colleagues. Occasional Sunday evening buffets at his gracious Brookline home for his departmental staff and students helped to weld his small group into a strong unit. His friendship with Karl Taylor Compton is discussed elsewhere in this article. He was a close friend of Vannevar Bush (then vice president of MIT and Dean of Engineering, and later Director, Office of Scientific Research and Development during World War 11). Among his friends in food technology, he was close to Roy Newton, vice president of Swift and Co.; Victor Conquest, vice president of Armour and Co.; Fred Blanck of the H. J . Heinz Co.; H. C. Diehl of TRRF; Arnold T. Thompson, then president of the Merchant’s Cold Storage & Warehouse Co. of Providence; Milton Rosenau, the distinguished sanitarian of Harvard; William V. Cruess, University of California; and his students, particularly Emil M. Mrak and Maynard Joslyn. Carl Fellers at the University of Massachusetts was a friend of many years’ standing, as were many others in the world of food science, including Tom Rector, head of research at General Foods Co., Clarence Birdseye, and Lawrence Burton, editor of Food Industries (later called Food Engineering). One of his closest friends, until he died, was William Lyman Underwood, with whom Prescott did his first and perhaps best known research. Underwood and Prescott fished together for many years, and their friendship was deep and warm. Underwood was a pioneer in photography and an avid naturalist. Together, they gave many lectures. Underwood served on the faculty of the Department of Biology as a lecturer from about 1900 until his death in 1928. During the last seven years of Prescott’s life, he and George Seybolt (later president, chairman, and chief executive officer of the William Underwood Com pany) developed a deep friendship. It was in large measure because of Seybolt’s realization of the importance to the canning industry of the pioneering researches of Underwood and Prescott, and the significant role of university-industry linkages, as well as his deep respect for Dean Prescott, that the first endowed chair in food technology in the United States was established at MIT, endowed by

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FIG. 7. Prescott, Emerson, and Peakes in the coffee research laboratory (circa early 1930s).

American food and allied industries (with much of the endowment coming from the William Underwood Company). Sedgwick, Bunker, Horwood, and Clare Turner (the public health writer and teacher) were also among Prescott’s close circle of friends. Closer yet were the friends he made among his former students and, in turn, their students. Chief among these were Bernard Proctor, Mrs. Proctor, Earle A. Griswold and his brother Hugh, Milton E. Parker, Gerald A . Fitzgerald (all of the MIT class of 1923, except Hugh Griswold), Philip K. Bates and his wife Eleanor, and James R. Killian, former president of MIT and earlier, editor of the TechnologyReview. Later came Cecil G. Dunn, with whom he wrote the book Industrial Microbiology (1949), John T. R. Nickerson, and the writer of this article, who had an office adjacent to Dean Prescott’s for the last decade of Prescott’s life and who heard firsthand of much of Prescott ’s early work with Underwood.6

b1 hope that errors or omissions in this list of Prescott’s close friends will be deemed unintentional and will be brought to my attention.

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FIG. 8.

XI.

Dean Prescott setting up a coffee distillation (circa 1935).

PRESCOTT'S HONORS, AWARDS, AND PUBLIC SERVICE

In addition to the two highly prized awards of the Institute of Food Technologists mentioned above, Dean Prescott received two honorary doctorates. The citation from Bates College, where he received a Doctor of Science degree (hon. caus.), summed up his career well to that point (1923): Pioneer in the science of Sanitary Bacteriology and foremost living authority on Food Technology; practical administrator who rendered invaluable service to his country during the Great

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War as Major in charge o f food dehydration; patient investigator whose researches in the field of microbe organisms have made incessant warfare upon invisible but relentless enemies of mankind.

He also was awarded an honorary Doctor of Science degree from Lehigh University in 1947. He served as president of the Society of American Bacteriologists in 1919; the American Public Health Association in 1927; and the Institute of Food Technologists, as founding president, in 1940-1941. He was founding co-editor of Food Research,’ a journal established in 1935, until 1952. His book with Professor B. E. Proctor, Food Technology (McGraw-Hill Book Co., 1937), was a classic in its field for over a decade, as was his book Industrid Microbiology (written with Professor C. G . Dunn, 1940). Two of his other books are Sedgwick’s Principles of Sunitar?,Science and Public Health, with M. P. Horwood, 1935, and Water Bacteriology, with C. E. A. Winslow and M. H. McCready (6th edition, 1946). He also published privately a book of his poems. To these accomplishments should be added his voluntary leadership in the National Research Council in Washington, and in a host of other organizations. Lastly, he served as a founding member of the infamous Bug Club (the Boston Bacteriological Club). This was a club unique in that it had no officers, no constitution, and no bylaws; it had only a secretary. In a Lawrence (Massachusetts) saloon, in the early part of this century, a group of young microbiologists formed a club where they could discuss “pipe dreams and wild theory about bacteria (then a young science less than two decades old) and let the other fellows shoot the foolishnessout of them if they can.”* Meetings were held in Lawrence at Sullivan’s Saloon, at the Hayward on Hayward Place, and later at the Copley Square Hotel. (Once they also met at Jake Wirth’s.) Regular attendants in the early years were Prescott, Winslow, Gage, Phelps, and Kendall of Lawrence; Slack of the Boston Board of Health Laboratories; and Underwood, the naturalist with whom Prescott did his early work. Once in a while, the “Chief,” Sedgwick, dropped in. Theobald Smith, d’Herelle, Whipple, and other distinguished scientists spoke at meetings. John W. M. B ~ n k e r , ~ then a senior at Brown University, presented the first paper on chlorination of swimming pools in America. He was made a member on sight. The club still was active while the present writer was a graduate student and was elected a member. He had the pleasure of driving Dean Prescott to many meetings. The club died out sometime in the 1960s. ’NOWJournul (JJ Food Science. “Quoted from a talk given by Prescott at the third meeting of the Society of American Bacteriologists in Boston, in 1930. ”Bunker later became Professor of Biochemistry at MIT and subsequently the second Dean of the Graduate School at MIT.

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FIG. 9. MIT’s “Big Three” in mid-1930s at MIT “frosh” summer camp (circa 1935). From left to right: Dr. Karl Taylor Compton. president of MIT, 1930-1949; Samuel Cate Rescott, dean of School of Science; Dr. Vannevar Bush, vice president of MIT and dean of School of Engineering (also co-founder, Raytheon C o . ) .

XII.

PRESCOTT AND MIT

Dr. Prescott became head of the Department of Biology at MIT in 1922 after Sedgwick passed away. He became MIT’s first Dean of Science in 1932, while remaining active as head of the Department of Biology and teaching microbiology (the present writer was in one of his last classes in microbiology in 1937 and 1938).

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Word has it that the late Dr. Karl Taylor Compton, who had become president of MIT in 1930, smelled coffee brewing, followed the scent up two and one-half stories above his office to Prescott’s coffee laboratory, and then joined him almost daily (when both were in Cambridge) for coffee and a talk. Thus, as the story goes (the writer will not vouch for its verisimilitude), Compton learned about Prescott, and Prescott became MIT’s first Dean of Science, a post he filled with distinction for a decade. At age 7 5 , he began writing a history of MIT, and in 1954, at age 82, he

FIG.10. Dean Prescott and one of his distinguished ‘‘former students,” MIT class of 1923, Dr. Bernard E. Proctor (circa 1935).

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published When M.1.T. Was Boston Tech, royalties of which were donated to the MIT Alumni Fund. This is the only history of MIT ( 186 1 - 19 16) written to date.I0 For the last 30 years of his life, Prescott presided over a table at Walton's Restaurant across the street from MIT 's main buildings on Massachusetts Avenue. The group that met at this table was known as the Tranquility Club, and among its members were Ernest Huntress, Professor of Organic Chemistry; Nicholas Milas, Professor of Organic Chemistry (and synthesizer of vitamin A); Professor Hamilton, analytical chemist; Professor Bertram Warren, X-ray crystallographer; Hans Mueller, a physics professor who was voted the most dynamic lecturer of his era at MIT; James Holt, Professor of Mechanical Engineering; and many others.

XIII.

IN CONCLUSION

For some people, work itself is a diversion. So it was for Dr. Prescott. In addition to over 100 original papers (with real pioneering stuff therein!), he published books on water bacteriology, enzymology, sanitary science, food technology and industrial microbiology. The breadth of Prescott 's interests and researches-his seminal work with William Lyman Underwood in thermal processing; his researches on milk microbiology; his work on dehydration and refrigeration; his pioneering studies on the chemistry of coffee; his founding and direction of the United Fruit Laboratories in Costa Rica; his founding and direction of the Boston Biochemical Laboratory and the problem solving carried out there; his formulation of the first formal subjects on food preservation and later on food technology and industrial microbiology; his textbooks on enzymes, food technology, industrial microbiology, principles of sanitary science, and water bacteriology, and his role in establishing degree courses at MIT in industrial biology (in the first quarter of this century) and later in food technology; and the key role that he played in the first two Food Technology Conferences held at MIT, from which the Institute of Food Technologists was born-all serve as conclusive evidence that he should rightfully be called the father of food science and technology. He was in large measure responsible for the development of these subjects as academic disciplines. Dean Prescott was a patriot in the finest sense of the word. In the 1920s, he submitted an essay in a competition run by the then Registrar of Motor Vehicles of the Commonwealth of Massachusetts, Registrar Goodwin. Dean Prescott 's essay won first prize. The reward was the Massachusetts license plate 1776, which he '"The writer's copy is inscribed, "Specially signed for my young fellow technologist"-The Sam.

Elder

SAMUEL CATE PRESCOTT

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FIG. I I . Samuel Cate Prescott (about 1955) scanning his last book: “When MIT was Boston Tech. 1861-1916” (taken in his office in the Dorrance Building, Room 16-319).

used proudly on his car and with equal pride passed on to his elder son Robert later, when he ceased driving. As a teacher he was an inspiration and became a lifelong friend to many students. He was a fisherman and poet. He loved science and he loved people; he had a tremendous memory and excelled in his recollection of names, especially of former students. Statistics was a hobby of his. He knew the population of every city of over 5000 people that he had visited. In 1935, he took an active part in the revision of the entire legal structure of the health laws of Massachusetts. In 1910, Dean Prescott married Alice Chase of Deny, New Hampshire, the daughter of an MIT alumnus. Their union resulted in two sons, Robert Sedgwick and Samuel Chase (both alumni of MIT) and a daughter Eleanor (Clemence), and four grandchildren. He and Mrs. Prescott attended meetings of the Institute of Food Technologists almost until Mrs. Prescott’s death in the mid-1950s. Dean Prescott was a gifted writer and speaker, and a wonderful poet. With the hope that his scientific accomplishments described here have sufficiently illumi-

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nated that part of his character, it seems fitting to close with his last ChristmasNew Year’s Greeting, 1961-1962. A Christmas-New Year’s Greeting, 1961 -1962 Ceaseless as ocean tides. years come and go, Drawn from the vast eternal seas of time; The seasons are but waves that ebb and flow, Leaving their transient trails in every clime. Thus our New England cycles bring their trainThe quickening springtime, pledge of life anew; The summer’s wealth of growth and ripening grain; The autumn’s wondrous glory, hastening through Till winter brings its shortening days again.

So comes this Christmas, when though days are chill, Deep in my heart warm fires of friendship glow, And I would wish for you, in all good will. The blessings I’ve been privileged to know. Distant or near, old friends will understand That years cannot inhibit my desire That I once more might give the welcoming hand With cordial greetings long-time thoughts inspire When Christmas breathes its spirit o’er our land.

SAMUEL. C. PRESCOTT

Thus “spake” Samuel Cate Prescott, scientist and humanist.

REFERENCES Bitting, A. W. 1937. “Appertizing or the Art of Canning; Its History and Development.” The Trade Pressroom, San Francisco, California. Goldblith, S. A. 1971. A condensed history of the science and technology of thermal processing. Part 1 . Food Techno/. 25 (12). 44-46, 48-50. Goldblith, S. A. 1972. A condensed history of the science and technology of thermal processing. Part 2. Food Techno/. 26 ( I ) , 64-69. Prescott. S. C. 1902. “Enzymes andTheir Applications,” Vol. 1 (by Jean Effront, translated from the French by author). Wiley. New York. Prescott, S. C. 1919. Dried vegetables for army use. Am. J. Physiol. 49, 573-578. Prescott, S. C. 1920. Some bacteriological aspects of dehydration. J. Bucteriol. 5, 109-125. Prescott, S . C. 1930. A brief sketch of the history of the “bug club”: Boston Bacteriological Club. Presented at the 3rd Boston Meeting of the Society of American Bacteriologists. Prescott, S . C. 1943. Dried apples-I943 model. Techno/. Rev. 45 (3). 3-7. Prescott, S. C. 1944. “A Survey of Rationing and Subsistence in the United States Army 17751940.” N.D.R.C., Office of Scientific Research and Development, Washington, D.C.

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Prescott, S. C. 1951. “Retrospects and Prospects: Remarks on Receiving Stephen M. Babcock Award.” Inst. Food Technol. Prescott, S . C. “Spring Fever and Other Verses Relating to Spring Fishing and the Call of the Wilds” (To these is added a small sheaf of Christmas Greetings). Printed with Pleasure by the Murray Printing Co., Cambridge, Massachusetts. Prescott, S. C. 1954. “When M.I.T. was Boston Tech., 1861-1916.” MIT Press, Cambridge, Massachusetts. Prescott, S. C., and Dunn, C. G. 1949. “Industrial Microbiology,” 2nd ed. McGraw-Hill, New York. Prescott, S . C., and Geer, L. P. 1936. Observations on food poisoning organisms under refrigeration conditions. Refrig. Eng. 32, (Oct.),21 1-213. Prescott, S. C., and Honvood, M. P. 1935. “Sedgwick’s Principles of Sanitary Science and Public Health.” MacMillan. New York. Prescott, S. C., and Proctor, B. E. 1936. Refrigeration in public health. Refrig. Eng. 32 (Nov.), 1-3. Prescott, S . C., and Proctor, B. E. 1937. “Food Technology.” McGraw-Hill. New York. Prescott, S . C., and Winslow, C. E. A . 1904. “Water Bacteriology.” Wiley, New York. Prescott, S. C., Winslow. C. E. A , , and McCready, M. H. 1946. “Water Bacteriology,” 6th ed. Prescott. S . C., Nichols, P. F., and Powers, R. 1922. Bacteria and molds in dehydrated vegetables. Am. Food J . 17 (6). 11-16, Prescott, S. C., Bates, P. K . , and Highlands, M. E. 1932. Numbers of bacteria in frozen foods stored at several temperatures. A m . J. Public Heulth 22, 257-262. Prescott, S. C., Emerson, R . L., and Peakes, L. V. 1937a. The staling of coffee. I . Food Res. 2 , 1-20. Prescott, S . C., Emerson, R. L., Woodward, R. B., and Heggie, R. 1937b. The staling of coffee. 11. FoodRes. 2 , 165-175. Sedillot, M. C. 1878. De I’influence des decouvertes de M. Pasteur sur les progks de la Chirurgie. C . R . Hehd. Seances Acud. Sci. 86, 634-640. Sizer, I . W. 1943. Effects of temperature on enzyme kinetics. Adv. Enzymul. 3, 35. Sizer, I. W., and Josephson, E. S. 1942. Kinetics as a function of temperature of lipase, trypsin, and invertase activity from -70” to 50°C. Food Res. 7 , 201. Ukers, W. H. 1935. “All About Coffee,” 2nd ed., p. 408. The Tea and Coffee Trade Journal Co., New York. Ukers, W . H . , and Prescott, S . C. 1951. Coffee and tea. I n ”Food and Food Products” (by M. B. Jacobs). Vol. I I , Chapter XXXI, pp. 1656-1705. Interscience. New York.

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ADVANCES IN FOOD RESEARCH, VOL.

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MECHANICALLY DEBONED RED MEAT RAY A . FIELD Division of Animal Science, University of Wyoming, Laramie. Wyoming

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . , , . 11. Potential Yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Regulations Governing Mechanically Deboned Meat IV. Economic Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Composition of Mechanically Deboned Meat ... ..... . A. Protein, Fat, and Moisture Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Mineral Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Safety Aspects of Mechanically Deboned Meat .................. A. Mineral Toxicity , , . . , , . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . B. Bone Particle Size ..................................... C. Microbiological Properties . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

VII. Functional Properties of Mechanically Deboned Meat A. Color . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Emulsion Characteristics C. Water-Holding Capacity ........................... VIII. Nutritional Value of Mechani eboned Meat . . . . . . . . . . . . . . . . . . . . . . A. Protein Quality . . . . . . , . . . . . , . B. Lipid Makeup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Calcium . . . . . . . . . . . . . . . . . . . . . . D. Iron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX . Palatability of Mechanically Deboned Meat . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Oxidative Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . B. Other Sensory Characteristics and Use Levels.. . . . . . . . . . . . . . . . . . . . . X . Additional Research Needs . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. INTRODUCTION Lean that is removed from bones by machines is termed mechanically deboned meat. The lean removed is that left on irregularly shaped bones from the vertebral column during hand-boning operations. The cost of producing this meat, in terms of labor and other resources required, is the same as that of meat sold in the 23 Copynght @ 1981 by Academic k s s . Inc. All rights of reproduction in any form reserved. ISBN &12-016427-2

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market, so it is in everyone’s best interest to recover as much of it as possible before the bones are rendered for nonfood use. In addition to recovering meat left on bones from hand-boning operations, some mechanical deboners are also capable of separating bones and lean from whole carcasses or from carcass parts. Although mechanically deboned beef, pork, lamb, turkey, chicken, and fish are the terms most often found in the literature, mechanically deboned meat is called by many different names. The term “mechanically separated meat” was adopted at the 10th Session of the Codex Committee on Processed Meat and Poultry Products in Copenhagen, November 20-24, 1978, and is being used in some countries. “Mechanically processed (species) product”-for example, mechanically processed beef product-is the name used by the Food Safety and Quality Service of the United States Department of Agriculture (USDA) (1978b) for red meat. Nevertheless, the Food Safety and Quality Service continues to call chicken and turkey “mechanically deboned poultry” (U.S. Department of Agriculture, 1979). “Mechanically recovered meat” is a still different name used in some European countries. Perhaps the most controversial name is “tissue from ground bone,” which was proposed by the Food Safety and Quality Service of the USDA (U.S. Department of Agriculture, 1977). The name was widely circulated by the popular press and by consumer activist groups that opposed acceptance of the process, but it was never accepted by government or industry in any country. Nevertheless, the name led many to believe that mechanically deboned meat was ground bone, not meat removed from bone. Equipment manufacturers who make deboners for recovering meat from whole bones vigorously objected to the name because their machines removed meat from bones that were not ground. Manufacturers whose equipment require bone to be broken or coarsely ground prior to separation also objected to the name, stating that changing the name “mechanically deboned meat” to “tissue from ground bone” would be like changing the name of T-bone steaks to “tissue from sawed bone,” since both mechanically deboned meat and the meat surface of T-bone steaks contained some bone powder when sold at retail. Objections to the name were also raised because mechanical deboning could be used to remove meat from whole carcasses and carcass parts just as it could to remove meat from bones. Since the name tissue from ground bone was misleading to consumers, technically inaccurate, and degrading to a wholesome, nutritious, and highly palatable product, it was not adopted and is not in use today. Throughout this report, “mechanically deboned meat,” the name that is most common in scientific and popular literature, will be used. The discussion will center on mechanically deboned red meat and will leave mechanically deboned fish and poultry for another review. Most mechanically deboned meat is produced by commercially available machines, which force whole, broken, or coarsely ground bones with meat attached against the screened or slotted surface of the deboner. The size of the opening through which the meat is forced varies by make and model of the deboning equipment. According to Noble ( 1 976), one deboner forces meat and

MECHANICALLY DEBONED RED MEAT

25

bone against microgrooves on a cylinder that will not allow particles of meat or bone larger than 0.001-0.010 mm to pass through. Other deboners use rotating augers to force red meat continuously through orifices approximately 0.46 mm in diameter. Rectangular slots 1.3 x 1 mm are used by machines that press batches of bone against a stationary slotted surface. The successful production of mechanically deboned meat throughout the world has encouraged the development of red meat deboners, and new patents for meat and bone separation are continually being granted. This review will focus on mechanically deboned red meat produced by commercially available machines. Mechanically deboned meat from deboners that are in developmental stages or are not widely used, will not be covered, although it is recognized that new processes and improvements in current deboners are continually being made. Current types of meat-deboning equipment have been discussed and analyzed by Gorbatov et al. (1977), who presented design alternatives aimed at improving deboners. Equipment for mechanical separation of meat from bones has also been reviewed by Bakker (1978) and Winter (1978). One modified method aimed at improving the present methods of obtaining meat from bones by mechanical means has been described by Neuhauser (1977). The method utilizes a bone pre-breaker and a vertically operating hydraulic press. Refrigerated flat bones for processing are passed through a flaming tunnel to reduce bacterial count, broken into 5-cm pieces in a pre-breaker, and pressed twice. Product from the second pressing has elevated amounts of bone powder, which is removed by a decanter that separates the bone powder from the mechanically deboned meat. Therefore, the procedure described by Neuhauser (1977) further processes mechanically deboned meat with liquid to reduce bone powder. Liquid separation of meat from bone has been tried in the past and is still the subject of active research. Gimel'fard and Zharinov (1977) described a rotating drum-type system, which tumbles bones with residual meat attached in water. After the meat is freed from the bone, water and fat are removed from the resulting meat emulsion with a centrifuge. Relatively little protein is lost into the water in this process. Liquid separation of bone is also accomplished by coagulation of protein from alkaline extracts of beef bones by acidification, heating, and freeze-thawing (Jelen e t a / . , 1979; Golan and Jelen, 1979). Protein recovery of more than 90% is made possible by heating the extracts to 3 80°C at pH 5.0-6.0, but the coagulum is gritty and uncohesive. Lower yields are obtained by mild heating to 60"C, by acid coagulation at pH 5.0 and 5.5, and by rapid freezing to -30°C and thawing of the pH-adjusted extracts. The protein efficiency ratio (PER) values for low-temperature alkaline extracts from beef bones are comparable to values for lean beef, but a flavor loss is noticed after heating. Other reports on the technical feasibility of meat protein recovery from boning-room wastes by aqueous extraction are available (Duerr and Earle, 1974; Hamilton and Richert, 1976; Gorbatov et ul., 1977). The aqueous extraction methods of recovering meat protein have not met with wide commercial acceptance. Since

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aqueous-extracted products differ greatly from mechanically deboned meat recovered when meat is forced through small holes or slots, these products will not be discussed further in this review. Mechanically deboned meat produced by forcing meat through holes or slots to increase meat yield and decrease waste have been developed over the last 30-40 years (Institute of Food Technologists, 1979). The first successful machines were for fish, and they appeared in the late 1940s. Some of the more popular of these machines are still in use. Several important factors encouraged the development of methods for deboning fish. Mechanical deboning provided an economic means for obtaining higher yields of edible flesh; and there was a growing demand for fish sticks and other speciality items made from machine-deboned fish. As the demand for boneless fish fillets increased, the demand for saving fish flesh left on the bones increased, and this demand was met by deboners-typically a wide belt wrapped around a perforated drum. As the drum rotated, the fish bones were brought between the belt and the drum, which forced the meat through the perforations while the bones remained on the outer surface of the drum and were eventually scraped off. Mechanical deboning of chicken began in the late 1950s, when fast-food establishments and consumers shopping at retail stores began to show a strong preference for buying pieces of chicken instead of whole birds. Preference for the more demanded parts made it necessary to find ways to utilize backs and necks, which were not in demand. Also, dry soup mixes, poultry hot dogs, boned turkey rolls, and baby foods provided a ready market for deboned poultry meat. Mechanical deboning of red meat developed in the 1970s, as equipment for poultry and fish was modified and new equipment was designed especially for red meat. During the 1970s boxed beef replaced carcass beef in meat-processing centers and in retail butcher shops in many parts of the world. Breaking carcasses in a central location and shipping boneless meat in boxes in place of shipping carcasses increased cutting and handling efficiency, decreased weight loss, improved meat quality, and made tons of bones with residual meat attached available for mechanical deboners. Mechanically deboned meat is now approved for use in most major meat-producing countries and is being used in sausages and other ground and processed meat items. The regulatory and technological restrictions on its use, along with its potential yield, composition, nutritive value, palatability, functional properties, economic impact, and safety are the subjects of this review.

II. POTENTIAL YIELD

The potential yield of mechanically deboned meat can best be estimated by first understanding what proportion of the carcass is bone and what proportion of the bone is lean and marrow, which mechanical deboners can recover. Attached

MECHANICALLY DEBONED RED MEAT

27

lean and marrow, which cannot be economically removed by hand, is the major raw material for mechanical deboners. A review of the literature on bone content of beef, pork, and lamb carcasses will help make it possible to estimate weight of bone available for mechanical deboners. The percentage of the carcass that is bone varies with animal weight, breed, sex, plane of nutrition, and closeness of trim. Kelly et al. (1968) reported that bone cleaned of all visible lean varied from 12. I to 27.6% of the beef carcass weight. Price and Yeates (1969) found that carcasses from 363-kg Hereford bulls and steers on a high plane of nutrition had 15.7 and 14.6%bone, respectively, whereas Hereford bulls and steers on a low plane of nutrition had 20.6 and 14.8% bone. Bulls and steers slaughtered at 340-430 kg had 16-18% of the carcass weight as bone, according to Robertson et al. (1969). Charlet (1969) reported that about 14% of the carcasses from Charolais bulls and steers slaughtered at 373 kg and 15 months of age was cleaned bone. Swedish Friesian bulls and steers slaughtered at 17- 18 months (carcass weight 189-279 kg) had approximately 17%of the carcass as cleaned bone (Brannang, 1966). Other values for percentage of separable bone in carcasses of marketweight cattle range from 12 to 17% (Champagne et al., 1969; Hedrick et al., 1969; Nichols et al., 1964; Epley et al., 1971; Guenther et al., 1965; Waldman et a / ., 197 I ) . The average figure for bone percentage of all market-weight cattle in the references cited is 14.7%. The percentage of the lamb carcass that is cleaned bone varies in a manner similar to that for cattle. Bell et al. (1967) and Munson et al. (1964) found that different breeds of lambs slaughtered at approximately 50 kg ranged from 15.4 to 17.4% bone on a carcass-weight basis. Kemp and Barton (1969) reported that bone in the carcass varied by grade (13.1-18.3%), with the leanest lambs possessing the highest percentage of bone. Ewe and wether carcasses both contained 14.3%bone, as reported by Judge et al. (1966); however, others have reported some differences between the sexes (Kemp and Barton, 1969; Barton and Kirton, 1958; Fourie et al., 1970). Carcass bone percentage also varies considerably as weight changes (Fourie et al., 1970). Other researchers have reported 13.917.7% bone in carcasses of market-weight lambs (Kemp et al., 1970; Field et al., 1963, 1967; Craddock et al., 1974; Rouse et al., 1970). When all references cited are included, the average bone content of market-weight lamb carcasses is 15.7%. Because pigs usually have greater amounts of backfat than do cattle and sheep, the bone percentage in pork carcasses is often lower. Richmond and Berg (1971) reported bone percentages of 13.2, 10.5, 9.9, and 9.2 for carcasses of pigs weighing 23, 68, 91, and 114 kg, respectively. In their study, differences due to breed and sex were not significant ( P < 0.05). Cleaned bone as a percentage of market-weight pork carcasses has been reported at 11.3%(Brooks et al., 1964), 14.6%(Hankins and Ellis, 1945), 1 1 .O% (McMeekan, 1941), 10.9%(Pomeroy, 1941), and 9.5%(Cuthbertson and Pomeroy, 1962).

28

RAY A. FIELD

Bone content of carcasses is higher when the carcasses are completely boned under commercial plant conditions than when the bones are carefully scraped by hand as is done in studies to determine clean bone percentages. Gorbatov et al. (1975) found that bone yield from mutton carcasses conventionally deboned ranged from 24.3% of the carcass weight in fat carcasses to 40.5% of the carcass weight in very lean mutton carcasses. Field (1975) removed all visible lean from beef and pork bones destined for rendering. Sow loin bones contained 46.7% of their weight as lean; ham, picnic, and Boston butt bones separated under commercial plant conditions had 25.1, 14.3, and 18.7%, respectively, of their weight as lean (Table I). Beef neck bones had 41.3% of their weight as lean, and bones from the rib, rump, and shortloin had 26.2, 18.1, and 25.6% of their weight as lean. Field (1975) also found that bones from smaller

TABLE I MEAT YIELD AND MEAT TEMPERATURE FROM BONES THAT ARE NORMALLY RENDERED"

Temperature of mechanically deboned meat ("C)

Yield ( % ) b

Bone source Butcher hogs Ham Picnic Boston butt sows Loin Veal Shoulder Frames Backs Cow beef Rib, plate Rump Short loin Choice beef Neck Plate

'' From Field

No.'

Hand

Mechanical

Out of grinder

out of deboner

4 5 13

25. I 14.3 18.7

27.5 21.7 22.6

5 2 2

II 20 13

3

46.7

51.0

I

15

4 3 3

16.7 37.0 42.0

36.2 60.8 63.0

-I

0

0 -1

3

3 4

26.2 18.1 25.6

32.9 26.3 34.0

5 8 I

20 38 20

41.3 26.9

48.4 28.8

4 4

14 13

4

3 3

0

et a / . ( 1976). Weight of boneless product divided by fresh meat and bone weight. Each number represents more than one bone; that is, 4 indicates that all bones from four different hams of four different hogs were cleaned of all fat, lean, and tendon.

MECHANICALLY DEBONED RED MEAT

29

carcasses (veal bones) had a higher proportion of lean attached than did bones from larger carcasses (beef bones). In addition to lean attached to bone, the amount of marrow within bone is also important in estimating potential yield because marrow, like lean, is soft and can be forced through the screened or slotted surface of the deboner to become part of the mechanically deboned meat. According to Maximow and Bloom (1957), bone marrow forms 2-3% of the whole body weight, which would convert to 4-6% of the carcass weight. Carcasses from red meat animals contain about 15% clean bone when marrow is included. If we use the figures of Maximow and Bloom (1957), 4-6% of the carcass weight or 25-40% of the bone weight is marrow, and much of this marrow can be harvested by mechanical deboners. Although few data on marrow weight in meat animals are available, data from other species support the conclusion that marrow makes up a significant proportion of the body. Reich (1946) stated that bone marrow is the largest organ in the body, constituting 4.5% of the total body weight. Winthrobe (1974) estimated that marrow weight in humans accounted for 3.4-5.9% of the body weight. He stated that the marrow weight is roughly equal in amount to the weight of the liver. Ellis (1961) is also in agreement with other workers who have shown that marrow makes up approximately 4.5% of the human body. Work by Gong and Arnold (1965) showed that 49% of the skeleton volume in dogs was marrow, and Gong and Ries (1970) found that 51% of the volume of the rabbit skeleton was marrow. The volume of marrow was based on densities for the various chemical components of bone and marrow, and the figures may not apply directly to bones of beef, pork, and lamb. The marrow volume figures, like those for man, make 25-40% marrow in beef, pork, and lamb bones seem like a reasonable figure. From the figures of Field (1979, which show that 14.3-46.7% of bone weight is lean when the bones are removed commercially, and the figures of Maximow and Bloom (1957), which when interpolated indicate that bone is 25-40% marrow, it seems safe to predict that on the average at least 30% of the weight of beef, pork, and lamb bones removed commercially is recoverable lean and marrow. The figure of 30% is similar to that given by Goldstrand (1975), Field (1 9741, and Noble (1 976) for yield of mechanically deboned meat from bones suitable for mechanical deboning. If 30% is used as the proportion of the bone from commercial operations that can be recovered as mechanically deboned meat, and if bone with its residual lean attached makes up 20% of the carcass weight, carcass-weight figures compiled for 59 meat-producing countries can be used to estimate the potential yield of mechanically deboned meat. However, the 20% bone figure must be cut in half, because not all bones are suitable for mechanical deboners. Bones from the vertebral column, ribs, and sternum are most suitable for mechanical deboners because they usually have more lean attached and because their red bone marrow is high in protein. Unpublished data

30

RAY A. FIELD

of the author at the University of Wyoming show that the vertebral column, ribs, and sternum from USDA Good grade beef carcasses trimmed under plant conditions make up about 10% of the carcass weight. The 0s coxae and scapula are the second most suitable group of bones for mechanical deboners. They have less lean attached than do the bones from the vertebral column (Field, 1975). These bones trimmed under plant conditions account for 2.7% of the beef carcass weight. The round bones are least suitable for mechanical deboners. Although they account for 6.5% of the beef carcass weight when trimmed under plant conditions, they have very little lean attached, and the marrow is mostly fat. According to the USDA (U.S. Department of Agriculture, 1980), 77 million metric tons of beef, pork, lamb, mutton, goat, and horse meat on a carcassweight basis were produced commercially in 1979 in 59 countries where data were available. If all red meat carcasses were hand-boned and only the most suitable bones were saved for mechanical deboners, 10% of the weight or 7.7 million metric tons of bones with marrow and residual lean attached would be available for mechanical deboners. If 30% of the weight of these bones could be recovered as lean and marrow by mechanical deboners, 2.3 million metric tons of mechanically deboned meat, which has not been part of the world food supply in the past, could be made available. The figures are in line with other estimates that have been made for potential production of mechanically deboned meat in the United States. Total red meat production on a carcass-weight basis in the United States was 17 million metric tons in 1979 (U.S. Department of Agriculture, 1980). From the same figures of 10%for bones in the carcass that are suitable for mechanical deboners and 30% yield from these bones, the potential yield of mechanically deboned meat in the United States in 1979 was 510,000,000 kg. The figure is in good agreement with the USDA (U.S. Department of Agriculture, 1977) statement that there is a potential for the eventual use of 453,592,370 kg of mechanically deboned meat yearly in the United States. Goldstrand (1975) has stated that 5.8-7.5 kg of mechanically deboned beef and 1.4-1.9 kg of mechanically deboned pork per carcass could be saved with mechanical deboners. If these maximum figures are multiplied by the number of pork and beef slaughtered per year and if other red meat species are accounted for, the total is close to the 510 million kg estimated from the above bone and residual meat figures. Goldstrand (1979) later revised his figures for potential total beef and pork production in the United States to 340,194,277 kg of mechanically deboned meat per year. This lower figure is a more realistic estimate of the amount of mechanically deboned meat that could actually be produced. Not all bones suitable for mechanical deboning will be available for recovery of meat. The value of some bones, like pork neck bones and spareribs, is often too high to make deboning profitable. We shall continue to eat T-bone steaks, lamb loin chops, and center-cut pork chops, all of which contain bone. In addition, many bones

MECHANICALLY DEBONED RED MEAT

31

will continue to be removed in small plants and in retail stores where the bones will not be under the supervision of government inspectors and will therefore not be eligible for use. Williams (1979) estimated that about 50% of the commercial hog production and 70% of the boxed beef production in the United States would be mechanically deboned if regulations were changed, Combined beef and hog production based on Williams’ estimates was 288.8 million kg of deboned meat per year in the United States. Government regulations become very important in estimating the amount of mechanically deboned meat that will actually be produced. According to Goldstrand ( 1979), labeling requirements have effectively eliminated mechanically deboned meat as an ingredient of consequence in branded meat products in the United States. The year after United States regulations required any product containing mechanically deboned meat to be labeled with the qualifying phrase “with mechanically processed (species) product, contains up to -% powdered bone,” only 232,192 kg of beef and 906 kg of pork were recovered mechanically (Goldstrand, 1979). This is in a country where over 90.6 million kg of mechanically deboned poultry meat is produced annually without labeling regulations (Institute of Food Technologists, 1979). Other countries without special labeling requirements for products containing mechanically deboned meat are increasing production rapidly. In the Netherlands, an estimated 2.5-5 million kgof mechanically deboned pork is produced each year (Bijker et al., 1979). Worldwide, there is a potential for 2.3 million metric tons of mechanically deboned meat to be added to the food supply. The meat has been wasted in the past and much of it is being wasted at present because a large proportion of the world’s meat production is sold in boneless form and the bones with residual meat attached are rendered for inedible uses. When bones are sold as part of retail cuts, they are removed by consumers after the meat is cooked. These bones often have more meat discarded with them than they would if the bones were removed by professional meat cutters. The proportion of the 2.3 million metric tons that will actually be recovered for food in the future will depend upon changes that will take place in meat processing and upon government regulations.

Ill. REGULATIONS GOVERNING MECHANICALLY DEBONED MEAT

When mechanically deboned meat, fish, and poultry began to be produced commercially, regulatory agencies in most countries looked on the product in the same way they did hand-boned meat. The meat was inspected to assure wholesomeness, sanitary preparation and handling, and freedom from disease, adulteration, and misbranding. No special regulations regarding its composition and use and no special labeling requirements were written. In the United States early regulations for poultry simply called the product “boneless poultry” (Murphy et

32

RAY A. FIELD

1979). Final regulation defining boneless poultry products and imposing a 1% limit on their bone content occurred in 1969 (U.S. Department of Agriculture, 1979), approximately 10 years after the first commercial use of mechanical deboners for recovery of poultry meat. No usage levels, labeling requirements (other than the name listed in the ingredient’s statement), or regulations regarding proximate analysis or nutritional data were required, and none are in effect at the time of this writing. Denmark, a country with a reputation for high-quality processed meat, was one of the first to use mechanically deboned red meat. After deboners in meatprocessing plants were tested over a period of several years, general directions for production were set up in 1974 (Wermuth and Madelung, 1974). Only fresh chilled bones were to be used. The mechanically deboned meat had to be cooled down to 3°C or lower, either while being processed in the deboner or by cooling in immediate connection with the deboner. The product had to be used within 24 hr or be frozen and held at - 18°C or lower. Mechanically deboned meat could be used up to an amount of 2% of the finished product without declaration. Anything over this amount was to be declared. The Australian government (Wilson, 1974) also issued standards for mechanically deboned meat for export in 1974. Mechanically deboned meat was defined as an emulsion derived from boning-room bones by using a mechanical method of separation. Acceptable materials could include any part derived from a carcass that had been inspected and passed for human consumption provided that part was handled and processed in a hygienic manner. Bones for mechanical deboning had to be kept cold prior to deboning. If the bones with residual meat attached were to be held longer than 36 hr they had to be frozen. Mechanically deboned meat was reduced to a temperature below 7°C within 2 hr of production and used within 24 hr or frozen. Finished product for export was required to carry the term “edible mechanically deboned meat/beef/mutton” (etc., as appropriate) and a statement of the maximum calcium content, the maximum moisture content, and the minimum protein content. Up to 25% of the contents of finished products could be edible, mechanically deboned meat without declaration on the label, and finished products could contain mechanically deboned meat at any level over 25% of the finished product with declaration on the label. Declaration on the label meant that the words mechanically deboned meat must be included in the list of ingredients so that the name of the ingredient in the highest proportion appeared first, the name of the ingredient of which the processed meat contained the next highest proportion appeared next, and so on. The maximum calcium content of the finished product also appeared after the list of ingredients. Wilson (1974) stated that in 1974 no overseas requirements in respect to acceptance of edible mechanically deboned meat were known. Neither Denmark or Australia placed limitations on bone content, composition, or nutritional value of mechanically deboned meat. Nevertheless, Dutch regulations (Bijker rt d.,1979) re-

(I/.,

MECHANICALLY DEBONED RED MEAT

33

quire mechanically deboned meat intended for export to contain a maximum of I % bone and 0.25% calcium, with no bone particles being larger than 1 mm. Regulations governing mechanically deboned meat in the United States have been characterized by interim regulations, suits and withdrawals, proposals, hearings, and thousands of comments from consumers, industry, and scientists to regulatory agencies. The first official action by the United States was publication of a bulletin entitled “Preparation of Mechanically Deboned Meat and Mechanically Deboned Meat Fatty Tissue” (U.S. Department of Agriculture, 1974). Standards for raw bones and adhering meat from chilled carcasses and standards for mechanically deboned meat including composition, bone particle size, protein efficiency ratio, and percentage of essential amino acids and labeling were outlined. Immediately after publication, over 40 meat-processing companies in the United States made large investments in equipment. A meat trade magazine, The National Provisioner, devoted its April 1975 issue to mechanical deboning and declared that “mechanical deboning is here.” The USDA approval in 1974 was understood to be preliminary, and in 1976 the USDA proposed more detail standards for mechanically deboned meat. The USDA (U.S. Department of Agriculture, I978b) has summarized the historical background on regulations regarding mechanically deboned meat between 1976 and 1978, when a final rule was published. Essential parts of that summary are given in the following paragraphs. On April 27, 1976, the USDA published a notice of proposed rule-making titled “Definition of Meat and Classes of Meat, Permitted Uses, and Labeling Requirements. That proposed rule-making included, among other things, a proposal for the permitted manufacture of “Mechanically Deboned Meat, “Mechanically Deboned Meat for Processing, and “Mechanically Deboned Meat for Rendering.” As proposed, these materials were defined as the product resulting from the mechanical separation and removal of most of the bone from attached skeletal muscle tissue. Nutritional parameters were proposed for “Mechanically Deboned Meat and “Mechanically Deboned Meat for Processing.” Limits for “Mechanically Deboned Meat” were to be lower for calcium and fat and higher for protein than were the parameters for the other two classes. “Mechanically Deboned Meat for Processing” was to have a requirement for protein quality equal to the requirement for “Mechanically Deboned Meat” as measured by the protein efficiency ratio (PER) or by the proportion of essential amino acids to total protein. No nutritional parameters were to be set for “Mechanically Deboned Meat for Rendering,” which was not to be used as such in the formulation of meat food products. No limitation on the amount used in products was proposed for “Mechanically Deboned Meat, but “Mechanically Deboned Meat for Processing” was to be limited to 20% of the total meat. Also on April 27, 1976, an interim regulation that included standards for the ”

”

”

”

”

34

RAY A. FIELD

use of mechanically deboned meat was published. The interim regulation was to remain in effect pending final rule-making on the proposal, unless rescinded before rule-making was completed. The interim regulation was considered necessary in order to develop data, previously unavailable except on an experimental basis, for determining if the analytical parameters were effective in assuring nutritional quality of the products. The interim regulation was challenged in the United States District Court for the District of Columbia by a coalition of consumer-oriented organizations and the Attorney General of Maryland. On September 10, 1976, a preliminary injunction was issued by the court enjoining the Secretary of Agriculture from using the provisions of the interim regulation with respect to mechanically deboned meat. In the court’s opinion, the USDA had not adequately assessed the potential health hazards of mechanically deboned meat. Following the court order, the USDA ordered discontinuation of the placement of the official mark of federal inspection on all mechanically deboned meat, which in effect stopped production and use. After production of mechanically deboned meat in the United States stopped, a panel of government scientists was convened to study questions that had arisen concerning health and safety aspects. Many of the panel’s findings as they relate to composition, nutritional value, and safety are discussed in other parts of this review. The overall finding of the panel was that no health hazards from production and consumption existed (Kolbye and Nelson, 1977a). As a result of the panel’s recommendations and conclusions and the controversy surrounding mechanically deboned meat, a revised proposal was published (U.S. Department of Agriculture, 1977). According to the USDA (U.S. Department of Agriculture, 1978b), a total of 4537 comments were received on the new proposal. After reviewing all the comments, the information presented in the public hearing, and the findings of the government scientists, the USDA concluded that mechanically deboned meat is wholesome and safe and should be permitted, except in baby, junior, and toddler foods (U.S. Department of Agriculture, 1978b). The product was named “mechanically processed (species) product” [MP(S)P], and the regulations that were published in 1978 are in effect today. The regulations regarding the controversial labeling requirement are summarized in the following paragraphs. The USDA concluded that, since MP(S)P is unique and not an expected ingredient in a product, a qualifying statement should be added to the names of those products containing MP(S)P. Accordingly, all labels of products containing MP(S)P are required to qualify the product name with the phrase “With Mechanically Processed (Species) Product. In order that such qualifying statements are likely to be read and understood by consumers, the USDA determined that they shall be at least one-half the size of the product name. Many consumer groups and the select panel indicated that the labeling should ”

MECHANICALLY DEBONED RED MEAT

35

clearly indicate the presence of bone, as some individuals must limit their intake of foods containing calcium. The USDA believed this health problem to be an important consideration. Therefore, labels of products containing MP(S)P must bear the additional qualifying statement ‘‘Contains Up To -% Powdered Bone” under the first qualifying statement. The percentage of powdered bone represents the amount of hard dry bone in the finished product. Also, in order that such a qualifying statement is likely to be read and understood by consumers, such statement qualifying the product has to be at least one-fourth the size of the product name. Other parts of the regulation published by the USDA in 1978 state that MP(S)P must be limited in the finished product to 20% of the meat portion. Requirements of no less than 14% protein, no less than 2.5 PER or 33% essential amino acid content, and no more than 30% fat are also in effect. Mechanically deboned meat that fails to meet the protein, PER, or fat limit can still be used provided it is labeled “Imitation MP(S)P. ” Ninety-eight percent of the bone particle cannot exceed 0.5 mm in any dimension, and the largest particles cannot exceed 0.85 mm in dimension. Bone in MP(S)P is limited by calcium content, which cannot exceed 0.75%. Time and temperature requirements ensuring proper handling and chilling of bones with residual meat as well as MP(S)P are also part of the regulations. Since 1978, the United States meat industry, because of its fear of negative consumer reaction to the labeling requirements, has not used equipment it has on hand for mechanical deboning. Additionally, mechanically deboned meat often exceeds the 30% maximum fat content and often fails to contain the 14% minimum protein content required by United States regulations. The potentially vast amounts of recoverable meat protein in the United States lie untapped because of government regulations. A revision of the restrictive government regulations was being considered by the U.S. Department of Agriculture at press time for this article. Because of limited space and the fact that regulations are changing or are still in the developmental stages, standards in other countries will not be discussed. The reader is referred to reports by Tandler (1978) and Winter (1978) for requirements and regulations regarding mechanically deboned meat in some other countries. Many countries that produce mechanically deboned meat do not distinguish meat separated by machine from that separated by hand; these countries have not felt a need for special regulations governing mechanically deboned meat. IV. ECONOMIC IMPLICATIONS

Objections to mechanically deboned meat by some farmers and ranchers and some in the labor force have been largely economic in nature (U.S. Department of Agriculture, 1978b). The objections are based on the belief that prices of live animals would be depressed as the result of using mechanically deboned meat

36

R A Y A . FIELD

and that some meat cutters would lose their jobs because of the use of machines. There are also opinions that the process is not needed because of an oversupply of meat. In contrast, the Presidential Commission on World Hunger (1979) and numerous other groups throughout the world have as their objective reduction in world hunger and malnutrition. Ironically, mechanically deboned meat, with a potential for 2.3 million metric tons of wholesome and nutritious product annually, has not been encouraged by those who must lead the fight against hunger and malnutrition by increasing food production. Economic evaluations of potentials through mechanical deboning of red meat are lacking. Studies by McNiel (1977) and by Williams (1979) have been completed. According to Dr. Williams, production of mechanically deboned red meat would virtually assure a significant increase in the nation's supply of red meat and reductions or smaller increases in the retail prices of meat without reducing returns to producers; it would also provide another effective tool in the fight against inflation. It was concluded that prices paid to producers for livestock would be reduced little, if any, and that they might increase. He described as forced economic waste the current practice in the United States of using cuts from fed beef to supply ground meat for a hamburger society. The practice resulted from decreased cow slaughter and a continued high demand for ground beef, part of which could be met by mechanically deboned beef if government regulations were altered. Authorization of mechanical deboning on a practical and economic basis would have important secondary effects. It no longer would be necessary to ship huge tonnages of bone to consumers, a practice that increases transportation costs and wastes energy. The residual meat attached to the bone also represents a massive loss of income to meat packers. According to Williams (1979). several different views can be taken when mechanically deboned beef is compared with beef imports. If net imports were maintained, mechanically deboned beef could represent an increase in supply approximately equal to 50% of net imports. Another view is that, if the full potential of mechanical deboning were exploited, it would be possible to maintain supplies at recorded levels while reducing imports substantially, thereby improving the nation's balance of payments. Other significant statements relating to mechanically deboned meat are found in the impact statement of the USDA (U.S. Department of Agriculture, 1978a). Excerpts from the statement are quoted as follows: USDA Costs: There will be no significant cost impact on USDA upon final implementation of this regulation as federal meat inspectors are already located in meat processing plants and will assume the monitoring of MPP (mechanically deboned meat) as pan of their responsibility.

Impact on Major Purposes: The utilization of "mechanically processed (species) product" will promote efficient utilization of the total carcass. Industrial efficiency and productivity will be enhanced and the supply

MECHANICALLY DEBONED RED MEAT of high protein beef and pork products will increase in both domestic and world markets. The safety, wholesomeness, nutritional integrity and proper labeling of “mechanically processed (species) product” will be assured and processors will be allowed to resume production. [While much of this is correct, processors, of course, have been forced to halt production.]

Cost Impact: Industry: Prior to the September, 1976 restraining order, the industry employed 50 mechanical deboning machines, each with a capacity of producing 6804 kg of product per day. If this machinery has not been salvaged and if it meets new specifications, initial cost outlays for plants who own deboning equipment will be minimized. However, if new equipment is required, outlays will run $50,000-$150,000 per plant. Installment of an entire mechanical deboning system (conveyors, refrigeration, etc.) will cost an additional $75.000-$225.000 per plant. “Mechanically processed (species) product” is estimated to cost 44 cents per kg to process. Production of this product will prove possible as returns will exceed costs. ...In 1977, 10.6 billion kg of beef and 5.7 billion kg of pork were prepared for consumption. As stated above, utilization of “mechanically processed (species) product’’ may add 1.4-2.3 kg of additional pork product and 3.2-6.8 kg of additional beef product to a carcass. Assuming 3.6 kg of additional beef product and I .8 kg of additional pork product is obtained per carcass, utilization of ‘MP(S)P’could have increased beef food products by 152 million kg and pork food products by 140 million kg in 1977. Meat product supplies would have increased by 1.8% in 1977, with maximum utilization of MP(S)P. Incorporation of MP(S)P into traditional meat products would reduce processing costs and consumer prices. Since the demand elasticity of beef products is estimated around I , increased supply is expected to result in a commensurate decrease in price; and total returns to the beef industry should remain fairly constant. The price reduction effect would be concentrated in processed meat products, utilizing mechanically processed products. This would tend to reduce the price of beef utilized for processing through increased meat product yields per carcass, and should tend to mitigate such effects on slaughter prices. Price increases associated with current incipient cattle shortages may, therefore, be slightly moderated by the utilization of “mechanically processed beef product. ”

Consumers: Utilization of “mechanically processed (species) product’’ will decrease the price of products which incorporate it. Price increases attributed to increases in the cost of living will be slowed and the consumer will benefit.

Other Significant Economic Effects: US imports of beef and pork comprise approximately 7 and 3% of our domestic consumption, respectively. Approximately 86% of the imported beef and 4-8% of the imported pork is incorporated into processed foods, hence utilization of “mechanically processed (species) product” may reduce our demand for imports and concomitantly improve our terms of trade. Employment is not expected to be significantly affected. Although mechanical deboning will reduce demand for hand deboners, employment vacancies and mechanical deboning will prompt hand deboners to acquire the necessary skills to operate mechanical deboning equipment, mitigating any short-term unemployment that may occur.

37

38

RAY A . FIELD

Other Significant Social Effects: The production of ‘mechanically processed (species) product’ will increase the availability of low-cost meat food product. As a result, more high protein meat food products will be available to people on limited budgets. The select panel noted that the additional calcium available in “mechanically processed (species) product” should be valuable to most individuals as calcium consumption in the normal diet is often deficient.

While meaningful economic data are difficult to obtain because factual information on the amount of mechanically deboned meat that will actually be produced is lacking, it is clear that there is overwhelming economic justification for its production. McNiel ( 1977) estimated that the annual welfare gain to consumers in the United States would be $1.1 billion. In addition, comments received as a result of the USDA notice (U.S. Department of Agriculture, 1979) and solicitation of information on mechanically deboned poultry make it clear that poultry producers object to any change in the regulations that would decrease the amount of mechanically deboned poultry because it would result in lower prices. Labor also has a clear lesson to be learned from the poultry industry. Mechanical deboning of poultry has required additional labor for handling, packaging, boxing, and warehousing. Numerous processed poultry items have been made possible as a result of mechanical deboning, and the industry continues to grow at a rapid rate, creating even more jobs. Some in the labor force recognize the potential for increased jobs as well as the potential for making work easier. Comments sent to the USDA on mechanically deboned meat included one remark from a meat cutter paid by piecework, who indicated that he would earn more money if deboning machines were used because he would not be slowed by work on hard-to-bone cuts such as necks (U.S. Department of Agriculture, 1978b). Economic considerations, which may delay mechanically deboned meat production, are not related to cost, availability of product, labor, use, or any of the other traditional economic factors. The economic consideration delaying production in the United States is the labeling requirement as described under the regulations section of this review. A consumer survey by Homemaker Testing Corp. (1977) has shown that consumers will not buy a product that carries the word “bone” on the label. Questions were raised regarding safety and nutritional value, and the great majority of the consumers stated that a product containing bone was repulsive and unappetizing and that they did not want bone in their meat. Other unpublished meat industry studies on the labeling requirements for mechanically deboned meat have drawn similar conclusions. Millions of kilograms of mechanically deboned poultry, processed by the same machine capable of deboning red meat, are sold in the United States each year,

MECHANICALLY DEBONED RED MEAT

39

and the volume continues to grow. The increase in growth for mechanically deboned poultry and the lack of growth for mechanically deboned red meat are due to labeling regulations that create misleading connotations of product inferiority for red meat. Since the labeling regulations do not apply to poultry, they discriminate against red meat.

V.

COMPOSITION OF MECHANICALLY DEBONED MEAT

According to Watt and Merrill (1963), the total edible portion of the beef carcass ranges from 18% fat in the Utility grade to 41% fat in the Prime grade, and individual trimmed Choice grade T-bone steaks range from 37. I % fat in the edible portion to 8 . I % fat in the separable raw lean. Other cuts of beef and cuts of pork and lamb have ranges of fat similar to those listed for beef. Mechanically deboned meat is obtained from carcasses of all different grades as well as from different anatomical parts that have been cut and trimmed by a variety of different methods. Therefore, it is not surprising that the composition of mechanically deboned meat varies in the same amount as do the carcass parts from which the mechanically deboned meat originated. A.

PROTEIN, FAT, AND MOISTURE CONTENT

The composition of mechanically deboned meat from bones that are normally rendered is found in Table 11. The highest protein percentages were in meat from sow loin bones (14.01%), veal frame bones (17.57%). veal back bones (15.98%), and beef neck bones (17.18%). These bones yielded 37-46.7% of their weight as hand-boned meat (Table I). In contrast, picnic and rump bones yielded 14.3% and 18.1% of their weight, respectively, as hand-boned meat. The protein content of mechanically deboned meat from picnic bones and from cow rump bones was 9.06% and 10.05%, respectively (Table 11). The highest fat percentages in the mechanically deboned product were found in meat from picnics (42.37%) and rumps (41.89%). As would be expected, those bones that had the most meat adhering to them yielded the most protein. Bones that had the least meat adhering to them yielded mechanically deboned meat with the least protein and the most fat. Goldstrand ( 1975) reported the composition of mechanically deboned pork neck bone meat as 14.2-15.1% protein, 24.7-29.9% fat, and 53.7-60.3% moisture. These figures are in agreement with those of field (1974) shown in Table 111. Several different makes of mechanical deboners are on the market. The make of the machine has very little influence on protein, fat, or moisture content (Goldstrand, 1975). Mechanically deboned meat from beef neck bones (espe-

40

RAY A . FIELD TABLE I1 COMPOSITION OF MECHANICALLY DEBONED MEAT FROM BONES THAT ARE NORMALLY RENDERED"

Bone source

Dry matter"

Fat"

Crude protein"

Ash"

Calcium"

54.81 55.59 43. I5

39.02 42.37 26.04

10.21 9.06 13.50

4.07 3.68 2.71

I .39 1.22 0.73

46. I5

29.53

14.01

1.77

0.41

26.27 26.64 24.21

7.56 6.79 5.81

12.85 17.57 15.98

5.36 2.59 2.21

I .76 0.71 0.54

50.33 58.06 50.97

3 I .87 41.89 33.38

12.98 10.05 I I .62

4.57 4.35 4.35

I .55 I .55 ISO

35.13 49.88

13.76 32.70

17.18 11.43

3.43 4.35

1.06 1.49

Butcher hogs

Ham Picnic Boston butt sows Loin Veal Shoulder Frames Backs Cow beef Rib, plate Rump Short loin Choice beef Neck Plate "

From Field er al. (1976) expressed as a percentage of fresh weight.

!' Data

TABLE 111 TYPICAL ANALYSES OF MECHANICALLY DEBONED MEAT FROM NECK AND BACK BONES OF BARROWS AND GILTS","

Item

Range

Original hone temperature ("C) Temperature of meat out of dehoner ("C) Yield of meat (%) Fat content of dehoned meat (%) Protein content (%) Moisture content (%) Ash content (%) Calcium content (%)

0-3 10-15 30-40 20-30 14-17 50-60 2-3 0.5-0.8

"

From Field (1974).

!' Holes in the cylinder of the Beehive mechanical dehoner were 0.46 mm in diameter.

MECHANICALLY DEBONED RED MEAT

41

cially from lean animals) is high in protein and low in fat. Goldstrand (1975) reported that mechanically deboned meat from beef neck bones was 16-17% protein and 9.9-24.4% fat. The data in Table I1 and other data collected at the University of Wyoming show that mechanically deboned meat from beef neck bones is often near 10-15% fat. In contrast, mechanically deboned meat from beef plates trimmed under commercial conditions is often 40-50% fat, 9-12% protein, and 30-45% moisture. Mechanically deboned meat from the brisket is often fatter than that from the plate. These figures vary considerably, depending upon the amount of lean and fat on the bones prior to mechanical deboning. The trend toward more roughage and less concentrate in cattle and sheep rations will increase the proportion of bone in the average carcass and result in leaner bones, which will produce mechanically deboned meat with less fat. Goldstrand ( 1975) gives the chemical composition of mechanically deboned meat from ham bones as 10.0% protein, 42.3% fat, and 44.6% moisture. These data are close to the figures for mechanically deboned meat from ham bones listed in Table 11. Kruggel and Field ( 1977)evaluated 63 commercial mechanically deboned beef samples from flat bones of mature cows and bulls in three regions of the United States and mechanically deboned pork from two different regions. Beef samples from older, thinner animals contained from 10.8 to 22.6% fat and from 56.4 to 67.6% moisture. Pork samples averaged 23.4% fat and 57.7% moisture. Low fat levels for mechanically deboned beef and pork indicate that under production conditions meat processors process bones from those parts of the carcass that contain less fat. Meat processors often buy and sell processed meat ingredients on a guaranteed maximum for fat and a minimum for protein. Therefore, mechanically deboned meat with a low fat content is worth more to meat processors, and price encourages production of mechanically deboned meat with less fat. Some information is also available on mechanically deboned meat from whole carcasses or carcass parts. Mechanically deboned meat from wholesale lamb breasts contains 38. I % fat, 15.5% protein, and 45.7% moisture (Field and Riley, 1974), whereas low-quality mutton carcasses contain 19.1% fat, 19.6% protein, and 59.6% moisture when they are mechanically deboned (Field et u l . , 1974b). Slightly less fat and more moisture are present in meat from mutton carcasses that are mechanically deboned hot off the slaughterhouse floor. Mechanically deboned meat from young and old goat carcasses contained fat, protein, and moisture values that were comparable to those for mechanically deboned meat from low-quality mutton carcasses (Marshall et ul., 1977). Higher yields of mechanically deboned meat are often associated with higher levels of fat. Field et (11. (1974~)found that, as the yield of mechanically deboned mutton increased from 52% to 84%, fat increased from 8.6% to 24.9%.

42

RAY A . FIELD

Neuhauser (1977) used a different make of mechanical deboner, and the result was the same. When bones from the beef forequarter were pressed a second time to increase the yield of mechanically deboned meat, fat content rose from a maximum of 26.7% on the first pressing to a maximum of 40.6% on the second pressing. Other studies that list data on fat, protein, and moisture content of mechanically deboned meat include those of Cross et al. (1977), Kunsman et a / . (1978), Chant et a f . ( 1977), and Field et al. (1979a). Species, grade, age of animal, anatomical location, yield, amount of lean left on the bones, bone marrow content, and length of time between slaughter and mechanical deboning are all items that can influence protein, fat, and moisture content of mechanically deboned meat. B.

MINERAL CONTENT 1.

Calcium and Ash

Calcium is the chemical means by which bone content of mechanically deboned meat is determined; calcium content is closely associated with ash content. Calcium makes up approximately 37% of the bone ash in bones from all species, all ages, and all anatomical locations (Taylor et al. , 1960; Field et al., 1974a). Nevertheless, ash and calcium content of bone increase with age during the calcification process (Posner, 1969; Doyle, 1979). Prediction of bone content in mechanically deboned meat by changes in calcium content must therefore be based on the calcium content of the bones that have the same degree of calcification as those furnishing the mechanically deboned meat. Lean and fat mixtures also vary in calcium content. Lean that is free from bone contains 12 mg of calcium per 100 gm and 1.2% ash, whereas fat contains 3 mg of calcium per 100 gm and 0.2% ash (Watt and Merrill, 1963), resulting in slight changes in the amount of ash and calcium in meat with changes in the lean-to-fat ratio. Because calcium in bone and calcium in lean-to-fat mixtures varies, estimation of bone content from the calcium present in mechanically deboned meat is at best an approximation. The method is still in use for poultry (U.S. Department of Agriculture, 1969), where bone content based on calcium analysis is limited to 1%. The USDA (U.S. Department of Agriculture, 1978b) limits calcium in mechanically deboned red meat to 0.75% and avoids any approximations of bone content from calcium. With this system fatter samples and samples of mechanically deboned meat from younger animals could have a slightly higher bone content than leaner samples or samples from older animals and still be within the 0.75% calcium limit. Ash and calcium content for mechanically deboned meat from a continuous-type deboner that rotates meat and bone against a screened surface are found in Tables I1 and 111. The make of mechanical

MECHANICALLY DEBONED RED MEAT

43

deboner does influence the calcium content of mechanically deboned meat (Goldstrand, 1975). Field et al. (1974~)used a continuous-type machine and found that the calcium content of mechanically deboned meat from trimmed lamb necks increased from 0.21 to 0.37% calcium when the yield was increased from 32 to 69%, whereas Neuhauser (1977) increased the yield but more than doubled the calcium content when beef and pork bones were pressed a second time to remove additional lean with a batch machine. Maturity of bone also affects the calcium content of mechanically deboned meat. Grunden and MacNeil(l973) stated that the higher degree of calcification of more mature bones caused more fragmentation when the bones were passing through the deboner, resulting in an increased level of bone particles. Field et al. (1979a) concluded that costal cartilage in lamb breasts was completely removed by a mechanical deboner. The amount of meat left on bones affects the calcium and ash content of mechanically deboned meat. Calcium and ash percentages in mechanically deboned meat were lowest when the meat was obtained from sow loin and veal back bones (Table II), and these bones had the highest amount of lean attached (Table I). Since bones from small animals have a greater surface area per unit weight on which lean is attached than do bones from larger animals, there is an opportunity for the lean-to-bone ratio to be higher and the calcium and ash content to be lower when mechanically deboned meat from small bones is compared with mechanically deboned meat from large bones. Diluting bone with more meat does not reduce the weight of calcium or ash extracted from a given bone. It merely decreases the percentages because of more meat present (Field, 1976b). Other factors that influence the calcium and ash content of mechanically deboned meat include the size of the plate through which the meat and bone were ground prior to mechanical deboning, deboning machine settings and maintenance, and the condition of the bones from which residual lean is to be removed. Field ef al. (1974b) showed that mutton machine-boned cold contained more calcium and ash than did mutton machine-boned hot off the kill floor, even though the meat yield from hot-boned carcasses was higher. 2 . Phosphorus

The most comprehensive analyses of minerals in mechanically deboned meat is that of Djujic et al. (1979). They obtained product from a stationary, batchtype machine and compared it with meat separated by hand from the same bones of pork (Table IV) and beef (Table V). The phosphorus content of handseparated and mechanically deboned meat was similar in beef and pork; this result is surprising in view of the fact that phosphorus makes up 10-13% of dry

TABLE IV CONTENT OF MINERAL COMPONENTS IN PORK AND MECHANICALLY SEPARATED PORK"

MEAT Component

e

Ash (5%) Phosphorus (%) Calcium (mg 5%) Magnesium (mg 70) Sodium (mg 70) Potassium (mg 76) Iron (mg 5%)

Zinc (mg/kg)

Mechanically separated meat

Leg

Shoulder

Head

Leg

Shoulder

Head

0.89-1.02 0.96 0.2 19-0.223 0.220 15.02-48.98 26.10 23.45-27.42 25.44 35.6 I - 43.24 39.32 358.20-505.90 401.12 I .39-1.70 1.55 18.76-35.19 24.64

0.94-1.04 0.98 0.194-0.280 0.208 22.20-35.78 26.75 20.84-24.32 22.60 40.12-55.41 46.21 398.81-572.77 475.75 1.07-2.00 1.65 22.74-37.74 33.08

0.6 1-0.94

0.89-1.49 1.10 0.198-0.235 0.229 85.25- I 57.75 121.00 21.24-28.38 24.39 109.30- 190.41 153.53 256.38-383.82 342.24 5.25-7.19 6.07 I 3.46- 16.41 14.88

0.96-1.42 1.15 0.167-0.253 0.209 139.10-190.8n 151.85 17.81 -32.37 24.96 120.04-214.38 158.68 299.18-465.20 336.69 5.68-9.88 7.35 12.OO-24. I 1 18.86

I . 14-1.77 1.40 0.157-0.292 0.227 87.22-291.18 182.63 13.75-18.16 16.82 149.30-240.02 174.90 252.37-406.87 327.81 4.54-6.44 5.38 12.19- 16.05 14.63

0.81

0.084-0.141 0.122 58.00-100.04 77.75 9.39-16.20 13.42 74.59-91.52 87.45 196.19-445.45 359.75 1.14-2.98 2.26 12.40-22.38 18.93

Nickel (mg/kg) Cobalt (mglkg) Copper (mg/kg)

Tin (mg/kg) Lead (mg/kg) Cadmium (mg/kg) Antimony (mg/kg) Selenium (mg/kg)

& Arsenic (mg/kg) Mercury ( m a g ) Water (%)

" From

0.42-0.55 0.49 0.02-0.20 0.04 0.36-2. I 1 1.19 0.84- 1.36 1.11 0.00-0.72 0.18 0.00-0.08 0.02 0.00-0.37 0.21 0.00-0.12 0.03 0.00-0.82 0.28 0.00-0.12 0.02

0.23-0.72 0.49 0.00-0.10 0.04 0.2 1-0.72 0.48 0.12-1.48 0.95 0.00-0.41 0.16 0.00-0.04 0.02 0.00-0.62 0.17 0.00-0.10 0.04 0.00-0.70 0.30 0.00-0.09 0.03

0.24-0.79 0.50 0.00-0.23 0.12 0.86-1.05 0.96 0.74-1.44 I .04 0.00-0.77 0.43 0.00-0.06 0.03 0.00-0.14 0.06 0.00-0.14 0.09 0 .OO-0.77 0.33 0.00-0.13 0.05

0.20-0.43 0.21 0.07-0.33 0.21 0.18-0.98 0.60 1.34-1.92 I .61 0.00-1.10 0.59 0 .00-0. 07 0.02 0.00-0.73 0.33 0.00-0.06 0.03 0.00-0.42 0.18 0.00-0.07 0.03

0.1 1-0.40 0.18 0.05-0.25 0.17 0.32-1.00 0.75 0.74-1.16 1.03 0.00-1.20 0.71 0.00-0.04 0.01 0.00-0.54 0.38 0 .00-0.06 0.04 0.00-0.37 0.19 0.00-0.08 0.04

0.17-0.68 0.35 0.03-0.23 0.09 1.20-3.25 2.16 0.64- 1.46 1.14 0.00-1.05 0.45 0.00-0.02 0.01 0.00-0.22 1.75 0.00-0.08 0.06 0.00-0.45 0.14 0.00-0.15 0.07

65.26-75.71 72.57

69.52-72.89 71.96

4 1.48-59.27

43.86-65.05 54.28

43.29-65.98 57.16

58.64-69.71 06. I6

DjujiC et al. (1979).

53.19

TABLE V CONTENT OF MINERAL COMPONENTS IN BEEF AND MECHANICALLY SEPARATED BEEF"

MEAT Component

Mechanically separated meat

Leg

Shoulder

Head

Leg

Shoulder

Head

0.92-1.07 0.99 0.207-0.234 0.224 16.82-30.14 28.16 20.3 1-26.83 24.72 33.2 1-40.48 37.86 318.17-514.13 414.20 I .40-3.04 2.23 23.11-28.04 25.67

0.98-1.12 I .04 0.184-0.227 0.209 23.77-34.32 30.08 18.24-2 I .67 20.14 39.93-48.98 45.14 398.47-572.88 482.16 I .22-2.56 2.16 26.12-34.79 31.98

0.88-0.99 0.92 0.102-0. I56 0.124 136.29-2 14.13 189.20 8.36-13.47 10.89 78.16-90.02 83.61 283.36-462.72 364.12 I .42-3.57 2.86 15.32-24.36 19.84

I . 12-1.35 I .20 0.193-0.235 0.226 204.32-306.2 I 269.17 23.35-27.07 25.68 107.84-198. I2 141.79 356.45-475.30 464.28 6.73-9.21 8.32 17.03-24. I I 20.34

1.26-1.67 1.48 0.180-0.229 0.218 289.15-41 8.76 369.17 22.33-25.97 24.18 129.65-172.17 143.24 335.62-448.38 386.20 6.40-8.71 8.09 19.22-22.96 20.78

1.34-2.36 2.04 0.165-0.241 0.220 398.42-621.54 508.20 15.96- 18.25 16.62 109.56- 198.32 162.I I 267.34-398.92 341.28 5.11-8.32 6.78 11.53-18.36 15.25

m P Ash (%) Phosphorus (8) Calcium (mg 9%) Magnesium (mg Q) Sodium (mg 8 ) Potassium (mg %) Iron (mg %) Zinc (mg/kg)

Nickel (mg/kg) Cobalt (mg/kg) Copper (mg/kg) Tin (mg/kg) Lead (mdkg) Cadmium (mg/kg) Antimony (mg/kg) Selenium (mg/kg)

5

Arsenic (mg/kg) Mercury (mg/kg) Water (%)

I'

0.47-0.64 0.56 0.03-0.22 0.06 0.61-2.62 2.08 0.91-2.06 1.32 0.00-0.80 0.30 0.00-0.12 0.02 0.00-0.42 0.29 0.00-0.15 0.03 0.00-1.06 0.34 0.00-0.12 0.03 69.58-73.14 71.86

From DjujiC et al. (1979).

0.26-0.70 0.48 0.01-0.12 0.04

0.34-1.08 0.82 0.34-1.66 1.26 0.00-0.69 0.22 0.00-0.09 0.03 0.00-0.64 0.22 0.00-0.1 I 0.05 0.00-0.88 0.37 0.00-0.12 0.04 68.12-71.03 69.14

0.25-0.80 0.47 0.00-0.26 0.09 0.81 - 1.84 I .34 0.82-1.58 1.16 0.00-0.84 0.56 0.00-0.08 0.03 0.00-0.22 0.09 0.00-0.20 0.10 0.00-0.89 0.41 0.00-0.14 0.07 43.2 1 -5 1.14 46.74

0.22-0.49 0.32 0.14-0.51 0.3 I 0.79-3.08 I .90 I .22-3.02 I .96 0.00- 1.48 0.72 0.00-0.06 0.02 0.00-0.89 0.40 0.00-0.10 0.04 0.00-0.50 0.25 0.00-0.09 0.04 64.11-67.82 65.57

0.18-0.66 0.30 0.10-0.49 0.24 0 . 4 4 1.24 0.79 0.82-2.46 I .54 0.00- 1.5 1 0.69 0 .00-0.05 0.02 0.00-0.78 0.42 0.00-0. I 1 0.06 0.00-0.51 0.27 0.00-0.11 0.06 56.02-59.74 58.43

0.22-0.75 0.41 0.06-0.24 0.14 0.99-2.24 1.58 0.79-2.38 I .62 0.00- 1.22 0.58 0.00-0.06 0.03 0.00-1.46 0.83 0.00-0.16 0.07 0.00-0.39 0.16 0.00-0.20 0.09 56.56-59.28 57.10

48

RAY A . FIELD

fat-free bone. The data in Tables IV and V are not adjusted for differences in moisture, fat, or protein, and mechanically deboned meat in the tables is lower in moisture, which would indicate a higher fat content. Lean meat contains approximately 200 mg of phosphorus per 100 gm, whereas fat contains 10 mg of phosphorus per 100 gm (Watt and Memll, 1963). It is obvious that the additional phosphorus in the bone powder in mechanically deboned meat was diluted by additional fat, which is low in phosphorus. Samples of mechanically deboned meat that are higher in bone content than those in Tables IV and V, or samples that contain the same fat, protein, and moisture as hand-separated meat, are higher in phosphorus. 3 . Magnesium After calcium and phosphorus, magnesium is the most common mineral in dry fat-free bone, constituting approximately 0.45% of the bone weight (Doyle, 1979). Beef and pork contain 18 mg of magnesium per 100 gm of lean, according to Rice (1971), and these figures are close to those listed for beef and pork in Tables IV and V. Therefore, the distribution of magnesium in bone and meat is similar to that of phosphorus, and magnesium follows a pattern similar to that of phosphorus in mechanically deboned meat. 4 . Sodium

Sodium, like magnesium and phosphorus, is found in meat and bone, and the amount in mechanically deboned meat increases as the amount of bone ash increases (Tables IV and V). Forbes and McCoord (1963) concluded that sodium in rat cortical bone increases throughout growth and that the increase is most pronounced in early life when growth in body size is rapid. The sodium content of bone on a fresh weight basis ranged from 0.095 meq/gm in 13- to 14-day-old rats to 0.254 meq/gm in 360-day-old rats. The latter figure for sodium content in bones from 360-day-old rats is similar to the values for cortical bone from humans, cats, pigs, and rabbits, when compared on the basis of bone water content (Forbes, 1960). It is evident that mechanically deboned meat from older animals will have higher levels of sodium than will meat from younger animals at similar levels of bone content. 5 . Potcissium

Values for potassium content of meat from leg and shoulder in Tables IV and V are similar to others for lean (Watt and Merrill, 1963). Potassium content of bone is much less than that of lean at approximately 0.01% in dry fat-free bone (Armstrong and Singer, 1965; Taylor et al., 1960). Therefore, the lower potas-

MECHANICALLY DEBONED RED MEAT

49

sium values for mechanically deboned meat, when compared with those for hand-separated meat, are not surprising. 6 . Iron

The iron content of mechanically deboned meat has been reported by,several researchers (Kolbye and Nelson, 1977a; Kunsman et al., 1978; Field et al., 1979a,c; Kruggel and Field, 1977). The values are in good agreement with those in Tables IV and V, which show that mechanically deboned meat has two to three times as much iron as does hand-boned meat. According to Eastoe (1961), bone contains less than 0.01% iron, but red bone marrow present in mechanically deboned meat is high in iron. According to Garcia (1957), rat bone marrow from 150- and 250-day-old rats contains 8.8 mg and 1 1 .O mg of iron per 100 gm of marrow, respectively. The iron content of marrow varies considerably in bones from different anatomical locations. Field et al. (1980) has shown that cervical marrow from steers contains 23.0 mg of iron per 100 gm of fresh marrow, whereas lumbar marrow from the same animals contains 13.1 mg of iron per 100 gm. Therefore, the iron content of mechanically deboned meat varies according to the anatomical location from which it is obtained. Within anatomical locations for animals of the same age and species, those samples of mechanically deboned meat that have the highest marrow content will have the highest iron content, since iron content in fresh muscle is relatively constant at 2-3 mg per 100 gm for beef and 1-2 mg per 100 gm for pork (Jenkins, 1977; Watt and Merrill , 1963). 7. Zinc

Published values for zinc are 42 pg/gm in beef and 27 pg/gm in pork (Murphy et d.,1975); these values are in line with those for pork and beef in Tables IV

and V , respectively. The lower zinc values for mechanically deboned meat in Tables IV and V , when compared with values for hand-separated meat, are probably due to increased fat levels. Kolbye and Nelson (1977a) have concluded that zinc values for hand-separated meat are similar to values for mechanically deboned beef. Their values were 34-47 p g of zinc per gram in mechanically deboned beef and 23-27 p g of zinc per gram in mechanically deboned pork. 8 . Nickel, Cobalr. and Copper

Studies by Djujic et a/. (1979) and by Kolbye and Nelson (1977a) show traces of nickel, cobalt, and copper present in mechanically deboned meat. In both studies nickel and copper were found in similar amounts in hand-separated and mechanically separated meat, whereas cobalt levels were higher when mechanically deboned meat was compared with hand-separated meat (Tables IV and V).

50

RAY A . FIELD

Mean values for levels of cobalt in mechanically deboned meat reported by Kolbye and Nelson ( 1977a) were similar to those in Tables IV and V; they are far too low to have any public health significance. According to Underwood (1977), the amounts of cobalt needed to produce polycythemia and thyroid hyperplasia are 50-75 times the typical adult intake of 0.13-1.4 mg/day. 9.

Tin and Antimony

Levels of tin in hand-separated and mechanically separated samples were similar, but antimony values in mechanically deboned meat were slightly higher than those for hand-separated meat (Tables IV and V). In a biological sense, the low levels of both tin and antimony in mechanically deboned meat are nonsignificant. Tin has been reported in bone at a level of 0.5 mg/kg (Eastoe, 1961). 10. Lead

The lead content of mechanically deboned meat has been studied by several research workers because lead, like sodium, fluorine, and strontium, accumulates in bone (Forbes and McCoord, 1963; Snowden and Stitch, 1957). About 90% of the body lead can be accounted for in the skeleton (Ammerman et a l . , 1977). According to the review of Doyle (1979), lead concentrations in fresh bone of cattle, sheep, and swine vary from 0.3 to 8.0 pg/gm. Doyle and Spaulding (1978) found that lead content in meat from beef, pork, and sheep ranged from 0.20 to 0.34 pdgrn. The latter figures are in line with values of 0.25 pg/gm in hamburger and beef chuck roast (Kolbye and Nelson, 1977a), and with those listed for pork and beef in Tables IV and V . Fourteen mechanically deboned beef samples analyzed by Kolbye and Nelson (1977b) had 0.09 pg/gm; 16 mechanically deboned pork samples averaged 0.06 pg/gm. Michigan State University researchers (M.R. Bennink, personal communications) have not been able to detect lead in mechanically deboned beef or pork. The values for lead in mechanically deboned beef and pork, which are lower than those in some hand-separated meat samples, are not surprising in view of the variability that exists for lead in animal feeds in different regions (Ammerman et a l . , 1977). The higher values for lead content of mechanically deboned pork and beef in Tables IV and V are undoubtedly a result of higher levels of lead in the animals feed. 11. Cadmium and Selenium

Cadmium and selenium contents of hand-separated and mechanically separated pork and beef shown in Tables IV and V are low, and no meaningful differences between hand-separated and mechanically separated meat are pre-

MECHANICALLY DEBONED RED MEAT

51

sent. The USDA analyses (Kolbye and Nelson, 1977a) for cadmium in mechanically deboned beef and pork showed no values high enough to reach the analytical detection limit of 0.01 pg/gm. Literature cited by Doyle and Spaulding (1978) lists values of
Kolbye and Nelson (1977a) did not determine arsenic in mechanically deboned red meat because their data for mechanically deboned poultry showed no measurable amounts of arsenic present. They reasoned that any difference between mechanically separated and hand-separated poultry should have surfaced in the poultry data because of the use of arsenicals in poultry feed. Data of DjujiC ct al. (1979) in Tables IV and V confirm Kolbye and Nelson’s reasoning. Arsenic levels were lower in mechanically deboned pork and beef than in handboned meat. Holm (1978) analyzed numerous meat samples from beef, pork, poultry, and game and found a mean arsenic level of S 0.01 ppm and a maximum level of 0.03 ppm. Mercury does not accumulate in bone; it is a soft tissue problem. Data in Tables IV and V show similar low levels of mercury in hand-separated and mechanically separated beef and pork. The low levels are of no biological significance. 13. Fluoride

Fluoride levels in mechanically deboned meat from commercial sources in different geographical regions in the United States are shown in Table VI. Mechanically deboned beef from the West and Midwest regions of the country had a significantly higher (P < 0.05) fluoride content than did pork from the same regions (Table VI). Differences in fluoride content between beef and pork are due to differences in age at slaughter (Waldbott, 1963; National Academy of Sciences, 1974) and to type of feed consumed. Concentrates consumed by pigs

52

RAY A . FIELD

have much lower levels of tluoride than does the forage consumed by mature cows and bulls. There is only a slight fluoride increase in the muscle or fat when animals are fed diets high in fluoride. Shupe et u1. (1963b) found insignificant amounts of fluoride in the soft tissues of cattle receiving up to 93 ppm of fluoride for 7.5 years. Singer et a / . (1967) found that the fluoride content of fresh muscle in two groups of rats with diets containing 0.5 ppm of fluoride and 100 ppm of fluoride were very similar (0.20 ppm and 0.21 ppm, respectively). Approximately 99% of the fluoride retained in the body is stored in the bone. According to Shupe et (11. (1962), bones from animals receiving 10 ppm of tluoride in hay for 588 days contained 328-528 ppm of fluoride on a dry fat-free basis. Pool and Thomas ( 1970) studied weanling rats fed 14, 28, and 56 ppm of fluoride for 341-397 days. Concentrations of fluoride in the skeleton varied almost linearly with the concentration ingested. Suttie et 01. (1958) reported that cattle cancellous bone was uniformly higher in fluoride than were compact bones from the same animals. This finding was supported by Suttie and Phillips ( 1959) and by Shupe e t a / . ( 1 963a), who found that cancellous bones such as the frontal, ribs, and vertebrae, and those of the pelvis, had a higher fluoride content than did the more compact metatarsal and metacarpal bones. It is apparent that the bones most suited for mechanical deboners (Field, 1976b) are also the bones highest in fluoride content. Since bones of animals contain a high and varying amount of fluoride, compared with that found in muscle and soft tissue, it may be concluded

TABLE VI LEAST-SQUARES MEANS" AND STANDARD ERRORS FOR COMPOSITION OF MECHANICALLY DEBONED MEAT FROM COMMERCIAL SOURCES BY GEOGRAPHIC REGIONSb,"

Beef Variable Fluoride (pg/gm) Ash ( % ) Calcium Magnesium (mg1100gm) Iron (mg1100gm) Ascorbic acid (gg/lW gm) Moisture ( % ) (Qm)

Fat ( % )

West ( N

=

26)

Midwest ( N

Pork =

12)

South ( N

=

25)

2

West and Midwest ( N

16.21b ? 2 . 1 6 2.50 i 0.18 0.48b 2 0.06

15.67b ? 3 . 2 2 2.22 ? 0 . 2 7 0 . 3 9 ~i 0.09

9.83b.c 1.97 0.40~

2.30 0.20 0.06

7 . 6 2 ~? 0 . 6 7 2.45 ? 0 . 2 4 0.50b +- 0.07

18.85 ? 1.01 5.65b -t 0 . 3 7

18.40 5 1.50 6.30b ? 0 . 5 5

15.57 -+ 1.07 4 . 2 6 ~ L 0.40

18.83 i 0.64 3 . 7 8 ~5 0.38

2 . 0 8 ~? 0.13 5 6 . 3 7 ~? 1.27 22.61h L 1.50

2.67b 2 0.19 67.11b ? 1.89 1 2 . 3 4 ~? 2 . 2 3

2.38c.b 2 0.13 67.60b 5 1.35 1 0 . 8 3 ~ 2 1.60

2.61b 2 0.18 5 7 . 7 6 ~+- 0.7X 23.45b 5 0 . 8 8

5

-t

Means on the same line bearing different letters differ significantly ( P < 0.05) All data expressed on a fresh weight basis. ' From Kruggel and Field (1977). 'I

"

= 18)

MECHANICALLY DEBONED RED MEAT

53

that fluoride found in mechanically deboned meat comes primarily from bone particles. Fluoride levels in mechanically deboned meat are comparable to those of Dolan et a / . (1978) for mechanically deboned beef and pork, but they are lower than the levels Dolan er a / . (1978) reported for mechanically deboned poultry. The mechanically deboned poultry was apparently from mature fowl, since fluoride in mechanically deboned poultry from young fowl is considerably lower (Murphy et al., 1979). The data of Kolbye and Nelson (1977b) also support those fluoride levels in Table VI for mechanically deboned beef and pork.

VI. SAFETY ASPECTS OF MECHANICALLY DEBONED MEAT The controversy surrounding mechanically deboned meat has centered on toxic and essential elements associated with the bone powder, on possible gastroenterological side effects from ingestion of bone particles, and on microbiological hazards that could result from machine deboning or from further processing. In order to respond to the questions on safety, an intensive analytical program aimed at developing data on amounts of potentially toxic substances in mechanically deboned meat was conducted in USDA laboratories (Kolbye and Nelson, 1977a). In addition, the controversy involved with toxic and essential elements in meat and bone has resulted in several excellent reviews (Doyle and Spaulding, 1978; Ammerman et a / . , 1977; Mahaffey, 1977b; Mertz, 1977; Ullrey, 1977; Doyle, 1979). Angelotti (1978) stated that the problem of residues in meat is a continuing one, and one on which increased emphasis is being placed by the Food Safety and Quality Service of the USDA. A.

MINERAL TOXICITY

The term “heavy metal” is often used to describe toxic elements. In the chemical sense the term refers to the detection of metal impurities by precipitation reactions. In the biological sense, particularly in considering elements requiring scrutiny and control, the term is misleading because it would not include such potentially toxic elements as selenium, fluorine, and cobalt. The following discussion on mineral toxicity in mechanically deboned meat will center on those potentially “toxic metals” that the review in Section V on mineral content showed are higher in mechanically deboned meat than they are in hand-boned meat. In addition, strontium-90 will be discussed because a preliminary court injunction (Kolbye and Nelson, 1977b) stopping the production of mechanically deboned meat in the United States was based in part on the court’s finding that “possible unduly high levels of strontium-90 which may be contained in bone particles in mechanically deboned red meat” had not been adequately assessed.

54

RAY A . FIELD

1. Leud

Mahaffey (1977b) listed six toxic elements of major interest to the Food and Drug Administration in Washington, D.C. They were mercury, lead, cadmium, zinc, arsenic, and selenium. Lead is the only one of the six that could be higher in mechanically deboned meat than it is in hand-separated meat because lead is known to accumulate in bone. However, mechanically deboned meat does not always have a higher lead content than hand-separated meat. Kolbye and Nelson ( 1977b) found 0.09 p g of lead per gram in mechanically deboned meat, which is lower than reported values of 0.20-0.34 pg/gm in hand-separated meat (Doyle and Spaulding, 1978). Because some lots of mechanically deboned meat contain higher levels of lead than does hand-separated meat, and because dietary lead intakes for infants and young children indicate that average levels of ingestion usually are between 75 and 120 p g of lead per day (Kolbye et ul., 1974), which approaches recommendations on permissible levels of lead intake of 150 p g of lead per day for children 6 months to 2 years of age (Mahaffey, 1977a), a close look at the intake of lead from mechanically deboned meat is justified. Kolbye and Nelson ( I977a) used the highest projected intakes for mechanically deboned meat and concluded that the amount of lead added to the diet would be less than 0.5 pg/day. Even under the most extreme assumptions for ingestion of mechanically deboned meat, the amounts of lead included in mechanically deboned meat are hard to document as an addition of lead to the diet. It was concluded that, relative to the magnitude of other environmental sources of lead for children such as air, soil, plants, and paint (Hankin, 1972; Wessel and Dominski, 1977), the amount of lead from mechanically deboned meat would be toxicologically insignificant. One additional assurance that lead in mechanically deboned meat is of no toxicological consequence is the presence of calcium. A protective effect of dietary calcium against lead toxicity has been known for many decades (Ammerman et u / . , 1977). Voluntary lead ingestion in weanling rats increased with calcium deficiency, suggesting that the latter condition contributes to lead pica (Snowdon and Sanderson, 1974). The beneficial effects of higher dietary calcium (0.7% vs I % Ca) were demonstrated in adult rats consuming 3 ppm or 200 ppm lead acetate in the drinking water for 10 weeks (Mahaffey et al.. 1973). Dietary calcium in the bone powder of mechanically deboned meat should help decrease the absorption of lead from the intestinal tract and decrease the retention and metabolism of lead in bone and in tissues (Quarterman and Morrison, 1975; Morrison et al., 1974).

2 . Fluoride Fluoride in mechanically deboned meat was the reason the USDA (U.S. Department of Agriculture, 1978b) recommended that such meat should not be

MECHANICALLY DEBONED RED MEAT

55

included in strained baby, junior, or toddler foods, since excess fluoride can cause mottling of teeth in young children. Foods containing higher levels of fluoride than would be found in frankfurters containing mechanically deboned meat include soft drinks, tea, tuna fish, salmon, sardines, and spinach (Kolbye and Nelson, 1977a). According to Labuza (1977), fluorine is found in almost all foods and dietary intake ranges 0.3-3.1 mg/day. He believes that communities should fluoridate their water to help overcome deficiencies of fluoride in the diet and that those who oppose fluoridation of water for political purposes are doing a disservice to the nutritional well-being of their children. Fluoride toxicity occurs if 20-80 mg are consumed per day for a period of several years, however, fluoride toxicity is an unusual dietary occurrence. Fluorine is an essential nutrient (Food and Nutrition Board, 1974). If mechanically deboned meat is included in hot dogs at the maximum level of 20%, and if high fluoride levels of 16 pg/gm are found in the mechanically deboned meat (Kruggel and Field, 1977), less than 1 mg of fluoride would be found in 0.45 kg of hot dogs. According to Knight and Winterfeldt (1977), beneficial intakes of fluoride could result from the use of mechanically deboned meat in areas where the intake might be low or where water is not fluoridated. This would include rural areas where additional nutrition from mechanically deboned meat is badly needed and where people with lower incomes normally buy lower-priced meat in which mechanically deboned meat could be included. Kolbye and NelTon (1977a) stated that the fluoride content of mechanically deboned meat posed no health problem for adults, but they felt that there was a lack of information on fluoride intake of children and that additional information should be obtained before recommendations to include fluoride in foods for infants were made. The recommendation is open to question, since the need to add fluoride to the water supply to prevent dental carries is well established and since years of experience with mechanically deboned poultry in infant foods containing up to five times the levels that would be allowed for red meat have not produced any documented cases of mottling of teeth in young children. 3 . Strontium-90

One of the issues in 1964 concerning mechanically deboned poultry was that of the presence of strontium-90, which accumulates in bone (U.S. Department of Agriculture, 1979). At that time the National Academy of Science was asked to evaluate the data accumulated on strontium-90 and advise the USDA if undesirable amounts would be added to the diet, especially for infants, through the consumption of mechanically deboned poultry. The Food Protection Committee of the National Academy of Science’s Food and Nutrition Board concluded that there was no significant health consequence associated with the amount of bone

56

RAY A . FIELD

left in mechanically deboned poultry as long as the ratio of strontium-90 to calcium in the product did not exceed that of cows’ milk. This ratio was far lower in mechanically deboned poultry, approximately one-third of that in cows ’ milk (U.S. Department of Agriculture, 1979). Mechanically deboned beef contains 0.097 pCi of strontium-90 per gram; mechanically deboned pork is lower at 0.029 pCi/gm (Kolbye and Nelson, 1977b). Michigan State University data (M. R. Bennink, personal communication) support the preceding low values for strontium-90 in mechanically deboned meat. When Bennink fed mechanically deboned meat and hand-separated meat to rats, no differences in strontium-90 content of the femurs were observed. He concluded that, since strontium-90 contamination in the environment is very low (Bruce et al., 1974), the intake from mechanically deboned meat would be minimal and would not present a health hazard. The conclusion reached was similar to that of Kolbye and Nelson (1977a), who used Federal Radiation Council data to determine the significance of an increase in strontium-90 from ingestion of mechanically deboned meat. They concluded that the use of mechanically deboned meat would result in approximately a 1% increase of strontium-90 in the diet and a 1% increase in millirems per year. Both of these increases were considered insignificant. The retention of strontium-90, like that of lead, is influenced by the amount of calcium in the diet. According to Kolbye and Nelson (1977a), Spencer has shown that adult males on high-calcium, high-phosphorus diets retain less strontium-90 than do those on low-calcium, high-phosphorus diets. Additional historical data on strontium-90 content in bone, milk, and vegetation in past years when strontium-90 was produced and disseminated by atomic explosions are reported by Comar et al. (1957) and MacDonald et al. (1955). B.

BONE PARTICLE SIZE

Much of the controversy relating to mechanically deboned meat has centered around the small quantities of bone powder that become part of the product during mechanical removal of meat from bone. The powder has been referred to as bone chips, bone fragments, and ground bone. Opponents (Bryant, 1976) have claimed that the bone particles are an adulteration of the meat and that possible gastroenterological side effects may result from their frequent ingestion. Potential hazards in terms of impact of bone particles on gums and teeth or between teeth have also been hypothesized. As was indicated in the discussion of calcium and ash, the amount of bone in mechanically deboned meat as estimated by calcium content can vary widely. Some countries limit the amount of bone present by limiting the amount of

57

MECHANICALLY DEBONED RED MEAT

calcium. Nevertheless, a low calcium limit for mechanically deboned meat does not limit the size of the bone particles. The size of the bone particles, in addition to total bone content, may affect the overall acceptability of the product. Since different makes of deboners have different-sized holes or slots through which the meat is forced when it is separated fromthe bone (see Introduction), it is logical that bone particle size in mechanically deboned meat from different machines varies. Therefore, bone particle size in mechanically deboned meat from a deboner with 0.46-mm holes in the screen and mechanically deboned meat from a deboner with 1 x 1.3-mm rectangular slots will be discussed. The data in Table VII were obtained on bone powder isolated from mechanically deboned meat from a machine with 0.46-mm holes in the screen. Bone was separated by papain digestion followed by separation in carbon tetrachloride : acetone mixtures as outlined by Hill and Hites (1968). Bone particles were isolated on the day the meat was deboned and after the meat was held for 2 wk at 0°C. The purpose of the 2-wk isolations was to determine if bone powder was solubilized in meat after a holding period. The calcium levels in mechanically deboned meat (Table VII) are typical of those previously reported for mechanically deboned meat from beef neck bones. The actual percentage of bone isolated from all five lots of mechanically deboned meat ranged from 2.8 to 4.1 % and was lower than would be expected. When the TABLE VII SIZE OF BONE PARTICLES IN MECHANICALLY DEBONED BEEF AT THE TIME OF DEBONING AND AFI'ER 2-WK STORAGE',b

Fresh MDM

MDM after 2 wk at 0" C

Diameter (pm) Lot no.

Calcium (%)'

Bone ( % ) d

Mean

S.D.

Range

I 2 3 4 5

1.04 0.76 0.91 0.83 0.92

4.0 2.9 3.9 3.3 3.5

111.7 76.6 83.2 79.4 100.6

49.1 37.4 45.9 37.3 56.3

10-430 10-450 10-420 10-360 10-370

" From Field

Diameter (pm)

Bone

(%)d

4.1 2.8 4.0 3.4 3.5

Mean

S.D.

Range

102.4 81.4 82.7 76.3 91.5

40.3 41.0 48.2 35.7 50.8

10-410 10-420 10-440 10-370 10-400

ef a / . (1977b). Mechanically deboned meat (MDM) from a Beehive mechanical deboner with 0.46-mm holes in the cylinder. Yield for all lots of beef ranged from 45 to 54%. Calcium in fresh MDM. " Based upon actual weight of dry, fat-free bone isolated from MDM by the Hill and Hites (1968) procedure. "

58

RAY A . FIELD

calcium values of 0.76- I .04% (Table VII) are multiplied by a factor of 5 (Field er al., I974a) to obtain the percentage of bone in mechanically deboned meat, the actual bone percentages in Table VII are lower than the calculated values by approximately one percentage point. Some solubilization of bone by the water in meat could occur (Rootare et al., 1962). Solubilization of bone by the 0.9% lactic acid in meat (Lawrie, 1974) is also possible. When bone particles isolated from mechanically deboned meat were added at the 5 or 10% level to hand-boned ground meat, only about 80% of the bone particles could be recovered. The 20% loss in bone particles may have been due to solubilization, but most of the loss was probably a result of failure to recover the bone particles during isolation. Evidence that bone in mechanically deboned meat is not solubilized is shown in Table VII. No differences were found in the amount of bone isolated from fresh mechanically deboned meat compared with the amount of bone isolated from mechanically deboned meat stored for 2 wk. Further evidence for the stability of bone particles in mechanically deboned meat is found in the similar diameters of bone particles from fresh mechanically deboned meat and mechanically deboned meat after 2-wk storage. In addition, pH values for mechanically deboned meat did not change during the 2-wk storage period (data not shown in tabular form). Changes might have been expected if the basic calcium phosphate from bone had gone into solution. High standard deviations and wide ranges for bone particle size are evident (Table VII). Mean bone particle size ranging from 76.6 to 1 11.7 p m was similar to the values given by Kolbye and Nelson (1977b) for bone meal tablets used as calcium supplements. The greatest diameter of the largest bone particles measured approaches the theoretical limits for largest bone particle size, since the holes in the cylinder through which the meat passes are 460 p m . If long slivers of bone particles were started through 460-pm holes of the deboner, they would be sheared into maximum lengths of 423 p m . The 423-pm figure is obtained by dividing the 10.16-cm flow of mechanically deboned meat per min through the cylinder holes by the 240 rpm of the auger on the inside cylinder wall. The flow rate is often slower than the 10.16 c d m i n obtained in this study, and the 240 rpm is slower than that on many other machines where the auger is set at 306 rpm, making the theoretical figure of 423 p m a maximum for bone particle length. A photomicrograph of bone particles from mechanically deboned meat in this study is shown in Fig. 1. Variability in bone particle diameter, as is shown by the high standard deviations (Table VII), is evident. Bone particles solubilized in a 0.037 M HCl solution leave a mineral deposit (Fig. 1B). This concentration of HCI is similar to that found in the gastric contents of the human stomach (White et al., 1954). Bone particles solubilized in 0.15 M HCl, which is

MECHANICALLY DEBONED RED MEAT

59

FIG. I . Photomicrograph of bone particles isolated from MDM ( A ) before and ( B ) after contact with 0.037 M HCI. From Field CI ul. (1977b).

60

RAY A . FIELD

similar to the concentration of HCl in gastric juice (Lehninger, 1970), were similar to those in Fig. 1 B. Visual changes were confirmed by atomic absorption spectrophotometry when the bone particles were placed in 0.15, 0.075, 0.037, or 0.018 M HCI. Bone completely dissolved in all concentrations of HCI in 5 min. Increasing concentrations of HCl or increased time in the HCl did not significantly change the amount of calcium in the solution. Only trace amounts of calcium were found in the pellets after centrifugation of the HCl solutions. Bone (calcium phosphate) is solubilized in HCI concentrations even lower than those used in our study. Johnson et al. (1970) used a 0.007 M HCl solution to leach mineral from the surface of bone. Bone residue remaining after the leachings was dissolved in 0.05 M HCI. Posner (1969) observed that nonreversible hydrolysis of bone readily occurs in aqueous media at physiological pH values. Davidson and Passmore (1963) also confirmed that calcium phosphate is in solution in the unneutralized HCI of human gastric juice. Therefore, bone is a good source of calcium in the diet (Tisdall and Drake, 1938; Drake et al., 1949). Our data show that bone particles from mechanically deboned meat are relatively stable in meat. Nevertheless, bone particles are readily solubilized in concentrations of HCl similar to those found in gastric juice or in the gastric contents of the human stomach. Since the size of the bone particles in mechanically deboned meat is determined mainly by the diameter of the filter openings, data for bone particles from machines with filter openings larger than the one discussed in the preceding paragraphs are shown in Tables VIII and IX. Most press machines in Table VIII produced mechanically deboned pork with less than 0.4% bone powder, but the bone particles were much larger than those discussed previously. Approximately 90% of the bone particles isolated from mechanically deboned meat from the press machines were 100-lo00 p m in diameter, 7.6% were 1000-2000 pm, 1 . 1 % were 2000-3000 pm, and 0.6% were > 3000 p m in diameter. Bijker et al. ( 1979) stated that microscopic examination of intact mechanically deboned meat samples supported the data in Table IX, which were obtained on isolated bone particles. The low values for total bone powder and the relatively large values for bone particle size in mechanically deboned meat from press machines are comparable to the values obtained by Anhalt et al. ( 1977) for 720 samples of hand-boned beef and pork. Sedimentation procedures gave bone contents from head and neck samples of 0.05-0.3 1%; bone content of residual beef and pork from forelimbs and hind limbs was lower at 0.01%. They concluded that the sedimentation methods of determining bone in meat were less sensitive than ash or calcium methods, but that ash and calcium methods required knowledge of the ash or calcium content in boneless meat and bones, as discussed in the section on calcium and ash. Bone particle size as well as bone particle content of mechani-

MECHANICALLY DEBONED RED MEAT

61

TABLE VIII HARD BONE RESIDUE IN 80 MECHANICALLY DEBONED PORK SAMPLES. TAKEN FROM 8 PLANTS, DETERMINED ACCORDING TO THE KOH METHOD“”

Meat plant

Type of machine

I I1 I1 111 IV IV V V VI VI VII VII VIII VIII

Protecon Protecon Protecon Protecon Protecon Protecon Hydrau Hydrau Protecon Protecon Protecon Protecon Soeren Soeren

Bone type

Number

Hard bone residue (%)

10 5 5 10 5 5 5 5 5 5 5 5 5 5

0.28 2 0.06 0.44 2 0.15 0.29 2 0.03 0.62 t 0.14 0.28 2 0.07 0.31 0.05 0.05 2 0.02 0.40 2 0.08 0.12 t 0.02 0.24 & 0.06 0.13 t 0.03 0.14 & 0.01 0.19 t 0.04 0.30 & 0.08

Ribs + backs Sows’bones (mixture) Mixture (porkers) Mixture

Ham Shoulder Boiled pigs’ heads (warm) Boiled pigs’ heads (cold) Ham + shoulder Ribs and backs Shoulder Ham Ham + shoulder Backs + ribs

*

,I From Bijker et al. (1979). Data presented as average percentages and standard deviation.

cally deboned meat from the press machines is comparable to that of hand-boned meat. Froning (1978) found bone particles in hand-boned turkey up to 1142 p m in diameter. Large bone particles in hand-boned or mechanically deboned meat are objectionable because of the gritty texture they impart to products in which they are incorporated and because in extreme cases with some hand-boned meat samples the bone particles have been large enough to cause chipped teeth. Bijker et al. (1979) considered the interests of the consumer and the producer as well as the harmless nature of the injested bone particles and suggested that hard bone residue, determined by the KOH method which they recommend, not exceed 0.4%, with 90% of the particles being less than 1 mm and none greater than 3 mm. Bone particles larger than 3 mm are common in minced fish, since larger openings in the screens or slots are used with fish. Even so, no problems with bone particles in minced fish have surfaced (Keay, 1976). Kolbye and Nelson (1977a) agreed that bone particles of the size found in mechanically deboned meat (12-840 pm) were of a harmless nature. They concluded that no impact of bone particles on gums and teeth or between teeth would occur and that perforation of the gastrointestinal tract by bone particles would be remote, since these problems are related to bone particles much larger in size than those produced by mechanical deboners. Lack of lesions in any of the tissues examined was supported by histological studies of M. R. Bennink (per-

62

R A Y A . FIELD

TABLE IX DISTRIBUTION, IN PERCENTAGE, OF DIFFERENT SIZES OF BONE PARTICLES FOUND IN MECHANICALLY DEBONED PORK"

Class ranges Number of samples

100-1000 p m

1000-2000 prn

2000-3000 p m

Meat plant

(%u)

(%)

(%)

(%i ' o )

I

10

11

5

91.3 91.6 92.6 90.7 91 .O 89.9 86.0 89.6 95.6 93.4 92.0 89.8 90.0 85.6

6.7 7.0 6.0 7.9 8.0 8.1 10.4 7.4 4.0

0.4 I .o I .2

7.0 9.0 8.4 11.2

0.7 1.3 2.6 1.6 0.4 0.6 0.8 0.8 1 .o 2.0

I .6 0.4 0.2 0.5 0.3 0.7

90.6

7.6

1.1

111

IV V

VI

5 10 5 5 5 5 5

5 VII

5 5 5 5

VIIl

I'

From Bijker

ef

5.8

1 .o

1

3000 Wm

1 .o

I .4 0.0 0.2 0.2 0.4 0.6 I .4 0.6

ul. (1979).

sonal communications) of Michigan State University, who demonstrated that bone particles in mechanically deboned meat are solubilized under physiological conditions and that the resultant minerals are available for absorption. Overall, it is generally agreed that the bone powder is digestible and nonirritating and that no injury is possible from bone particles in mechanically deboned meat (Childers ef ctl., 1979). On the contrary, bone particles dissolve in the stomach acid in a very short time and provide an additional source of calcium. C.

MICROBIOLOGICAL PROPERTIES

From a theoretical standpoint a positive or a negative view can be taken regarding the microbiology of mechanically deboned meat. The positive view is that mechanically deboned meat is usually produced by removing residual meat, which, up until the time of hand boning, did not have an exposed surface where microbial contamination could occur. Since the interior portions of muscle are usually sterile or contain extremely low numbers of microbes, the residual meat

MECHANICALLY DEBONED RED MEAT

63

on the bones, which becomes mechanically deboned meat, contains low microbial numbers. Another theoretical plus for mechanically deboned meat is a reduced potential for secondary contamination of the meat in the form of foodpoisoning microorganisms transmitted by humans, because the bones with residual meat attached would require shorter processing times with less human handling. Shorter processing times and less human handling result from the fact that it is not necessary for boners to remove as much of the meat as is economically feasible by hand when machines are available. On the negative side, one can hypothesize that the fine particle size of mechanically deboned meat accompanied by increased particle surface area, compared with that found in hand-boned meat, would be conducive to microbial growth. Improperly cleaned and maintained equipment and a slight to moderate temperature increase in mechanically deboned meat over that in hand-boned meat also create the potential for elevated microbial counts. In addition, pH values of mechanically deboned meat are higher than those of hand-boned meat because of incorporation of marrow into the meat. Ostovar et a/. (1 97 1) has pointed out that the microbiological quality of deboned meat can be a problem if the bones or carcass parts are not kept cold and deboned immediately upon removal from the carcass. Documentation of lax standards of hygiene during the commercial production of mechanically deboned pork in the Netherlands has been made by Bijker et a/. (1980). Improvements that would reduce bacterial numbers were recommended for the work rooms and for the collection and cooling of fresh products. When strict limitations on time, temperature, and storage conditions are followed, the microbiological quality of mechanically deboned meat compares very favorably with that of hand-boned meat (Goldstrand, 1975; Field et al., 1974b; Field and Riley, 1974; Meiburg et af., 1976). Neuhauser ( 1977) concluded that bacterial counts are not changed during mechanical deboning but that hand boning results in a significant increase in total aerobic bacteria and enterobacteriaceae. Schothorst et al. (1979) conducted studies at four meat-processing plants on the bacteriological quality of mechanically deboned meat from pork bones from three types of deboners. They studied total aerobic counts, gram-negative bacilli, enterobacteria, lactic acid bacteria, yeasts, molds, and staphylococci and concluded that the microbiological quality of mechanically deboned meat was similar to that of hand-boned meat. Little increase in microbial counts occurred during a 24-hr storage period at 4"C, but counts increased by a factor of 2 lo" during storage for 24 hr at 20°C. In the same study the authors compared the microbiological quality of mechanically deboned meat with that of meat comminuted in a bowl chopper. The results showed the microbiological quality of the mechanically deboned meat to be superior to that of meat comminuted in a bowl chopper. Similar conclusions were reached by Linke et al. (1 974), who compared the microbial condition of meat

64

RAY A . FIELD

obtained by mechanical and manual methods. The bacterial counts of mechanically recovered meat did not exceed those from conventionally recovered meat, and the number of gram-negative organisms was markedly lower than that found in manually recovered meat when salt and nitrite were added. Clostridia spores observed in a number of the mechanically deboned samples did not appear capable of multiplying at + 15°C. Linke et al. ( 1974) concluded that, because of the small number of spores (100 per gram), this contamination was of no practical significance. A most significant finding was the observation that gramnegative organisms did not increase at + 10°C in the samples of mechanically deboned meat, whereas these organisms showed a distinct increase in the manually produced meat samples. The present interest in hot boning as a method of saving energy makes the study of Field et al. (1974b) significant. They compared microbial counts in mechanically deboned meat from chilled mutton carcasses with those from hot mutton carcasses right off the kill floor. Bacterial counts per gram of meat immediately after boning were much lower in hot-boned than in cold-boned carcasses. This result would be expected, since bacterial growth on the hot carcasses did not have time to occur. The study also supports the work of others, who have shown that differences in the microbial content of hand-boned and mechanically deboned meat from chilled carcasses are not significant. Nevertheless, a temperature rise of 1 - 10°C does occur during grinding, with the greatest rise occurring in bones that contain the least amount of meat (Field et al., 1976). In some deboners there is an additional 10-15°C temperature rise of the meat during deboning, making rapid chilling of the meat after deboning a necessity. In practice, those meat-processing plants that have the best success in keeping bacterial numbers low in mechanically deboned meat blast-freeze the meat in layers 5-7 cm thick in boxes or trays, as it is removed from the deboner, or the meat is incorporated into cooked processed meat items as soon as it is obtained. Various types of cooling equipment in addition to blast freezers are also successfully operated in meat-processing plants with deboners. Joseph et al. (1978) studied total plate counts and psychrotrophic counts in cooked salami possessing 0, 10, 20, and 30% levels of mechanically deboned beef after 0, 14, 28, 42, and 56 days of storage at 6°C. Psychrotrophic bacterial counts showed a sizable increase after 14 and 28 days of storage in all treatments. Counts in salami slices containing 30% mechanically deboned beef were lower than those found for other treatments after 14, 28, and 42 days of storage. These studies on the microbiology of mechanically deboned meat make it clear that, when good manufacturing practices and strict quality control programs are employed, no microbiological problems exist. This finding supports a similar conclusion reached by Kolbye and Nelson (1977a), and it helps explain why, with the exception of France, no country has established microbial guidelines for mechanically deboned meat (Gola et al., 1977).

MECHANICALLY DEBONED RED MEAT

65

D. NUCLEIC ACIDS Kolbye and Nelson (1977~)in their study of the health and safety aspects of mechanically deboned meat did not include nucleic acid or purine content. Nevertheless, when standards for tl-e production of mechanically deboned meat were published, the USDA (U.S. Department of Agriculture, 1978b) expressed concern that, if the bone marrow content of mechanically deboned meat were significant, the nucleic acid content might be higher than that found in handboned meat, and that this increase could have serious health implications for gout sufferers. The nucleic acid content of any tissue varies with age, sex, species, and nutrition of the animal (Davidson, 1947). In addition, discrepancies between different methods of estimating RNA and DNA are very large (Hutchison and Munro, 1961; Schneider, 1946). When a factor of 9.8 is used to convert DNA phosphorus values to milligrams of DNA per gram of tissue, and 9.4 is used to convert RNA phosphorus values to milligrams of RNA per gram of tissue (Schmidt and Thanhauser, 1945), marrow from round bones of adult rats has 9.26 mg RNA and 15.61 mg DNA per gram of fresh marrow (Lutwak-Mann, 1951). Other values are 13.40 mg RNA and 13.27 mg DNA per gram for rat femur and tibia marrow (Rambach et al., 1952). Arasu (1980) reported a mean of 24.4 mg DNA per gram of fresh cervical marrow from steers, heifers, and cows, and a mean of 16.1 mg DNA per gram of fresh marrow from the lumbar region of the same animals. Differences in DNA content between cervical and lumbar regions were inversely related to the amount of fat in the marrow, which helps explain why DNA values for marrow from the vertebrae are higher than those from the round bones. Values of 2.54 mg RNA per gram for marrow from the cervical region and 1.35 mg of RNA per gram for marrow from the lumbar region were much lower than the values for DNA cited previously. The data of Arasu (1980) support the conclusion of Seitz (1969) that the DNA content of bone marrow is high, whereas the RNA content of bone marrow is less than that in many other tissues. Munro and Gray (1969) found 0.47 mg RNA and 0.30 mg DNA per gram of fresh muscle from bullocks; Ezekewe and Martin (1975) reported 0.38 mg DNA and 0.72 mg RNA per gram in pig muscle. The gastrocnemius muscle of sheep decreased in nucleic acid content with increasing age (Johns and Bergen, 1976). At a 35-kg body weight, 4.5 mg RNA and 1.5 mg DNA per gram of fresh muscle was present, while muscle from 45-kg sheep contained 3.5 mg RNA and 1.2 mg DNA per gram. Longissimus muscle of cattle also changes in DNA content with changes in weight, from 0.49 mg DNA per gram at 410-kg live weight to 0.58 mg DNA per gram at 230 kg (Guenther et al., 1979). The average DNA value of 0.35 mg/gm and the average RNA value of 0.36 mg/gm of fresh muscle as reported by Trenkle et al. (1978) does not change with breed type. According to

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LaFlamme et al. (1973). neither castration nor breed type has any effect on DNA concentration in muscle. Lipsey et al. (1978) reported values of 0.27-0.39 mg DNA and 2.28-2.92 mg RNA per gram in fresh muscle from 555-kg cattle. Arasu (1980) found 1.4 mg DNA and 0.37 mg RNA per gram in longissimus muscle from cattle. The only data available for nucleic acid content of mechanically deboned beef are those obtained by Arasu (1980) on 20 different lots of mechanically deboned meat, which averaged 2.95% ash. Approximately one-half of these samples exceeded the 0.75% calcium limit established by the USDA (U.S. Department of Agriculture, 1978b). An average of 7.6 mg DNA and 0.73 mg RNA per gram of fresh mechanically deboned meat that contained 24.6% fat indicates that, although RNA values for mechanically deboned beef are in the same range as those reported for muscle, DNA values are higher in mechanically deboned meat than they are in hand-boned meat. If an average value of 20.3 mg DNA per gram of vertebral marrow and an average value of 1.4 mg DNA per gram of muscle as reported by Arasu (1980) are used, one can calculate that 33% marrow and 67% muscle would be needed to make a mixture of the two that would contain 7.6 mg DNA per gram and be similar to the level of DNA in mechanically deboned meat. The ratio of muscle to marrow in mechanically deboned meat as calculated from DNA values is higher, but in the same general range as the estimated ratio for muscle to marrow in mechanically deboned meat using iron or pigment values (see Section VII1,B). Furthermore, Arasu (1980) recorded average pH values of 7.34 for marrow, 5.8 for muscle, and 6.2 for mechanically deboned meat. A linear relationship between pH and marrow content in muscle : marrow mixtures was evident. Therefore, the pH values indicate that the mechanically deboned meat evaluated by Arasu (1980) was approximately 31% marrow. It is believed that 31% marrow in mechan cally deboned meat is higher than that normally found in commercial samples of mechanically deboned meat from auger-type machines in the United States (Field et al., 1979c). One way to judge the significance to health of an increase in dietary DNA due to marrow content in mechanically deboned meat is to relate this increase to nucleic acid values that have been agreed upon as safe. Waslien et al. (1968), the Protein Advisory Group (1970), and Scrimshaw (1975) have suggested that 2 gm nucleic acid per day added to the usual mixed diet is the upper safe limit for healthy young adults. According to the American Meat Institute (1979), consumption of processed meat items that could contain mechanically deboned meat averages approximately 28.6 gm per person per day. Since the meat block is approximately 85% of processed products, actual consumption is 24.3 gm per person per day. If the maximum limit of 20% mechanically deboned meat containing 7.6 mg DNA (Arasu, 1980) were used in all processed meat products, 75 mg nucleic

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acids could be consumed daily, in contrast to the 43 mg nucleic acids per person that is being consumed in processed meat that does not include mechanically deboned meat. Therefore, 32 mg more of nucleic acids would be consumed if all processed meat contained the maximum level of mechanically deboned meat. Thirty-two mg is only 1.6% of the 2 gm/day additional intake that has been suggested as the safe upper limit. Clifford et ul. (1976) investigated the metabolism of individual purines and found that adenine, and to a lesser extent hypoxanthine, had pronounced effects on blood uric acid levels. The purine content of foods, in particular adenine, would therefore be of immense nutritional significance. Davidson (1976) states that the molar proportions of bases in DNA from different tissues in a particular species are essentially the same, and that there are equimolar proportions of purines and pyrimidines in DNA. In terms of the molar proportions of bases in bovine thymus, purines equaled 56.1% of DNA (Arasu, 1980). The purine content of mechanically deboned meat was calculated at 56.1 % X 7.6 mg DNA per gram, or 4.2 mg/gm, while Mattice (1950) reported 0 . 6 mg of purines per gram in beef muscle. Therefore, purine intake per person per day from 24.3 gm of processed meat would approximate (20% X 4.2 mg purinedgm MDM X 24.3 gm) (80% x 0.6 mg purinedgm muscle x 24.3 gm), or 32.1 mg of purines if the processed meat contained the maximum level of mechanically deboned meat allowed. Krause and Hunscher (1972) reported that the average daily intake of purines in the normal diet ranges from 600 to 1000 mg; the purines derived from processed meat products containing mechanically deboned meat would not substantially increase this level. Furthermore, processed products with mechanically deboned meat [(20%MDM of 85% meat block X 4.2 mg purines/gm) (80% muscle of 85% meat block X 0.6 mg purines/gm)] contain 1.1 mg of purines per gram and would be categorized with hand-boned meat as a moderate-level purine food. According to Howard and Herbold (1978), moderate-level purine foods contain 0.5-1.5 mg of purines per gram. Murphy et a / . (1979) studied the purine content of mechanically deboned poultry and found that the total purine content of hand-boned poultry does not differ from that of mechanically deboned poultry. They concluded that mechanically deboned poultry does not pose any increased health hazard in regard to its purine content. Their judgment was based in part on the data of Clifford ef a / . (1976), who showed that foods high in nucleic acids or total purines were not necessarily high in the purines adenine and hypoxanthine, which are especially conducive to elevation of serum uric acid levels. Data of Murphy et al. (1979) showed that hypoxanthine was actually lower in mechanically deboned poultry than it was in hand-boned poultry, and adenine in mechanically deboned poultry did not differ from that in hand-boned poultry. Other USDA data (Young, 1980) show that, per gram of protein, mechanically deboned poultry contains some

+

+

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purines in amounts higher than those found in hand-boned products. Nevertheless, the values are not high enough to be of concern in the health of the consumer. Finally, monitoring the dietary intake of purines in the treatment of gout is being de-emphasized because uric acid is produced from endogenous and exogenous purines. Lamb (1973) reported that medicines 'are currently the mainstay in treating gout and elevated uric acid levels. However, Howard and Herbold (1978) still suggest that dietary purines be maintained at 100 mg/day or less during acute gout attacks, since diet does influence the total metabolic pool of uric acid. Processed meats containing mechanically deboned meat should present no problem in this regard. Meat is recognized as a food that contains moderate amounts of purine (50-150 mg per 100 gm), and processed meat containing the maximum of 20% mechanically deboned meat would still fall within this range. Tarladgis (1967) considers purines to be desirable curing agents because these heterocyclic molecules help retain a stable red color in processed meat, and they also retard lipid oxidation. Therefore, purines in marrow found in mechanically deboned meat could be beneficial from the standpoint of color and flavor.

VII.

FUNCTIONAL PROPERTIES OF MECHANICALLY DEBONED MEAT A.

COLOR

A brighter red color in mechanically deboned meat, compared with that found in hand-boned meat, is due to the addition of heme pigments from red bone marrow and to the elimination of connective tissue, which is devoid of pigments. According to Sanchez (1979), total heme pigment in marrow and hemoglobin in marrow are synonymous, since the only heme pigment identified in marrow is hemoglobin. Fresh marrow from the cervical vertebrae of veal, steers, and cows contained 30-36 mg of hemoglobin per gram, with the highest values present in veal marrow, whereas fresh muscle attached to the cervical vertebrae contained values of 1.5, 3.9, and 6.7 mg total pigment (myoglobin and hemoglobin) per gram in veal, steers, and cows, respectively. Lumbar marrow from veal and steers contained amounts of hemoglobin similar to those for cervical vertebrae, but the hemoglobin content of lumbar marrow from cows was considerably lower than those from veal and steers. Lower hemoglobin values obtained from lumbar marrow of cows, when compared with values for veal and steers, were due to reduced physiological activity of the cow marrow. Therefore, the color intensity of mechanically deboned meat from the lumbar region will be lower than that of

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mechanically deboned meat from the cervical region, provided both meat samples contain the same amount of marrow. Mechanically deboned meat from regions of the carcass where little red marrow is present possesses color traits similar to those found in hand-boned meat from the same region. In addition to the age of the animal and the anatomical location, the amount of lean left on the bones, the type of deboner, the fat content, the freshness of the bones, the temperature of meat at the time of deboning, and the conditions under which the mechanically deboned meat is chilled and stored all influence the color of the product. Pisula and Rejt (1979) recorded dominant wavelength, purity of color, lightness of color, and stability of color of mechanically deboned meat from pork backbones recovered with a press machine and compared the values with those for pork semimembranosus and mixtures of semimembranosus muscle and mechanically deboned meat in which 10% or 20% of the muscle protein was exchanged for mechanically deboned meat protein. The color measurements were recorded on fresh samples after heating the samples in cans to 70°C. Replacing 10%and particularly 20% muscle protein with mechanically deboned meat protein raised values for dominant wavelength and purity of color in fresh meat but had little influence on the same measurements in heated meat. Values for lightness of color and stability of color were lowered in fresh meat, and lightness of color values were lowered in cooked meat with the addition of 10 or 20% mechanically deboned meat. When Pisula and Rejt (1979) compared fresh semimembranosus muscle containing 26% fat with fresh mechanically deboned meat containing 25.3% fat, values were 581.4 nm and 592.8 nm for dominant wavelength, 27.9 and 29.8 for purity of color and 46.0% and 17.4% for lightness of color, respectively. Values for cooked semimembranosus muscle vs cooked mechanically deboned meat were, for dominant wavelength, 580.1 and 583.5 nm; for purity of color, 20.2 and 19.3; for lightness of color, 46.4 and 26.8%;and for stability of color, 1578 and 1426, respectively. Color intensity was closely associated with pH when different mixtures of muscle and mechanically deboned meat were compared. The relationship is logical, since marrow (pH 7.0-7.4) in mechanically deboned meat raises the pH and also increases the color intensity. Goldstrand (1975) stated that the color level of mechanically deboned pork and beef from fabrication room bones is 25-35% higher than that of pork and beef trimmings of similar protein and fat levels. Goldstrand’s subjective scores for increased color are supported by the work of Sanchez et al. (1978), who found 7-10 mg of total pigment per gram of mechanically deboned meat in contrast to 1.5-6.7 mg of total pigment per gram in hand-boned meat (Sanchez, 1979). The color of mechanically deboned meat is a bright red when fresh, properly chilled, vacuum-mixed products are evaluated, but it is a dull brownish red if pigment

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oxidation has occurred. Carbon dioxide pellets are often used to chill mechanically deboned meat rapidly, and this method of chilling may produce a slightly duller color than other methods (Uebersax et af., 1978). Data showing that increased levels of mechanically deboned meat increase the color intensity are not always consistent because of differences that exist in lots of mechanically deboned meat. Marshall et al. (1977) concluded that the color of frankfurters was not affected when 10, 25, or 40% mechanically deboned meat from whole goat or mutton carcasses was added. Hemoglobin from marrow in mechanically deboned meat from whole carcasses would make up a very small portion of the total pigment present, and when it was diluted further with handboned meat it would not be expected to have a significant effect on color. In contrast, Seideman et al. (1977) added 10, 20, or 30% mechanically deboned beef from beef bones with residual meat attached and found that higher levels of mechanically deboned meat produced ground beef with a brighter red color than was found in hand-boned beef. The brighter red color associated with most lots of mechanically deboned meat from bones with residual lean attached is usually considered a plus in ground meat, fresh sausage, and some processed meats. Nevertheless, sausage makers who prefer a pale or white sausage find the additional pigment in mechanically deboned meat troublesome. Since wide variability exists in the amount of red marrow present in mechanically deboned meat, sausage makers who purchase this ingredient would do well to determine the total pigment present in each lot, utilizing the rapid methods outlined by Franke (1973) and Warriss (1976).

B . EMULSION CHARACTERISTICS Emulsion stability and emulsifying capacity of mechanically deboned meat are affected by the same factors that affect emulsion stability and emulsifying capacity of hand-boned meat. In addition, the amount and type of marrow present in mechanically deboned meat can influence emulsion characteristics because red marrow has a higher pH than muscle and is higher in minerals such as iron, copper, and maganese, whereas fatty marrow is low in minerals but higher in unsaturated fats than is muscle. Pisula and Rejt (1979) studied characteristics of mechanically deboned meat from pork backbone produced by a press-type deboner. Mechanically deboned meat had a higher emulsifying capacity of the oil phase than did hand-boned pork semimembranosus muscle, but the emulsifying capacity was similar when 10 or 20% of the muscle protein was exchanged for mechanically deboned meat protein. In studies involving blends of semimembranosus and mechanically deboned meat, an increase in viscosity was observed with an increase in mechanically deboned meat in the formula. A marked increase in viscosity was noted in blends containing 20% mechanically deboned meat. Both sodium caseinate and soya isolate lowered the viscosity of blends, but

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the addition of 20% mechanically deboned meat in the case of the blends with sodium caseinate allowed the value for emulsifying capacity to equal that of muscle. Correlations were found between viscosity and pH of 0.75, and between viscosity and water-holding capacity of 0.91. Goldstrand (1975) indicated that the bind value of mechanically deboned meat from fabrication room bones compared favorably with that of beef and pork trimmings of similar protein levels. In his experience, no compensation for reduced bind was necessary in formulations of processed meat products at 10, 20, or 30% levels of addition. Field et al. (1974b) attributed an increase in emulsion stability of mechanically deboned mutton, when compared with that of hand-boned mutton, to lower connective tissue content and a higher pH of the machine-boned product. Other studies showing that emulsifying capacity and/or emulsion stability of mechanically deboned meat is at least equal to that of hand-boned meat include those of Anderson and Gillett (1974) with mutton, Vadehra and Baker (1970) with poultry, and Seideman et al. (1977) and Goltry (1976) with beef. According to Marshall et a1. (1977), frankfurters containing 10, 25, or 40% mechanically deboned pork, goat, or mutton did not differ from controls in degree of fattening-out, ease of peeling, or smokehouse shrinkage. They also found that the addition of 20% mechanically deboned beef increased texture desirability in beef patties when it was compared with that of hand-boned beef. Several studies have recorded the cooking loss of products containing mechanically deboned meat. Pisula and Rejt (1979) reported cooking losses of 17.8, 1 1.2, and 9.9% for meat blends containing 0, 10, or 20%mechanically deboned meat, respectively. Field et al. (1974b) found slightly lower cooking losses during heating when bologna made with mechanically deboned meat was compared with that made with hand-boned meat. The addition of mechanically deboned meat to restructured beef steaks did not change weight loss during cooking (Field et ul., 1977b). Cross et a1. ( 1977) reported that levels of mechanically deboned meat and cooking losses were not consistently associated. It is apparent from the above literature that emulsion stability, emulsifying capacity, and cooking losses of products containing mechanically deboned meat are not a problem, and under some conditions mechanically deboned meat may even improve these characteristics. Variability in emulsion stability, emulsifying capacity, and cooking loss would be expected to occur with variability in type of protein, amount of protein, denaturation, composition, chilling conditions, and storage. The amount of red marrow would also be expected to be a significant factor, since marrow is high in albumin and hemoglobin but devoid of myosin (Field et af., 1978), which has superior binding strength to other muscle proteins (Ford et al., 1978). Marrow, in spite of its theoretical decrease in binding strength, is high in some of the same proteins that are found in blood, and the pH of marrow and blood are similar. Dr. Robert Dudley of Geo. A. Hormel and Co. has described blood

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protein as the highest quality and most functional protein known to man, and one of the best emulsifiers (Anonymous, 1976). It is obvious that marrow, which is found in variable amounts in mechanically deboned meat, has some assets and liabilities in regard to emulsion characteristics. These characteristics may change with length of time in frozen storage, Meiburg et al. (1976) found a slight yet consistent decrease in the emulsifying capacity of mechanically deboned meat stored at -23°C and tested every 2 wk during an 18-wk period. Warmer freezing temperatures than the -23°C used by Meiburg et al. (1976) could have a greater influence on emulsifying capacity with increased storage time. C.

WATER-HOLDING CAPACITY AND pH

Water-holding capacity plays an important role in the formulation, processing, storage, cooking, and freezing of meat because it is related to weight loss and quality of the finished product. According to Hamm (1960), changes in waterholding capacity are influenced by the pH of the meat. He pointed out that, at certain pH values or in the presence of certain ions, muscle can take up in “immobilized” form 700-800 gm water per 100 gm of protein. At pH 5 the water-holding capacity of meat is at the minimum, corresponding approximately to the isoelectric point of actomyosin. When bases are added to muscle, the water-holding capacity of meat increases until a pH of 9 is reached and then continues to increase, but at a decreasing rate from pH 9-10 (Hamm, 1960). Nevertheless, within normal pH ranges for aged beef (5.4-5.8), pH and waterholding capacity are not correlated. Only at pH values >5.8 does water-holding capacity in aged beef increase significantly with increasing pH. In pork, factors that change the pH of meat within the normal 5.4-5.8 pH range also affect water-holding capacity, and this fact led Hamm (1960) to conclude that changes in pH affect the water-holding capacity of pork more than that of beef. It is well established that the pH of mechanically deboned meat is higher than that of hand-boned meat (Anderson and Gillett, 1974). Pisula and Rejt (1979) gave a pH value of 5.9 for pork semimembranosus muscle and 6.3 for mechanically deboned meat from pork backbone recovered by a press machine; Goltry (1976) reported pH values of 5.9 for hand-boned beef and 6.1 for mechanically deboned beef from an auger machine. Arasu (1980) reported that 20 lots of mechanically deboned beef had an average pH of 6.2, compared with a pH of 5.8 for hand-boned beef. Field (1976b) observed that pH values of 6.0-7.0 for mechanically deboned meat from fabrication room bones are common. Increases in the pH of mechanically deboned meat when compared with hand-boned meat are a result of the incorporation of red marrow, which has a pH range of 6.8-7.4. Arasu (1980) has shown a linear increase in pH with an increase in the marrow-to-muscle ratio up to mixtures that are 50% red marrow and 50% muscle. In beef muscle an initial pH value of 5.62 changed to 6.20

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when 50% beef muscle and 50% cervical vertebrae marrow were mixed together, and in muscle mixed 50:50 with marrow from the cervical vertebrae the initial pH of 5.90 became 6.48. Unpublished work from the University of Wyoming shows similar trends for lamb muscle when mixed with lamb cervical vertebrae. As would be expected, the increase in pH with increasing cervical marrow concentrations in the muscle-marrow mixtures was accompanied by a linear increase in water-holding capacity. Another factor, in addition to marrow, that could influence the pH of mechanically deboned meat is the basic calcium phosphate in bone powder found in mechanically deboned meat. Nevertheless, Field et al. (1977b) has shown that bone powder in mechanically deboned meat is relatively stable and that neither pH nor bone particle size in mechanically deboned meat changes with storage. Data on the water-holding capacity of mechanically deboned meat are minimal. Pisula and Rejt (1979) noted that the water-holding capacity of added water in blends of muscle and mechanically deboned meat improved as the amount of mechanically deboned meat in the blends increased. This phenomenon was observed although mechanically deboned meat itself showed a fairly low waterholding capacity. Kosiba and Jawarek (1979) found that mechanically deboned cooked meat had a very limited water-holding capacity when compared with mechanically deboned raw meat because of denatured proteins. However, mechanically deboned cooked meat reduced cooking losses when used in sausage production. The presence of calcium and magnesium from bone powder could also influence water-holding capacity if the ions were in solution. Swift and Berman (1959), using eight different muscles of the same beef animal, found a highly significant negative correlation between water-holding capacity and content of calcium, magnesium, and potassium. Gola ef al. (1977) also indicated that total iron, copper, and maganese contents influence the capacity of meat to bind with and retain water. Since iron and copper are found in higher concentrations in marrow than in muscle, increased amounts could detract from the water-holding capacity of mechanically deboned meat that contains marrow and could partially offset the beneficial effects of the increased pH attributed to marrow earlier in this section. Some published industry studies show that unfrozen mechanically deboned meat is superior to frozen mechanically deboned meat in water-holding capacity. The influence of freezing on water-holding capacity is related to the freezing rate and the pH of the meat. It is known that slow freezing results in more drip and a lower water-holding capacity than does quick freezing and that meat frozen in a state of high hydration with small amounts of lactic acid present has less drip loss than does meat in a low state of hydration (Hamm, 1960). The time of aging and storage before freezing, the temperature of freezing and storage, the duration and conditions of frozen storage, the rate and temperature of thawing, the fat and

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collagen content, and the addition of salt and nitrite all influence the waterholding capacity of meat. The addition of water, salt, and nitrite not only extracts myosin and improves water-holding capacity but also reduces bacterial growth. Overall, the water-holding capacity of mechanically deboned meat is comparable to that of hand-boned meat when the meat is produced and processed under optimum conditions. Practices that retain or improve the water-holding capacity of hand-boned meat are also effective for mechanically deboned meat.

VIII.

NUTRITIONAL VALUE OF MECHANICALLY DEBONED MEAT A.

PROTEIN QUALITY

Most of the literature on the protein quality of mechanically deboned meat comes from the United States and relates to USDA regulations (U.S. Department of Agriculture, 1978b) or to debate concerning mechanically deboned meat prior to the regulations that gave approval for its production. Questions regarding protein quality were based on the fact that beef, pork, and lamb bones have large amounts of connective tissue attached as they come from the fabrication room and on the fact that bone itself contains 20-30% collagen (Brown et a / . , 1972). Since collagen is high in proline, glycine, and hydroxyproline and low in many essential amino acids such as the sulfur-containing amino acids (Osborne rt a / . , 1971; Eastoe et al., 1973), the protein quality of mechanically deboned meat was hypothesized by some to be low. Hydroxyproline content, an indicator of the amount of connective tissue in hand-boned or mechanically deboned meat, has been reported by several investigators. Kolbye and Nelson (1977b) found an average of 0.45% hydroxyproline in 30 mechanically deboned meat samples. When the average hydroxyproline level of 0.45% is multiplied by a factor of 7.25 (Eastoe and Leach, 1958) to convert hydroxyproline content to collagen content, the figure becomes 3.26% collagen in mechanically deboned meat. The figure of 0.45% hydroxyproline is comparable to hydroxyproline figures for hand-boned meat of 0.57% for beef shank, 0.36% for beef chuck, 0.37% for beef plate, and 0.25% for pork shoulder (Gillett et al., 1976). Cross et al. (1978b) found 3.35% collagen in minor cuts from Choice beef and 2.99% in triangles from Utility beef. Chang and Field (1977) hand-trimmed all lean with its accompanying fat and tendon from beef bones from the vertebral column. The bones were from the same anatomical locations and contained the same amount of meat as those normally used in mechanical deboning operations. Collagen content in the lean averaged 16.49%, far exceeding the figure of 3.26% that Kolbye and Nelson (1977b) found in 30 lots of mechanically deboned meat. It is clear that much connective tissue, in addition to bone, is removed during mechanical deboning.

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This observation is supported by the work of Field and Riley (1974), who found higher levels of hydroxyproline in hand-boned lamb breasts than in machineboned breasts, and that of Satterlee et d . (1971), who found that the majority of poultry skin protein was passed out in the residue along with the bone. Chang and Field (1977) found some lots of mechanically deboned meat with collagen content ranging from 6 to 15%, but these lots contained 1.7-6.3% calcium, far exceeding the USDA maximum for calcium of 0.75% (U.S. Department of Agriculture, 1978b). Some of the lots were from bones that had all visible lean removed prior to mechanical deboning and hence were not comparable in any way to mechanically deboned meat, which is produced commercially. Decreases in connective tissue during deboning are noted because tendons, facia, cartliage, and ligamentum nuchae associated with bone are soft and pliable so that the tissue does not fragment during deboning, yet it is too large and tough to be forced through screens with deboned meat and must therefore be eliminated with the bone. One way to evaluate the quality of protein in mechanically deboned meat is to express the eight essential amino acids (isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine) as a percentage of the total amino acids present. When hand-boned beef, pork, and lamb are expressed on this basis, 35-40% of the total amino acids are composed of the eight essential amino acids (Rice, 1971; Happich et ul., 1975; Hendricks et al., 1977). The figure is lower if the meat analyzed is high in connective tissue. According to Field (1976b), the data show the following essential amino acid percentages for mechanically deboned meat from bones : beef plates 24-39%, lamb necks 38-39.6%, pork backs 35-39%, beef necks 32-39%, beef ribs 3436%, veal legs 37.6%, and pork necks 36-40%. The data are supported by Kolbye and Nelson (1977b). who found that the eight essential amino acids made up 36. I% (range 34.6-39.4%) of the total in mechanically deboned meat, and by Goldstrand (1975), who indicated that 34-42% of the total protein in mechanically deboned meat is made up of the eight essential amino acids. Bones with more meat attached generally produced mechanically deboned meat with higher essential amino acid percentages and less calcium, but the fat content of mechanically deboned meat does not influence the ratio of essential amino acids to total amino acids. Mechanically deboned meat that is high in calcium is lower in essential amino acid percentages because higher calcium levels indicate that more bone powder is present, and bone collagen, which makes up most of the protein in compact bone, has only 15.7% essential amino acids (Eastoe, 1955). Mello et al. ( 1 975) studied the amino acid content of whole bone, including the marrow, and found that 28.4% of the total amino acids were the eight essential amino acids. In addition to bone powder and connective tissue, the amount of red marrow present in mechanically deboned meat could also change the ratio of essential

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amino acids to total amino acids. Field et al. (1978) have shown that the protein quality of marrow is relatively constant, regardless of age or percentage of fat or protein present in the marrow. The variation in the amino acid composition of marrow within an animal age group or between animals of different ages was similar to the variation in the amino acid composition of whole blood (Altman and Dittmer, 1971). Hemopoietic marrow did have higher concentrations of cystine and methionine when compared with whole blood (Altman and Dittmer, 1971) or when compared with the globin or plasma fractions of blood (Tybor et al., 1975). Marrow, like blood, is an excellent source of lysine and leucine and is characterized by a relatively high level of histidine, which is required by human infants (Clark, 1965). The amino acid composition of meat has been reported by Rice (1971) and Happich et af. (1975). When compared with meat, hemopoietic marrow is low in isoleucine; this results from the fact that hemoglobin, a major protein in marrow (Field, 1978b), is devoid of isoleucine (White et al., 1968). Chang and Field ( 1977) reported that mechanically deboned meat from bones having the most lean attached had higher contents of isoleucine than did mechanically deboned meat from bones where very little lean was attached. Chant er af. (1977) evaluated the amino acid content of tissue mechanically separated from bones where all the visible lean had been removed. This tissue, which contained high amounts of hemopoietic marrow, was similar to mechanically deboned meat in amino acid composition, except for isoleucine, which was deficient in mechanically separated tissue. Since the protein efficiency ratios of red meat and commercially produced mechanically deboned meat containing marrow are often similar (Field, 1976b), it is apparent that marrow when diluted with sufficient amounts of meat ceases to be a problem with regard to isoleucine as a limiting amino acid. Overall, some connective tissue is removed by mechanical deboners, and some bone powder and bone marrow are added to the meat. The net result is that the essential amino acid content of mechanically deboned meat is very similar to the essential amino acid content of hand-boned meat. This is not true of the bone residue discarded by mechanical deboners. Bone discard contains approximately 28% essential amino acids and has a protein efficiency ratio (PER) of less than 0.5 (Field, 1976b). The quality of protein in mechanically deboned meat has been evaluated according to the PER because the USDA (U.S. Department of Agriculture, 1978b) requires the product to have a minimum PER value of 2.5 or be labeled as imitation. Growth response, food intake, and PER for rats fed mechanically deboned meat at the 9% protein level are presented in Table X. Lamb neck bones produced mechanically deboned meat that was similar in PER to beef semimembranosus muscle. Lamb neck bones, because of their smaller size, have more surface area per unit weight of bone on which lean meat is attached after hand

MECHANICALLY DEBONED RED MEAT

77

TABLE X PROTEIN EFFICIENCY RATIOS FOR RATS FED MECHANICALLY DEBONED MEAT, HAND-BONED BEEF, AND CASEIN'

Protein sources Choice beef plates Lamb neck bones Cow rib bones Veal bones Beef semimembranosus Pork neck bones (reg) Pork neck bones (close trim) Casein (control)

Total PER food Total (gm gainedgm Initial Final Total consumed protein protein weight (gm) weight (gm) gain (gm) eaten)b (gm) eaten (gm) I .90'

56. I

110.6''

54.0'

61.9"

151.8"."

90.0"*' 344.8"*f~J 3 l.Od*'

2.90"

57.4'1." 56.4",'

99.8' 153.9'

42.4u 97.Y."

324.9"-' 404.4"

29.2' 36.4'

1.44' 2.67"

55.9""

147.0"."

91. I".'

347.4".'

3 1.3".'

2.91"

56.6".('

143.3"

85.7"

364.5"

32.8"

2.60"

58.1"

109.5"

51.4'

285.7'

25.7'

2.00'

54.6"

155.8"

101.2"

377.4r.d

34.0'."

2.9IL'

313.9'.'

28.4'J

~~

From Field er a / (1979~) Ten rats per group Rats were fed for 5 weeks " f J s Means within columns having a common superscnpt letter are not significantly different (P S 0 05) "

boning. The higher proportion of lean on lamb neck bones when compared with the other bones listed in Table X is undoubtedly related to the higher PER. Veal bones and untrimmed pork neck bones are next to lamb neck bones in terms of the amount of lean left on the bones, and they are also next in line in terms of PER. Although the PER for mechanically deboned meat from beef bones in Table X is low, beef bones often yield meat with a high PER of 2.3-3.0 (Kolbye and Nelson, 1977b). A high PER in mechanically deboned meat is usually obtained by keeping the calcium content below the maximum limit of 0.75% (U.S. Department of Agriculture, 1978b) or by leaving greater amounts of lean on the bones prior to mechanical deboning. The effect of the amount of lean on the bone in relation to PER values is illustrated by the PER of 2.60 in mechanically deboned meat from regular pork neck bones compared with the PER of 2.00 in mechanically deboned meat from closely trimmed pork neck bones. Bone residue and connective tissue discarded by the mechanical deboner when meat was separated failed to promote growth in rats. The PER values

78

R A Y A. FIELD

of S 0 for bone residue are expected, since bone residue is high in collagen, which lacks the essential amino acid tryptophan in addition to being low in most other essential amino acids. When collagen is fed to rats as the only protein source, the PER is always S O . Nevertheless, one protein expert has commented that he is not concerned about the collagen content of mechanically deboned meat because collagen is supplementary to other proteins and increases the nutritional value overall (Kolbye and Nelson, 1977b). Equations for estimating PER values in mechanically deboned meat from amino acid content or from iron and calcium present have not been highly successful (Field et al., 1979~).However, lots of mechanically deboned meat with low PER values can be easily identified by determining hydroxyproline content. It must be pointed out that the PER as a measure of protein quality is questionable (Hegsted and Chang, 1965; Pellett, 1978), and alternative methods for determining protein quality in mechanically deboned meat should be considered. Perhaps the best approach would be to drop all PER and amino acid requirements, since the protein quality in products containing mechanically deboned meat is consistently high. The calcium limitations of 0.75% and use limitations of 20% of the meat block specified by the USDA (U.S. Department of Agriculture, 1978b) assured products high in protein quality. Another reason for dropping all PER and amino acid requirements for mechanically deboned meat in the United States is related to the large amounts of high-quality protein that are presently consumed, making a deficiency in protein quality in the diet quite unlikely. Animals presently supply 70 gm of protein daily per capita in the United States, and with the additional 32 gm of available plant protein the total i s 102 gm available per capita (Pimentel et al., 1980). The United Nations Food and Agriculture Organization ( 1973) has recommended a combined animal and plant protein intake of 41 gm/day per capita. Recent recommendations of the Food and Nutrition Board (1980b) have increased this amount to 56 gm of protein per day for a 70-kg man and 44 gm per day for a 55-kg woman. Clearly, consumption of enough high-quality protein in the United States is not a problem. The regulation on protein quality in mechanically deboned meat is not necessary from a health standpoint, and it is inflationary because of the added costs involved in determining amino acid content or PER values.

B. LIPID MAKEUP Much of the lipid in mechanically deboned meat is the same as that found in hand-boned meat, because fat as well as lean is forced through the small openings that separate lean from bone. Lipids found in bone make up an insignificant amount of the lipid in mechanically deboned meat, since the bone content of mechanically deboned meat is low at approximately 0.5-3 .O%, and the lipid

MECHANICALLY DEBONED RED MEAT

79

content of dry compact bone is 0.06-0.10% (Leach, 1958; Spector, 1956). Therefore, differences between the lipid content of hand-boned meat and that of mechanically deboned meat are limited to minor changes, which could result from forcing subcutaneous and intramuscular fat through small openings during mechanical deboning, and to changes resulting from the incorporation of bone marrow lipid into the meat during the deboning process. Lipid from bone marrow has a different spectrum composed of more polyunsaturated fatty acids, more phospholipids, and more cholesterol than does lipid from subcutaneous or intramuscular fat (Mello et al., 1976; Lund et a / . , 1962; Meng et al., 1969; Moerck and Ball, 1973); it would be expected to make a significant contribution to differences between hand-boned and mechanically deboned meat. Studies by Field et a / . (1979b) using variation in total pigment to estimate marrow content show that the marrow content in mechanically deboned meat from an auger-type machine ranges from 16 to 30%. The figures for the percentage of marrow in mechanically deboned meat are in good agreement with other calculations. Kolbye and Nelson (1977b) reported that 16 samples from beef averaged 4.09 mg of iron per 100 gm of mechanically deboned meat. If 1.81 mg of iron per 100 gm of muscle from young steers (Watt and Merrill, 1963; Jenkins, 1977) and 12.48 mg of iron per 100 gm of red marrow (Blum and Zuber, 1975; Garcia, 1957; Seitz, 1969) are used, 79% muscle and 21% marrow would be needed to make a mixture of the two that would contain 4.09 mg of iron per 100 gm and be similar to mechanically deboned meat. The muscle and marrow percentages would need some slight adjustment to account for the small amount of bone present in mechanically deboned meat. Nevertheless, it is evident that the percentage of muscle and marrow in mechanically deboned meat, when based on the iron content of muscle and marrow, is in the same range as that found by Field et al. ( 1979b). Estimating marrow percentages in mechanically deboned meat from actin bands in acrylamide gels also shows marrow percentages of about the same magnitude (Field et al., 1978). Red marrow, which is found in bones most often mechanically deboned, varies considerably in fat content, and it increases as the animal ages. Cervical marrow contains 6.6, 16.2, and 36.5% fat and lumbar marrow contains 8.4, 46.4, and 47.8% fat in veal, steers, and cows, respectively (Field et al., 1980). The preceding figures on the percentage of marrow in mechanically deboned meat and the percentage of lipid in red marrow make it clear that marrow lipid could be a significant proportion of the lipid in mechanically deboned meat. Nevertheless, the amounts of marrow in mechanically deboned meat and of lipid in marrow are highly variable. Additional variability in marrow depending on the make of mechanical deboner would be expected. In general, the press machines can recover meat from bones with less residual meat attached than can the auger machines and still keep the calcium content low. As a result, press machines

80

RAY

A. FIELD

would be expected to have proportionally higher levels of marrow in the recovered product than would auger machines. In auger machines, greater amounts of residual lean would dilute the marrow. Perhaps the most extensive analyses of the lipids in mechanically deboned beef, pork, and lamb from an auger-type machine are those of Kunsman and Field (1976). The total lipid content of mechanically deboned lamb and pork was 28%, whereas that of the beef neck bone samples was 8.8% (Table XI). Differences in lipid content among the species reflected differences in age, grade, and anatomical location. The mechanically deboned meat from older, lower grading cows came from cervical vertebrae only, whereas thoracic vertebrae and ribs were included with neck bones in mechanically deboned pork and lamb. Both the lamb and pork lipids contained 95% nonpolar and only 3% phospholipids, whereas the nonpolar lipid contents of beef samples were reduced to 83% with a comparable increase in the phospholipid content. The higher phospholipid content in mechanically deboned beef, when compared with that in pork and lamb, can be partially explained on the basis of a higher percentage of hemopoietic marrow present, the latter being rich in cellular membrane phospholipids. Mechanically deboned beef contains more hemopoietic marrow because beef neck bones are larger than the pork and lamb bones. The larger beef bones have less surface area per unit weight and as a result have less meat and fat per unit weight than do smaller pork and lamb bones. The smaller amount of attached fat and meat on beef bones did not dilute the bone marrow in mechanically deboned beef as much as did the larger amounts of meat and fat found on pork and lamb bones. Combined cholesterol and cholesterol esters, as reported by Kunsman and Field (1976) in Table XI, are 2.9, 1.9, and 1.9% of the total lipid in mechanically deboned beef, lamb, and pork, respectively. Since the lipid content was 8.8, 28.4, and 28.0%, one can calculate that mechanically deboned beef, lamb, and pork contained 260, 540, and 530 mg cholesterol and cholesterol esters per 100 gm, respectively. The figures are considerably higher than those of Kolbye and Nelson (1977a), who reported a range of 28-27 mg of cholesterol per 100 gm of mechanically deboned beef and pork. They concluded that the cholesterol content of mechanically deboned meat was in the same range as cholesterol in hand-boned meat. Additional unpublished data from the University of Wyoming on cholesterol content in 20 lots of mechanically deboned beef gives an average value of 153 mg of cholesterol per 100 gm. Our data show that the amount of spinal cord and, to a lesser extent, the amount of marrow present in mechanically deboned meat can greatly alter the amount of cholesterol present. It is believed that the higher values of Kunsman and Field (1976) in Table XI are a result of weight analysis from thin-layer chromatography when they are compared with values obtained by gas-liquid chromatographic analyses of cholesterol, which were used to obtain the lower value. Moerck and Ball (1973) reported that

MECHANICALLY DEBONED RED MEAT

81

TABLE XI LIPID CONTENT AND COMPOSITION OF MECHANICALLY DEBONED MEATa

Lipid fraction Total lipid Nonpolar Triglyceride Free fatty acid Cholesterol Cholesterol ester Dig1yceride Monoglyceride Sugar lipid Unknown Phospholipid" Phosphatidylcholine Sphingomyelin Phosphatidylethanolamine Phosphatidylinositol Cardiolipid Phosphatidylserine

Percent of total lipidb Beef'

Lamb

Pork'

8.8 83.3 79.0 0.8 2.7 0.2 0.3 0.3 3.4 Tr 13.2 7.5 1.7 2.2 1.7

28.4 95.9 91.8 0.7 1.6 0.3 0.7 0.9 0.5 0.4 3.3 1.2 0.4 I .o 0.6 0.1 Tr

28.0 95.0 90.1 1.5 1.6 0.3

Tr Tr ~~

1 .o

0.5 0.6 0.7 3.6 1.8 0.4 1 .o

0.4 Tr

~~

From Kunsman and Field (1976). Percent of total lipid except for total lipid which is percent of wet weight. Average from two separate lots. 'I All phospholipid values represent duplicate determinations. In the case of beef and pork, the values represent duplicate determinations run on two separate lots. "

mechanically deboned poultry contained 700 mg of cholesterol per 100 gm when weight analysis from thin-layer chromatography was used, but Murphy et ul. (1979) used gas-liquid chromatography and reported the range in mechanically deboned poultry to be 97-140 mg of cholesterol per 100 gm. The lower values are similar to those of Newkirk et af. (1978), who reported cholesterol values of 86-100 mg per 100 gm in chicken frankfurters made with mechanically deboned meat. According to Murphy et ul. (1979), daily increases in cholesterol consumption from use of mechanically deboned poultry would be negligible on a per capita basis. The cholesterol content of mechanically deboned beef, pork, and lamb is limited, but persons in the United States consuming products containing mechanically deboned red meat in place of hand-boned meat would be expected to have smaller increases in dietary intake of cholesterol than those consuming poultry, since mechanically deboned meat is limited to 20% of the meat block. The nutritional significance of higher levels of cholesterol in some kinds of mechanically deboned meat can be determined once the controversy regarding

82

RAY

A . FIELD

dietary cholesterol has been resolved. The Food and Nutrition Board (l980a) has concluded that small increases in dietary cholesterol cannot be considered nutritionally hazardous. Mechanically deboned beef has a phospholipid spectrum similar to that of hamburger (Table XI). Keller and Kinsella (1973) reported a phospholipid content in hamburger of 0.3-0.5%, and Hornstein et al. (1967) found several beef muscles containing 0.6-0.9% phospholipid. Expressing the phospholipid data from mechanically deboned beef on a wet weight basis reveals a phospholipid content of 0.9%. Data on the phospholipid classes present in bovine muscle are limited. Keller and Kinsella (1973) found that 53-57% of the phospholipids present in various grades of hamburgers was phosphatidylcholine (PC) and 24% was phosphatidylethanolamine (PE), with lesser quantities of lysophosphatidylcholine (LPC), sphingomyelin (SP), phosphatidylinositol (PI), phosphatidylserine (PS), and cardiolipin (CAR). A similar distribution of phosolipids in mechanically deboned beef is shown in Table XI. Phospholidylcholine represented 56% of the total phospholipid present, and PE made up 17% of the phospholipids. Kuckmak and Dugan ( 1963) reported on the presence of phospholipids in four pork muscles. The major phospholipids were PC (58-63%) and PE (28-34%), with lesser quantities of PS and SP. Data for mechanically deboned pork are quite similar to these muscle values (PC 51%, PE 27%, and lesser quantities of SP and PS). Empirical data on the phospholipids of lamb muscle are lacking. This is unfortunate, since mechanically deboned lamb appears to have a much lower PC content than do the other two red meats, and thus it would be interesting to speculate on whether this is caused by the lean or bone marrow lipid. The differences do suggest that mechanically deboned lamb can be stored for longer periods than beef and pork without oxidation occurring. Table XI1 lists the polyunsaturated fatty acid content of the various fractions of mechanically deboned meat. As expected, the total lipid from each of the three species was low in polyunsaturated fatty acids (acids with two or more double bonds). The total lipid fatty acid spectrum resembled that reported for each species in animal fats (Price and Schweigert, 1960). The fatty acid spectrum of the total lipid was quite similar to both the nonpolar lipid and the triglyceride fractions. The free fatty acids were always the most polyunsaturated fractions of the nonpolar lipids. Keller and Kinsella ( 1973) reported the polyunsaturated fatty acid content of PC from hamburger to be 29.8%. This is in excess of the amount found in mechanically deboned beef, as is the polyunsaturated fatty acid content of the PE (54.3% for hamburger, 38.6% for mechanically deboned beef). However, the polyunsaturated fatty acid content of PE from hamburger is swelled by the enormous amount of arachidonic acid present (39.0%). As is shown in Table XIII, the arachidonic acid content of PE from mechanically deboned beef ex-

MECHANICALLY DEBONED RED MEAT

83

TABLE XI1 POLYUNSATURATEDFATTY ACIDCONTENTOFTHE LIPID FRACTIONS FROM MECHANICALLY DEBONED MEAT"

Percent of fatty acids as polyunsaturatesb Lipid fraction Total lipid Nonpolar Triglyceride Free fatty acid Cholesterol ester Diglyceride Monoglyceride Sugar lipid Phospholipid Phosphatidylcholine Sphingomyelin Phosphatidylethanolamine Phosphatidylinositol ~~

~

Beef'

Lamb

Pork"

6.I 3.8 4.2 13.2 6.7 3.6 3.4 30.8 26.7 19. I 6. I

5.9 5.3 3.4 15.6 2.2 4.4 8.2 22. I 24.7 27.0 18.2

11.1 10.4 10.0 27.8 15.0 17.9 9.3 37.1 34.1 27.1 23.4

38.6 19.2

16.1 17.0

41.4 19. I

~~

From Kunsman and Field (1976). Two or more double bonds. ' Average from two separate lots. "

"

ceeded that found in the other phospholipid fractions but did not approach the hamburger figure. Terrell e r a / . (1968) reported a polyunsaturated fatty acid level of 27.5% for beef longissimus muscle, whereas O'Keefe er a/. (1968) reported levels of 40.46%,43.88%,and 41.80%for the polyunsaturated fatty acid content of beef longissimus, semitendinosus, and triceps brachii muscles, respectively. Mechanically deboned pork contains the most polyunsaturated fatty acids of the three species. Values for phospholipids of mechanically deboned pork d o not differ greatly from those reported by Kuckmak and Dugan (1963) for four pork muscles (22.6-45.4%polyunsaturated). It is evident that mechanically deboned pork, like hand-boned pork, would turn rancid much faster during storage than would mechanically deboned beef or lamb. The mechanically deboned lamb was the most saturated of the three species; however, even the mechanically deboned lamb is seen to have a significant amount of polyunsaturated fatty acids when one observes the phospholipid fractions.

84

RAY A . HELD TABLE XI11

FA'lTY ACID CONTENT (AREA PERCENT) OF SEVERAL IMPORTANT LIPID FRACTIONS FROM MECHANICALLY DEBONED MEAT"

Beef" Chain length

TL"

C13:O C14:O 1.9 C15:O 0.8 CI5:Br C I6:O 20.2 C16:I 2.2 C17:Br c I8:O 24.8 18:l 43.7 C18:2 4.2 C18:3 1.1 C21:l c22:o 0.1 C23: 1 C24: 1 C204 0.8 C22:4 C20:5 C22:5 -

'I

SL

Lamb

PC

PE

TL

0.6 0.4 1.4 20.2 24.7 0.9 13.9 4.9 34.9 46.7 30.5 1 1 . 1 0.3 3.9 0.7 0.5

0.7 0.5 3.2 4.3 4.7 2.9 15.0 27.7 8.8 9.9 2.1

2.9

SL

-

24.1 2.4

PC

PE

0.9 22.2 36.1 -

-

-

-

18.1 46.5 4.5 1.4

-

19.6 8.4 36.2 27.1 22.1 20.9 2.8 0.4

-

-

-

2.9 0.2

-

0.5

-

0.6

11.5 3.0 2.4 3.2

TL

PC

PE

3.5 0.5 22.3 25.7 8.7 1.7

0.3 33.4 0.6

11.7 13.7 34.8 47.4 10.6 9.8 3.1 1.1

14.9 8.2 41.1 29.3 37.1 25.0 - 0.6

0.4 0.5 2.0 5.6 6.6 I .8 17.8 22.5 11.0 5.0

-

_

SL

-

-

Pork"

-

-

-

-

-

-

2.6 0.4 2.4

-

1.2

0.9

-

-

1.2

-

-

-

0.3

-

3.3 -

-

-

-

16.6 8.4 -

-

-

-

-

-

-

1 .o

From Kunsrnan and Field (1976)

' Average from two separate lots. ' TL mine.

=

total lipid; SL = sugar lipid; PC

=

phosphatidylcholine: PE = phosphatidylethanola-

Overall, mechanically deboned meat has more polyunsaturated fatty acids, more phospholipids, and more glycolipids than have been reported for handboned meat (Kunsman and Field, 1976; Kunsman et al., 1978). C. CALCIUM Factors that influence the calcium content of mechanically deboned meat have been discussed in Section V , and bone particle size from which calcium is derived has been discussed in Section IV. It has been shown that calcium content of mechanically deboned meat ranges from 0. I to 2.0% and that the USDA limits calcium in mechanically deboned meat to 0.75% and restricts the amount of mechanically deboned meat to 20% of the meat block in products where it is allowed.

MECHANICALLY DEBONED RED MEAT

85

The addition of calcium to the diet via the small amount of bone powder in mechanically deboned meat must be considered a nutritional plus, since retention of calcium from bone sources is high (Forbes et al., 1921; Mitchell et al., 1937) and since many diets are low in calcium (Walker, 1972; Lutwak, 1975). The additional calcium intake would be particularly beneficial for persons who have osteoporosis and for those who receive long-term treatment with medications that induce a loss of calcium (Kolbye and Nelson, 1977a). Data on calcium intakes in the United States show that men and women 35 years and older consume only two-thirds of the recommended dietary allowance (U.S.Department of Agriculture, 1972), and children and mothers primarily of low-income groups have calcium intakes below two-thirds of the recommended dietary allowance (Abrahams et al., 1977; Lee and Johnson, 1977). In view of the high levels of protein and phosphorus provided by most diets, the Food and Nutrition Board ( 1980b) has recommended 800 mg of calcium per day for adults and 1200 mg of calcium per day during gestation. Those individuals who consume calcium greatly in excess of the recommended dietary allowance do not absorb a large part of the calcium ingested, and as a result no detrimental effects for healthy individuals who consume excess amounts of calcium are known. Excessively high levels of calcium in the serum and urine, or calcification of soft tissues, are found in such conditions as idiopathic hypercalcemia of infancy, hypercalcinuria, hyperparathyroidism, and certain instances of renal stones. There is no adequate evidence that high calcium intakes per se are a primary causal factor (Food and Nutrition Board, 1980b). High intakes may contribute to the difficulty, as indicated by the fact that low calcium intakes are often important aspects of therapy (Hegsted, 1973). The suggestion that bone be used to supply calcium in human diets is not new. According to Drake et al. (1949), man in the past, in common with other carnivora, was dependent on animal and fish bones for a large portion of his calcium intake. Today many aboriginal people still depend on bone as their chief source of calcium. Bone also supplies many other minerals essential for normal nutrition (Posner, 1969). In the past, according to Drake et al. (1949), a large proportion of the canned meat products prepared in Canada contained 15% of cooked ground bone. In 1949, all flour sold in Newfoundland, where the consumption of milk was low, contained 0.5% bone meal, and for many years bone meal was an ingredient of Pablum (infant cereal). It was for this reason that Drake et al. (1949) undertook a study of the comparative availability of the calcium in bone and in milk. They concluded that the availability of calcium in bone meal appeared to be. in the same range as that in milk. They also reported that the retention by young rats of calcium from whole cooked ground bone was approximately 90% of the retention from whole dried milk. An earlier study by Tisdall and Drake (1938) showed that the percentage of the added calcium retained was essentially the same, regardless of whether the

86

R A Y A. FIELD

calcium added to the human diet was in the form of calcium carbonate, calcium chloride, dicalcium phosphate, calcium gluconate, calcium lactate, whole milk powder, casec, or Pablum (infant cereal with bone added). These findings are not surprising, in view of the fact that nonreversible hydrolysis of bone readily occurs in aqueous media at physiological pH values (Posner, 1969). Davidson and Passmore (1963) also confirmed that calcium phosphate (bone mineral) is in solution in the unneutralized hydrochloric acid of human gastric juice. The above authors and numerous others make it clear that bone in the human diet can be beneficial (Knight and Winterfeldt, 1977). Other benefits may result from increased levels of calcium in the diet. Fleischman et al. ( 1966) elucidated the hypocholesterolemic and hypotriglyceredemic action of dietary calcium in rats. They found that blood lipids decreased as dietary calcium increased. The major decrease occurred at the 0.2% calcium level, a level that is attainable in sausage products with 20% mechanically deboned meat containing I % calcium. Knox (1973) has correlated the regional frequency of various major diseases in Britain with the intakes of nutrients as measured by the British National Food Survey. He observed that ischemic heart disease was inversely related to intake of calcium and of vitamin C. Therefore, the observation of Fleischman et al. (1966) with rats may also apply to man. The calcium present in mechanically deboned meat would be of special benefit to those persons who have low calcium intakes because they have a lactase deficiency and cannot tolerate milk. Lactase is the enzyme in the intestinal tract that aids in the digestion of lactose (Newcomer, 1979). When lactose is poorly digested, gastrointestinal discomfort occurs. The incidence of this affliction varies. From 15 to 30% of adults from northern Europe or of European extraction exhibit lactase deficiency, whereas from 50 to 100% of adults from far eastern Asia, the Pacific, and Africa have .been reported to exhibit the deficiency (Johnson et al., 1974). Similarly, Eskimos, American Indians, and Negroes have a high incidence of lactase deficiency. Mechanically deboned meat as a source of calcium is not accompanied by lactase, and low-cost processed meats that could contain mechanically deboned meat are consumed by many of the above groups. One additional benefit of calcium in the diet has been discussed under lead toxicity. The protective effect of dietary calcium on lead toxicity has been known for many decades.

D. IRON According to the Food and Nutrition Board (1980b), there are four situations in which iron intake is frequently inadequate in the United States: (a) in infancy, because of the low iron content of milk and because the endowment of iron at birth is usually not sufficient to meet needs beyond six months; (6) during the periods of rapid growth in childhood and adolescence, because of the need to fill

MECHANICALLY DEBONED RED MEAT

87

expanding iron stores; (c) during the female reproductive period, because of menstrual iron losses; and ( d ) in pregnancy, because of the expanding blood volume of the mother, the demands of the fetus and placenta, and blood losses in childbirth. The Food and Nutrition Board (1980b) recommended 10 mg iron per day to provide for the necessary retention of 1 mg iron per day in adult males and postmenopausal females. The allowance for women of childbearing age was set at 18 mg iron per day. According to Eastoe (1961), bone contains less than 0.01% iron. Nevertheless, iron is higher in mechanically deboned meat than in hand-boned meat because of the presence of bone marrow. According to Field et al. (1980), cervical marrow ranges from 16.0 mg iron per 100 gm in veal to 23.0 mg iron per 100 gm in steers. Their values for marrow for the bovine lumbar region ranged from 10.3 to 13.5 mg iron per 100 gm of marrow and were similar to those reported by Garcia (1957) for rat bone marrow. Iron content in marrow varies with age, anatomical location, and species (Schultze et al., 1936; Kerr, 1957; Blum and Zuber, 1975). Iron content of hand-boned meat is much lower than iron content of marrow. Veal muscle from the cervical contains 1.19 mg iron per 100 gm, steers 1.98 mg iron per 100 gm, and cows 2.77 mg iron per 100 gm (Field et al., 1980). Muscle from the lumbar region contains slightly higher values for iron from all three age groups. The values for iron in beef are in general agreement with those of Sanchez et al. (1978), Monsen et al. (1978), and Jenkins, 1977). Because mechanically deboned meat contains marrow that is high in iron, approximately twice as much iron is present in commercial samples of mechanically deboned meat as is present in hand-boned meat (Field, 1976b). Kruggel and Field ( 1 977) found that commercial samples of mechanically deboned beef from different regions of the United States differed in iron content. Samples from the West, Midwest, and South contained 5.65, 6.30, and 4.26 mg iron per 100 gm, respectively. These differences in iron content could have been a result of different muscle-to-marrow ratios, which could be caused by variations in the amount of meat left on the bone, or they could reflect differences in the age of the animals slaughtered. The South traditionally slaughters younger animals when compared to other regions of the United States and the younger animal would be expected to produce mechanically deboned meat with less iron. Mechanically deboned beef contains twice as much iron as hand-boned (Kruggel and Field, 1977). Kolbye and Nelson (1977a) considered the iron in mechanically deboned meat to be nutritionally advantageous and concluded that bone powder would not interfere to any extent with iron absorption. Monsen et al. (1978) concluded that calcium and phosphate salts such as those found in bone can decrease iron absorption. On the other hand, ascorbic acid enhances iron absorption, and bone marrow is high in this vitamin. In rat bone marrow about 24 mg of ascorbic acid was found per 100 gm (Lutwak-Mann, 1952), and human bone

88

RAY A. FIELD

marrow cells contain 13- 15 mg of ascorbic acid per 100 cells (Cox et u l . , 1960). According to Kruggel and Field (1977), mechanically deboned beef produced commercially contains 2-3 mg of ascorbic acid per 100 gm. Ascorbic acid is easily oxidized, and more work on the ascorbic acid content of mechanically deboned meat is needed before its content in mechanically deboned meat and its influence on iron absorption in mechanically deboned meat are known. Monsen et al. (1978) believe that the amount of absorbable iron must be based upon the amounts of heme and nonheme iron in a meal because of their different availability and susceptibility to influence from other dietary ingredients. Although the proportion of heme iron in meat and marrow varies, it amounts to an average of 40% of the total iron. The remaining 60% of the iron in meat and all the iron in vegetables are nonheme iron, which is absorbed at lower rates than is heme iron. In animals, iron in excess of the needs for hemoglobin and myoglobin synthesis and other essential cell functions is predominantly stored in the form of two nonheme compounds, ferritin and hemosiderin, with high concentrations present in bone marrow (Underwood, 1971). Nevertheless, Blum and Zuber (1975) found that no hemosiderin could be observed in some bone marrows from younger animals. Nonheme iron, when found in substantial amounts in meat or marrow, may be more readily absorbed than nonheme iron from some other sources. Hazel1 et al. ( 1978) believe that the high availability of iron in meat may be due not to the heme proteins per se but rather to the nature of their degradation products formed by digestion within the meat environment. Their data show that much of the nonheme iron within the meat environment is also utilized. Variability in source of marrow in mechanically deboned meat and variation in bone powder could influence the amount of iron absorbed. Nevertheless, the increased content of hemoglobin from marrow in mechanically deboned meat over that in hand-boned meat, along with potential increases in ascorbic acid present, make it clear that mechanically deboned meat is an excellent source of readily absorbable iron, which is needed in many diets.

IX.

PALATABILITY OF MECHANICALLY DEBONED MEAT A.

OXIDATIVE STABILITY

Low flavor scores in mechanically deboned poultry that has been stored have been a result of oxidative rancidity (Dimick et al., 1972; Maxon and Marion, 1970; Froning et u l . , 1971). Lipid oxidation in mechanically deboned poultry is enhanced by the addition of unsaturated fatty acids from bone marrow (Moerck and Ball, 1974), increases in meat temperature during the deboning process (Field, 1974), and incorporation of air and heme pigments. Since mechanically deboned red meat is produced in the same manner as is mechanically deboned

MECHANICALLY DEBONED RED MEAT

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poultry, it also is susceptible to lipid oxidation. Nevertheless, the extent of lipid oxidation in red meat is much less than that in poultry, and differences between red meat species do exist. Meiburg et al. (1976) determined TBA values of mechanically deboned beef, pork, and mutton at 2-wk intervals throughout an 18-wk storage period. No changes in TBA values for mechanically deboned beef and mutton were observed, but large changes in mechanically deboned pork occurred during frozen storage. The large changes in pork were closely correlated with a rapid decrease in taste acceptability, while beef and mutton maintained uniformly high levels of taste acceptability throughout the 18-wk storage period. Only in cooked salami containing 10, 20, or 30% mechanically deboned beef have slightly high TBA values in test samples been reported (Joseph et al., 1978). Goldstrang (1975) observed that the TBA values for 54 mechanically deboned pork samples were somewhat higher than the values usually found in pork, and the pork TBA values were higher than the values for mechanically deboned beef. Unpublished work of the author at the University of Wyoming shows differences in oxidation between mechanically deboned pork and beef. In our study, peroxide values and monocarbonyl content of pork were intermediate between low values for beef and high values for turkey during frozen storage. The rate of lipid oxidation was closely associated with the total polyunsaturated fatty acid content of mechanically deboned beef, pork, and turkey. Increased levels of polyunsaturated fat were present in samples with the highest levels of lipid oxidation. The lack of oxidation of mechanically deboned beef during frozen storage or during processing has been documented by Kunsman et al. (1978) and by Cross et al. (1978a). Kunsman et al. (1978) measured oxidative changes in mechanically deboned beef and hand-boned ground beef by the disappearance of fatty acids from polar and nonpolar lipids and by the production of monocarbonyls during storage. Only minor differences existed between the slopes of the regression lines for oxidation of each polyunsaturated fatty acid in mechanically deboned meat vs ground beef, and no differences were noted in the monocarbonyl content of mechanically deboned beef when compared with ground beef. The molar ratios of polyunsaturated fatty acid to hemoprotein in all mechanically deboned beef samples were in the area of 90: 1, where hemoproteins act as antioxidants. Lee et a / . (1975) studied the effect of added hemoprotein concentrations on the oxygen uptake rate catalyzed by mechanically deboned chicken meat homogenates. They found that at high molar ratios of linoleate to hemoprotein of 500 : 1, oxygen uptake was maximum. This high oxidation rate was applicable also at ratios of 350 : 1. However, as the ratio decreased below these levels, the oxidation rate declined. At a ratio of 180 : 1, the oxygen uptake was only one-third of the maximum level. As the ratio approached 90: 1, the oxygen uptake fell to one-thirtieth of the maximum level. From these data the authors suggested that at high linoleate-to-hemoprotein ratios the hemoproteins act as

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pro-oxidants, but as the ratio falls below 350 they become increasingly antioxidant. In fact, at low ratios of 90: 1 the hemoproteins seem to exert strong antioxidant activity. The 90: 1 ratio reported by Lee et al. (1975) for maximum antioxidant activity is similar to the ratio for maximum antioxidant activity reported by Kendrick and Watts (1969). The drastic difference between the polyunsaturated fatty acid-heme pigment ratio of mechanically deboned chicken and that of mechanically deboned beef is partially due to the higher heme pigment content in beef. Mechanically deboned beef contains 0.61 pmol of heme pigments per gram, whereas chicken contains only 0.26 pmol/gm. Change in the ratio of unsaturated fatty acids to hemoprotein is undoubtedly responsible for the difference in storage characteristics between mechanically deboned poultry and mechanically deboned beef. Although mechanically deboned beef is less stable than hand-boned meat, the large decrease in flavor during storage as noted by Dimick ef al. (1972) for mechanically deboned poultry is not present in deboned beef. Cross et ul. (1978a) found no evidence that ground beef formulations containing mechanically deboned beef are any different in storage properties from hand-boned beef. Misock el ul. (1979) made bologna with hand-boned beef as a control and compared it with bologna in which 20% of the beef was replaced with mechanically deboned beef. The bologna was evaluated for palatability characteristics and monitored for decreases in the fatty acids of the lipid fractions and for production of monocarbonyls. The addition of mechanically deboned beef to bologna had no effect on fatty acid loss during storage, and the production of monocarbonyls in bologna containing mechanically deboned beef was similar to that in the control bologna. Flavor scores were similar in both bolognas, and both showed a slight decrease with 60 days of storage. Therefore, the findings of Misock et al. (1979) with processed meat support other studies showing that mechanically deboned beef does not have problems with oxidative rancidity when it is properly produced, chilled, and stored. Two studies (Mast er a / . , 1979; Uebersax et al., 1978) indicate that mechanically deboned poultry has higher TBA values when CO, cooling following deboning is practiced and that taste panelists score products made from C02-chilled poultry lower. Although no comparisons for CO,-chilled mechanically deboned beef vs other methods of chilling are available, it is unlikely that CO, chilling would increase TBA values in mechanically deboned beef. B.

OTHER SENSORY CHARACTERISTICS AND USE LEVELS

Flavor not associated with rancidity, texture, or juiciness in products containing mechanically deboned meat can vary depending upon the amount of red bone marrow in the mechanically deboned meat and upon the amount of mechanically deboned meat in the finished product. According to Souron (1975). the gustatory

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qualities of bone marrow are well known for purposes such as preparation of soup stock. Marrow in bones used for soup is removed when the bones are heated in liquid. The characteristics of marrow are such that palatability is improved if mechanically deboned meat that contains marrow is used in proper amounts, but the flavor is objectionable if too much marrow is incorporated (Council for Agricultural Science and Technology, 1980). Likewise, a more pleasing texture, which gives fresh sausage an extra bite, is often present when 5-10% mechanically deboned meat is added, but a grainy or gritty texture may be present in products with more than 30% mechanically deboned meat (Field, 1976a). According to Gola et u l . (1977), the proportion of marrow plus bone powder in processed products should be less than 10%. Field (1976a) observed that panels have not been able to detect differences in juiciness or flavor when 10% mechanically deboned meat is added to formulations. Chant et al. (1977) compared control bologna with bologna containing 30% marrow and bone powder from bones that were hand-cleaned of all lean prior to mechanical deboning. Triangle tests showed significant differences for grittiness and flavor. Of 28 correct answers for flavor, 15 preferred the control while 13 preferred the bologna containing marrow and bone powder. The finding that approximately half of the panel members preferred the flavor of bologna with marrow and bone powder was surprising in light of a statement by Carpenter (1975), who associated excess iron from marrow in mechanically deboned meat with an off-taste described as a “liver” taste. It is probable that experienced panel members would have preferred the control bologna because the flavor was more typical of traditional bologna. Perhaps larger consumer tests comparing control bologna and bologna containing mechanically deboned meat should be conducted to ascertain if the change in flavor is objectionable to consumers as a whole. Misock et a l . (1979) used an untrained panel to score the flavor of control bologna and bologna made with 20% mechanically deboned beef containing 1.5% calcium. The mean flavor scores for control bologna and bologna containing mechanically deboned beef were not significantly different. Relatively minor changes in flavor acceptability of beef frankfurters occurred with storage times up to 60 days. The findings support the work of Meiburg et al. (1976) with beef and mutton, but their study shows that flavor acceptability of frankfurters made with mechanically deboned pork decreased with time in storage. Marshall et al. ( 1977) have shown that frankfurters containing 40% mechanically deboned meat from whole carcasses of old goats, young goats, or mutton were significantly more desirable in flavor than were control frankfurters. The mechanically deboned meat was low in calcium (0.09-0.13%) and in marrow, owing to the dilution effect of meat from whole carcasses. They also evaluated the flavor of frankfurters containing 10, 25, and 40% mechanically deboned pork from neck bones, vertebrae, and ribs. Frankfurters containing 10 and 25% mechanically

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deboned pork (0.48% calcium) were rated higher for flavor than were the controls, but samples containing 40% mechanically deboned pork had lower flavor scores. Panel scores for juiciness, texture, and overall satisfaction followed those for flavor. The addition of 10% mechanically deboned goat, mutton, or pork to frankfurters equaled or improved juiciness, texture, and overall satisfaction, whereas frankfurters made with 40% mechanically deboned pork had undesirable scores for juiciness, texture, and overall satisfaction. The improved texture scores with the addition of 10% mechanically deboned meat probably resulted from the removal of connective tissue from meat during the deboning process, whereas lower scores for texture in products containing 40% mechanically deboned meat resulted from frankfurters that were mushy. Not one of the panelists detected grittiness in the frankfurters made with 40% mechanically deboned pork, even though the auger-type deboner had 0.79-mm holes through which the meat was forced (Marshall et al., 1977). The study makes it clear that mechanically deboned meat from whole carcasses is decidedly different from mechanically deboned meat from bones which have been removed by hand. It is clear that the amount of mechanically deboned meat that can be added to a product will depend on the amount of marrow and bone powder in the meat. Proper amounts of marrow can enhance palatability attributes, whereas excessive amounts detract from flavor, juiciness, texture, and overall satisfaction. Joseph et al. (1978) evaluated cooked salami with 0, 10, 20, and 30% levels of mechanically deboned beef at 0, 14, 28, 42, and 56 days of storage. A consumer panel, a descriptive attribute panel, and a flavor profile panel found less desirable flavor, juiciness, tenderness, and texture in salami containing 20 or 30% mechanically deboned beef, but salami containing 10% mechanically deboned beef seldom differed from the control. Addition of mechanically deboned beef to ground beef and to restructured steaks has shown results similar to those for frankfurters, bologna, and salami. Cross et al. (1977) characterized the palatability of cooked beef patties from seven formulations of ground beef patties containing 0-30% mechanically deboned beef. Mean panel ratings for overall acceptability were greater for patties containing 5 , 10, 15, and 20% mechanically deboned beef from necks, backs, ribs and pelvic girdles of U.S. Utility beef than for hand-boned beef from Utility triangles and Choice plates. Patties containing 25 and 30% mechanically deboned beef had lower flavor and acceptability ratings than the control did. As the percentages of added mechanically deboned beef increased, panel ratings for tenderness and juiciness increased. The amount of detectable connective tissue decreased significantly as the percentage of added mechanically deboned beef increased. Control and ground beef patties containing 10, 20, or 30% mechanically deboned beef were compared by Seideman et al. (1977). Panel scores for texture, flavor, and juiciness were similar for control patties made with 100%

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hand-boned beef and patties containing 10% mechanically deboned beef, but scores for patties containing 20 or 30% mechanically deboned beef from a commercial supplier were lower than those of the controls. Field ef al. (1977b) suggested that mechanically deboned meat from beef necks added at the 10% level can improve the quality of restructured beef steaks because cooked steaks containing 10%mechanically deboned beef have a softer, more acceptable outer surface than the controls, and their flavor, juiciness, and texture was similar to those of the controls. Steaks containing 5% mechanically deboned meat were similar to controls for all traits studied. Restructured steaks containing 15 or 20% mechanically deboned meat had a softer outer surface when broiled than the controls did, but there was a tendency for these steaks to have mushier interiors. It is evident from the above findings that mechanically deboned meat that has been produced according to good manufacturing practices is acceptable for use in ground and processed meat, and in some instances at the 10%to 20% levels can enhance palatability traits. Nevertheless, mechanically deboned meat that has been abused with regard to storage time and temperature, or mechanically deboned meat from some machines that has not been properly chilled can reduce the palatability of the product in which it is used. For example, O’Palka (1973) found that cysteine destruction in mechanically deboned poultry was closely correlated with methyl mercaptan production. This was unexpected, because Casey et al. (1965) and Grill er al. (1967) have shown methionine to be a precursor of methyl mercaptan . The formation of volatile sulfur-containing compounds in the mechanically deboned meat was undoubtedly related to bacterial growth, which would decrease the palatability of the mechanically deboned product.

X. ADDITIONAL RESEARCH NEEDS It has been shown that mechanically deboned meat is similar to hand-boned meat except for the small amounts of bone powder and the variable amounts of red bone marrow that are incorporated into the product during the mechanical deboning process. Much research has been done on the composition of bone (Doyle, 1979; Field er al., 1974b; Posner, 1969; Eastoe, 1961) and its use as a mineral supplement in human foods (Tisdall and Drake, 1938; Drake et al., 1949; Kolbye and Nelson, 1977a,b), and it is clear that the bone powder will not create a palatability or safety problem provided the particle size is kept small. Additional work involving the bone powder in mechanically deboned meat should center on the amount and variability in the trace elements that are found in bone and on the availability of trace elements when ingested. Fluoride is an example of one trace element in bone on which there is dis-

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agreement. Kruggel and Field (1977) and Knight and Winterfeldt (1977) feel that beneficial intakes of fluoride could result from the use of mechanically deboned meat in areas of the United States where the intake might be low or where water low in fluoride is not fluoridated. However, Kolbye and Nelson (1977a) recommended that mechanically deboned meat not be incorporated into baby and junior foods because infant intake of fluoride is known to be high. They emphasized that the fluoride content of mechanically deboned meat poses no health problem for adults but that fluoride in children needs to be controlled to avoid mottling of teeth, which, according to Labuza (1977), would be an unusual dietary occurrence. Kolbye and Nelson (1977a) emphasized that their recommendation not to include mechanically deboned meat was based upon lack of information rather than evidence of a hazard and should be subject to further evaluation as data are gathered. The Food and Nutrition Board (1980b) has recommended a total fluoride intake of 1.5-4 mg/day for adults and up to 2.5 mg/day for younger age groups in order to reduce the incidence of tooth decay. They have suggested that these levels of fluoride in the diet will be difficult to achieve in areas where the fluoride content of drinking water is low. Since bone powder in mechanically deboned meat has the potential of furnishing about the same amount of fluoride when it is used in processed meat as the Food and Nutrition Board (1980b) suggests be furnished in drinking water, and since many individuals do not have access to drinking water that contains fluoride, it is suggested that additional research on the benefits of fluoride in mechanically deboned meat is needed. Other questions on the content of trace minerals in bone powder relate to their influence on lipid oxidation of fat. In addition calcium, phosphorus, and magnesium in bone powder, as well as the trace minerals, could impart a flavor of their own to mechanically deboned meat. No work in this area has been accomplished. In contrast to bone powder, where much information regarding its composition and nutritional value is'available, very little is known about the composition and nutritional value of the marrow in mechanically deboned meat. Red marrow contains up to 50% lipid, but information on the lipid composition of bone marrow from animals that could furnish mechanically deboned meat is almost nonexistent. Information is needed concerning specific lipid patterns in marrow, which becomes part of mechanically deboned meat. Work on triglyceride types and cis-trans fatty acids and their relationship to palatability and health would be of particular interest, since data reviewed in the discussion of the lipids show that major differences do exist between the composition of bone marrow lipid and lipid in subcutaneous and intramuscular fat. Tompkin (1978) found that cured products made from beef hearts showed no botulinal inhibition; turkey thigh meat and beef rounds were more inhibitory, and pork ham, turkey breasts, and veal were the most inhibitory. In his study the degree of botulinal inhibition did not appear to be related to residual nitrite level,

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but it was related to the iron content of the meat. Since mechanically deboned meat contains more hemoglobin than hand-boned meat does, a possible loss of botulinal inhibition in products containing mechanically deboned meat needs to be investigated. Much more work is needed in the area of microbiology as it relates to bone marrow characteristics. The vitamin content of mechanically deboned meat may differ from that of hand-boned meat because of the red marrow present. For example, limited data indicate that red marrow is high in ascorbic acid (Lutwak-Mann, 1952; Cox rt al., 1960), but muscle is almost devoid of ascorbic acid. The vitamin content of other organs such as the liver, which is high in vitamin A , is quite different from that of muscle, and there is reason to expect that red marrow may have a vitamin content closer to that of liver than it does to that of muscle. No information on the vitamin content of mechanically deboned meat is available. Another area of needed research concerns flavor. How much red marrow is the optimum for flavor enhancement, and does the enhancement come from the lipid, protein, or mineral fraction? Mechanically deboned meat is a wholesome, nutritious, highly palatable product with a bright future as a food, but more information on its composition and use is needed. New methods of recovering residual meat and marrow will be developed, so continual collection of information is required. In view of the many new types of mechanical deboners already on the market, it appears that basic research involving m g o w and bone, which become a part of mechanically deboned meat, will be more fruitful than studying the characteristics of mechanically deboned meat, which will change as new deboners are developed.

REFERENCES Abraham, 0.. Schaefer. M., Kohrs, M. B.. O’Neal, R., Smith, D., and Eklund. D. 1977. Nutritional status of preschool children in Missouri. Fed. Proc., Fed. Am. SOC.Exp. Biol. 36, I182 (abstr.). Altman, P. L., and Dittmer, D. S. 1971. “Blood and Other Body Fluids,” p. 73. Fed. Am. SOC. Exp. Biol., Bethesda, Maryland. American Meat Institute. 1979. “Meat Facts-A Statistical Summary About America’s Largest Food Industry.” AMI, Washington, D. C . Ammerman, C. B., Miller, S. M . , Fick, K . R., and Hansard, S. L. 1977. Contaminating elements in mineral supplements and their potential toxicity: A review. J. h i m . Sci. 44, 485. Anderson, J. R.,and Gillett, T. 1974. Extractable-emulsifying capacity of hand and mechanicallydeboned mutton. J. Food Sci. 39, 1147. Angelotti, R . 1978. Dr. Angelotti, USDA’s “Chief Inspector,” examines the meat we eat. Employee News/. 37, I .

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Anhalt, G.. Bruning-Oeltzin, A., and Wenzel, S. 1977. Bone content in manually deboned residual meat from pork and beef. Arch. Lehensrnittelhyg. 28 (6), 202. Anonymous. 1976. Franks of the future. Meut I n d . 22, 26. Arasu, P. 1980. The influence of age, sex and anatomical location on the nucleic acid content of bovine bone marrow, and its relationship to mechanically deboned meat. Master’s Thesis, University of Wyoming, Laramie. Armstrong, W. D., and Singer, L. 1965. Composition and constitution of the mineral phase of bone. Clin. Orthop. Relut. Res. 38, 179. Bakker, A. F. 1978. Equipment far mechanical separation of meat from bones. Fleischerei 29 (7). 12. Barton, R. A , , and Kirton, A. H. 1958. The leg and the loin as indices of the composition of New Zealand lamb and mutton carcasses. N. Z. J. Agric. Res. I , 783. Bell, T. D.. Orme, L. E., Hodgson, C. W., Christian, R. E., and Everson, D. 0. 1967. Weanling and carcass characteristics of purebred and crossbred Rambouillet, Targhee and Panama lambs. Res. Bull.+duho, Exp. Stn., 75. Bijker, P. G. H., Gerats, G . E., VanLogtestijn. J. G . , Koolmees, P. A,, and Fransen, T. 1979. Methods to determine the bone content and the size of the bone particles in mechanically deboned pork. Proc. E u r . Meet. Meat Res. Work., 25th, I Y 7 Y p. 845. Bijker. P. G . H., Scholten, J . I . M., Fransen, T. and Koolmees, P. A. 1980. Hygienic aspects in the production of mechanically deboned pork. Tijdschr. Diergeneeskd. 105, 433. Blum. J. W., and Zuber, U. 1975. Iron stores of liver, spleen and bone marrow, and serum iron concentrations in female dairy cattle in relationship to age. Res. Vet. Sci. 18, 294. Brhnnang, E. 1966. The effect of castration and age of castration on the growth rate, feed conversion and carcass traits of Swedish Red and White Cattle. Lunthrukshoegsk. Ann. 32, 329. Brooks, C. C., Thomas, H. R . , Kelly, R. F., Graham, P. P., and Allen, L. E. 1964. Body composition and feed efficiency changes in swine. Vu. Polytech. Inst.. Agric. Exp. S t n . . Tech. Bull. 176. Brown, R. G., Aeschbacker. H. U.. and Funk, D. 1972. Connective tissue metabolism in swine IV: Growth dependent changes in the composition of long bones in female swine. Growth 36,389. Bruce, R., Downs, W . , and Harris, W. 1974. Contamination of diet with radioactive fallout from nuclear explosions. Annu. Rep.-ARric. Res. Counc. Letcornhe Lab. p. 25. Bryant, A. B. 1976. United States District Court for the District of the District of Columbia. Civil Action No. 76-1585. Filed Sept. 10. Carpenter, J. A. 1975. Mechanically deboned meat in sausage. Proc.-Annu. Meat Sci. Inst. 17, 9 . Casey, J. C., Self, R., and Swain, T. 1965. Factors influencing the production of low-boiling volatiles from food. J . Food Sci. 30, 33. Champaigne, J . R . , Carpenter, J. W., Hentges, J. F., Jr., Palmez, A. Z., and Koger, M. 1969. Feedlot performance and carcass characteristics of young bulls and steers castrated at four ages. J. Anim. Sci. 29, 887. Chang. Y. 0.. and Field, R. A . 1977. Protein utilization of mechanically deboned meat by growing rats. J . N u t r . 107, 1947. Chant, J . L., Day, L., Field, R . A,. Kruggel, W. G . , and Chang, Y . 0. 1977. Composition and palatability of mechanically deboned meat and mechanically separated tissue. J. F a o d Sci. 42. 306. Charlet, P. 1969. The production of meat from male animals in France. In “Meat Production from Entire Male Animals” (D. N . Rhodes. ed.). p. 143. Churchill, London. Childers, A. B., Heidelbaugh, N. D., Carpenter, Z. L . , Smith, G. C., and White, S. 1979. Technologic advances in the food industry: Their influence on public health. J . Am. Vet. Mrd. Assoc. 175 (12). 1291. Clark, H. E. 1965. Utilization of essential amino acids by man. In “Newer Methods of Nutritional Biochemistry” (A. A. Albanese, ed.), Vol. 2, p. 123. Academic Press, New York. I

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Clifford, A. J., Riumello. J. A., Young, V. R., and Scrimshaw, N. S. 1976. Effect of oral purines on serum and urinary uric acid of normal, hyperuricemic and gouty humans. J. Nurr. 106 (3), 428. Comar, C. L., Scott, R. R., and Wasserman, R . H. 1957. Strontium-calcium movement from soil to man. Science 126, 485. Council for Agricultural Science and Technology. 1980. “Foods from Animals, Quantity, Quality and Safety,” Rep. No. 82. C.A.S.T., Ames, Iowa. Cox, E. V., Mattews, D. M., Meynell, M. J . , Cooke, W. T., and Gaddie, R. 1960. Cyanocobalamin, ascorbic acid and pteroylglutamates in normal and megaloblastic bone marrow. Blood 15, 376. Craddock, B. F., Field, R . A,, and Riley, M. L. 1974. Effect of protein and energy levels on lamb carcass composition. J. Anim. Sci. 39, 325. Cross, H. R., Stroud, J., Carpenter, 2. L., Kotula. A. W . , Nolan, T. W., and Smith, G. C. 1977. Use of mechanically deboned meat in ground beef patties. J. Food Sci. 42, 1496. Cross, H. R., Kotula, A. W., and Nolan, J. W. 1978a. Stability of frozen ground beef containing mechanically deboned beef. J. Food Sci. 43, 281. Cross, H. R., Berry, B. W., Nichols, J. E., Elder, R. S., and Quick, J. A. 1978b. Effect of desinewing versus grinding on textural properties of beef. J. Food Sci. 43, 1507. Cuthbertson, A., and Pomeroy, R. W. 1962. Quantitative anatomical studies of the composition of the pig at 50, 68 and 92 kg carcass weight. I I . Gross composition and skeletal composition. J. Agric. Sci. 59, 215. Davidson, J. N . 1947. Some factors influencing the nucleic acid content of cells and tissues. Cold Spring Hurhor Symp. Quunr. Biol. 12, 50. Davidson, J. N., Leslie, I . , and Waymouth, C. 1947. The distribution of nucleic acids in bone marrow cells. Biochem. J. 41, Proc. xxvi. Davidson, S., and Passmore, R. 1963. “Human Nutrition and Dietetics,” Vol. 1, 2nd ed., p. 157. Williams & Wilkins, Baltimore, Maryland. Davidson, J. N . 1976. “The Biochemistry of the Nucleic Acids,’’ 8th ed., pp. 69-76. Academic Press, New York. Dimick, P. S . , MacNeil, J. H., and Grunden, L. P. 1972. Poultry product quality. Carbonyl composition and organoleptic evaluation of mechanically deboned poultry meat. J. Food Sci. 37, 544. Djujic, I . , Djordjevic, V . , Mihajlovic, B., and RadoviC, N. 1979. Mineral components of mechanically separated meat. Proe. Eur. Meet. Meat Res. Work., 25th. 1979 p. 859. Dolan, T.. Legette, L., McNeal, J., and Malansoki, A. J. 1978. Determination of fluoride in deboned meat. J. Assoc. Of.A n d . Chem. 61, 982. Doyle, J. J. 1979. Toxic and essential elements in bone-A review. J. Anim. Sci. 49, 482. Doyle, J. J . , and Pfander, W. H. 1975. Interactions of cadmium with copper, iron, zinc and maganese in ovine tissues. J . Nurr. 105, 599. Doyle, J . J . . and Spaulding, J. E. 1978. Toxic and essential trace elements in meat-A review. J. Anim. Sci. 47, 398. Drake, T. G . H., Jackson, S . H., Tisdall, F. F., Johnstone, W. M., and Hierst, L. M. 1949. The biological availability of the calcium in bone. J. Nurr. 37, 369. Duerr, P. E. and Earle, M. D. 1974. The extraction of beef bones with water, dilute sodium hydroxide and dilute potassium chloride. J. Sci. Food Agric. 25, 121. Eastoe. J . E. 1955. The amino acid composition of mammalian collagen and gelatin. Biorhem. J. 61, 589. Eastoe, J. E. 1961. The chemical composition of bone. In “Biochemists’ Handbook” (C. Long, ed.), pp. 7 15-720. Van Nostrand-Reinhold, Princeton, New Jersey. Eastoe, J . E., and Leach, A. A. 1958. A survey of recent work on the amino acid composition of vertebrate collagen and gelatin. In “Recent Advances in Gelatin and Glue Research” (G. Stainsby, ed.), p. 173. Pergamon, Oxford.

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Sanchez, L. R. 1979. Chemical composition of marrow and muscle from the lumbar and cervical regions of the bovine by age groups. Master’s Thesis, University of Wyoming, Laramie. Sanchez, L. R., Kunsman, J. E., Field, R. A., and Kruggel, W. G. 1978. Determination of red marrow in muscle/marrow mixtures. Proc. Annu. Meet.-Am. Soc. Anim. Sci., West. Sect. 29, 139. Satterlee, L. D., Froning, G. W., and Janky, D. M. 1971. lnfluenceof skincontent oncomposition of mechanically deboned poultry meat. J . Food Sci. 36, 979. Schmidt, G . , and Thanhauser, S . J. 1945. A method for the determination of DNA, RNA and phosphoproteins in animal tissues. J . Eiol. Chem. 161, 83. Schneider, W . C. 1946. Phosphorus compounds in animal tissues. 111. A comparison of methods for the estimation of nucleic acids. J. Eiol. Chem. 164, 747. Schothorst, M., Leusden, F. M., and Naracka, U. 1979. Hygienic aspects of mechanical deboning machines. Voedingsmeddelenrechnologie 12 (7). 1 I . Schultz, M. 0.. Elvehjem, C. A , , and Hart, E. B. 1936. Studies on the copper and iron content of tissues and organs in nutritional anemia. J . Eiol. Chem. 46, 93. Scrimshaw, N . S. 1975. Single-cell protein for human consumption. In “Single-cell Protein” (s.R. Tannenbaum and D. I. C. Wang, eds.), Vol. 11, p. 24. M.I.T. Press, Cambridge, Massachusetts. Seideman, S. C., Smith, G. C., and Carpenter, Z. L. 1977. Addition of textured soy protein and mechanically deboned beef to ground beef formulations. J. Food Sci. 42, 197. Seitz, J . F. 1969. “The Biochemistry of the Cells of Blood and Bone Marrow,” p. 220. Thomas, Springfield, Illinois. Shupe, J. L., Miner, M. L., Harris, L. E., and Greenwood, D. A. 1962. Relative effects of feeding hay atmospherically contaminated by fluoride residue, normal hay plus calcium fluoride, and normal hay plus sodium fluoride to dairy heifers. A m . J . Vet. Res. 23, 777. Shupe, J. L., Harris, L. E., Greenwood, D. A,, Butcher, J. E., and Nielson, H. M. 1963a. The effect of fluorine on dairy cattle. 5 . Fluorine in the urine as an estimator of fluorine intake. Am. J . Vet. Res. 24, 300. Shupe, J. L., Miner, M. L., Greenwood, D. A,, Harris, L. E., and Stoddard. G . E. 1963b. The effect of fluorine on dairy cattle. 2. Clinical and pathologic effects. Am. J . Vet. Res. 24, 964. Singer, L., Armstrong, W. D., and Vogel, J. J. 1967. Role of the skeleton in regulation of tissue fluoride content. Isr. J . Med. Sci. 3, 714. Snowden, E. M., and Stitch, S . R. 1957. Trace elements in human tissue. Eiochem. J. 67, 104. Snowdon, C. T., and Sanderson, B. A. 1974. Lead pica produced in rats. Science 183, 92. Souron, Y. M. F. 1975. Preservation process for bone marrow. London Patent 1,409,856. Spector, W. S. 1956. “Handbook of Biological Data,” p. 53. Saunders, Philadelphia, Pennsylvania. Suttie, J . W., and Phillips, P. H. 1959. Studies of the effects of dietary sodium fluoride on dairy cows. 5 . A three-year study of mature animals. J. Dairy Sci. 42, 1063. Suttie, J . W., Phillips, P. H . , and Miller, R. F. 1958. Studies of the effects of dietary sodium fluoride on dairy cows. 3. Skeletal and soft-tissue fluorine deposition and fluorine toxicosis. J . Nutr. 65, 293. Swift, C. E., and Berman, M. D. 1959. Factors affecting the water retention of beef. I. Variations in composition and properties among eight muscles. Food Technol. 13, 365. Tandler, K . 1978. “Hard separator meat” and “soft separator meat” regulations and recommendations for manufacture and processing. Fleischwirrschafr 58 (4), 535. Tarladgis, B. G . 1967. Treatment of cured meats with electron-donating compounds. U. S. Patent 3,360,381. Taylor, T. G . , Moore, J. H., and Hertelendy, F. 1960. Variations in the mineral composition of individual bones of the skeleton of the domestic fowl. Er. J. Nutr. 14, 49.

106

RAY A. FIELD

Terrell, R. N., Suess, G . G . , Cassens. R . G., and Bray, R. W. 1968. Broiling, sex and interrelationships with carcass and growth characteristics and their effect on the neutral and phospholipid fatty acids of the bovine longissimus dorsi. J. Food Sci. 33, 562. Tisdall, F. F., and Drake, T. G. H. 1938. The utilization of calcium. J. Nurr. 16, 613. Tompkin, R. B. 1978. The role and mechanism of the inhibition of C. Botulinium by nitrite - is a replacement available? Proc. Recip. Meat Con$ 31, 135. Trenkle, A,. DeWitt, D. L.. and Topel, D. G. 1978. Influence of age, nutrition and genotype on carcass traits and cellular development of the longissimus muscle of cattle. J . Anim. Sci. 46, 1597. Tybor. P. T., Dill, C. W., and Landmann, W. A. 1975. Functional properties of proteins isolated from bovine blood by a continuous pilot process. J . Food Sci. 40, 155. Uebersax. K . L., Dawson, L. E., and Uebersax, M. A. 1978. Storage stability (TBA) and color of MDCM and MCTM processed with CO, cooling. Poulr. Sci. 57, 670. Ullrey, D. E. 1977. Analytical problems in evaluating mineral concentrations in animal tissues. J. Anim. Sci. 44, 475. Underwood, E. J. 1971. “Trace Elements in Human and Animal Nutrition.” 3rd ed. Academic Press, New York. Underwood, E. J. 1977. “Trace Elements in Human and Animal Nutrition,” 4th ed.. pp. 153-154. Academic Press, New York. United Nations Food and Agriculture Oragnization. 1973. “Energy and Protein Requirements,” Joint FAO/WHO Ad Hoc Expert Committee, F A 0 Nutr. Meet. Rep. Ser. No. 52. FAO, Rome. U.S. Department of Agriculture. 1969. Inspection of poultry and poultry products. Labeling requirements, standards of composition and definitions. Fed. Regist. 34, 13, 991. U.S. Department of Agriculture. 1972. “Food and Nutrient Intake of Individuals in the United States, Spring 1965,” Household Food Consumption Survey 1965-66, Rep. No. I I . Agriculture Research Service, USDA, Washington, D. C. U.S. Department of Agriculture. 1974. Preparation of mechanically deboned meat and mechanically deboned meat fatty tissue. U S . , Dep. Agric., Meat Poult. Inspecr. Program Bull. 865. U.S. Department of Agriculture. 1977. Standards and labeling requirements for tissue from ground bone. Fed. Regis:. 42, 54437. U.S. Department of Agriculture. 1978a. Impact analysis, mechanically processed (species) product. U S . . Dep. Agric., Meat and Poultry Inspection Program. Food Safety and Quality Service, Washington, D. C. U.S. Department of Agriculture. 1978b. Mechanically processed (species) product: Standards and labeling requirements. Fed. Regis:. 43, 26416. U.S. Department of Agriculture. 1979. Notice and Solicitation of Information: Mechanically Deboned Poultry. Fed. Regis!. 44, 37965. U.S. Department of Agriculture. 1980. FLM 2-80. USDA, Foreign Agriculture Service, Washington, D. C. Vadehra, D. V., and Baker, R . C. 1970. Physical and chemical properties of mechanically deboned poultry meat. PcJu/:.Sci. 49, 1446 (abstr.). Waldbott, G . L. 1963. Fluoride in food. Am. J. Clin. Nutr. 12, 455. Waldman, R. C., Tyler. W. I . , and Brungardt, V. H. 1971. Changes in the carcass composition of Holstein steers associated with ration energy levels and growth. J. h i m . Sri. 32, 611. Walker, A. R. P. 1972. The human requirement of calcium: Should low intakes be supplemented? Am. J. Clin. Nutr. 24, 518. Warriss, P. 1976. The quantitative determination of hemoglobin in ovine muscles. Anal. Biochetn. 72, 104. Waslien, C. I., Calloway, D. H . , and Margen, S. 1968. Uric acid production of men fed graded amounts of egg protein. Am. J. Clin. Nurr. 21, 892.

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RED MEAT

107

Watt, B. K., and Merrill, A. L. 1963. “Composition of Foods. Raw, Processed, Prepared,” USDA Agric. Handb. No. 8. US Govt. Printing Office, Washington, D. C. Wermuth, N . , and Madelung, 1. 1974. Circular regarding mechanical deboning of minced meat. Dan. Minisr. Agric. J . No. 10.1.3-8/74. Wessel, M . A., and Dominski, A. 1977. Our children’s daily lead. Am. Sci. 65, 294. White, A,, Handler, P.. Smith, E. L., and Stetten, D. 1954. “Principles of Biochemistry,” p. 737. McGraw-Hill, New York. White, A , , Handler, P., and Smith, E. L. 1968. “Principles of Biochemistry,” 4th ed., p. 140. McGraw-Hill, New York. Williams, W. F. 1979. “Economic Evaluation of Potentials through Mechanical Deboning of Red Meats,” TARA, Inc., Lubbock, Texas. Wilson, J. P. 1974. “Edible Meat Fractions and Mechanically Deboned Meat for Export.” Australian Government, Department of Primary Industry, Canberra. Winter, F. F. 1978. Production and processing of mechanically-separated meat. Fleischerei 29 (7). 9. Winthrobe, M. W. 1974. “Clinical Haematology,” 7th ed. Lea & Febiger, Philadelphia, Pennsylvania Young, L. L. 1980. Evaluation of four purine compounds in poultry products. J . FoodSci. 45, 1064.

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ADVANCES I N FOOD RESEARCH, VOL.

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MECHANICAL DEBONING OF POULTRY AND FISH G . W. FRONING Depurtmenr of Animal Science, University of Nebraska, Lincoln, Nebraska

1. Introduction 11.

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110

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Ill

Protein Quality ..................................... Fatty Acid and Content . . . . . . . . . . ......... Bone Content . . . . . . . ............................... Heme Pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

117 117 119

Ill. A. Structural Characteristics

.......... D. E. F. G.

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IV.

B . Storage .............................

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127

A. Effect of Composition . . . . . . . . .................. B. Effect of Processing Variables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Effect of Food Additives.. . . . . . . . . ................. VII . Utilization of Bone Residue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..................... VIII.

129 132 134

V.

v1.

I37 138

IX . X. References . . . . . . . .

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109 Copyright @ 1981 by Academic Rcss. Inc. All rights of npmduction in any form reserved. ISBN 0-12-016427-2

110

G. W . FRONING

I.

INTRODUCTION

The process of mechanical deboning of fish and poultry meat has received increasing attention during the last 30 years. Mechanical deboning of fish began in Japan in the late 1940s. Its development has enabled the fish processing industry to recover meat from numerous underutilized species of fish. A potential source of 40 million tons of underutilized fish exists, which may be mechanically deboned to produce minced fish (Food and Agriculture Organization, 1971). Martin (1976) indicated that 46 different species of fish and shellfish were currently being mechanically deboned for use in further processed products. Additional minced fish could also be obtained by trimming from other species presently used for fillet production. Miyauchi and Steinberg (1970) reported yields of 37-60% from trimming from various species, and 25-30% from conventional filleting techniques. Martin ( 1976) listed several potential products that may utilize minced fish, including fish sticks, spreads, pastes, fish sausage links, fish cakes, and stuffings. Mechanical deboning of poultry began in the late 1950s and early 1960s. The increased popularity of cut-up chicken and processed turkey meat provided con-

Turkey c a r c a s s

Chicken broiler

A c u t up

or cut up

Packaged

Breast, thigh meat

Frame, back, neck, drumsticks, wings, etc.

Packaged parts

Roasts, rolls, steaks, etc. deboned

Bone residue

I

Animal feed, protein isolate FIG. 1 ,

Meat paste for emulsified products (frankfurters, bologna, salami, etc.)

Deboning of poultry meat.

MECHANICAL DEBONING OF POULTRY AND FISH

111

siderable quantities of parts suitable for mechanical deboning. This mechanically deboned poultry meat supply became available for use in a variety of new products. Mechanically deboned poultry has been used with success in frankfurters, bologna, salami, turkey rolls, and soup mixes. A typical product flow for mechanical deboning of poulty is shown in Fig. 1. Yields of mechanically deboned poultry meat from various parts range from 55.0 to 70.0%, depending on which part is deboned. Whole carcasses give a somewhat higher yield than do parts such as necks, backs, or wings. More than 200 million pounds of mechanically deboned poultry are now being produced in the United States yearly. This quantity of mechanically deboned poultry adds approximately $60-75 million to the economy of the poultry industry. The mechanical deboning process offers a method of harvesting meat from poultry and fish parts and from underutilized species of fish that would otherwise be wasted. In view of the world food shortage, this technique provides another means for helping to feed the world. Tremendous strides have been made in research to improve the utilization of mechanically deboned poultry and fish. There is still more potential for further improvement and development of this important process.

II. TYPES OF MECHANICAL DEBONERS Mechanical deboning basically involves grinding meat and bone together and forcing the meat through a fine screen or slotted surface of a mechanical deboner. Bone particles are left behind, becoming a part of the waste residue. Several types of mechanical deboners have been used for separation of fish and poultry meat (Martin, 1972; Froning, 1976). Basically, deboners may be categorized into two different groups. One design forces the meat from the outside to the inside of a perforated drum, leaving the bone residue on the outside of the drum. The other design separates the meat from the bone by forcing the meat from the inside to the outside of a perforated cylinder, leaving the bone residue on the inside (Fig. 2).

Ill.

COMPOSITION AND NUTRITIVE PROPERTIES A. STRUCTURAL CHARACTERISTICS

The shearing action of the mechanical deboning process causes considerable cellular disruption. Poultry meat deboned mechanically results in a finely ground, pastelike product in which the myofibrils are heavily fragmented.

FIG. 2.

Deboning head on the Beehive Deboning Machine

MECHANICAL DEBONING OF POULTRY AND FISH

113

Mechanically deboned fish has a somewhat coarser texture, depending on the size of the deboner screen. Schnell et al. (1974) studied the ultrastructure of mechanically deboned poultry meat using a deboner with screen sizes of 0.1575, 0.1016, and 0.0508 cm. The characteristic size of the myofibrils was destroyed by the smaller screen size. Breaks were observed at the Z or M bands, indicating that the shearing process affects the length of the fibrils. The resulting particles tended to be spherical or oval in shape. Heating appeared to affect sarcomere length, which was attributed to contraction. Wong et al. (1978) investigated the influence of various perforation sizes of mechanical deboners on the texture of minced fish flesh. Texture was measured as the resistance to compression by means of an Ottawa Texture Measuring System. Resistance to compression appeared to increase with an increase in flesh particle size in most instances. Minced rockfish and pollock flesh exhibited somewhat higher force values when a 7-mm screen was used than were obtained with a 2-mm screen. A decrease in force (texture) was observed in herring pressed through a drum with 7-mm perforations, compared with hemng obtained from 5-mm orifices. These data suggest that for smaller species of fish, such as herring, the optimum screen size may be somewhat different from those used for larger fish species. Miyauchi (1 972) compared coarse-minced fish flesh with finely minced fish flesh. A sensory panel judged the texture of samples that were battered, breaded, and deep-fried. The texture of the coarsely minced fish flesh was preferred. Thus, mechanically deboned fish is usually separated by using somewhat larger screen sizes than those used for separating mechanically deboned poultry meat. The ultrastructure of mechanically deboned poultry meat is altered considerably more than that of fish flesh.

B . PROXIMATE COMPOSITION The mechanical deboning of poultry or fish affects the proximate composition. Considerable quantities of lipid and heme componets are released from the bone marrow and subsequently accumulate in the mechanically separated product. These fractions from the bone marrow dilute the protein fraction and increase the lipid fraction in the mechanically deboned tissue. Table I presents proximate composition data for mechanically deboned poultry meat. Considerable variability is found, related to several factors such as age of the bird, bone-to-meat ratio, cutting methods, skin content, and possible protein denaturation. Values for chicken backs and necks vary according to different workers; these differences may be related to skin content or deboner settings. The skin content of the ingoing meat source, for example, markedly influences the fat content of the deboned product (Satterlee et al., 1971). Turkey frames come

114

G . W . FRONING

TABLE 1 COMPOSITION OF MECHANICALLY DEBONED POULTRY MEAT FROM DIFFERENT SOURCES

Protein

Moisture

Fat

Source

(%)

(%)

(o/.)

Reference

Chicken backs and necks Chicken backs and necks Chicken backs and necks Chicken backs Skinless necks Turkey frame Turkey frame Turkey frame Spent layers Spent layers Cooked spent layer frame

14.5 9.3 13.4 13.2 15.3 12.8 12.8 15.5 14.2 13.9 18.3

66.6 63.4 72.2 62.4 76.7 70.7 73.7 70.6 60.1 65.1 63.2

17.6 27.2 14.4 21.2 7.9 14.4 12.7 13.5 26.2 18.3 16.5

Froning ( 1970) Grunden P I a / . (1972) Essary ( 1979) Froning (1970) MacNeil e t a / . (1978) Froning et u / . (1971) Grunden et a / . (1972) Essary ( 1979) Grunden er d.(1972) Froning and Johnson (1973) Babji ef a / . (1979)

from birds of a wide range of ages, which could influence fat content. Turkey frames also differ substantially in the amount of muscle adhering after hand deboning. Variability in the fat composition of mechanically deboned spent fowl meat is probably due to differences in the amount of abdominal fat adhering to the carcass prior to deboning. Deboner settings may also markedly influence yields, as well as fat, protein, and ash content. If deboners are set for exceptionally high yields, the fat and ash or bone content may be substantially increased. Improper deboner settings may also increase the temperature of the meat excessively, thereby denaturing the protein and possibly resulting in some protein loss into the residue. All sources in Table I are from raw carcasses with the exception of the mechanically deboned, cooked spent layer frame meat. Spent fowl is usually cooked, and most of the meat is removed by hand deboning, leaving some meat adhering to the frame. The frame is then mechanically deboned. Mechanically deboned, cooked spent layer frame meat is generally higher in protein and lower in fat. Spent fowl bones are more calcified, which lessens the quantity of bone marrow extruded into the meat, thereby minimizing the protein dilution effect. Cooking also causes gelatinization of the collagen, which probably increases the protein content. The solubilized collagen may separate into the meat fraction rather than being expelled with the residue. Satterlee et al. (1971) observed that collagen was expelled with the residue when raw poultry meat was deboned. Crawford et al. (1972) reported compositional data on several different species of mechanically deboned fish (Table 11). Fat levels varied from 1.9 to 8.2%, which is a somewhat lower range than that observed in mechanically deboned poultry. Differences in moisture content were generally in proportion to

MECHANICAL DEBONING OF POULTRY AND FISH

1 I5

TABLE I1 PROXIMATE COMPOSITION OF MECHANICALLY SEPARATED FISH FLESH FROM DIFFERENT SPECIES‘’

Moisture Fish

(%I

English sole Petrale sole Orange rockfish Yellowtail rockfish True Cod Ling cod

83.44 77.12 76.94 73.21 82.90 79.73

I‘

Fat

2.28 8.16 7.66 7.69 1.90 4.28

Protein

Ash

(%)

(%)

11.84 13.52 14.50 14.48 14.06 14.93

2.14 1.34 I .62 2.03 1.52 I .30

From Crawford et a / . (1972)

the variance in fat content. Mechanically separated flesh from English sole appeared to have a slightly lower protein content than that observed in flesh from other species. Webb et at. (1976) compared mechanically separated fish tissue with hand-separated tissue from Atlantic croaker (Table 111). Differences in moisture and fat were attributed to the exposure of hand-processed fillets to water during rinsing by commercial-type processing ‘and to a higher concentration of fat from skin and bone marrow during machine separation. The shearing action of the mechanical deboner was believed to remove more fatty tissue from the skin than was obtained by hand skinning. Also, bone marrow apparently contributed to the increased fat content of the mechanically separated fish tissue. The protein content of mechanically deboned poultry and fish appears to be quite similar. The fat content of mechanically deboned fish is considerably lower than that found in poultry. The ash content of mechanically deboned fish is higher than that found in mechanically deboned poultry meat. Many of these compositional differences may be related to screen sizes used during the deboning process. Fish deboners generally have a substantially larger screen size than do poultry deboners. C.

MINERAL COMPOSITION

The mineral content and composition of mechanically deboned poultry and fish have been a concern of consumers. The mechanical separation process, as with hand separation techniques, may leave small amounts of powdered bone. Considerable research has been conducted to determine the types and quantities of minerals found in mechanically deboned poultry. Calcium content has generally been utilized as a measure of bone present in mechanically deboned meat. The poultry industry has used the modified EDTA

G . W. FRONING

116

TABLE 111 PROXIMATE COMPOSITION OF HAND AND MECHANICALLY SEPARATED FISH MUSCLE TISSUE” ~

Treatment

Moisture (%)

Hand Machine

80.2 77.5

”

Protein (a) 19.5 19.2

Fat (%)

Ash (%)

I .2 3.6

I .o 1.1

From Webb e r a / . (1976).

titration method of Steagall (1966) for monitoring calcium content. This method is fully described in a Beehive technical bulletion entitled, “Raw Meat Deboner” (Beehive Machinery, Inc., Sandy, Utah). The U.S. Department of Agriculture requires that the bone content not exceed I % . Murphy et al. (1979) and Grunden and MacNeil(1973) observed higher levels of calcium in mechanically deboned spent fowl than were found in other types of poultry. Calcium present in mechanically deboned poultry meat should be beneficial in the diet of many individuals. Several other minerals have been studied because of their health and safety aspects. Those of particular concern include arsenic, fluoride, cadmium, strontium 90, selenium, iron, nickel, copper, lead, and zinc. Murphy et al. (1979) summarized reports on each of these minerals; they concluded that none of these minerals presented any basic health hazard in mechanically deboned poultry. Fluoride content was high in the mechanically deboned spent fowl, possibly requiring some limitations on its use, especially in baby foods. This report further recommended that kidneys from mature chickens not be allowed in mechanically deboned poultry, since they may contribute unwanted cadmium to the product. Essary (1979) also analyzed several minerals from various types of mechanically deboned poultry. The levels detected were concluded to be safe for human consumption. Crawford et al. (1972) studied the mineral composition of machine-separated flesh from various species of fish. Levels of phosphorus, calcium, strontium, manganese, boron, and chromium were much lower in the separated flesh fractions than those observed in the whole carcass wastes. Concentrations of potassium, sodium, and iron were higher in the machine-separated flesh than in whole carcass waste. Calcium-to-phosphorus and potassium-to-sodium ratios were generally lower in machine-separated flesh than those observed in whole carcass flesh. Their work indicated that the mineral composition of machineseparated flesh was considerably upgraded nutritionally, compared with that of whole waste.

MECHANICAL DEBONING OF POULTRY AND FISH

D.

117

PROTEIN QUALITY

If mechanically deboned poultry and fish are to replace hand-deboned sources in various products, the protein quality must be maintained. Several studies have investigated the protein quality of mechanically deboned poultry and fish. Essary and Ritchey (1968) observed that the amino acid composition of mechanically deboned turkey meat was similar to that found in hand-deboned sources. More recently, MacNeil et al. (1978) reported PER (protein efficiency ratio) values of 2.65 for skinless mechanically deboned broiler necks and 2.45 for a combination of skinless mechanically deboned necks and backs. Hsu et al. (1978) assayed protein quality of mechanically deboned turkey meat, using the computed PER and rat PER techniques. Computed PER values were 2.7, whereas rat PER values were 2.6. Babji et al. (1980) also reported that computed PER values of mechanically deboned poultry were similar to rat PER values, and protein quality was comparable to that of the casein control (Table IV). Crawford et al. (1972) observed excellent PER values of 3.2 for various species of fish, compared with a PER value of 2.9 for casein. Overall, mechanically deboned poultry and fish have a protein quality quite comparable to that of hand-deboned sources. Thus, mechanically deboned sources of poultry and fish can be used in products without sacrificing protein quality. E.

FATTY ACID AND CHOLESTEROL CONTENT

During the mechanical deboning process, lipid components from the bone marrow are incorporated into the mechanically separated product. Moerck and Ball (1973) reported that chicken bone marrow from 8- to 9-week-old broilers contained 46.5% lipids, of which 98.4% were neutral lipids. The lipid fractions of the bone marrow from the femur are shown in Table V. On a weight percentage basis, bone marrow contained a higher percentage of phospholipids and cholesterol than did other broiler tissue. The significance of cholesterol in the diet is a much debated issue among scientists. The fatty acid composition of chicken bone marrow lipids and mechanically deboned poultry was found to be similar to that of breast, thigh, and skin tissue (Moerck and Ball, 1973, 1974). Murphy et al. (1979) reviewed previous work and indicated that substitution of mechanically deboned poultry for hand-deboned sources did not affect the linoleic acid content of the final product but might slightly increase the content of oleic acid. Mai and Kinsella (1979) studied changes in lipid composition of minced carp that was cooked by baking and deep-fat frying and then stored at 18°C for periods

118

G. W . FRONING TABLE IV COMPARISON OF COMPUTED PER AND RAT PER OF VARIOUS TYPES OF MECHANICALLY DEBONED POULTRY MEAT"

Sample

Rat PER

Computed PER

Casein Mechanically deboned chicken back and neck meat Cooked mechanically deboned fowl meat Mechanically deboned turkey frame meat

2.50 2.34

2.50 2.44

2.41

2.41

2.59

2.76

'I

From Babji et al. (1979). TABLE V LIPID FRACTIONS OF THE BONE MARROW FROM THE FEMUR OF 8- TO 9-WEEK-OLD CHICKEN

BROILERS"

Lipid fractions

Femur (%)

Triglycerides Phospholipids Cholesterol Diglycerides Free fatty acids Monoglycerides Cholesterol ester Glycolipids

94.6 1.7 1.4

"

0.8

0.7 0.6 0.3 Trace

From Moerck and Ball (1973) TABLE VI

AVERAGE PERCENTAGE OF BONE SOLIDS FOR MECHANICALLY DEBONED POULTRY MEAT"

Source

Spectrophotometer method

EDTA method

Young male turkey racks Mature male turkey racks Broiler necks and backs Broiler backs Spent layer carcasses

0.32 0.55 0.70 0.79 1.21

0.41

" From Grunden and MacNeil (1973)

-

0.79 0.82 1.44

MECHANICAL DEBONING OF POULTRY AND FISH

119

up to 8 weeks. Phospholipid levels decreased while free fatty acids increased in all samples during frozen storage. The carbonyl content fluctuated during storage, but no specific pattern was observed. There was no significant change in fatty acid composition during storage. F.

BONE CONTENT

Consumer groups have expressed concern over the possible inclusion of bone fragments in mechanically deboned poultry meat. Murphy et al. (1979) reported that bone particles in mechanically deboned poultry will not present any health hazard because of size or hardness, provided that bone particle size is controlled. The bone content of mechanically deboned poultry meat is limited to 1% by the U.S. Department of Agriculture. Grunden and MacNeil (1973) determined the bone content of various sources of mechanically deboned poultry, using the modified EDTA method and an atomic absorbance spectrophotometric method (Table VI). Their data indicated that preslaughter age is an important factor affecting the bone content of mechanically deboned poultry meat. Mechanically deboned meat from spent fowl had the highest bone content, running in excess of the 1 % limit. Bone content appeared to be related to increased bone calcification in older birds. The EDTA and atomic absorbance spectrophotometric methods gave comparable results. Froning ( 1979) isolated bone particles from hand-deboned turkey meat, sawed turkey carcasses, and mechanically deboned turkey meat, using the papain digestion procedure of Hill and Hites (1968). The largest bone particles were separated from hand-deboned turkey meat (average diameter 513 pm). Bone particles from mechanically deboned turkey meat (average diameter 233 pm)were substantially smaller than those from hand-deboned sources. Bone particles from sawed carcasses were similar in size to those found in mechanically deboned turkey meat. These date indicate that bone particles from mechanically deboned poultry present no hazard. Patashnik el al. (1974) examined the bone particle content of commercial blocks of minced fish, using a gravity-flotation method and a sensory technique. Bone particle counts per pound of product varied widely. Variability appeared to be a function of processing methods and raw material, but not of species. Sensory data indicated that panel members could detect only a small fraction of the total bone particles present. It was postulated that the granular, crusty nature of cooked batter and breading combined with the chewy flesh apparently concealed the presence of soft, pliable bone particles. These workers recommended straining the minced flesh to reduce bone particle content to acceptable limits. Wong et al. (1978) studied the effect of various mechanical deboner perforation sizes on bone content. Bone and scale contents were considerable lower in minced flesh separated through drums with 2- and 3-mm perforation ranges.

G . W. FRONING

120

G.

HEME PIGMENTS

The mechanical deboning process releases substantial quantities of hemoglobin from the bone marrow, which subsequently is extruded with the mechanically deboned poultry. Froning and Johnson (1973) observed that mechanical deboning of whole fowl carcasses tripled the hemoglobin content of the resultant mechanically deboned fowl meat. Myoglobin content was not influenced by the mechanical deboning process. Froning et ul. (1973) noted even higher increases in hemoglobin content of mechanically deboned broiler back meat. The difference was likely due to less calcification of the bone marrow from younger birds, which should contribute more hemoglobin to the mechanically deboned product. Silberstein and Lillard ( 1978) measured the concentrations of hemoglobin, myoglobin, and total pigments found in samples of hand-deboned and mechanically deboned mullet (Table VII). The ratio of hemoglobin to myoglobin was markedly increased in the mechanically deboned mullet, compared with ratios observed in hand-deboned sources. These data indicated that mechanical deboning increased the amount of hemoglobin in the mechanically deboned mullet but had very little effect on the myoglobin content of the mechanically deboned fish. The higher hemoglobin and myoglobin content of sample B of the hand-deboned fish was postulated to be due to a higher content of dark meat than that observed in sample A of the hand-deboned fish.

IV. A.

FLAVOR STABILITY

EFFECT OF PROCESSING VARIABLES

As mentioned earlier, the mechanical deboning process causes considerable cellular disruption and release of heme components from the bone marrow into the mechanically deboned product. The mechanical deboning process, therefore, may speed up the development of rancidity, particularly where the variables of the deboning process are not carefully controlled. Lee and Toledo (1977) examined several processing factors to determine how they may affect flavor stability of mechanically deboned fish. Parameters included contact with iron surfaces, mechanical stress applied to the muscle during deboning, temperature of the deboning drum, and washing of the deboned fish flesh. The degree of stess applied during deboning did not significantly affect thiobarbituric acid (TBA) values, whereas increased temperature of the deboning drum adversely affected flavor stability. Contact of flesh with iron parts of the deboner also increased TBA values. When stainless-steel parts were used, no increase in TBA values were noted. Washing of the mechanically deboned fish and filtering the excess

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121

TABLE VII HEME PIGMENT CONCENTRATION OF DEBONED FISH"

Deboning method

Myoglobin concentration (mdgm)

Hemoglobin concentration (mdgm)

Total pigment (mdgm)

Ratio Hb: Mb

Hand (A) Hand (B) Mechanical (C) Mechanical (D)

2.08 3.48 2.59 2.74

1.13 2.14 3.14 3.08

3.21 5.62 5.73 5.82

0.54 0.61 1.21 1.19

"

From Silberstein and Lillard (1978).

water through a cheesecloth significantly improved product quality when the washed product was stored for extended periods. Cellular disruption in fish flesh during mechanical deboning has been reported to lead to the breakdown of trimethylamine oxide (TMAO) to trimethylamine (TMA) or dimethylamine (DMA) (Babbitt et al., 1974a). Trimethylamine has been linked to fishy odors and flavors. Dimethylamine may be a good quality index for frozen fish. Babbitt et a / . (1972, 1974a) observed that TMA and DMA levels were two to four times as high in mechanically deboned minced muscle as in intact fillets immediately after deboning. Froning and Johnson (1973) studied the effect of centrifugation of mechanically deboned fowl meat on subsequent lipid stability. Centrifugation significantly lowered TBA values in mechanically deboned fowl meat. It was postulated that commercial-scale continuous centrifugal separators may be adaptable to improving the flavor characteristics of mechanically deboned poultry meat. Baker et 01. (1974) investigated the effect of chopping times on the acceptability of frankfurters made from mechanically deboned poultry meat from backs and necks of chicken fryers. Chopping times had little effect on taste acceptability. Taste panel results showed, however, that frankfurters made from flesh separated on two types of deboners differed in overall acceptability. Formulation variables have been studied to determine their influence on flavor characteristics. Froning et al. (1971) investigated the effect of incorporation 15% mechanically deboned turkey meat into red meat frankfurters. Difference flavor tests, preference flavor tests, and TBA values indicated that frankfurters containing 15% mechanically deboned turkey meat were comparable to all-red-meat frankfurters in flavor stability if fresh, mechanically deboned turkey meat was used. Baker and Darfler ( 1975) studied the effect of type and level of fat and level of protein on flavor characteristics of frankfurters made from mechanically deboned turkey frame meat. Taste panel scores showed little difference between frankfur-

122

G . W .FRONING

ters made with chicken fat and pork fat at equivalent levels, but those made with cottonseed oil were less flavorable. Protein levels did not significantly influence flavor characteristics. Lyon et al. (1978a.b) reported that structured soy protein fibers at levels of 16-25% produced significant off-flavors in chicken meat patties made from mechanically deboned poultry meat. Off-flavors were weak to moderate in intensity. These workers theorized that the structured soy protein fibers imparted a flavor that differed from typical all-meat flavor.

B.

STORAGE

The mechanical deboning process has been found to affect storage stability. Considerable research has been conducted to further our understanding and control of flavor change during storage. Miyauchi P t al. (1975) observed that minced muscle blocks of black rockfish had a storage life at - 18°C of less than 4 months, as indicated by rancid odors. Washing of the minced muscle gently in ice water and allowing it to drain for several hours through a close-knit nylon mesh bag substantially improved storage stability. The washing reduced the water-soluble heme proteins (hemoglobin and myoglobin), thereby improving the lipid stability of the product. Babbitt et al. (1974b) reported that incorporation of shrimp in a fish-shrimp portion greatly enhanced the acceptability of mechanically deboned minced fish muscle. Babbitt er al. (1976) investigated the feasibility of utilizing this concept with minced rockfish, which has poor storage stability. The addition of shrimp to minced rockfish markedly improved the shelf life of the minced fish. This was directly related to decreased formation of malonaldehyde and peroxides. They postulated that the beneficial effects of incorporating shrimp with minced fish was due to substance(s) extractable in ethanol that exhibited antioxidant properties. Nakayama and Yamamoto (1977) monitored TBA values of several species of frozen stored minced fish. Surface TBA values were much less stable than those observed in core samples. The TBA values increased most rapidly at the surfaces of blocks prepared from dogfish flesh; the values for pollock and grey cod increased at a much slower rate. In all frozen samples tested, surface TBA values revealed a tendency to increase steadily rather than to decline after 4 or 5 months of storage at -20°C. Taste panel results did not exhibit a clear-cut relationship with TBA values. Panel evaluation indicated a marked loss in quality during the second month of storage and another pronounced drop in quality during the fourth month. Several studies have dealt with flavor changes in mechanically deboned poultry during storage. Dimick et uI. (1972) observed that keeping quality, as measured by carbonyl concentration and organoleptic evaluation, is maintained up to

MECHANICAL DEBONING OF POULTRY AND FISH

123

6 days at 3°C. In general, mechanically deboned turkey neck meat was the least stable in storage, followed in order by mechanically deboned spent fowl and mechanically deboned chicken backs and necks. Holding parts for 5 days at 3-5°C prior to deboning did not influence flavor stability. Froning et al. (1971) found that mechanically deboned turkey meat that had undergone 90 days of storage at -24°C was an inferior product, as indicated by flavor evaluation and TBA values. Dhillon and Maurer (1975a,b) indicated that, even though TBA values were higher in mechanically deboned poultry after 6 months of storage, summer sausages formulated from the same meat sources were highly acceptable. Johnson er a / . ( 1 974) reported that flavor and aroma scores of mechanically deboned turkey were constant through 10 weeks of storage but lower from 12 weeks on. The TBA values increased with both time and temperature of storage and followed patterns that were closely analogous to flavor and aroma scores. Attempts have been made to modify the composition of deboned poultry through centrifugation. Froning and Johnson (1973) found that centrifugation to remove excess heme and lipid components improved the storage stability of mechanically deboned fowl meat. Dhillon and Maurer ( 1975b) investigated the effect of centrifugation on the storage stability of mechanically deboned chicken meat and mechanically deboned turkey meat and observed no advantage. The flavor stability of mechanically deboned poultry and fish during storage is related to several variables, which are reflected in the differing results from the studies reported. Factors such as meat type, composition, and age prior to deboning, deboner settings, and temperature of deboning are important variables that influence the flavor stability of the mechanically deboned product. C.

INTERACTION WITH HEME PIGMENTS

Heme pigments in mechanically deboned meat are known to interact with intramuscular fat, causing flavor problems. The heme portions of the various heme pigments act as catalysts in the autoxidation of lipids in meat (Tappel, 1955). High-pressure conditions necessary for mechanical deboning may create significant increases in the temperature of the separated meat unless precautions are taken. Contact with the metal of the deboner and higher temperatures may result in increased oxidation of both heme and lipid components. Janky and Froning (1975) observed the oxidation rate of heme and lipid components in mechanically deboned turkey meat. Heme oxidation decreased as storage temperature was reduced from 30°C to - 10°C. Oxidation rates for heme and lipid components were virtually identical at temperatures below - 10°C. The oxidation rate constant for heme at 30°C was 0.047 hr-I. The rate constant of lipid oxidation was the same (0.23 mg of malonaldehyde per kilogram of meat per hour) at all temperatures examined above 20°C. Oxidation rates of heme and

G . W .FRONING

124

lipid components were quite similar at temperatures between 10 and 15"C, indicating more pronounced interaction at these higher temperatures. Lee et al. (19%) attempted to characterize the mechanism of lipid oxidation in mechanically deboned chicken meat. The catalytic effect of a mechanically deboned chicken meat homogenate was most active at neutral and alkaline pH. When heme proteins were destroyed by prior treatment of the homogenate with hydrogen peroxide, the catalytic function was decreased to less than 10% of the original activity. It was concluded that the heme proteins were the predominant catalysts of lipid oxidation. The relative concentration ratio of polyunsaturated to heme protein was also reported to be in the range where heme-catalyzed oxidation would occur at close to the maximum rate. Lee and Toledo (1977) investigated lipid oxidation in mechanically deboned fresh mullet. The lateral tissue (red muscle) along the visceral cavity and the bone marrow exudate appeared to be most susceptible to oxidative rancidity. The rate of change in TBA value was very rapid when these muscles had contact with iron surfaces. Silberstein and Lillard ( 1978) studied the pro-oxidant effect of hemoglobin, myoglobin, and total heme and nonheme pigments in mechanically deboned mullet. Studies on oxygen uptake, with oleic acid as a substrate, indicated that the heme protein content influenced pro-oxidant activity of the buffer extracts of deboned fish. Myoglobin was noted to have a greater catalytic effect than hemoglobin. It was concluded that, in addition to the concentration of total heme pigments, the hemoglobin-to-myoglobin ratio should be considered in ascertaining the influence of heme proteins on the oxidative stability of deboned fish. When total heme pigments were kept constant and ratio of hemoglobin to myo-

TABLE VIll OXYGEN UPTAKE OF OLEIC ACID AS INFLUENCED BY DIFFERENT CONCENTRATION OF HEME PIGMENTS'

Myoglobin

Mg 0.04 0.2 0.4 0.6

'I

Oxygen uptake (nmoledmin)

26.8 52.4 69.4 93.2

Hemoglobin

Mg 0.04 0.2 0.4 0.6

From Silberstein and Lillard (1978)

Oxygen uptake (nmoledmin)

13.5 24.4 45.2 53.0

Heme pigment mixture

Hb (mg)

Mb (mg)

Oxygen uptake (nmoleshin)

0.40

0.20 0.25 0.30 0.35 0.40

55.6 63. I 71.8 75.1 84.9

0.35

0.30 0.25 0.20

MECHANICAL DEBONING OF POULTRY AND FISH

125

globin was varied, the rate of oxidation uptake increased as the concentration of myoglobin increased (Table VIII). D.

EFFECT OF ANTIOXIDANTS

Since lipid oxidation in mechanically deboned poultry and fish is a major problem, several researchers have investigated the use of antioxidants to maintain desirable flavor attributes. Moledina et al. (1977) studied the effectiveness of a combination of antioxidants, chelating agents, and polyphosphates in retarding the development of rancidity in mechanically deboned flounder meat. The most effective technique to maintain desirable flavor was a 1-min dip of the racks, prior to deboning, in a pH 4.5 solution of 0.5% each of ascorbic and citric acids and 0.2% of Na, EDTA and polyphosphates, followed by a postdeboning addition of 0.3% each of ascorbic and citric acids and 0.2% each of polyphosphates and Na, EDTA. Moms and Dawson (1979) observed that FreezGard (NaCI, Na tripolyphosphate, Na erythorbate) (Calgon Corporation) was an effective antioxidant for frozen mechanically deboned sucker flesh stored for 12 months at -18°C. FreezGard was a more effective antioxidant than the phenolic antioxidants Tenox A, Tenox 11, or Tenox PG. The presence of tripolyphosphates and sodium erythorbate in FreezGard was probably a factor. It was postulated that tripolyphosphates may sequester metal ions and that sodium erythorbate may interfere with heme-catalyzed oxidation (Watts, 1950). The lack of effectiveness of phenolic antioxidants may have been due to their poor solubility for adequate distribution throughout the minced flesh (Watts, 1961). Mai and Kinsella (1979) investigated the effectiveness of the antioxidants BHA and TBHQ on storage stability of minced carp tissue that was baked and deep-fat-fried and held at -18°C for up to 8 weeks. Samples treated with antioxidants gave significantly higher values for free fatty acids than did the controls. The TBA values were higher in the cooked samples than in the raw samples. Samples containing antioxidants were observed to have lower TBA values than did control samples after storage. Both BHA and TBHQ were effective antioxidants, with TBHQ being slightly better. Antioxidants were more effective in baked samples than in deep-fat-fried samples. It was postulated that deep-fat frying reduced concentration of antioxidants by volatilization and leaching. Froning (1973) chilled spent fowl carcasses in 6% polyphosphate in ice slush overnight prior to mechanical deboning. Polyphosphate-treated mechanically deboned fowl meat was observed to have significantly lower TBA values than were found in the controls after storage at -29°C for two months. Polyphosphate treatment prior to mechanical deboning apparently protected the product against oxidative changes during the critical deboning cycle and subsequent frozen storage.

126

G . W. FRONING

MacNeil et al. (1973) noted that rosemary spice extractives, polyphosphates, and BHA + citric acid were effective antioxidants in simulated mechanically deboned poultry meat. Both sensory tests and TBA values were utilized to assess antioxidant capabilities. Moerck and Ball (1974) observed that the addition of Tenox I1 at 0.01% by weight of fat present extended the induction period for fat oxidation and the keeping quality of mechanically deboned poultry held at 4°C. The TBA values for samples treated with Tenox I1 remained below the rancidity threshold of 1. E. EFFECT OF CO, AND NZ COOLING The mechanical deboning process can generate considerable heat, thereby initiating lipid oxidation and rancid flavor. Heat buildup may be minimized through the use of heat exchangers and application of cryogens such as CO, or Np. Processors have used C02 or N, to chill and freeze mechanically deboned poultry meat (Cunningham and Mugler, 1974). Uebersax et al. (1977) precooled mechanically deboned chicken meat with “CO, snow,” using a tumbling process. The cooling of mechanically deboned chicken meat with C 0 2 snow activated lipid oxidation, resulting in higher TBA values. Whether the activation was due to the chemical reaction with CO, snow or to the mixing of additional air by the tumbling action was not determined. Vacuum packaging of mechanically deboned chicken meat significantly improved storage stability as measured by TBA values. Uebersax el al. (1 978) precooled mechanically deboned chicken meat and mechanically deboned turkey meat in a CO, cooling chamber attached to a deboner. The highest TBA values were observed in meat samples cooled with CO, . Mixing without CO, was investigated, but it did not appear to be a major factor for increased TBA values. These researchers recommended, however, that CO chilling for mechanically deboned poultry should not be eliminated, since CO, chilling does have a definite microbiological advantage. Uebersax et al. (1978b) further observed that meat mixed in air and CO, showed higher TBA values than were found in the unmixed control meat or in meat mixed under nitrogen Mast et al. (1979) also noted that CO, snow may be a contributing factor in the development of oxidative rancidity in mechanically deboned poultry meat as demonstrated by elevated TBA and peroxide values. However, the incorporation of small amounts of CO, snow into the meat did not have a detrimental effect on sensory acceptability of the product, whereas large amounts of CO, did detract from the acceptability of mechanically deboned poultry meat. These studies indicate that CO, chilling or freezing may not be advisable because of its apparent triggering of oxidative rancidity. Use of liquid N, as a cryogen may be a feasible option, since it apparently does not affect TBA values as drastically as does CO,,

,

MECHANICAL DEBONING OF POULTRY AND FISH

V.

127

COLOR STABILITY

A. INFLUENCE OF PROCESSING VARIABLES AND STORAGE Color abnormalities (brown, green, and gray) are known to be a problem in mechanically deboned poultry and fish. Processing and storage parameters have been major factors considered in studies of these abnormalities. Conditions of mechanical deboning have been shown to be involved in the color characteristics of mechanically separated meat tissue. Froning et al. (1973) studied the effect of skin content prior to deboning on the color of mechanically deboned chicken back meat. Higher skin levels generally increased Gardner L values, decreased Gardner a l , values, and increased bl, values of the mechanically deboned chicken meat. The fat from the skin apparently diluted the heme pigments, thereby producing a mechanically deboned product that was lighter in color, less red, and more yellow than meat with no skin prior to deboning. Froning and Johnson (1973) attempted to modify the color characteristics of mechanically deboned fowl meat by centrifugation. Centrifugation significantly reduced Gardner L values and significantly increased Gardner ul, values. The higher trend for a,, values was believed to be associated with differences in total pigment concentration. It was postulated that Gardner values may be used as an index of heme pigment concentration and perhaps of pigment destruction during storage. Dhillon and Maurer ( 1975b) noted less redness in centrifuged mechanically deboned turkey and chicken broiler back and neck meat than in their noncentrifuged controls. This is the opposite effect of that reported by Froning and Johnson (1973). Perhaps the discrepancy can be partially explained by the fact that Froning and Johnson used mechanically deboned fowl in their centrifugation studies. Use of cryogens during chilling and freezing has been shown by some workers to influence color characteristics. The C0,-mixed meat is darker (decreased L values) and redder (increased uI,values) than the unmixed controls (Uebersax et a l . , 1977; Cunningham and Mugler, 1974). Generally, mechanically deboned poultry meat, whether C0,-chilled or not, became darker and grayer during storage (Uebersax et al., 1978; Mast et al., 1979). Variables in the processing formulation, as related to color, have been investigated by numerous workers. Higher levels of structured soy protein fibers in patties containing mechanically deboned poultry meat generally resulted in an increase in Gardner L values (lightness) and a decrease in Gardner a L values (redness) (B. G. Lyon et al., 1978; C. E. Lyon et a l . , 1978a,b). Dhillon and Maurer ( 1975a) formulated summer sausages with various combinations of mechanically deboned turkey meat and mechanically deboned chicken meat with ground beef. Appearance scores were quite satisfactory for 50/50 mixtures of mechanically deboned turkey meat and beef and for 50/50 mixtures of mechanically deboned chicken meat and beef. The highest sensory color scores were observed in sausage made from a 50/50 mixture of mechanically deboned

I28

G . W . FRONING

chicken and beef. Hunter color values were quite similar for all formulations. Froning et ul. (1971) found that red meat frankfurters containing 15% mechanically deboned turkey meat exhibited slightly lower a,, (less redness) values than did frankfurters formulated with 100% red meat. The effect of storage on the color stability of mechanically deboned poultry has been the subject of considerable research in recent years. Froning ef ul. (1971) noted that frankfurters containing 15% turkey meat exhibited lower a,, values than the control 100% red meat frankfurters when stored under dark refrigerated or dark frozen conditions. Color fading occurred in both frankfurter formulations after 60 days of storage, but this fading would probably not be noticed by the average consumer. Dhillon and Maurer (l975b) compared the color stability of mechanically deboned chicken and turkey meat with that of ground beef after a 6-month storage period at -25°C. The intensity of redness was highest in ground beef, but all meat sources exhibited some color fading after prolonged storage. The color stability of mechanically deboned fish has been found to be influenced by processing and storage variables. Miyauchi et a / . (1975) washed minced muscle from black rockfish with chilled water to reduce the amount of blood and flesh pigments; both color stability and appearance were improved. Moledina et al. (1977) found that postdeboning addition of 0.1% citric acid, 0.2% Kena (A mixture of sodium polyphosphates supplied by Merck and Company, Rahway, New Jersey) and 0.25% Na, EDTA with either 0.1% ascorbic acid or 0.1% sodium erythorbate to mechanically deboned flounder meat improved both the initial Hunter L color value and subsequent values during storage. When pH values were below 5.8-6.0, a chalky colored product was noted. Post-deboning additions of 0.3% each of ascorbic acid and citric acids and 0.2% each of Kena and Na, EDTA were effective in minimizing color deterioration in mechanically deboned flounder meat during frozen storage without giving a chalky appearance. Nakayama and Yamamoto (1977) studied the color stability of deboned flesh from several underutilized fish species after 6 months of storage at -20°C. During frozen storage, the deboned flesh of pollock became less dark, whereas the flesh of the shortspine thornyhead, turbot, and dogfish revealed a gradual shift to more “yellowness. ’ ’ After cooking, color was somewhat darker with extended storage. Cooked turbot flesh was lightest in color of all species examined. B.

CHANGES IN MYOGLOBIN DUE TO MECHANICAL DEBONING

During the mechanical deboning process, the state of myoglobin is probably affected, thereby influencing color changes. Oxygen is mixed thoroughly with the meat, converting myoglobin to oxymyoglobin. Oxidation at the surface during storage may also form metmyoglobin, giving the product an undesirable brown color (Janky and Froning, 1975). Brown et al. (1957) indicated that

MECHANICAL DEBONING OF POULTRY AND HSH

129

TABLE 1X ISOELECTRIC POINTS (PI) OF TURKEY MYOGLOBIN EXTRACTED FROM MECHANICALLY DEBONED TURKEY MEAT"

Isoelectric point (PI)

Intact muscle

Mechanically deboned turkey muscle

Mb I Mb I

7.99 7.88

7.58 7.41

MbII Mb I1 Mb 11

7.42 7.01 6.79

7.02 6.99

Mb 111 Mb 111

6.47 6.38

6.38 6.12

Component

"

-

From Janky and Froning (1975).

brown discolorations in whitefish were due to conversion of oxymyoglobin or oxyhemoglobin to metmyoglobin and methemoglobin. Furia (1972) reported that high concentrations of metals in fish, together with amines, sulfhydryl, and phenolic compounds, may catalyze undesirable color changes. Janky and Froning (1975) compared the nature of myoglobin purified from mechanically deboned turkey meat with that from hand-deboned sources. Electrophoretic patterns for myoglobin extracted from mechanically deboned turkey meat were similar to those previously described by other workers for myoglobin extracted from intact muscle. Three distinct fractions were detected electrophoretically. Six fractions isolated by isoelectric focusing exhibited lower isoelectric points than did the fractions from intact muscle (Table IX). It was postulated that this phenomenon was due to binding of anions by the heme complex and was possible related to the amount of contact the meat had with the metal surface of the mechanical deboner. Another source of anions may have been calcium and phosphorus, which may act as catalytic agents in heme oxidation.

VI.

FUNCTIONAL CHARACTERISTICS A.

EFFECT OF COMPOSITION

Mechanically deboned fish and poultry have been utilized in a number of formulated products. Greatest usage has occurred in emulsified products. As discussed earlier in this review, the mechanical deboning process does affect the composition of the product. Functional attributes are known to be closely related

130

G. W .FRONING

to many components in the meat system, such as myofibrillar and connective tissue proteins. Several studies have attempted to further define functional and compositional interrelationships. Webb et al. (1976) compared mechanically separated fish muscle tissue with hand-separated tissue to determine the relative effects of the deboning processes on the functional properties and texture of the final products. Mechanically separated tissue had higher quantities of sarcoplasmic and nonprotein nitrogen than did hand-separated tissue. Myofibrillar protein content, as determined by sodium dodecyl sulfate (SDS) extraction, was similar, although hand-separated tissue had a slightly higher content of the myofibrillar fraction. When NaCl was used for extraction of myofibrillar proteins, an insoluble sol was sedimented as a gel by centrifugation, thereby giving somewhat lower myofibrillar protein yields for both separation techniques. Stroma (connective tissue) protein was somewhat higher in hand-separated tissue than in mechanically separated tissue. Emulsifying capacity and cooking stability were comparable for the two treatments. The texture of a comminuted product prepared from hand-separated tissue was somewhat better than that of a product prepared from mechanically separated tissue. The insoluble sol formed by NaCl extraction may have resulted in the firmer texture for the hand-separated product, since the hand-separated tissue had a higher level of this insoluble gel fraction. It was further postulated that the shearing action of the mechanical separator produced stress on the myofibrillar proteins, resulting in denaturation. Cheng er al. ( 1979b) investigated protein functionalities in comminuted fish gels from mechanically deboned fish tissues of different species after storage at -29°C for periods up to 12 months. Panel members were able to detect significant differences in gel texture between species. Gels made from tissues stored for longer periods were less firm and springy. In uncooked fish gels, texture was not related to protein solubility, but it appeared to be closely related to the waterholding capacity and protein solubility of cooked gels. SDS-polyacrylamide gel electrophoresis indicated that proteolytic degradation of tropomyosin and other protein probably occurred in some of the fish gels during thermal processing. Cheng et al. (1979a), in a related study, noted that degradation of tropomyosin and myosin in cooked fish was highly related to textural properties. Their study also suggested that changes in muscle proteins during heating were caused by a proteolytic factor(s) in the sarcoplasmic fraction. McMahon and Dawson (1976) determined the amount of salt-soluble proteins in hand-deboned turkey and mechanically deboned turkey meat. The percentage of salt-soluble proteins was somewhat lower in mechanically deboned turkey meat than in hand-deboned sources. Emulsification capacity was superior in hand-deboned turkey meat, on both a meat and a protein basis. On the other hand, water-holding and water-binding capacities were higher in mechanically deboned turkey meat than in hand-deboned meat. Some studies have related various formulation changes in mechanically de-

MECHANICAL DEBONING OF POULTRY AND FISH

131

boned sources to changes in functional performance. Baker and Darfler (1975) varied the type and level of fat and the level of protein in products made from mechanically deboned turkey frames. In general, higher fat levels produced firmer frankfurters with more viscous emulsions. The type of fat had little effect on shear values, but pork fat produced a more viscous emulsion. Increasing the protein level of frankfurters increased shear values and emulsion viscosity, but lowered the taste panel scores for tenderness and juiciness. In a similar study, Mayfield et al. ( I 978) utilized a temperature-controlled, capillary extrusion viscometer to compare meat batters prepared from mechanically deboned poultry meat. Protein levels of 12% produced more viscous batters and less gel-water release and fat release during emulsion stability tests than did levels of 11% protein. The viscosity of meat batters increased with higher levels of fat, but less stable batters were encountered at higher fat levels. Other aspects of formulations have been studied. M. A. Uebersax et al. ( I978a) evaluated turkey loaves containing various levels of mechanically deboned turkey meat. Cooked yield and loaf size inproved with increased levels of mechanically deboned turkey meat. Slices possessed less binding strength and were more tender with increased amounts of mechanically deboned turkey meat. The textural properties of patties made from mechanically deboned turkey meat have been improved by adding structured soy protein fibers (B. G. Lyon et al. 1978; C. E. Lyon et ul., 1978a,b). The relationship between the source and type of mechanically deboned poultry parts and functional performance has been studied. Grunden er al. (1972) investigated the composition of mechanically deboned broiler backs and necks, mechanically deboned spent layers, and mechanically deboned turkey backs as related to apparent viscosity. Deboned meat from female turkey backs was less viscous than other products. No significant correlation existed between viscosity and moisture, viscosity and protein, or viscosity and pH. Orr and Wogar (1979) reported that mechanically deboned chicken necks and backs from different sources exhibited significantly different values for emulsifying capacity, waterholding capacity, emulsion stability, percentage of fat, and percentage of moisture. Froning et al. (1973) studied the influence of skin content of mechanically deboned poultry on various functional characteristics. As the level of skin content prior to deboning was increased, there was a significant decrease in emulsion stability and emulsion capacity, reported as milliliters of fat emulsified per 2.5 gm of meat. The changes in emulsifying stability and emulsifying capacity (meat basis) were closely related to the higher fat content at increased skin levels. When emulsifying capacity was reported as milliliters of fat emulsified per milligram of protein, there was no significant change in emulsifying capacity with increasing skin levels. Schnell et al. (1973) observed that increasing the amount of skin added to a frankfurter formula increased the fat content, increased organoleptic tenderness and viscosity, and decreased emulsion stability.

132

G . W . FRONING

Lee and Toledo (1979) studied process requirements and formulations necessary to prepare a coarse-textured smoked fish sausage. Sausages were prepared from Spanish mackerel using two comminution processes and different levels of shortening, soy protein fiber, and added ice. The addition of 12 gm of vegetable shortening per 100 gm of fish muscle and soy protein fiber significantly improved the taste panel ratings on texture relating to the structure of material and increased juiciness. The texture and general acceptability of products prepared from mechanically deboned fish were comparable to those of products made from filleted fish if moisture content and bone residue were carefully controlled in the raw material. B.

EFFECT OF PROCESSING VARIABLES

Processing conditions during deboning and modifications thereafter influence the textural characteristics of mechanically deboned fish and poultry. The texture of mechanically deboned flesh is somewhat different from that of hand-separated flesh. Mechanically deboned poultry has a very fine emulsion-like consistency, whereas mechanically deboned fish may have a texture varying from a fine emulsion-like consistency to one of larger meat particle sizes. Miyauchi (1974) studied the effect of the particle size of minced fish on texture. Coarse minced flesh was obtained by using a separator having a drum with 7-mm-diameter holes, and finely minced strained flesh was taken from a Bibun deboner with 1 .Cmm-diameter holes. The coarse minced samples received the higher textural scores. Textural scores did not appear to be affected by storage. Wong et al. (1978) compared the textures of mechanically deboned fish flesh from deboners with various perforation sizes. Texture (resistance to compression) was measured by using at Ottawa Texture Measuring System. The texture of minced flesh generally increased with larger particle size. Minced rockfish and pollock flesh showed the most prominent differences for samples obtained through 2-mm and 7-mm holes. There was a decrease in texture of herring deboned in a drum with 7-mm perforations, compared with the texture of herring obtained through 5-mm orifices. These data suggest that smaller species of fish, such as hening, may require a different optimum orifice size than do larger species. These workers further observed that minced fish flesh that had been washed and dewatered had a somewhat firmer texture. Keay and Hardy ( 1 974) reported on the feasibility of changing the textural characteristics of comminuted fish flesh by extrusion techniques. They reported that, by judicious addition of calcium ions to alginate solution, precipitation of gelatinous hydrolyzed calcium alginate takes place, and the viscosity of the resultant jelly is stable and highly controllable. The jelly was stable to both heating and freezehhaw variables. Fish sticks were prepared by layering, thereby simulating the flake-like appearance of whole fish.

133

MECHANICAL DEBONING OF POULTRY AND FISH

Acton ( 1973) texturized mechanically deboned chicken neck meat with and without skin, using an Osterizer food grinder. The meat was forced along the grinder path (cutting blade omitted) through a plate with a single 4-mm orifice. Texturization was accomplished by heating at 100°C for 1 , 3 , 5 , 7 . 5 , and 10 min. Table X presents some of the attributes of the texturized meat strands. As heating time increased, there was a significant increase in shear resistance, indicating that the degree of firming of the tissue can be controlled by varying the exposure to heat. Skin generally increased shear resistance. Binding strength showed an increase in tensile strength as process time was lengthened. Samples processed for 3 min had very poor binding strength. Skin did not change binding characteristics. Although heating resulted in a significant loss of extractable protein, the meat tissue's ability to hydrate a 0.6 M NaCl solution increased. The emulsion stability of the extruded strands was enhanced by longer heating times, with no differences attributed to skin content. Froning and Johnson (1973) attempted to modify mechanically deboned chicken fowl meat by centrifugation at 48,200 g for 15 min. Emulsion stability was significantly improved by the centrifugation technique. Dhillon and Maurer ( 1975b) reported also that centrifugation improved the water-holding and emulsifying capacity of mechanically deboned chicken and turkey meat. It appears that centrifugation may enhance the functional attributes of mechanically deboned poultry meat. Commercial-scale centrifugal separators are available and possibly could be adapted to this purpose. Froning (1970) investigated the effect of chopping time and temperature on mechanically deboned poultry meat. Mechanically deboned chicken and turkey meat showed good emulsion stability when chopped at temperatures of 7.212.8"C. Mechanically deboned poultry meat chopped at temperatures higher than 12.8"C had inferior emulsion stability. Hand-deboned chicken broiler meat TABLE X SHEAR FORCE, BINDING STRENGTH, WATER-HOLDING CAPACITY, AND EMULSION-STABILIZING CAPACITY OF TEXTURIZED BROILER NECK MEAT STRANDSO

Shear force (kg/gm)

Binding strength Water-holding capacity Emulsion stability

Minutes at 100°C

With skin

Without skin

With skin

Without skin

With skin

Without skin

With skin

Without skin

0 3 5 7.5 10 12

-

2.6 3.2 4.7 5.6 6.3

-

3.7 4.1 4.6 5.4 6.5

2.2 3.8 4.7 6.2 7.4

3.6 5.2 7.6 8.2 8.4

17.7 11.4 16.6 41.1 53.4 65.1

16.0 10.3 15.4 36.0 53.1 63.7

12.4 12.2 7.0 6.2 7.8 4.0

11.0 13.5 10.5 4.5 3.2 3.5

" From Acton (1973).

134

G . W. FRONING

possessed good emulsion stability when chopped at temperatures above 12.8"C. Histological examination showed that mechanically deboned poultry meat has less of a protein matrix available for emulsion formation than do hand-deboned sources. The lack of a protein matrix may be due to protein loss from heat denaturation during the deboning cycle. It was indicated that a combination of hand-deboned and mechanically deboned poultry is desirable to improve the stability of emulsified products. Baker et al. (1974) found that mechanically deboned poultry from some machines is affected more by overchopping than is poultry from other machines. These workers suggested that overchopping should not be a problem as long as chopping temperatures are kept below 12.8"C. Angel et al. (1974) noted that emulsion formation is complete for mechanically deboned poultry meat after 1%-3 min of chopping. Lee and Toledo (1976) studied the effect of extensive chopping on comminuted fish muscle. As chopping was intensified, mechanical strength diminished, and the concentration of extractable myosin in the muscle homogenates decreased. Formulations with higher moisture contents were more susceptible to adverse changes brought about by mechanical treatment such as chopping. Young and Lyon ( 1 973) investigated heat treatment of mechanically deboned chicken meat at 65°C to reduce bacterial load. In general, frankfurters were satisfactory when they contained up to 30% heated meat, but higher levels of heated meat produced inferior frankfurters. As the percentage of heated meat increased, the frankfurters were less firm and less juicy. C. EFFECT OF FOOD ADDITIVES The altering of the textural and binding characteristics of mechanically deboned tissue by use of various food additives and binders has been investigated by several researchers. Since mechanically deboned tissue generally has a lower protein and a higher fat content, binding and water-holding capacities are sometimes quite different from those observed in intact muscle. Improvement of minced fish has been emphasized in some studies. Teeny and Miyauchi (1972) developed a modified minced fish block. Sodium chloride and sodium tripolyphosphate were utilized to partially solubilize the muscle protein in order to bind the particles of coarse-minced muscle into a cohesive block. Miyauchi et ul. (1975) washed minced muscle from black rockfish to remove water-extractable constituents and added a binder mixture to improve the textural attributes of minced muscle (Table XI). The binder improved the cohesiveness, the water-holding capacity, and the succulence of minced rockfish muscle. Lee and Toledo (1976) investigated the effect of sodium chloride and polyphosphates on the textural characteristics of comminuted fish muscle. Textural characteristics were evaluated objectively by using as an index compression

135

MECHANICAL DEBONING OF POULTRY AND FISH TABLE XI FISH BINDER ADDED TO 100 POUNDS OF MINCED MUSCLE“

Ingredients”

Weight (Ibkach 100 Ib of minced muscle)

Fish muscle Sodium chloride

2.5 I .o

Sodium tripolyphosphate

0.15

sugar Monosodium glutamate Corn oil

I .o 0.3 I .o

Ice water

5.0

~~

Function Improves textural properties Stabilizes protein for textural properties; flavor Solubilizes protein; improves water-holding capacity Taste Flavor intensifier Carrier for oil-soluble antioxidants Carrier for water-soluble antioxidants

~~

From Miyauchi et a / . (1975). The ingredients were homogenized for about 2 minutes until the mixture became “sticky” or “tacky. ” “

strength, modulus of elasticity, resilience, and shear strength. In the absence of salt, textural strength was extremely weak with respect to shear and compression strength. Low compression strength was due to lack of cohesion between particles. When 0.3% polyphosphates were added in addition to 2% sodium chloride, samples showed significant increases in compression strength, modulus of elasticity, and resilience when compared with samples containing sodium chloride alone. These workers found that polyphosphates increased the solubilization of muscle protein and improved the water-binding characteristics of comminuted fish muscle. Moledina et al. (1977) dipped flounder racks, prior to deboning, in a pH 4.5 solution of 0.5% each of ascorbic acid and citric acids and 0.2% each of Na, EDTA and Kena, followed by postdeboning addition of 0.3% each of ascorbic acid and citric acids and 0.2% each of Kena and Na, EDTA. This treatment decreased the emulsifying and water-holding capacities of mechanically deboned flounder meat but did not produce a chalky appearance or granular texture. Taste panel evaluation indicated that the treated mechanically deboned flounder meat was “tougher” and “drier” than the untreated control. Cobb and Yeh ( 1974) discussed the effect of several additives on the texture of minced Atlantic croaker. Sodium chloride increased the firmness of croaker patties, with 2% salt content being preferred for both taste and texture. The addition of CaHP0, to croaker patties had a positive effect on firmness but was less effective than sodium chloride. A flat taste was observed if CaHPO, ex-

I36

G. W . FRONING

ceeded 3% in the patties. Sugar improved the texture slightly. Egg albumen at the 2% level was the most effective additive in improving the texture of croaker patties. The addition of starch was beneficial in water-washed croaker, reducing the problem of “rubbery” texture. Froning and Janky ( I 97 1 ) investigated the feasibility of modifying mechanically deboned poultry meat through pH adjustment and salt preblending prior to freezing. Such modification markedly improved the emulsifying stability of the products. It was concluded that salt preblending could be used in conjunction with pH adjustment to control the variability of emulsifying characteristics. Schnell et a / . (1973) studied the effect of selected additives on frankfurters prepared from mechanically deboned chicken meat. The addition of 3% acid whey, 0.5% Kena, or I .75% sodium chloride to the meat prior to mechanical deboning had little effect on organoleptic acceptability of frankfurters. Sodium caseinate (3%) produced a less juicy frankfurter, but other additives had little effect on juiciness. The viscosity of the emulsions prepared from mechanically deboned chicken meat was definitely affected by several of the additives. Sodium caseinate and Kena both significantly increased the viscosity of the frankfurter emulsions. Adding salt or acid whey prior to deboning had little effect on viscosity. Froning (1973) chilled spent fowl in 6% polyphosphate (Kena) overnight prior to mechanical deboning. After chilling, the whole carcasses were mechanically deboned. Polyphosphate treatment significantly increased emulsification stability as measured by fat and gel-water release. The polyphosphate chilling treatment also improved the emulsification capacity of the mechanically deboned fowl meat. McMahon and Dawson (1976) observed the effects of salts and polyphosphates (Kena) on the functional properties of hand-deboned and mechanically deboned turkey meat. A combination of 0.5% phosphate and 3% sodium chloride increased the amount of extractable protein from both meat systems. Waterholding capacity, water-binding capacity, and emulsifying capacity were improved when salt was added, and a synergistic effect was observed when both salt and phosphates were used. However, water-holding capacity, water-binding capacity, and emulsifying capacity of mechanically deboned turkey meat were improved by 0.5% phosphate addition to a greater extent than by either 3% sodium chloride alone or a combination of 3% sodium chloride and 0.5% phosphate. Some researchers have investigated the use of structured soy protein in combination with mechanically deboned poultry meat. Janky et UI. (1977) observed that the addition of structured soy protein decreased emulsion stability. However, B . G . Lyon et a / . (1978) and C. E. Lyon et ul. (1978a,b) noted that structured soy fibers generally improved the textural properties of patties made from mechanically deboned poultry meat.

MECHANICAL DEBONING OF POULTRY AND FISH

VII.

137

UTILIZATION OF BONE RESIDUE

The bond residue from the mechanical deboning process has been investigated as a source of protein and minerals for animal and human diets. Since there is a world shortage of protein, this residue cannot be ignored. Also, the utilization of bone residue would be helpful in controlling pollution from processing plants. Crawford et al. (1972) studied the composition of bone-skin residue from the mechanical separation of fish flesh. Table XI1 presents proximate composition of the bone-skin fractions. These data indicate considerable variation with respect to the different species. Nevertheless, the residue contains considerable protein and ash, which may have excellent potential either as a source for a protein isolate or as a mineral source in animal diets. Young (1975) outlined a scheme for obtaining a protein isolate from mechanically deboned poultry meat. Young (1976) observed that this concept could also be applied to bone residue. The procedure is outlined in Fig. 3. One part by weight of bone residue was mixed with three parts by volume of 0.1 M sodium maleate at pH 7.0. Sodium chloride was added to adjust the ionic strength to 0.5. The protein isolate contained 60-65% protein, 23-25% lipid, 5-10% ash, and 4-6% moisture. Solubility and emulsifying characteristics of the isolate were improved at elevated pH (above pH 7.5) and increased ionic strength in the presence of polyphosphates. The author concluded that the protein isolate prepared from bone residue may have some utilization as a food ingredient. Although this procedure was developed with mechanically deboned poultry, it could be adapted with some modification to bone residue from mechanically separated fish. Wallace and Froning (1979) investigated the protein quality of bone residue from mechanically deboned chicken back and neck meat. The residue was finely ground and lyophilized. Fat was extracted by using an ether extraction. Two fractions were obtained, one being rather fibrous in nature and the other being the . TABLEXII PROXIMATE COMPOSITION OF BONE-SKIN RESIDUE'

Fish English sole Petrale sole Orange rockfish Yellowtail rockfish True cod Ling cod "

From Crawford et d.(1972)

74.8 67.8 62. I 61.9 74.9 69.7

3.0 7.2 5.3 6.4 I .2 3.9

15.5 16.7 19.2 19.6 17.9 19. I

7.2 9.0 14.6 12.6 7.0 8.0

G . W. FRONING

138

Buffer pH 7.0, ionic strength 0.5 3 parts

Bone residue I part

I--

Precipitate

Supernatant

I

Filter

-1

Residue

Filtrate

I

Dilute to ionic strength 0.2

-1

Supernatant (discard)

Precipitate Wish

k-

Supernatant (discard)

Centrifuge

I I

Precipitate Wash

-1

Supernatant (discard)

Centrifuge

I

Precipitate (dry)

FIG. 3 .

Preparation of protein isolate from bone residue. From Young (1976)

final bone residue by-product. The final product contained 42.0% protein, 6.2% moisture, 0.10% fat, and 50.7% ash, with a computed PER value of 1.78. This PER value was somewhat lower than the casein-adjusted PER of 2.5. Although the PER value was low, these authors indicated that it compared favorably with that of many other proteins such as yeast protein concentrate, corn distiller’s protein concentrate, and high-protein wheat bran flour. It is likely that the PER value of protein from bone residue is low because of the skin collagen, which is largely found in the residue fraction. Nevertheless, this by-product could be an excellent animal feed protein and mineral supplement.

VIII.

MICROBIAL QUALITY

The process of mechanical deboning causes considerable maceration of the tissue, as discussed earlier in this review. Microbial contamination may be easily blended throughout the deboned tissue. Also, heating during the deboning cycle

MECHANICAL DEBONING OF POULTRY AND FISH

139

may enhance bacterial growth. Several studies have been conducted on bacterial proliferation in mechanically deboned flesh. Ostover et a / . (197 1) examined mechanically deboned meat from broiler necks and backs, whole fowl, and turkey racks for total aerobic counts, fecal coliforms, salmonellae, Clostridum perfringens, coagulase-positive staphylococci, and psychrotolerant microorganisms. The meat was deboned immediately after the birds were processed (conventional processing) or after they had been held in the plants at 35°C for 5 days (delayed processing). Aerobic counts increased during storage at 3°C after both treatments, but were higher in delayed processed meat than in conventionally processed meat. Fecal coliforms were high in all samples and remained high during storage at 3°C. Freezing substantially reduced fecal coliforms. Six out of fifty-four samples were contaminated with salmonellae, and four showed the presence of C . perfringens, but none were contaminated with Staphylococcus aureus. Pseudomonas, Achromohacter, and Flavohacterium were the predominant psychrotolerant genera found in this study. Maxcy et al. (1973) studied microbial quality of ground hand-deboned poultry and mechanically deboned poultry. Several kinds of bacteria were noted, with Bacillus species accounting for the largest percentage. These workers indicated that the microbial load in either hand-deboned ground poultry or mechanically deboned poultry was comparable to that found in similar types of red meat products. Lillard (1977) analyzed mechanically deboned chicken backs and necks for levels of aerobic organisms and for incidence and levels of C. perfringens vegetative cells and spores before and after 46 weeks of storage at -23°C. Frozen storage significantly reduced the incidence and levels of vegetative cells and spores but did not affect levels of aerobic organisms. These findings indicated that C . perfringens should pose no hazard in mechanically deboned poultry if good manufacturing practices are followed and the frozen products are properly handled. The shelf life of fresh mechanically deboned poultry has been a problem, leading to investigations of techniques for prolonging shelf life. MacNeil et a / . ( 1973) observed that rosemary spice extract, butylated hydroxyanisole (BHA), and citric acid lowered bacterial counts in mechanically deboned poultry meat, thereby extending the shelf life. Mast and MacNeil (1975) noted that heat pasteurization of mechanically deboned poultry meat increased shelf life, as measured by total bacterial counts. Raccach et a / . (1979) found that the shelf life of mechanically deboned poultry meat could be extended by 2 days by using the resting cells of the starter cultures Pedincoccus cerevisiae (Accel) and Lactobacillus plantarum (Lactacel D.S.). Fluorescent psychrotrophic colonies were not detected in the treated samples but were observed in the controls. This study indicated that lactic acid bacteria may be used to control spoilage and pathogenic microorganisms in poultry meat products. Raccach and Baker (1978) noted that mechanical deboning of several tra-

140

G. W . FRONING

ditional and underutilized fish species increased the microbial count tenfold. The shelf life of minced fish was 5 days at 2°C and 3 days at 12°C. Bacterial and coliform counts showed little change during frozen storage. These studies indicate that mechanically deboned fish and poultry are similar microbiologically to hand-deboned red meat sources. If good quality control is practiced, mechanically deboned fish and poultry meat will generally have excellent shelf-life capabilities. The use of raw material with low bacterial numbers and a cool environment during the deboning cycle will usually assure a highquality deboned product from a microbiological standpoint.

IX. REGULATIONS The U.S. Department of Agriculture (USDA) regulates the production of mechanically deboned poultry meat, and the Food and Drug Administration (FDA) regulates fish and fishery products. The National Marine Fisheries Service of the Department of Commerce also works closely with the FDA in developing standards for minced fish products. A.

NAME AND LABELING

Presently, poultry is named mechanically deboned turkey meat, mechanically deboned chicken meat, etc., depending on the kind of poultry used in the product. The label need only state chicken, turkey, etc., without indicating that the source of the product was processed through a mechanical deboner. Label requirements are currently under review by the USDA. “Minced fish” has been established by the National Marine Fisheries Service as the name for mechanically deboned fish flesh. The FDA has also established this label for foods made from this product. An example would be “Fish Sticks, made from minced fish,” with the second phrase in type at least half the size of the product name. B.

LIMITS

Ryan (1974) reported on proposed United States standards for grades of minced fish blocks. Several aspects and guidelines were mentioned, including texture, color, flavor, additives, microbiology, and bone particle size. Recently, the USDA reviewed the health and safety aspects of the use of mechanically deboned poultry meat (Murphy et al., 1979). The report concluded that mechanically deboned poultry is a safe product, but recommendations were made to establish certain limits in monitoring the product. At the time of review, comments were still being received. Therefore, final labeling recommendations

MECHANICAL DEBONING OF POULTRY A N D FISH

141

are still forthcoming for mechanically deboned poultry meat. Highlights of the health and safety report are discussed below. I . Calcium

The calcium content of mechanically deboned poultry meat was found to be within safe limits. Calcium-to-phosphorus ratios were reported to be similar to those found in hand-deboned sources. The report suggested that mechanically deboned poultry should be appropriately labeled for those individuals who must limit their calcium intake. 2 . Bone Particles The health and safety report indicated that bone particles should not present any health hazard because of size or hardness. The product should be monitored for bone particle size, and a standard procedure should be developed. The alcoholic KOH procedure currently is the best method for monitoring bone particle size. The enzyme digestion method causes agglomeration of bone particles, thereby giving erroneously higher numbers of larger particles. 3 . Fluorine

The fluorine content of mechanically deboned young chicken or turkey meat was quite similar to that of hand-deboned sources. Mechanically deboned fowl meat, however, had a higher quantity of fluorine. Apparently, the fluorine content of fowl meat may be influenced by feed sources and by the cumulative effect of the increased age of the bird. Therefore, the USDA recommended that mechanically deboned fowl meat be limited to 20% in a poultry product and be prohibited in strained, junior, and toddler foods. 4 . Protein and Protein Quality

Protein efficiency ratios were reported to be comparable to that of casein, which is a high-quality protein. Generally, the protein quality of mechanically deboned poultry is similar to that reported for hand-deboned sources. Mechanically deboned cooked poultry meat may have slightly more collagen, but the protein quality appears to be little affected. Standards for protein quality of mechanically deboned poultry have not been established at this time.

5 . Lipid and Cholesterol Content The cholesterol content of different types of mechanically deboned poultry

G . W .FRONING

I42

meat was reported to be approximately double that of hand-deboned sources. The fat content of mechanically deboned poultry meat was higher than that of handdeboned sources. The USDA recommended that mechanically deboned poultry be identified to aid those people who must restrict their colesterol intake. Also, limitations were recommended on the fat content of mechanically deboned poultry. 6 . Formulation

The current report made no specific recommendations as to formulation limitations with the exception of establishing guidelines for the use of mechanically deboned fowl meat in baby foods and poultry products.

X.

RESEARCH NEEDED

Since the advent of mechanical deboning of fish and poultry, considerable progress has been made in improving the processing parameters and the functional characteristics of the product. Research has led to many of these improvements. Advanced technology in deboning machines and quality assurance programs have greatly improved the quality of the product. Nevertheless, several areas need further investigation if we are to realize the full potential of utilizing mechanically deboned fish and poultry. 1. Texturization and extrusion techniques require further study. If the texture of mechanically deboned fish and poultry more nearly resembled that of intact muscle, more efficient use of these products would be possible. The present pastelike texture is acceptable only in emulsified products. 2. The limited storage stability of mechanically deboned fish and poultry still hampers efficient handling. Deboning techniques that would minimize the inclusion of heme and lipid components from the bone marrow should be investigated. Deboning machines that would minimally affect muscle integrity would be instrumental in improving flavor and color stability. 3. Utilization of the bone residue has excellent potential. Further research is necessary to obtain a protein isolate from the bone residue that can be used to improve the functional characteristics of food systems. Data are needed on solubility, emulsifying characteristics, gelling properties, and binding properties. 4. The fluoride content of mechanically deboned fowl meat is quite variable. Several interacting factors that should be studied include the influence of the age of the bird, dietary feed sources, and geographical influences. 5. Improved methodology should be developed to monitor product quality and composition of mechanically deboned fish and poultry-for example, rapid and less expensive methods for measuring protein efficiency ratios (PER). PER val-

MECHANICAL DEBONING O F POULTRY AND FISH

143

ues computed from amino acid profiles offer some promise, but their true relationship to rat PER values should be established. 6. Basic research is needed to determine the effect of the mechanical deboning process on the myofibrillar proteins. This research would also provide information on the effect of deboning on the water-holding capacity and overall textural characteristics of mechanically deboned fish and poultry.

REFERENCES Acton, J. C. 1973. Composition and proterties of extruded, texturized poultry meat. J. FoodSci. 38, 571-574. Angel, S . , Darfler, J . M., Hood, L. F., and Baker, R. C . 1974. Frankfurters made from mechanically deboned poultry meat (MDPM). 2. Microscopy. Poulr. Sci. 53, 166-174. Babbitt, J . K., Crawford, D. L., and Law, D. K . 1972. Decomposition of triethylamine oxide and changes in protein extractability during frozen storage of minced and intake hake (Merluccius productus) muscle. J. Agric. Food Chem. 20, 1052-1054. Babbitt, J . K . . Crawford, D. L., and Law, D. K . 1974a. Quality and utilization of minced deboned fish muscle. I n “Mechanical Recovery and Utilization of Fish Flesh” (R. E. Martin, ed.), pp. 32-43. Babbitt, J . K . , Law, D. K., and Crawford, D. L. 1974b. Acceptance of-a fish-shrimp portion utilizing machine-separated minced fish flesh. J. Food Sci. 39, 1130. Babbitt, J. K., Law, D. K . , and Crawford, D. L. 1976. Improved acceptance and shelf- life of frozen minced fish with shrimp. J . Food Sci. 41, 35-37. Babji, A. S., Froning, G . W., and Satterlee, L. D. 1980. The protein nutritional quality of mechanically deboned poultry meat as predicted by C-PER assay. J. Food Sri. 45, 441-443. Baker, R. C., and Darfler, J. M. 1975. Acceptability of frankfurters made from mechanically deboned turkey frames as affected by formulation changes. Poult. Sci. 54, 1283-1288. Baker, R. C . , Darfler, J. M., and Angel, S. 1974. Frankfurters made from mechanically deboned poultry (MDPM). I , Effect of chopping time. Poulr. Sci. 53, 156-161. Brown, W. D.. Venolia. A. W., Tappel, A. W., and Stansby, M. E. 1957. Oxidative deterioration in fish. Commer. Fish Rev. 19, 27. Cheng, C. S., Hamann, D. D., and Webb, N. B. 1979a. Effect of thermal processing on minced fish gel texture. J. Food Sci. 44, 1080-1086. Cheng, C. S.. Hamann, D. D., Webb, N. B., and Sidewell, V. 1979b. Effects of species and storage time on minced fish gel structure. J. Food Sci. 44, 1087-1092. Cobb, B. F., and Yeh, C.-p. 1974. Improvement of texture, taste and shelf-life in minced Atlantic croaker. I n “Mechanical Recovery and Utilization of Fish Flesh” (R. E. Martin, ed.), pp. 44-75. Crawford, D. L., Law, D. K., and Babbitt, J. K . 1972. Nutritional characteristics of marine food fish, carcass waste and machine-separated flesh. J. Agric. Food Chem. 20, 1048-1051. Cunningham, F. E., and Mugler, D. 1 . 1974. Deboned fowl meat offers opportunities. Poult. Meur 25, 46-50. Dhillon, A. S . , and Maurer, A. 1. 1975a. Quality measurements of chicken and turkey summer sausages. Poult. Sci. 54, 1263-1271. Dhillon, A. S., and Maurer, A. J. 1975b. Stability study of comminuted poultry meats in frozen storage. Poult. Sci. 54, 1407-1414.

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Dimick, P. S., MacNeil, J . H. and Grunden, L. P. 1972. Poultry product quality carbonyl composition and organoleptic evaluation of mechanically deboned poultry meat. J . Food Sci. 37, 544-546. Essary, E. 0. 1979. Moisture, fat, protein and mineral content of mechanically deboned poultry meat. 1.Food Sci. 37, 544-546. Essary, E. 0. 1979. Moisture, fat. protein and mineral content of mechanically deboned poultry meat. J . Food Sci. 44, 1070-1073. Essary, E. O . , and Ritchey, S. 3. 1968. Amino acid composition of meat removed from boned carcasses by use of a commercial boning machine. Poult. Sci. 47, 1953. Food and Agriculture Organization, 1971. In “The Fish Resources of the Ocean” (J. A . Gulland, ed.). Fishing News (Books) Ltd., London. Froning, G . W . 1970. Poultry meat sources and their emulsifying characteristics as related to processing variables. Poult. Sci. 49, 1625-1631. Froning, G . W. 1973. Effect of chilling in the presence of polyphosphates on the characteristics of mechanically deboned fowl meat. Poult. Sci. 52, 920-923. Froning, G. W. 1976. Mechanically deboned poultry meat. J . Food Technol. 30, 50-63. Froning. G. W . 1979. Characteristics of bone particles from various poultry meat products. forth. Sci. 58, 1001-1003. Froning, G . W., and Janky, D. M. 1971. Effect of pH and salt preblending on emulsifying characteristics of mechanically deboned turkey frame meat. Poult. Sci. 60, 1206-1209. Froning, G. W . , and Johnson, F. 1973. Improving the quality of mechanically deboned fowl meat by centrifugation. J. Fond Sri. 38, 279-281. Froning, G . W . , Arnold, R . G . , Mandigo, R. W . , Neth, C. E., and Hartung, T. E. 1971. Quality and storage stability of frankfurters containing 15% mechanically deboned turkey meat, J. Techno/. 36, 974-978. Froning, G. W . , Satterlee, L. D., and Johnson. F. 1973. Effect of skin content prior to deboning on emulsifying and color characteristics of mechanically deboned chicken back meat. Poult. Sci. 52, 923-926. Furia, T. E., 1972. Sequestrants in food. I n “Handbook of Additives” (T. E. Furia, ed.), pp. 289-312. Chem. Rubber Publ. Co., Cleveland. Ohio. Grunden, L. P., and MacNeil, J . H. 1973. Examination of bone content in mechanically deboned poultry meat by EDTA and atomic absorption spectrophotometric methods. J . Food Sci. 38, 7 12-7 13. Grunden, L. P., MacNeil, I . H.. and Dimick. P. S. 1972. Poultry product quality: Chemical and physical characteristics of mechanically deboned poultry meat. J. Food Sci. 37, 247-249. Hill, R . M.. and Hites. B. D. 1968. Meat and meat products. Determination o f small bone particles in meat. J . Assoc. Ofl.Anal. Chern. 51, 1175-1 177. Hsu, H. W., Sutton, N . E., Banjo, M. O., Satterlee, L. D., and Kendrick. J. G. 1978. The C-PER and T-PER assays for protein quality. Fond Technol. 32( 12). 69-73. Janky. D. M., and Froning, G. W. 1975. Factors affecting chemical properties of heme and lipid components in mechanically deboned turkey meat. Poult Sci. 54, 1378- 1387. Janky. D. M., Riley, P. K . , Brown, W. L., and Bacus, J . N. 1977. Factors affecting the stability of mechanically deboned poultry meat combined with structural soy protein emulsions. Poult Sci. 56, 902-907. Johnson, P. G., Cunningham. F. E., and Bowers, J . A. 1974. Quality of mechanically deboned turkey meat: Effect of storage time and temperature. Poult. Sci. 53, 732-736. Keay. J. N.. and Hardy, R . 1974. The application of extrusion techniques to the utilization of comminuted fish flesh. I n “Mechanical Recovely and Utilization of Fish Flesh” ( R . E. Martin, ed.), pp. 88-10.

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Lee, C. M., and Toledo, R. T. 1976. Factors affecting textural characteristics of cooked comminuted fish muscle. J. Food Sci. 41, 391-397. Lee, C. M., and Toledo, R. T. 1977. Degradation of fish muscle during mechanical deboning and storage with emphasis on lipid oxidation. J. Food Sci. 42, 1646-1649. Lee, C. M., and Toledo, R. T. 1979. Processing and ingredient influences on texture of cooked comminuted fish muscle. J. Food Sci. 44, 1615-1618. Lee, Y. B., Hargus, G . L., Kirkpatrick, J. A., Berner, D. L., andForsythe, R. H. 1975. Mechanism of lipid oxidation in mechanically deboned chicken meat. J. Food Sci. 40, 964-967. Lillard, H. S. 1977. Effect of freezing on incidence and levels of Clostridum perfringens in mechanically deboned chicken meat. Poult. Sci. 56, 2052-2055. Lyon. B. G., Lyon, C. E., and Townsend, W. E. 1978. Characteristics of six patty formulas containing different amounts of mechanically deboned broiler meat and hand deboned fowl meat. J. Food Sci. 43, 1656-1661. Lyon, C. E., Lyon, B. G., and Townsend, W. E. 1978a. Quality of patties containing mechanically deboned broiler meat, hand deboned fowl meat and two levels of structured protein fiber. Poult. Sci. 57, 156-162. Lyon, C. E., Lyon, B. G., Townsend, W. E., and Wilson, R. L. 1978b. Effect of level of structured protein fiber on quality of mechanically deboned chicken meat patties. J. Food Sci. 43, 15241527. McMahon, E. F., and Dawson, L. E. 1976. Effects of salt and phosphates on some functional characteristics of hand and mechanically deboned turkey meat. Poult. Sci. 55, 573-578. MacNeil, J. H., Dimick, P. S., and Mast, M. G. 1973. Use of chemical compounds and rosemary spice extract in quality maintenance of deboned poultry meat. J. Food Sci. 38, 1080-1081. MacNeil, J. H., Mast, M. G . . and Leach, R. M. 1978. Protein efficiency ratio and levels of selected nutrients in mechanically deboned poultry meat. J. Food Sri. 43, 864-865. Mai, J . , and Kinsella, J . E. 1979. Changes in lipid composition of cooked minced carp (Cyprinus carpio) during frozen storage. J. Food Sci. 44, 1619-1624. Martin, R. E. 1972. Mechanical recovery and utilization of fish flesh. In “Mechanical Recovery and Utilization of Fish Flesh” (R. E. Martin, ed.). pp. 1-270. Martin, R. E. 1976. Mechanically deboned fish flesh. Food Techno/. 30(9), 6 4 7 0 . Mast, M. G . , and MacNeil, J. H. 1975. Heat pasteurization of mechanically deboned poultry meat. Poult. Sci. 54, 1024-1030. Mast, M. G., Jurdi, D., and MacNeil, J. H. 1979. Effects of C0,-snow on the quality and acceptance of mechanically deboned poultry meat. J. Food Sci. 44, 346-354. Maxcy, R. B., Froning, G. W., and Hartung, T. E. 1973. Microbial quality of ground poultry meat. Poult. Sci. 52, 486-491. Mayfield, T. L., Hale, K . K., Rao, V . N. M., and Angelo-Chacon, I. A. 1978. Effects of levels of fat and protein on the stability and viscosity of emulsions prepared from mechanically deboned poultry meat. J. Food Sci. 43, 197-201. Miyauchi, D. 1972. Progress report on minced fish studies at National Fisheries Service. In “Mechanical Recovery and Utilization of Fish Flesh” (R. E. Martin, ed.), pp. 35-48. Miyauchi, D. 1974. Storage studies on minced rock fish and the use of carp in minced products. In “Mechanical Recovery and Utilization of Fish Flesh” (R. E. Martin, ed.), pp. 22-31. Miyauchi, D., and Steinberg, M. 1970. Machine separation of edible flesh from fish. Fish Ind. Res. 6, 165-171. Miyauchi. D., Patashnik, M., and Kudo, G . 1975. Frozen storage keeping quality of minced black rockfish improved by cold water washing and use of fish binder. J. Food Sci. 40, 592-594. Moerck, K. E., and Ball, H. R., Jr. 1973. Lipids and fatty acids of chicken bone marrow. J. Food Sci. 38, 978-980.

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Moerck, K. E., and Ball, H . R . . Jr. 1974. Lipid autoxidation in mechanically deboned chicken meat. J. Food Sci. 39, 876-879. Moledina, K . H., Regenstein. J . M., Baker, R. C., and Steinkraus, K . H. 1977. Effects of antioxidants and chelators on the stability of frozen stored niechanically deboned flounder meat from racks after filleting. J. Food Sci. 42, 759-764. Moms, D. M., and Dawson. L. E. 1979. Storage stability of mechanically deboned sucker (Catostomidae) flesh. J. Food Sci. 44, 1093-1096. Murphy, E. W., Brewington, C. R., Willis. B. W., and Nelson, M .A. 1979. “Health and Safety Aspects of the Use of Mechanically Deboned Poultry.” Food Safety and Quality Service, U.S. Department of Agriculture. Washington, D. C. Nakayama. T . , and Yamamoto, M. 1977. Physical, chemical and sensory evaluations of frozenstored deboned (minced) fish flesh. J. Food Sci. 42, 900-905. Orr, H. L., and Wogar, W. G. 1979. Emulsifying characteristics and composition of mechanically deboned chicken necks and backs from different sources. Poult. Sci. 58, 577-579. Ostovar, K., MacNeil, J . H . , and O’Donnell, K. 1971. Poultry product quality. 5. Microbiological evaluation of mechanically deboned poultry meat. J . Food Sci. 36, 1005-1007. Patashnik. M.. Kudo, G., and Miyauchi, D. 1974. Bone particlecontent of some minced fish muscle products. J. Food Sri. 39, 588-591. Raccach, M.. and Baker, R. C. 1978. Microbial properties of mechanically deboned fish flesh. J . Food Sci. 43, 1675-1677. Raccach, M.. Baker, R. C., Regenstein, J. M.. and Mulnix, E. J. 1979. Potential application of microbial antagonism to extended storage stability of a flesh type food. J. Food Sci. 44, 43-46. Ryan, J. 1974. Draft proposed U . S . standards for grades of minced fish blocks. / t i “Mechanical Recovery and Utilization of Fish Flesh” (R. E. Martin, ed.), pp. 208-216. Satterlee, L. D., Froning, G . W., and Janky, D. M. 1971. Influence of skin content on composition of mechanically deboned poultry meat. J. Food Sci. 36, 979-981. Schnell, P. C., Nath, K . R.. Darfler, J. M., Vadehra, D. V., and Baker, R. C. 1973. Physical and functional properties of mechanically deboned poultry meat as used in the manufacture of frankfurters. Poult. Sci. 52, 1363- 1369. Schnell, P. G . , Vadehra, D. V., Hood, L. R., and Baker, R. C. 1974. Ultra-structure o f mechanically deboned poultry meat. Poult. Sci. 53, 416-419. Silberstein, D. A., and Lillard. D. A. 1978. Factors affecting the autoxidation of lipids in mechanically deboned fish. J . Food Sci. 43, 764-766. Steagall, E. F. 1966. Titration ofcalcium and magnesium. J. Assoc. ON. A n d . Chem. 49, 287-291. Tappel, A. L. 1955. Unsaturated lipid oxidation catalyzed by hematin compounds. J. B i d . Chetn. 217, 72 1-733. Teeny, F. M., and Miyauchi. D. 1972. Preparation and utilization of frozen blocks of minced black rockfish. J. Milk Food Technol. 35, 414-417. Uebersax, K . L., Dawson, L. E., and Uebersax, M. A. 1977. Influence of “COL-snow”chilling on TBA values in mechanically deboned chicken meat. Poult. Sci. 56, 707-709. Uebersax. K . L., Dawson, L. E.. and Uebersax, M. A. 1978. Storage stability (TBA) and color of MDCM and MDTM processed with CO, cooling. Poult. Sci. 57, 670-675. Uebersax, M. A., Dawson, L. E., and Uebersax, K . L. 1978a. Physical and chemical composition of ineat loaves containing mechanically deboned turkey meat. Poult. Sci. 57, 660-669. Uebersax, M. A., Dawson, L. E., and Uebersax, K. L. 1978b. Evaluation of various mixing stresses on storage stability (TBA) and color of mechanically deboned turkey meat. Poult. Sci. 57, 924-929. Wallace, M. J. D., and Froning. G . W. 1979. Protein quality determination of bone residue from mechanically deboned chicken meat. Puulr. Sci. 58, 333-336.

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Watts, B. M. 1950. Polyphosphates as synergistic antioxidants. J. Am. Oil Chem. Soc. 27, 48-51. Watts, B. M. 1961. The role of lipid oxidation in lean tissues in flavor deterioration of meat and fish. In “Proceedings of the Flavor Chemistry Symposium,” p. 83. Campbell Soup Company, Carnden, New Jersey. Webb, N . B., Hardy, E. R., Giddings, G. G., and Howell, A. J. 1976. Influence of mechanical separation upon proximate composition, functional properties and textural characteristics of frozen atlantic croaker muscle tissue. J. Food Sci. 41, 1277-1281, Wong, J., Lau, Y. C.. and Yarnamoto, M. 1978. Mechanical fish deboners: Influence of various perforation sizes on bone content and texture of minced fish flesh. J. Food Sci. 43, 807-809. Young, L. L. 1975. Aqueous extraction of protein isolate from mechanically deboned poultry meat. J . FoodSci. 40, 1115-1118. Young, L. L. 1976. Composition and properties of an animal protein isolate prepared from bone residue. J. Food Sci. 41, 606-608. Young, L. L.. and Lyon, B. G . 1973. The use of heat treated meat in chicken frankfurters. Poult. Sci. 52, 1868-1875.

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A D V A N C E S I N FOOD RESEARCH, VOL.

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NATURALLY OCCURRING FOOD TOXICANTS: PHENOLIC SUBSTANCES OF PLANT ORIGIN COMMON IN FOODS VERNON L. SINGLETON Depariment of Viticulture and Enology, University of Culifornia. Davis, California

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11. Origins and Types of Plant Phenols . . . . . . . . . . . 111. Evolutionary Considerations . . . . . . . . , , . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . A. Phenols from Plants.. . . . . . . . . . . . . . . . . . . . . . . . . . . .

B. Animal Adaptation ............................. C. Animal Metabolism o ................................... IV. Examples of Common Plant Phenols with Actual or Potential Significance in Animal Consumption (Toxic or Beneficial) . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Small Phenols, Pyrolysis Products . . . . . . . . . . . . . . . . . . B. Phenolic Nutrients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Hydroxycinnamates . . . . . . . , . . , . , . . . . . . . . . . . . . . . . . . . . . . E. Flavonoids .................................... F. Tannins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..................... G. Lignins . . . . . . . . . . V . Mechanisms of Toxicity by Phenols , . . . . . . . . . . . . . . . . . . . . . . . A. Mimic-Interaction with Normal Phenol Metabolism . . . . . . . . . . . . . . . . . B. Nutritional Load . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........... C. Penetration Effects . . . . . . . . . . . . . . . . . . D. Protein Binding ............................ . . . . . . . . . F. Mutagenicity, Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Miscellaneous Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V1. Conclusions and Assessment of Risks ................... VII. Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................... References . . . . . . . . . .

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162 163 166 167 170 173 191 208 210 211 212 214 215 216 216 218 218 22 1 22 1

INTRODUCTION

The risks to humans of serious poisonous effects caused by natural phenols present in normal foods consumed under usual circumstances seem vanishingly 149 Copyright @ 1981 by Academic Press. Inc. All rights of reproduction in any form rcservcd.

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small, as determined by experiment as well as by common sense and long-term observation. Why, then, consider the matter further? Phenolic substances are naturally present in essentially all plant material, including food products of plant origin, and sometimes in sizable amounts. A few phenols occurring in nature are potent toxins; some have caused serious outbreaks of toxic dietary reactions in animals. In a few instances, deleterious effects in humans appear to be the result of an abnormal consumption of plant phenols or consumption of phenols abnormal to the diet. On the basis of tests with experimental animals, phenolic derivatives found to be carcinogenic or otherwise seriously toxic at any level-safrole and coumarin, for example-have been banned as food additives. Common flavonols recently have been found to be bacterial mutagens. Public agencies are demanding extensive testing if not summary elimination from food of any molecule that is not necessary for life. Phenols, with a few specific exceptions, are considered to be secondary metabolites (nonessential for life), even in plants. On the other hand, some kinds of plant phenols appear to have desirable physiological effects in diets or as drugs. Processing advantages, such as the antioxidant activity of certain phenols, can be important not only in maintaining a continuity of supply of nutritious and inexpensive food, but also in providing a protective effect against photocarcinogenesis through lipid oxidation (Logani and Davies, 1980). In many countries, particularly the economically disadvantaged ones, the diet is almost exclusively vegetarian, with a consequent increase in plant phenol intake. The current enthusiasm for vegetarian diets in the United States is partly a fad, but economics and population pressures are likely to reinforce the trend. The phenol content will increase in proportion to the substitution of plant for animal products in the diet. If exotic plant products or additional contaminants are taken into the diet as a result of emergencies or other factors, unusual phenols may be introduced as well-gossypol or aflatoxins, for example. Singleton and Kratzer (1973) made a case for the idea that common plant phenols are nontoxic only because of efficient animal metabolism of them under normal circumstances. In view of these considerations, an objective sought in this review is to place the common phenolic substances of the plant kingdom in perspective as real or potentially toxic or physiologically active substances in human and animal diets. An attempt will be made to focus on the compounds as phenols or derivatives of phenols rather than the more usual practice of singling out only certain classes of phenols (e.g., anthraquinones, coumarins, flavonoids, lignins, tannins), certain effects (e.g., carcinogen, estrogen, photosensitizer, vitamin antagonist), or certain sources (e.g., angiosperms, fruits, grains, legumes, mycotoxins). Absolute completeness is impossible and coherent synthesis is difficult because of the voluminous, disciplinarily diverse, and scattered literature from the nutritional, medical, pharmacological, physiological, toxicological, biochemical, chemical, and agricultural fields that relate to the topic. No individual, it is

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freely acknowledged, can be thoroughly expert and up-to-date in all these interrelationships. The information to be presented has been drawn from participation in cooperative research on inhibition by natural phenols of growth in chicks (Kratzer et a / . , !975) and in the preparation of two reviews (Singleton and Kratzer, 1969, 1973). Literature searching was based on that done for the previous research and on reviews plus references obtained in an effort to remain currently aware of developments in the field. More intensive manual searching of Chemical Abstructs in 1977 and 1978 was supplemented by a computerized search of Biological Abstracts (BIOSIS) files for specific phenol-animal interactions reported in reviews since 1969 and in individual papers from 1977 to October 1980. About 2000 papers were read or scanned and their bibliographies compared in an effort to include all important references. The references cited are limited, generally, to the most recent reviews or papers. Further references may be obtained from these cited articles by those desiring more background. Much of the discussion will be on specific examples of the toxicity of common phenols of plant origin. It is important to note, however, that toxicity and favorable therapeutic effects can overlap, and the way a compound is viewed may depend on how it was first discovered and tested. For example, dicoumarol was discovered as the cause of a bleeding death in cattle, and the discovery led to a useful rat poison. Development of dicoumarol and its derivatives as useful drugs to prevent unwanted clotting in cases of threatened embolisms and strokes was somewhat impeded by the negative image. In other cases, useful therapeutic activity was discovered first and serious toxicity later, with the result that usage has been tolerated more than in the reverse situation (diethylstilbestrol may be an example). Therefore, physiological effects as well as toxicity will be considered.

II.

ORIGINS AND TYPES OF PLANT PHENOLS

The phenols of nature are mostly biosynthesized via one of two major pathways or their combination (Hahlbrock and Grisebach, 1975; Harborne, 1980). One mechanism typically produces rn-trihydroxyphenols resembling phloroglucinol by cyclization of polyketides derived from linking at least three twocarbon units equivalent to activated acetate. The second mechanism synthesizes phenols via the shikimic acid pathway to phenylalanine and thence to C,& phenols such as p-hydroxycinnamic acid and tyrosine. Phenols from this mechanism typically have an hydroxyl para to the three-carbon side chain and often have additional hydroxyl groups or derivatives thereof adjacent to the first. Flavonoids are produced by a combination of the two mechanisms so that the A-ring is “acetate”-derived and has meta-hydroxylation and the B-ring is shikimate-derived with 4‘ or more hydroxylation. Typical flavonoids are malvidin, an anthocyanin, and quercetin, a flavonol (Fig. 1). Other biosynthetic path-

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Hod VERNON L. SINGLETON

@

I:

OUERCETIN

OH S AF R 0 LE

CATECHIN

HO UMBELLIFERONE

.-+ ,:

H&C :H3

OH MALVl DIN

THYMOL

FIG. I . Examples of phenols common and uncommon in foods from plants: common-uercetin (flavonol). catechin (flavan-3-ol), malvidin (anthocyanidin), sinapic acid (hydroxycinnamic acid); uncommon-safrole (4-allylphenol, methylenedioxy ether), umbelliferone (coumarin), and thymol (terpenoid phenol).

ways modify the phenols produced and in some instances such as certain terpenoid phenols, create other less common types of phenols. Terpenoid phenols like thymol can usually be recognized by their isoprenoid subunits. Flavonoids (anthocyanins, flavan-3-ols, flavones, flavonols, etc.) differ by the degree of oxidation of the heterocyclic ring formed from the three-carbon side chain of the phenylalanine part and one hydroxyl and two carbons of the “phloroglucinol” part. Chalcone, aurone, isoflavonoid, and neoflavonoid derivatives are generally included along with the flavonoids, owing to their similar origins and structures. Coumarin is considered a shikimate-derived phenol, since it is the lactone of 2-hydroxycinnamic acid (o-coumaric acid). Similarly, methoxy and methylenedioxy derivatives (e.g., safrole, Fig. 1) and any other linkage that is clearly biochemically or chemically derived from phenols-quinones, for example-are commonly included as phenols. A few exemplary structures are given in Fig. 1 . Sinapic acid (a cinnamic acid), safrole, and umbelliferone (a coumarin) are shikimic derivatives. Quercetin and (+)-catechin (a flavan-3-01) are probably the two flavonoids most widespread in nature. The structures of

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most other natural phenols are readily available from many good books and reviews (Harborne, 1975, 1980; Anonymous, 1976; Dean, 1963; Robinson, 1980). Further important natural phenols include lignans and lignin, which are dimers and polymers, respectively, of C,-C, units of the hydroxycinnamate type. Tannins are large polyphenolic molecules that are either condensed tannins (dimers and larger of flavan-3-ols, commonly) or hydrolyzable tannins (commonly glucose or quinic acid esterified with gallic acid, 3-digallic acid, or hexahydroxydiphenic acid). When released by hydrolysis, hexahydroxydiphenic acid (a dimer of gallic acid) forms the dilactone ellagic acid; hence the hydrolyzable tannins are also called gallotannins or ellagitannins. For the purposes of our discussion alkaloids are excluded, even though many of them are phenolic as well as nitrogen-containing (e.g., capsaicin, colchicine, mescaline, morphine). Each of these general types of natural phenols can be subdivided into members of common, uncommon, or botanically rare occurrence. Most of the phenols noted for toxicity or physiological potency in animals not only are extremely limited in botanical distribution, but also are unique or highly unusual in their chemical structure when compared with common plant phenols (Singleton and Kratzer, 1973).

Ill. EVOLUTIONARY CONSIDERATIONS Conclusions involving evolution obviously are speculative, but some putative relationships appear worthy of discussion to clarify the durability of animal life and the threat of plant toxins generally or of phenols particularly. Most of the food plants of agriculture are highly evolved and highly selected angiosperms. The common phenols within this restricted group include a few dozen substances. The total amounts of these common phenols can range from nearly zero in refined, plant-derived foods like table sugar, to 20% or so of the dry weight of the diet of some herbivorous animals. Given a free choice, animals in general and man in particular select food plant parts with a low phenol content from a lignin (toughness) or tannin (astringent taste) viewpoint, but perhaps a relatively high anthocyanin content (visibility, ripeness indicator). Particularly with agriculture, man has not only selected and modified by breeding the plants he cultivates for feed and food, but in a great part of the most fertile land he has completely changed the “ecology” in terms of the relative diversity and “commonness” of individual plant species and the resultant occurrence of various phenols. We therefore may have a warped and limited view of the possible risk of plant toxins. When domesticated animals were introduced by Europeans to unfamiliar flora in Australia, about one plant species

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in twenty was found capable of causing death (Culvenor, 1970). The ratio is a little more favorable in native North American or African flora, but if we allow for unrecognized, slow-developing, or subacute toxicity, it is quite clear that many plants are not entirely benign to animals. With the apparent exception of the grasses and grains of the Gramineae, few plants have adapted specifically to allow predation by animals without developing some form of active resistance, such as thorns or toxins (Culvenor, 1970). Fruit may be sacrificed to an animal in return for seed dispersal, but often the seeds themselves may be toxic or may at least resist digestion. Many plants have evidently evolved toxins to help protect them from pests, parasites, disease organisms, or predators. Animals have evidently evolved and survived by “learning” to avoid or withstand these toxins.

A.

PHENOLS FROM PLANTS

Phenols are probably the most important group of substances useful in chemotaxonomic differentiation among plant species (Harborne, 1975). The reasons for this include the fact that some phenols are found in nearly all plants, but the qualitative diversity among different plants is large. Not counting diversity caused by variation in the formation of derivatives, especially in glycosidation, at least 800 different phenolic aglycones from plants were known in 1968 (Singleton and Kratzer, 1969), and owing to a continuing rapid rate of discovery there are probably triple that number today (Harborne, 1979. 1980). For taxonomy, phenols of limited distribution are, of course, more useful than those common to most plants. A phenolic constituent unique to a single species is also not a very useful taxonomic key, but searching for it in related plants may show it or related compounds to be more widely distributed than was at first supposed. Although chemotaxonomy is still a developing field, considerable useful information is available from it and from chemical studies on lichen products, mold products, antibiotics, unusual plants, etc. In the most primitive plants, flavonoids are essentially absent, and the phenols of molds and lichens are mainly “acetate”-derived polyketides with metahydroxylation. They include, nevertheless, a large and diverse group of structures. Flavonoids are found in mosses, ferns, and gymnosperms-but generally in limited and less usual forms (Swain, 1975). They become more common and more diversified in angiosperms of increasingly evolved forms. Trees and vines, however, generally have a higher content and more different (but common) phenols than do the still more highly evolved herbaceous plants. The common phenols of the agriculturally predominant angiosperms include the cinnamic acid derivatives caffeic, p-coumaric, ferulic, and sinapic acids (respectively, 3,4-dihydroxy-, 4-hydroxy-, 3-methoxy-4-hydroxy-, and 3,5dimethoxy-4-hydroxycinnamicacids). These cinnamates usually occur as esters of quinic acid (chlorogenic acid and analogs) or other nonphenolic hydroxyl

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compounds (glucose, shikimic acid, tartaric acid, etc.). Gallic acid (3,4,5trihydroxybenzoic acid) is widely found in plants, and gallotannins and ellagitannins also are not uncommon, especially in certain woody species. Lignin is nearly universal in higher plants, although the level may be high or low, depending on woody versus herbaceous habit. Lignin precursors such as coniferyl alcohol and lignin fragments are also widespread, but usually low in amount. Certain flavonoids are common. The most primitive plants with flavonoids tend to lack 3-hydroxylation, but the usual pattern in higher plants is based on the 3,5,7-trihydroxyflavan structure. The common natural anthocyanins are glycosides of pelargonidin, cyanidin, peonidin, delphinidin, petunidin, and malvidin. The B-ring hydroxylation pattern is like that of the cinnamates or gallic acid. Other flavonoids considered very common in plants include the flavones apigenin and luteolin, the flavonols kaempferol and quercetin, the flavan-3-01s ( +)-catechin and ( -)-epicatechin, the flavanone naringenin, and the flavanonol taxifolin. The condensed or flavonoid tannins are dimers and larger polymers of the catechins. They are often called anthocyanogens, proanthocyanidins, or polymeric leucoanthocyanidins because when properly treated they usually produce cyanidin. They occur very widely, especially in woody plants. The common phenols, then, in terms of their consumption by animals, include a few dozen substances. Other phenolic derivatives-coumarins, for exampleand other substitution patterns are not necessarily rare, but they are less common, particularly in the usual unrestricted diet of animals and man. The remaining 90% or so of the known natural phenols are relatively rare among plant species and in animal diets. The functions of phenols in plants have been a continuing puzzle, but it is clear that one function can be protection via toxicity. Certain substances-many of the identified ones are phenols-called phytoalexins are produced in plant tissue upon injury by pathogens and are sufficiently inhibitory to the invader to produce resistance (Deverall, 1972; Kuc, 1976). Such inhibitory compounds can have a toxic effect in animals too. A series of furane derivatives (not phenolic) that are produced as phytoalexins by sweet potato tissue after attack by certain molds, yeasts, or bacteria have figured in repeated episodes of poisoning of farm animals (Wilson and Hayes, 1973). The general response to wounding and recovery in plants commonly involves production of a high level of phenols adjacent to the injury, along with oxidation of the phenols by polyphenoloxidase to quinones and phytomelanin brown polymer complexes. The oxidation products are often more toxic to potential invaders than are the original phenols, and the polymerization helps seal off the injured surface and begin the healing process. In some instances unusual phenols and other toxic compounds are clearly part of the normal resistance mechanism of a plant. Gossypol appears to confer resistance in cotton to some pests, for

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example, but specialist predatory insects often evolve to tolerate the toxins of a certain plant. Phenols even appear to be involved in hypersensitive self-toxicity reactions within a given plant and in allelopathic competition among plants (Muller and Chou, 1972). Summaries indicate the importance of phytotoxins, some phenolic, in the defense set up by plants against herbivores (Feeny, 1976; Harborne, 1977; Rhodes and Cates, 1976; Rosenthal and Jansen, 1979). Two classes of phytotoxins seem employed to combat herbivores. One class includes the generally and weakly inhibitory substances, notably tannin, which in larger amounts decreases the digestibility of the plant proteins or otherwise interferes with their consumption, owing to its disagreeable taste or other effects. The second group is more specifically toxic and requires less “investment” of photosynthate. Some plants in nature are readily found by herbivores (including insects) because they are conspicuous and long-lived. An oak tree, for example, is apt to depend on a high tannin level (and high lignification, etc.) to defend itself against all comers and to make a large biosynthetic investment in such defense. Such plants may also make specific toxins of higher potency. An ephemeral plant, inconspicuous and shortlived, is more likely to escape specialist predators and is not apt to make such a heavy investment of photosynthate in toxins. If toxins are made, they are usually “intended” for the generalist herbivore, but they are also likely to be more immediately toxic or repellent in low concentrations. Immediate cessation of feeding is most effective for plant survival, although a slowly acting poison eventually can be effective if it suppresses the predator population. Schoonhoven (1972) visualizes an evolutionary sequence resulting in an obligate association between certain insect herbivores (and their predators) and certain plants. A plant produces a new deterrent substance. An insect adapts to overcome the deterrent, thus avoiding competition. The specific plant’s unusual composition becomes an attractant, a feeding stimulant, and in some cases a requirement for such functions as oviposition. Ultimately in stepwise coevolution, an enormous diversification and specialization developed among plants and herbivorous insects. Apparently, the diversity that makes plant phenols especially useful in chemotaxonomy of species and races has, at least partly, resulted from efforts to escape phytophagy (Harborne, 1978). By agriculture’s homogeneous concentrations of relatively benign plants, man is inadvertently encouraging local population explosions of both specialist and generalist insect herbivores. There are a number of clear-cut examples of phenolic involvement in insect specialization (Schoonhoven, 1972; Harborne, 1979). Larvae of Torrrix viridunu inactivate oak leaf tannin by binding it in special cells in the gut wall. The silkworm, Bomhyx mori, is attracted to and stimulated to feed by the two major flavonoids, morin and quercetin-3-glucoside, of its host, mulberry, Morus ulbu. In fact, mulberry phenols are required as dietary constituents for proper development of the insect (Kato, 1978). Furthermore, other flavonoids with rela-

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tively small structural differences, such as quercetin-3-rhamnoside, will trigger avoidance and stop feeding (Schoonhoven, 1972; Harborne, 1979). Armyworms capable of tolerating 0.1% umbelliferone in the diet were killed by 0.1% xanthotoxin, a furanocoumarin biosynthesized from umbelliferone. This is an example of how plants escape from a generalist insect herbivore by producing a new secondary product (Berenbaum, 1978). An unusual combination of common plant phenols, quercetin-3-(2”galloylglucoside), produced 100% mortality in 12 hr in three species of snail fed at 10 ppm in leaves (Dossaji and Kubo, 1980). Examples of plants combating higher animals feeding on them are not so specific. Many studies with domestic animals and some with wild browsers show that high-tannin material is avoided. Milton ( 1979) found that herbivorous howler monkeys selected leaves with the highest protein and lowest fiber (lignin included) content available. Tannin or total phenol content appeared to affect choices among plant species, but not within species.

B . ANIMAL ADAPTATION Plants have evolved toxins to combat herbivores, but herbivores have evolved also to tolerate or avoid plant toxins. When a new toxin is initially contacted in small amounts, animal metabolism can often adapt in order to detoxify it in subsequent higher exposure (Feeny, 1976). The astringency of tannins and the bitterness of some phenols (and alkaloids, glycosides, etc.) are repellent to animals, at least at high intensity (Bate-Smith, 1972). The loss of tannin in the evolution of related plants is often associated with the development of alkaloids or other toxins or repellents. Animals differ by species, breed, and individual in food selection in relation to phenol content (Arnold and Hill, 1972). Coumarin, for example, is offensive to sheep (Harborne, 1977) and quite toxic (Singleton and Kratzer, 1969), whereas its odor is considered attractive to humans. Thus, it is quite clear that animals do adapt to phytotoxins and evolve differently, depending on exposure. Carnivores have the least exposure to plant phenols. Consequently, there appears to be a great toxicity of phenols in strict carnivores (and insectivores), less in omnivores, and least in strict herbivores (Singleton and Kratzer, 1969). That this is not due to the habitual microbial flora of the digestive tract, or at least not entirely, is indicated by similar relative toxicities observed after both parenteral and oral administration in the different species. Comparisons of the LDSo values collected from numerous sources so that such conclusions could be drawn are subject to many valid criticisms, not the least of which is the paucity of examples of the same phenolic preparation tested in a diverse range of animal species under comparable conditions. There are few species of animals that are inexpensive and convenient enough for extensive acute or chronic lethal toxicity studies to be made. Nevertheless, it is some comfort to humans that the higher

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primates are primarily vegetarians (herbivores) by nature, and man is the omnivore par excellence, with an herbivore-scavenger background only recently (geologically speaking) and regionally heavily carnivorous. Humans, therefore, should be moderately resistant to plant phenols and other phytotoxins-and this appears generally to be true. Furthermore, mammals in general seem to be adapted to angiosperms as their diet. Angiosperms gained ascendancy over gymnosperms and more primitive plants at about the same time that mammals were replacing reptilians. An interesting postulate is that the phytotoxins of the angiosperms were the reason or an additional reason for the demise of the dinosaurs (Swain, 1974; Rosenthal and Janzen, 1979). It is an intriguing idea, one that appears to be buttressed by the fact that the surviving reptilian species are seldom plant eaters. Fish and frogs tend to be quite sensitive to plant phenolic toxins (rotenoid fish poisons, gossypol in trout, and low LD,,, values for phenolic toxins in frogs come to mind). The high toxicity of aflatoxins to birds may be related to their reptilian connection. On the other hand, the surviving lichens, ferns, and gymnosperms often are quite toxic to mammals, and some of the toxins are phenols of unusual types. Perhaps the plants surviving from the primitive groups were the more toxic members, and the metabolism that enabled mammals to combat angiosperm phytotoxins was less successful. The microorganisms, which are primitive but highly adaptable forms, also remain, in some instances, as the producers of very potent toxins to animals, some phenolic. Arthropods, especially insects, sometimes sequester plant toxins and use them in their own defense (Rodriquez and Levin, 1976). The best known examples, such as the milkweed cardenolides in the monarch butterfly, are not phenols, but a few examples are phenolic o r related, such as the accumulation of phenol, hydroquinone, juglone, salicylaldehyde, hypericin, or mellein by various specialist insect herbivores (Rodriquez and Levin, 1976; Schoonhoven, 1972). Insects even make use of the “plant” resistance mechanism of phenol oxidation, polymerization, and tanning to harden and darken their “skin,” to inhibit pathogens and to seal lesions (Taylor, 1969). Again illustrating the evolutionary development of interacting toxins, some phenols from certain plants have a hormonal effect on insect feeders that forces them to remain juvenile (sesamin, a lignan from sesame) (Singleton and Kratzer, 1969) or converts them prematurely to adult forms (precocenes, chromenes from Ageratum houstianurn) (Ohta et ul., 1977). C.

ANIMAL METABOLISM O F PHENOLS

Higher animals cannot make compounds with benzenoid rings from aliphatic precursors, with very few exceptions (estrone and related phenolic steroids being one). Plants are the source of nearly all the phenols found in animals. Even the

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essential phenols for animals (tyrosine, catecholamines, vitamin E tocopherols, vitamin K menadiones, thyroxine) are drawn either directly or indirectly from plants or are modified from an essential plant precursor, usually phenylalanine. Tyrosinase, which is common to animals, is very similar to plant phenolases. Oxidation of phenylalanine to tyrosine and insertion of a second phenolic hydroxyl to form dopa, followed by oxidation to quinone derivatives and polymerization to melanins and melanoproteins or related adducts, show a facile management of phenolic compounds by animals. It is clear that animals are accustomed to efficient and rapid removal, “detoxication,” of excess phenols of these types, which are so potently active in the animal body. Control of nerve action depends on rapid removal of norepinephrine, for example, immediately after it has performed its nerve transmission function. If excess norepinephrine is injected, it is very toxic. Benzenoid compounds, not themselves phenols, are commonly hydroxylated in the animal body as an early step in their catabolism-phenylalanine, for example. Foreign (xenobiotic) phenols taken in by animals are metabolized and detoxified by mechanisms similar to those used for normal animal phenols. The toxicity of a given xenobiotic phenol in an animal involves conditions or structures that are unsatisfactorily managed by the animal’s detoxication system or that overload the system’s capacity. It is possible for “detoxification” to convert an innocuous compound into a toxic derivative. An example is seen in hydrocarbon carcinogens such as benzpyrene. The hydrocarbon itself is not toxic or carcinogenic, but a series of derivatives are produced in the body, some of which are toxic and carcinogenic. The derivatives are produced by enzymic epoxidation of certain double bonds followed by hydration of the epoxide to a trans-dihydrodiol and further dehydration to a phenol or oxidation to quinones (Heidelberger, 1972). Metabolic activation to a reactive electrophile is involved in producing the carcinogenicity (Cohen and Moore, 1977). However, of the at least five phenols, three quinones, three diols, several epoxides, and various conjugates produced (Yang et al., 19771, only a few are appreciably carcinogenic in various tests. Several phenolic derivatives were noncarcinogenic, but 2-hydroxybenz(a)-pyrene was highly carcinogenic on mouse skin (Wislocki et al., 1977). Quinoidal derivatives produced would also be toxic because of the continuous production of hydrogen peroxide and interference with normal oxidation-reduction systems (Lorentzen and Tso, 1977). Thus, detoxication and assessment of the active form of a toxicant can be complicated, and phenols may be involved in less obvious ways. The liver is considered the primary site of enzymic detoxication in animals, although the intestinal mucosa (Shirkey et al., 1979; Stohs et al., 1977), renal tubules (Fry et al., 1978), lung, skin, and placenta (Pelkonen and Moilanen, I979), and perhaps other tissues can be involved as well as nonspecific reactions and “external” factors, such as the microflora of the alimentary canal. The

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majority of plant phenols, like other xenobiotics, are metabolized in two phases: (I) conversion of the basic structure into a new form, usually more oxidized but sometimes reduced or hydrolyzed, and (11) conjugation with some natural constituent from the body. The conjugates are then excreted in the urine or the bile. Once conjugated, the product is invariably nontoxic, or at least much less toxic than before conjugation. Any one phenol may be incompletely absorbed (if orally introduced), excreted partially unchanged, or excreted partially in the form of one or more metabolites without conjugation; the rest is excreted with conjugation, with or without initial metabolism. Most substances produce more than one excretory product, and some produce many; therefore, a total inventory of the amount administered is frequently difficult. Other tissues may be important in conjugation, but the phase I metabolism is performed mostly in the liver. When the cells are experimentally disrupted, the endoplasmic reticulum membrane in the liver cells forms tiny sacs called microsomes, which have detoxication capabilities. The enzymes involved in detoxication are of low specificity, but they are highly inducible by exposure to a new foreign compound to increase greatly both in specific ability to metabolize that substance and in the amount of enzyme (the rate of processing) available for the task. Thus, preliminary or chronic exposure may raise the minimum toxic dose of a drug or toxin considerably. The fetus or infant is particularly vulnerable to drugs and toxins, owing to the late development and low initial adaptation of this system. The main enzymes involved in phase I are the liver microsomal mixedfunction oxidases. Considerable detail is known about these detoxication mechanisms. Useful references include Jenner and Testa (1978), Kappas and Alvares (1975), Parke and Smith (1977), Testa and Jenner (1976), and Williams (1959). Molecular oxygen is required, as is reduced nicotinamide adenine dinucleotide phosphate (NADPH). Cytochrome P-450 serves as the terminal oxidase. It is believed to function by ( a ) binding the foreign substance, ( h )being reduced from the ferric to the ferrous form (in the cytochrome heme) by interaction with NADPH and cytochrome P-450reductase, (c) accepting 02, and then ( d ) liberating the oxidized metabolite (one oxygen per molecule) of the foreign molecule with the ferric heme restored and the second oxygen atom, generally, appearing as water. Specific detoxications apparently may induce differences. The aryl hydrocarbon hydroxylase affecting benz( a)pyrene, for example, uses cytochrome P-448 rather than P-450(448 nm absorbance maximum of the carbon monoxide adduct) (Kappas and Alvares, 1975). Owing to the low specificity of these enzymes, induction by one foreign substance of efficient detoxication may serve as a protection against additional foreign substances not previously encountered by the animal. On the other hand, there is a finite capacity to adapt to a load of substances requiring detoxication, and an additional foreign substance may interfere with detoxication or may increase the toxicity of another.

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There are many conjugation reactions (Williams, 1967), and additional ones are still being discovered (Jenner and Testa, 1978). Those considered usually important in detoxicating phenols are methylation and conjugation with glucuronic acid, sulfate, or (for phenolic acids) glycine (hippuric acid formation). Conjugation of sulfate with a phenol to give the so-called ethereal sulfate (ester of sulfuric acid) is produced by means of activated sulfate, 3’phosphoadenosine-5‘-phosphosulfate(PAPS), and sulfokinase (Dodgson, 1977). The PAPS is formed by reactions consuming two units of ATP with the resultant loss of useful energy from other reactions. Glucuronic acid conjugation (Dutton, 1966, 1978; Williams, 1967) costs not only the energy necessary to produce the active transfer agent uridine diphosphoglucuronic acid (UDPGA), but also the potential energy and substance of the glucuronic acid. Similarly, an activated methyl group in the form of S-adenosylmethionine and the ATP consumed in its formation are lost from “normal ” metabolism, if they are consumed in detoxication. Glycine or glutamine conjugation would sacrifice the respective amino acids. In animals on a marginal diet, these costs of detoxication may be the difference between barely normal and abnormal health. No experimental animal adequately parallels man to serve as a satisfactory substitute in estimation of toxicity. Predictive values for toxicity in man have become more rather than less obscure as toxicity tests have become more detailed and have been done with more test species (Hayes, 1970). Qualitatively, the detoxication of a given substance by different animals may be similar, but quantitatively there are likely to be large differences not only among species, but among individuals of the same species under different conditions (Davies, 1977; Truhaut, 1978). The animal’s age, sex, diet, and environment affect toxicity and detoxication. Even the route of administration may be significant; in a study with hens, intramuscular injection of phenol gave 20 : 1 sulfate to glucuronoside, whereas oral and intraperitoneal administration gave less than 3 : 1 (Capel ef al., 1974). In an example illustrating species differences, phenol’s urinary metabolites were phenyl sulfate, phenyl glucuronoside, quinol sulfate, and quinol glucuronoside in man and the mouse, rat, guinea pig, jerboa, gerbil, hamster, and lemming, but only the phenyl glucuronoside was detected in the pig and the two sulfates in the cat (Capel et u l . , 1972). The urine of the dog, rabbit, ferret, and hedgehog lacked the quinol glucuronoside, and that of the squirrel monkey and capuchin lacked the quinol sulfate. About 12%of the urinary metabolites in sheep was phenyl phosphate (Kao el a / . , 1979). The cat’s very weak ability to make glucuronic acid conjugates may evolutionarily reflect its carnivorous habit (Capel et ul., 1972; Williams, 1967). The sulfate conjugation appears to be evolutionarily older than the glucuronide conjugation. Fish, aquatic turtles, and amphibia are usually weak in liver detoxication enzymes (Williams, 1967). High glucuronide conjugation relative to sulfate appears to be characteristic in the pig,

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a monogastric near-vegetarian, and it is also characteristic of New World monkeys. Old World monkeys and man, however, are highly capable in both sulfate and glucuronic acid conjugation (Smith and Caldwell, 1977). Nonprimates and prosimians convert arylacetic acids to glycine conjugates, but monkeys, apes, and man make glutamine conjugates of these compounds. Unlike the young of most laboratory animals, the human fetus and newborn infant are usually able to oxidize and detoxify a number of drug substrates. This ability seems not to be a result of exposure in utero, although that can have a role as well (Davies, 1977). It seems fair to conclude that man is relatively capable among animals of metabolically coping with ingested plant phenols and xenobiotics generally. The dose affects the pattern of conjugation. The capacity to conjugate sulfate is generally limited, and therefore, for man and other animals capable of both sulfate and glucuronic acid conjugation, glucuronosides become a higher proportion of the excretory products with higher dosage (Mehta et af ., 1978). Excretion of a relatively high portion of administered phenol as quinol sulfate in the cat is considered an indicator of slow conjugation and excretion, resulting in a longer residence time in the body and more metabolism of phenol to quinol (Mehta e t a l . , 1978). Detoxication is not complete until the substance is eliminated from the body. Excretion in the urine is the primary route for elimination of most water-soluble substances and conjugates. However, elimination via the bile can also be important (Griffiths and Barrow, 1972b; Hackett et a l . , 1979; Smith, 1973). Since the bile empties back into the intestine, enterohepatic recirculation of the substance usually occurs, with resultant slower elimination in the feces. The conjugate groups in bile excreta may be hydrolyzed off by intestinal microflora and allow reabsorption of the more toxic form. In general, the larger molecules are excreted in the bile (Hirom er a l . . 1976). In the molecular weight range of 350-450, there is usually extensive elimination in both bile and urine. For molecular weights below 350, biliary elimination is low even with renal obstruction. For molecular weights of 450-850, excretion occurs predominantly in the bile, and little appears in the urine, even if the bile duct is obstructed. Bile also increases absorption from the gut of fatty and lipid-soluble substances. Consuming phenols with lipid often increases their absorption.

IV.

EXAMPLES OF COMMON PLANT PHENOLS WITH ACTUAL OR POTENTIAL SIGNIFICANCE IN ANIMAL CONSUMPTION (TOXIC OR BENEFICIAL)

An effort will be made to include here all the common phenolic derivatives of plants having a specific history as toxic substances. Coverage will necessarily be as brief as possible, consistent with presenting a clear overall picture. Physiolog-

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ical activity in animals other than toxicity will be noted if it adds to the overall perspective of the phenols as dietary constituents. Groupings are somewhat arbitrary and overlapping. It has already been indicated that, owing to the evolution of efficient detoxication, the common dietary plant phenols should not be significant as toxicants under normal amounts and conditions, with the possible exception of tannins, which are seen as general deterrents to herbivore feeding. There are, however, limits to the amounts that can be tolerated, and a number of physiological effects have been reported. A.

SMALL PHENOLS, PYROLYSIS PRODUCTS

Phenol itself (Liao and Oehme, 1980), and other small phenols such as catechol, the cresols, guaiacol, orcinols, and pyrogallol, are commonly considered to be industrial chemicals. They may, however, be encountered as natural water constituents (from contact with shale, for example), and they constitute a sizable proportion of the solids of smokes, including smoke from tobacco. They are ingested by breathing smoke, but also by eating smoked foods. Most of the appeal of smoked foods is the flavor, the antioxidant, and the antimicrobial effect of the treatment, which is largely due to the phenols of the smoke. Pyrolysis of catechin (in gum catechu) and gallates was the historical source of catechol (pyrocatechol) and pyrogallol, respectively. The breakdown of lignin, hydroxycinnamates, and other natural phenols can give small volatile phenols (Singleton and Kratzer, 1973; Tress1 el a/., 1976). Both microbial breakdown and pyrolysis, for example, give 4-vinylguaiacol by decarboxylation of ferulic acid, and reduction or further reaction can produce the 4-ethyl and 4-methyl analogs and guaiacol. Pyrolysis in air gives vanillin and vanillic acid (Fiddler et d.,1967). Similar products can be produced from other natural cinnamates. Some of these "pyrolysis" products can be produced under relatively mild cooking conditions in foods and beverages. Pyrolysis of nonphenolic materials also produces phenols (Higman et a/., 1970). Sodium acetate, protein, or carbohydrate pyrolysis gives phenols, with 60-78% of the product of carbohydrate pyrolysis being phenolic. Not all the products of pyrolysis are simple phenols, of course. Benzpyrene and other arylhydrocarbons are produced, and napthoquinones, stilbenes, and more complex phenols have been found in smoke tars. Mutagens and comutagens are produced by charring of foods and pyrolysis of food constituents, and the mutagenicity is greater than can be attributed to benzpyrene (Sugimura and Nagao, 1979). However, the active substances do not appear to be phenols, and the pyrolysates of tyrosine and phenylalanine are among the least mutagenic of the amino acids (0.01 of the activity of tryptophan or serine, for example). Extensive studies in search of the best antiseptics among substituted small

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phenols have shown that microbial toxicity is general in phenols, but it varies from weak to strong (Singleton and Kratzer, 1969). Alkyl substituents on the ring tend to reduce animal toxicity and increase the antibacterial effect. Commonly, a second phenolic hydroxyl reduces both the antibacterial effect and animal toxicity. Many microorganisms can oxidize vicinal dihydroxyphenols to open the ring and produce aliphatic dicarboxylic derivatives subject to further metabolism (Dagley , 1967; Ribbons, 1965). This is probably one reason that considerable phenol may be unaccounted for after oral administration to animals. Phenol content estimated on the surface of smoked meat was about 37 mg/kg, but only about half was recovered, owing to losses in recovery and binding to the meat (Issenberg e t a / . , 1971; Singleton and Kratzer, 1973). Smoke components, probably the phenolic aldehydes such as sinapaldehyde, bind and thus cause considerable loss in dietarily available lysine (Chen and Issenberg, 1972). The toxicity of substituted small phenols in relation to the uncoupling of oxidative phosphorylation, bacterial inhibition, or phytoxicity can be predicted from physicochemical data on relative acidity and hydrophobicity (Fujita and Nakajima, 1969). The severity of nephrotoxicity of substituted hydroquinones and catechols injected in rats generally increased as their standard oxidationreduction potential decreased (Calder er al., 1975). Pyrogallol with a low potential was an exception that did not produce renal lesions under the test conditions. Interference with oxidation-reduction systems in the renal cells is the indicated mechanism, but quinone reaction with nucleophiles is an alternative. Pentachlorophenol, strictly an unnatural phenol, is a potent uncoupler of mitochondria1 oxidative phosphorylation, but it also interferes with microsomal detoxication reactions. The latter role appears to be more important, and it can have a synergistic effect with other toxins (Arrhenius et a / ., 1977). An oral dose of about 157 mg/kg was fatal to man; the oral LD,,, for rats is 125-210 mg/kg (Burger, 1966). Phenol, but generally not other monophenols, produced convulsions in experimental animals (Angel and Rogers, 1972). Catechol given intraperitoneally at 80 mg/kg caused tremors and epileptiform convulsions, even in the surgically anesthetized animal. Substituted catechols and certain hydroxynaphthalenes were active but less potent, and pyrogallol was inactive in these investigations (Angel and Rogers, 1972; Angel et a / . , 1977), but not in earlier studies (Angel and Rogers, 1968). The mechanism may involve catechol-0-methyltransferase and interference with the metabolism of catecholamines, but interference with acetylcholine metabolism is also indicated. The lethal dose for phenol in the adult human is 10-30 gm, and it is about the same for pyrogallol or resorcinol (Moeschlin, 1965). If phenol is spilled over a large surface of the skin, death may occur within 15 minutes. Detoxication of phenol by conjugation is very rapid even when the liver and gut are removed, and up to 600 mg of phenolic material of all types may be ingested per day in the normal human diet (Powell er al.. 1974). Feeding rats for 90 days with tenfold

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the estimated maximum human dietary content of 1,3-dimethoxybenzene ( 10 mg/kg per day) and guaiac wood oil (32 mg/kg per day) caused no adverse toxic effects (Oser et al., 1965). Phenol is nearly equally toxic whether given orally or subcutaneously (LD,,, 530 vs 400 for rats) (Singleton and Kratzer, 1969). Parenteral administration is usually ten times as toxic as oral (a rough estimate). Other small phenols are also similarly toxic regardless of the method of administration (e.g., for o-cresol the LD,,, for rats is 1350 orally, 650 subcutaneously). This is partly because these small phenols readily penetrate skin and other membranes. Phenol is a good solvent for lipids and proteins, and many membranes involve lipoproteins. Topical application to rat skin of phenol, o-cresol, m-cresol, p-cresol, dicresol, and 2,4-xylenol gave LD,,, values of 450,620, 1100, 750,825, and 1040 mg/kg, respectively (Uzhdavini and Gilev, 1976). Human skin is much less permeable than the skin of experimental animals (Idson, 1973). A balanced lipid-water solubility, a non-ionized state, and modest molecular dimensions are necessary for appreciable penetration of the skin. Penetration of phenol through the skin is speeded up about tenfold once the skin is damaged, and the minimum damaging concentration is much affected by the concentration and the solution in which the phenol is applied (Roberts and Anderson, 1975). The damaging phenol concentration on excised rat skin was 1.4% in liquid paraffin, 7% in water, 6.7% in peanut oil, and 80% in glycerol. Penetration is also shown by induction of liver enzymes after skin exposure only (Kappas and Alvares, 1975). The risk from small phenols as direct toxicants may not be the only risk involved, particularly in tobacco smoking. Cilia in the lungs sweep mucus and particulate contaminants from the lungs. Ciliary motion seems to be seriously and specifically impaired by smoke phenols (Bernfeld et a l . , 1964). Furthermore, phenols can serve as cocarcinogens, presumably by helping carry low-solubility carcinogens such as benzpyrene into target cells (Singleton and Kratzer, 1969, 1973). A single application of a carcinogen such as benzpyrene or multiple applications of a phenol solution from tea or from cigarette smoke did not produce significant skin cancer in experimental animals. A single application of benzpyrene, however, followed by repeated applications of phenol, did give a high incidence of cancerous skin tumors (Boutwell, 1967; Kaiser, 1967; Van Duuren et al., 1968). Similarly, increased lung cancer in exposed mice was associated with inclusion of the phenolic fraction in aerosols of tobacco tars (Tye and Stemmer, 1967). Certain phenols accelerate and others inhibit the absorption of benzpyrene into animal skin (Karu et al., 1972). Sugar solutions appear to have nearly equal effects in promoting benzpyrene induction of rat sarcoma, when compared with an aqueous extract of condensed cigarette smoke (Sydnor et al., 1972). Hydroquinone or quinol, a minor metabolite (in conjugated form) of phenol, decreased “takes” and increased the survival of mice receiving melanoma transplants (Chavin et al., 1980).

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B. PHENOLIC NUTRIENTS Tyrosine is considered a semi-indispensable amino acid for animals, since it spares the indispensable phenylalanine and is required in proteins and as a precursor for thyroxine, melanins, and related compounds. In general, an imbalanced excess of an indispensable or semi-indispensable amino acid is less well tolerated in the diet than an imbalance of one of the dispensable amino acids. Tyrosine at 3% or phenylalanine at 4% or more of the diet causes adverse effects in rats (Harper, 1973). Supplementation of a low-protein diet with 5% tyrosine gave rats external pathological lesions in a few days, but substitution of 4-hydroxyphenylpyruvate for the tyrosine gave no signs of toxicity in two weeks (Boctor and Harper, 1968). Low food intake was a symptom and not a cause of tyrosine toxicity, and addition of thyroxine increased the severity of tyrosine toxicity. These data have some value in suggesting maximum tolerance and detoxication levels for an essential constituent phenol. The water-soluble vitamins have generally been nontoxic, even at abnormally high levels in the diet. Fat-soluble vitamins are not always so benign (DiPalma and Ritchie, 1977; Hayes and Hegsted, 1973). Vitamin E (a-tocopherol, Fig. 2) appears to be relatively safe, however, even at levels higher than are possible with an unsupplemented diet. Consumption controlled to produce double the average plasma level in 28 adults over a period of 3 yr produced no definite bad or good effects. Allergy to vitamin E in an aerosol deodorant is the only known

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VITAMIN Kl

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FIG. 2. Structures of vitamin E (a-tocopherol) and vitamin K ( K , and menadione) active substances, tyrosine (the phenolic amino acid), and an uncommon cinnamylphenol.

PHENOLIC SUBSTANCES AS FOOD TOXICANTS

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toxic reaction to vitamin E in humans. A very high excess in animals can depress growth, thyroid function, mitochondria1 respiration rate, bone calcification, and hematocrit and sperm production, while increasing reticulocytosis and causing testicular atrophy. Deficiency symptoms include specific types of liver necrosis, muscular dystrophy, and respiratory deficiency. Adequate vitamin E helps protect against carbon tetrachloride hepatotoxicity, maintain embryonic growth, enhance certain immune responses, and prevent capillary permeability leading to exudative diathesis. The tocopherols, menaquinones, phylloquinone, ubiquinones, and plastoquinones are generally excreted in bile unchanged or in urine as glucuronoside conjugates with the side chain shortened to seven carbons and y-lactones formed (Wiss and Gloor, 1970). The natural vitamins K (K,, Fig. 2) prevent a hemophilia-like deficiency disease and are involved in respiratory chain electron transport. When injected, K, may cause localized nerve paralysis (Boctor and Harper, 1968). Premature infants have been killed by 10 mg of menadiol sodium diphosphate given daily for 3 days (Hayes and Hegsted, 1973). Most of the reports on toxicity in the vitamin K series have been on the synthetic water-soluble menadione (Fig. 2) as a hemolytic oxidant in the body at high doses relative to the dietary K intake. It leads to hemolysis when the natural fat-soluble menaquinones like K, do not. It is now known that vitamin K is part of a membrane-bound system that carboxylates glutamate residues in the peptide chains of a number of vitamin K-dependent proteins (Olson and Suttie, 1977; Suttie, 1979). The best known, bovine prothrombin, has 10 of 43 glutamic acid residues carboxylated on the y-carbon. Dicoumarol interferes with vitamin K in an unknown manner, so that fewer prothrombin glutamates are converted to y-carboxylglutamate, and the defective prothrombin does not function properly in blood clotting (Suttie, 1979). Dicoumarol can be considered an antivitamin or vitamin antagonist for vitamin K. Vitamin E antagonists of unknown structure exist in beans. Both vitamins E and K can be antagonized by vitamin A under some circumstances (Somogyi, 1973). In no case are common natural phenols from plants known to be active antagonists of vitamin E or K, and the exact mechanisms of antagonism are not yet clear. C. HYDROXYCINNAMATES The common phenolic cinnamates, particularly chlorogenic acid, are not generally present at high levels (coffee beans being an exception at about 3-8%), but they are present in nearly all tissues of higher plants in amounts up to 100 mg/kg fresh weight (Herrmann, 1967). In the course of processing, caffeic acid derivatives (the predominant natural hydroxycinnarnate) may be increased; defatted sunflower seed meal has about 2.4% (Mikolajczak et al., 1970). Chlorogenic acid administered orally to cats and rabbits at 1-2 g d k g or to mice and rats at

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4-5 gm/kg caused no symptoms of damage (Chassavent, 1969). The lethal dose

given intraperitoneally to mice was 3.5 gmlkg, but this appeared to be due to acidity, because the sodium salt at the same level was well tolerated. The common phenolic cinnamic acids do not ordinarily occur free in plants, but are acylated to quinic acid or other hydroxylic substances. Quinic acid and shikimic acids are among the rare nonbenzenoids that can be converted to benzene derivatives in animals (Brewster et al., 1977). It appears, however, that they are first converted to the saturated cyclohexane carboxylic acid by the intestinal microorganisms of man, mouse, rabbit, guinea pig, or hen (but not ferret-a carnivore) and then metabolized by the liver to the benzoic acid conjugate hippuric acid. The responsible components in the proven carcinogenicity of bracken fern are still uncertain (Wogan and Busby, 1980). Other fractions are definitely active, and the suggestion that part of this activity is due to shikimic acid seems to have been refuted by further studies (Miller and Miller, 1979; Wogan and Busby, 1980). Chlorogenic acid has been claimed to be allergenic, particularly in coffee; however, it now appears that this was incorrect and that the contamination of chlorogenic acid with coffee protein was the cause (Brewster et al., 1977; Layton et al., 1968). Considering its widespread occurrence, chlorogenic acid must have little allergenic potential, if any. At 1 gm/liter it has been reported to slow digestion by pepsin (Brewster et ul., 1977). A mixture of phenolic acids containing caffeic, p-coumaric, and ferulic acids (plus vanillic, protocatechuic, p-hydroxybenzoic, and rn-hydroxybenzoic acids) equivalent to the amount contributed by 5% distillers’ dried solubles in the diet increased the growth rate of chickens significantly (Dixon and Couch, 1970). In roughages fed to sheep, such as timothy hay and sawdust, vanillic acid, ferulic acid, p-coumaric acid, acetovanillone, p-hydroxyacetovanillone, and p-hydroxybenzaldehyde were present at about 10, 1.5, 1.5, 0.5, 0.5, and 0.005 mg/kg of lignin content, respectively (Fahey el al., 1980). These substances were partly modified or absorbed, since similar values in the feces fell about 10-50%. Phenolic antioxidants added to foods, notably butylated hydroxyanisole and butylated hydroxytoluene, inhibit the action of some chemical carcinogens when administered before or with the carcinogen. Similar effects have been shown for p-coumaric and o-coumaric acids (Wattenberg, 1980). The effect is apparently due partly to the enhancement of microsomal-detoxifying enzymes. Caffeic acid and other antioxidant vicinal dihydroxyphenols can also combat toxic reactions by scavenging free radical intermediates. Rao and co-workers have reported (1978) that nitrite in human saliva or rat diet produced nitrosation of added piperazine, oxytetracycline, and aminopyrine. Caffeic acid inhibited these nitrosations by 10-60%. Ferulic acid had a distinct antiandrogenic activity on the rat prostate, but not on other male accessory sex organs, nor did it have any estrogenic effect on

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female rats or mice (Saito et al., 1979). Dihydrocaffeic acid, which is also a metabolite in animals from flavonoids, caffeic acid, and isoferulic acid, but not dihydrocoumaric acid, can substitute for adrenaline as a cofactor in the formation of prostaglandin E from arachidonic acid in an in vitro preparation of rat renal medulla (Baumann et al., 1978). Bile flow, stomach acid secretion, and intestinal motility increase on administration of chlorogenic acid to experimental animals at about 5-100 mg/kg (Brewster et al., 1977). Caffeic acid has generally a similar effect, but quinic acid has not. Injection and oral administration were about equally effective routes for enhancing bile secretion. Cynarin (a dicaffeoyl quinate from artichokes) and the cinnamic acid fraction from wine (caffeoyl and related tartrates, Singleton et al., 1978) have been recommended as useful stimulants of bile flow, thereby lowering blood cholesterol. This appears to be a property common to many phenolic substances and perhaps to many other substances excreted via the bile, but the lack of toxicity of the common caffeic acid derivatives makes them attractive (Czok and Schulze, 1974; Galecka, 1969; Giraldi et al., 1970; Masquelier and Laparra, 1967). The lipid mobilization associated with fasting was normalized by cynarin and an oat polyphenol preparation (Dorigo and Fassina, 1970; Shulyakovskaya and Stroevaya, 1972). Cynarin has also been claimed to promote urea excretion and favorably affect chemically induced nephritis in the rabbit (Sokolova et al., 1970). Caffeic acid (but not cinnamic nor ferulic acids) has an antithiamine effect in v i m . It seems to be involved in bracken fern toxicity, which can be cured with high thiamine dosage, and in the antithiamine effect of coffee (Somogyi, 1978; Somogyi and Nageli, 1976). However, in other tests caffeic acid did not impair animals with latent thiamine deficiency, and in rats with sufficient thiamine it enhanced the level of thiamine in the body (Schaller et al., 1977; Swanowska and Tautt, 1972). A reported stimulating effect of chlorogenic acid on the central nervous system is minor compared with that of caffeine (Brewster et al., 1977). An effect that increases the volume per pulse, but lowers the pulse rate and blood pressure, appears to be related to the acidity. Chlorogenic acid is rapidly converted in the animal body to its hydrolysis products caffeic and quinic acids; it was not detected unchanged in the blood stream (Czok et al., 1974). Chlorogenic or caffeic acid gives rise in man to the main metabolites m-coumaric acid glucuronide and m-hydroxyhippuric acid (Brewster et af., 1977). Animal excretory products include conjugated and unconjugated caffeic, dihydrocaffeic, ferulic, dihydroferulic, vanillic, m-coumaric, and 3-hydroxyphenylpropionic acids. The hydrogenation of the a,P double bond and the dehydroxylation reactions may be the result of gut microbe metabolism (Sheline, 1968). Ferulic acid was excreted by the rat following injection both unmodified and as m-hydroxyphenylpropionic acid, which involves removal of the 4-hydroxyl, removal of the methyl group from the

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3-hydroxy1, and hydrogenation of the side chain (Teuchy and Van Sumere, 1971). Ferulic was not respired to C 0 2 and little was incorporated, but it was excreted after intraperitoneal injection-68% in the urine and 19% in the feces. Intravenous administration of p-methoxycinnamate to the rabbit gave a half-life of 0.4 hour, and blood level peaked within an hour after oral administration (Woo, 1968). The excretory derivative was p-methoxybenzoate. Westendorf and Czok (1978) found that hydroxycinnamates in doses of the order of 5 mg/kg were absorbed from the rat intestine two to ten times as fast as they were eliminated. They were excreted mainly via the urine within 2 hr. The time for 50% elimination was 13-37 min after intravenous administration of about 1 mg/kg. Only about 3% of caffeic and p-coumaric acids were excreted in the bile, but excretion of ferulic acid was about 30%. These workers concluded that chlorogenic acid was unlikely to have an important role in enterohepatic circulation. The antibacterial action of the more usual cinnamates may be significant in some instances (Gupta and Banerjee, 1978; Masquelier and Delaunay , 1965). Unusual cinnamylphenols (obtusastyrene, Fig. 2 ) are potent antimicrobial compounds in nature, and synthetic analogs are being considered for potential uses, including food protection (Jurd er al.. 1973; King et d., 1972). D. GALLIC AND ELLAGIC ACIDS AND RELATED DERIVATIVES Gallic acid is widely distributed in plants, but the concentration is usually very low. Bound forms of gallic acid, notably epicatechin gallate or galloyl glucoses and tannic acids, are associated with and probably are the main source of free gallic acid in foods. Gallotannic acid is discussed under tannins, and epicatechin gallate is a flavonoid. The acute toxicity of gallic acid is very low; 4-5 g d k g given subcutaneously or intraperitoneally is required for a 50% lethal dose to mice or rats (Anonymous, 1976; Singleton and Kratzer, 1969). Prolonged consumption in amounts well above normal food levels is not known to produce untoward effects. After intraperitoneal administration of gallic acid, ethyl gallate, syringic acid, or 3,4,5,-trimethoxybenzoicacid (or veratric or vanillic acid) at I50 mg/kg per day for 4 days in rats, no effect on liver microsomal enzyme activity was seen, whereas tannic acid at 25 mg/kg was inhibitory (Gaillard et ul., 1974). The major urinary metabolite from gallic acid and gallates including tannic acid is 4-methylgallic acid in rats or rabbits (Booth et al., 1959). A second product is pyrogallol . Syringic acid and 3,4-dimethoxy-5-hydroxybenzoicacids were produced from 3-0-methylgallic acid. It is interesting that the animal enzyme appears to favor methylation of the 4-hydroxy1, whereas the plant enzymes methylate the 3 and 5 positions. This may be a methyl-group-sparing mechanism in the animal. Feeding of 1o/o gallic acid in a diet low in methionine and choline gave rats fatty livers, which were prevented by the addition of choline or methionine

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(Booth et ul., 1961). A portion of the gallic acid is excreted unchanged, but the glucuronoside is also formed, and the total excretion of glucuronic acid is increased tenfold upon oral intake of gallic acid by rabbits (Blumenburg and Dohrmann, 1960). Gallates potentiate adrenaline toxicity (Baraboi, 1967), a property shared with several other readily soluble phenols, presumably by interfering with adrenaline’s detoxication by methylation, since catechol 0-methyltransferase is inhibited (Dorris and Dill, 1977). Gallic acid complexes with iron to make blue-black inks, but in the diet gallic acid can increase the regeneration of hemoglobin in anemic rats, presumably by making the iron more available and reducing it to the ferrous form (Stoewsand and Hrazdina, 1972). Gallic acid (4%) strongly inhibited acid secretion from the perfused stomach of rats as stimulated by intravenous tetragastrin (Tani, 1978). Tannic acid behaved similarly, but required higher concentration (8%). Protocatechuic, p-hydroxybenzoic, and salicylic acids or aspirin stimulated the response to tetragastrin and sometimes enhanced the basal acid secretion. Gallic acid derivatives, particularly esters, have appreciable activity against bacteria and fungi (Carlson et al.. 1951). Along with related esters of p-hydroxybenzoic acid, they have been used to stabilize cosmetics and foods, but they can produce crystalline deposits owing to their low solubility (Hinnekens and Vermeulen, 1966-1967). Contact dermatitis of an allergic type is produced in certain individuals by the latter substances (Goldberg, 1970). Bradykinin, a vasoactive peptide which induces contractions in smooth muscle, is antagonized by gallic acid and its esters as well as by certain tranquilizers (Posati et ( I / . , 1970). Gallates have been reported to have about 3% of the anti-inflammatory activity of hydrocortisone; they have also been used to inhibit spasms, to treat swellings, and to protect against radiation damage (Chernetskii et d.,1967; Saeed et ul., 1974). Gallic acid suppressed the in vitro immune response to antigens through its effect on the thymus lymphocytes (Archer et ul., 1 977). Protocatechuic (3,4-dihydroxybenzoic) acid is metabolized in man to vanillic and isovanillic (3-methyl-4-hydroxy- and 3-hydroxy-4-methoxybenzoic) acid conjugates in the ratio of about 12.8 : 1 after oral consumption of I-gm doses (Price, 1969). If isovanillic acid is eaten, some 3,4-dimethoxybenzoic acid is produced. Vanillin orally administered to rats was recovered 47% as vanillic acid, 19% as vanillyl alcohol. 10% as vanilloylglycine, 8% as catechol, 7% unchanged, 2% as 4-methylcatechol, 0.6% as 4-methylquaiacol, and 0.5% as guaiacol, for a total recovery of 94% (Strand and Scheline, 1975). These metabolites occurred mostly as glucuronide and sulfate conjugates, and the glucuronide conjugates that were excreted in the bile were metabolized by intestinal bacteria to the toluene derivatives and decarboxylated products. Ethyl vanillate is an antimicrobial agent active against fungi and food spoilage organisms. Its LD,,,

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for rabbits is about 1.6 g d k g , for guinea pigs about 2.2 gm/kg, and the maximum tolerated by rats is about 3 g d k g per day for an 18-day period (Christie et a/.', 1951). The effects of toxic doses include generalized engorgement of the capillary bed and hemorrhage followed by necrosis of the mucosa in the upper gastrointestinal tract. Myocarditis, renal degeneration, and thickening of pulmonary alveoli are seen also. Ethyl vanillate has been used against progressive infection of humans by Histoplasma capsulatum and has been credited with saving the lives of 5 out of 12 terminally ill patients (Christie et al., 1951). The curative dose was only 25-30% below the toxic dose. Ellagic acid is very poorly soluble in physiologically tolerated solutions, and oral toxicity appears to be nil under practical conditions. Very low levels of ellagic acid M ) in vitro or injected intravenously produce a hypercoagulable state in blood by activating the Hageman factor XI1 of the clotting system (Botti et al., 1965; Girolami et al., 1977). This ordinarily does not lead to platelet aggregation, thrombosis, or embolism in the animal, but it can minimize traumatic bleeding, and it speeds clotting. The activated clotting effect decays rapidly, but injection of ellagic acid on the ninth or thirteenth day after wholebody irradiation decreased the intensity of hemorrhage, compared with that in control animals without ellagic acid (Pospisil et al., 1969). Ellagic acid at 1.2 mg/kg given intravenously to mice on the eighth or sixteenth day of pregnancy increased the incidence of abortion (Moe, 1971). Suppression by ellagic acid of anaphylaxis, hypersensitive immune reactions, and inflammation edema induced by carrageenin have been reported (Briseid et a / . , 1971; Schwartz and Zimmerman, 1971). On the other hand, injection of ellagic acid directly into joints produces inflammation, which is countered by aspirin (Van Arman et al., 1970). The effects of ellagic acid on experimental carcinomas have been variously promoting or inhibiting; they seem to be related to effects on coagulation and blood supply. However, the hypercoagulation effect requires the lactone bonds and is destroyed by acetylation of the four phenolic hydroxyls (Botti er ul., 1965). Taspine, an alkaloid with a methoxylated ellagic skeleton, is an anti-inflammatory agent and inhibits the reverse transcriptase, RNA-directed DNA polymerase, of several animal tumor viruses (Sethi, 1977). Ellagic acid administered orally to normal rats did not appear in the feces or urine, and only a small amount was recovered from the feces of germ-free rats (Doyle and Griffiths, 1980). About 10% of the dose was excreted in the urine as the product with two fewer hydroxyls and one fewer lactone function, 3,8dihydroxydH-dibenz( b,d)pyran-6-one. A second metabolite was found but not identified; traces of sulfate conjugation products were also observed. Injection gave an additional metabolite in the urine. Both metabolites produced by oral administration were not found in germ-free animals, but they were produced when ellagic acid was incubated with normal feces, indicating that they are products of microbial action.

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E. I.

173

FLAVONOIDS

General Nutritional Aspects and Toxicology

The qualitative flavonoid composition of foods, some data on the amounts present, and the roles of flavonoids in foods have been reviewed periodically (Bate-Smith, 1954, 1959; Harborne, 1979; Heintze, 1965; Henmann, 1959, 1976; Kiihnau, 1976; Swain, 1962). Common plant foods contain from traces to several grams of flavonoids per kilogram fresh weight. A few hundred milligrams per kilogram is common for many vegetables and fruits. The human dietary intake has been estimated at about 1 g d d a y , with nearly half of this as total biflavans (which we consider condensed tannins) and the rest about equally divided (200 mg/day each) among anthocyanins, catechins, and 4-oxoflavonoids (flavonols, flavanones, and flavones) (Kiihnau, 1976). Animals consuming leafy plants may have much higher proportional intakes. Buckwheat may be as high as 5% rutin, leaves of rue as high as 2.2% rutin dry weight, and Chinese lettuce leaves 5.6 g d k g , fresh, in flavonoids. Individual seedling plants and plant parts may have very different qualitative patterns and relative contents of the different types of flavonoids. Clonally propagated varieties often can be identified by their flavonoid pattern-chemotaxonomy carried to the ultimate. The flavonoids contribute color, flavor, and processing characteristics important in food. They are ordinarily of extremely low toxicity. Broiler chickens fed diets with 2.5% citrus flavonoids were unaffected; 5.0% caused a reduction in growth (Deyoe er al., 1962). Anthocyanin preparations from berries at 20 g d k g given orally to rats and mice caused no deaths. No abnormalities were produced in rats fed 6 g d k g daily for 3 mo (Pourrat et al. , 1967). The LD5,,values in mice and rats were 4.1 1 and 2.35 g d k g for intraperitoneal injection and 840 and 240 mg/kg for intravenous, respectively. No teratogenic effect was found over three generations of rats, mice, and rabbits. Enhanced capillary resistance was greater than it was with rutin, and intravenous injections at 200 mg/kg caused vasodilation 2% that of adenosine, transient reduction of blood pressure, a decrease in respiratory amplitude, and diuresis. Quercetin, rutin, hesperidin, and especially tricin stimulated the basal metabolic rate when fed to rats (Stelig and Qasim, 1973). Naturally occurring in Prunus japonica fruits, acetylated kaempferol 3-rhamnoglucoside was a strong purgative, but it lost most of its activity when the acetyl group was removed (Haruji et al., 1975). An earlier finding of cataract formation after administration of rutin was shown to be due to impurities in the preparation (Nakagawa et al., 1965); in fact, dietary flavonoids appear to protect against diabetigenic cataract. Quercitrin fed to Octodon degus rodents at 2.5 gmkg of diet inhibited aldose reductase and the buildup of lens polyols, which causes

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cataracts (Varma et NI. , 1977). Similarly, gossypetin-8-glucoside from hibiscus flowers given at 1.5% in a 30% galactose diet for rats inhibited the enzyme, decreased galactitol deposition, and delayed, but did not prevent, cataract formation (Parmar and Ghosh, 1979). Absorption or excretion of galactose, itself, was not affected. Chemically modified to increase solubility, derivatives of quercitrin had effects on cataract prevention similar to those of quercitrin and greater than those found with rutin analogs (Fauran et a f . , 1978; Varma et uf., 1978). Some promise for preventive therapy or nutrition appears to be offered. Perinatal fatalities in human infants have been caused by supradietary parenteral dosage of the pregnant mother with rutin. The fatalities appear to have been caused by the insolubility of rutin and its formation of concretions within the liver and gall bladder, and by nephrotoxicity (Pfeifer et af., 1969, 1970). The more soluble derivative, rutin sulfate, did not give concretions, but caused other toxic effects, while the soluble semisynthetic derivative of rutin with three (7,3 ‘ ,4’) phenolic hydroxyls converted to /3-hydroxyethoxy groups was well tolerated, even in high doses. A mixture of the latter substance at 150 mg/kg per day plus coumarin at 25 mg/kg per day, injected from the sixth to the thirtieth day of gestation in pigs, produced no effects attributable to the medication in the mother or the offspring (Grote et a f . , 1977). Other selected examples of low toxicity or of mild negative effects might be cited, as well as more observations on the positive effects of flavonoids, but it has been generally agreed that the common flavonoids have very low toxicity when given orally, and it is usually still quite low when they are given parenterally (Bauer, 1978). In fact, the low order of toxicity and the mild but diverse and reportedly favorable physiological and therapeutic effects of flavonoids have promoted considerable search for synthetic or partially synthetic analogs with useful drug action. 2.

Biojlavonoids and Flavonoids as Drugs

The history of flavonoids as dietary constituents has been marred by considerable and continuing controversy regarding their role in relation to vitamin C, scurvy, and capillary bleeding. In 1936 Szent-Gyorgyi and co-workers reported (Rusznyak and Szent-Gyorgyi, 1936) that flavonoid preparations from paprika and citrus peel (“citrin”) could restore complete health to scorbutic guinea pigs when vitamin C alone did not. Particularly, a tendency of the capillaries to ooze blood after minor injury was corrected by the flavonoids along with vitamin C, and not by ascorbic acid alone. The flavonoids were termed vitamin P (for permeability). These reports were soon followed by both contrary and corroborative findings and by many reports on different flavonoids and other applications. By 1959, over 1000 papers had appeared; a number of reviews interpret this history (Anonymous, 1969; Bohm, 1959, 1960; Hughes and Wilson,

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1977; Kefford, 1973; Kefford and Chandler, 1970; Orzechowski, 1962; Vogin, 1960). In 1950, the American Society of Biological Chemists and the American Institute of Nutrition proscribed the term vitamin P on the basis that true vitamin activity was not exhibited. The term is still used, particularly in Russian literature, but it has largely been replaced by “bioflavonoid.” Further confusion arises from the fact that some authors claim that other phenolic substances, such as certain coumarins (notably esculetin), have “bioflavonoid” effects. Other workers consider planar benzpyrones to be the active structure, but they include catechins. Poor interaction among chemists, biologists, statisticians, and clinicians has plagued this subject more than most. In 1968, the United States Food and Drug Administration withdrew approval for use of bioflavonoids as a drug, even though no serious side effects were known, because it was considered that there was no proven efficacy in man for any clinical purpose. Since then, clinical use and experimentation have been minimal in the United States, but they continue apace in other countries, particularly in France, Germany, Hungary, and Russia (Come1 and Laszt, 1972; Farkas et ul., 1975, 1977). It seems clear that only vitamin C is curative for acute scurvy, but it is also true that flavonoids can have a favorable effect on chronic, borderline vitamin C deficiency. This vitamin C-sparing activity was known before 1936, and the active flavonoids were named vitamin CB,a term still used in France (Farkas er a / . , 1975, 1977; Kiihnau, 1976; Masquelier et 01.. 1973). Flavonoids of all common classes have been reported to have bioflavonoid activity, including, in increasing order, flavanones, anthocyanins, flavonols, leucoanthocyanidins, and catechins. Quercetin and its derivatives and catechins have been most studied, since they are more cheaply available and are quite active. When bioflavonoids at 50 mg/kg per day are administered to guinea pigs with a suboptimal level ( 1 mg/day) of ascorbic acid in the diet, and the results are compared with those for the same diet devoid of flavonoids, the animals receiving the flavonoids have a higher ascorbic acid content, particularly in the adrenals, spleen, liver, kidney, and leukocytes (Wilson et al., 1976; Zloch, 1974). Increased accumulation and retention in the reduced form in guinea pigs were also found for the unnatural D-isoascorbic acid administered orally with rutin and epicatechin (Zloch and Ginter, 1979). The bioflavonoids have other effects, but they may spare ascorbic acid by helping to keep dehydroascorbic reduced to ascorbic acid (Hughes and Wilson, 1977). The ratio between the two acids in animal tissues does seem relatively reduced under the influence of bioflavonoids. Flavonoids also can have an antioxidant effect in the diet itself (Bate-Smith, 1954; Kiihnau, 1976) and can help conserve minimal levels of ascorbic acid. Quercetin and many other active flavonoids are good chelators of copper and other heavy metal ions that catalyze rapid destruction of ascorbic acid (BateSmith, 1954; Hughes and Wilson, 1977; Kiihnau, 1976). Cavallini et al. (1978)

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found that quercetin and taxifolin were remarkably active, quercitrin less so, in protecting rat liver microsomal and mitochondria1 preparations from peroxidizing systems by free radical scavenging and not by metal chelation. The effects of flavonoids in animals have probably been too closely and unnecessarily associated with vitamin C (Hughes and Wilson, 1977). There are many other physiological effects of flavonoids in animals receiving an adequate supply of ascorbic acid. Willaman listed 33 types of biological and biochemical activities reported for flavonoids in 1955, and Bohm listed 40 by 1960. Although a few of these are either very limited in occurrence or have not stood the test of time, others are continually being added to the list or examined in more detail (Bauer, 1978). Havsteen ( 1980) has reviewed some specific biochemical effects of flavonoids in relation to medical applications. The most studied effects of flavonoids are related to the vascular system, particularly the decrease in pathological capillary fragility or the increase in resistance of normal capillaries to trauma (Comkl and Laszt, 1972; Fairbairn, 1959; Farkas et al., 1975, 1977; Gabor, 1974; Gazave et ul., 1974; Martin and Szent-Gyorgyi, 1955). Response to a small local skin injury or irritation involves capillary dilation (reddening) and edema (swelling, leakage of plasma proteins into surrounding tissue). Bioflavonoids resist these changes, apparently by more than one mechanism. They tend to maintain the normal tensile strength of capillary walls. They may antagonize histamine’s effect in inducing capillary permeability (but they are not specific antihistamine drugs). They appear to close capillary sphincters and to restrict blood entry by prolonging catecholamine action. They may affect collagen formation. They appear to affect membrane integrity, permeability and some forms of active transport across cellular membranes. Exhaustive review of these areas is not possible, owing to voluminous and somewhat conflicting reports, but in order to relate physiological effect to potential toxicity, some discussion seems required. Catechin, reported as one of the most potent bioflavonoids, occurs as the free aglycone and is fairly water-soluble, but it is also relatively lipid-soluble and apparently can adsorb to cells, interact with membrane proteins and lipids, and affect permeability in both directions across the membrane (Farkas et al., 1977; Ring et al., 1976). Collagen is the major organic constituent of the blood vessel wall, and catechin stabilizes it in terms of shrinkage temperature, etc., apparently by an adsorptive tanning and cross-linking process (Come1 and Laszt, 1972). The fact that biflavans (small condensed tannins) are quite active in capillary strengthening, especially if injected, also suggests phenol-protein binding as part of the effect on collagen. Indirect effects may include sparing of vitamin C, since ascorbic acid has a clearly established role in the collagen (hydroxypro1ine)forming lysine-proline hydroxylation system (Farkas et al., 1977; Hughes and Wilson, 1977; Kiihnau, 1976). Flavonoids in the diet have a normalizing, protec-

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tive role against abnormal collagen formation in lathyrism, a poisoning with natural P-aminopropionitrile leading to aortal aneurism (Cetta el al., 1972; Gendre et al., 1977). Catechin appears to have a general protective effect on collagen and the connective tissue system (Farkas et al., 1977). This effect appears to be related to adsorption affinity of the protein-tannin type, but it also involves hydroxyproline synthesis. Blumenkrantz and Asboe-Hansen (1978) found that catechin in the medium was inhibitory to collagen biosynthesis in chick tibia cultures. Decreased collagen formation was due to decreased biosynthesis of the protocollagen, decreased activity of the hydroxylating enzymes, and chelation of ferrous ions. Catechin was recommended for testing in diseases involving increased collagen synthesis, such as scleroderma. Effects that may be related to normalizing collagen include findings by Hladovec ( 1977) that exfoliation of endothelial cells into blood was inhibited by parenterally administered flavonoids in rats. This exfoliation is enhanced in humans by cigarette smoking, and oral administration of hydroxyethylrutosides suppressed the effect (Prerovsky and Hladovec, 1979). Daily oral administration of a mixture of 30 mg of coumarin and 180 mg of a hydroxyethyl derivative of rutin for periods of 1-54 mo produced significant softening of the arm tissue in patients after mastectomy (Clodius and Piller, 1979). This mixture and similar preparations involving hydroxyethylrutins have been widely used for some time experimentally and clinically, and in general medicine in Europe, with few undesirable effects noted (Scholten, 1979; Voelter and Jung, 1978). In experimental acute pancreatitis in dogs, only those that received injections of tris-0-hydroxyethylrutins plus coumarin survived, and both pancreatic edema and necrosis were reduced by the treatment (Bartos et al., 1979). Robinin and hyperin injections lengthened the average survival time of mice subjected to bilateral nephrectomy (Sokolova et al., 1978). Rats on a flavonoid-free diet showed prolonged effects from caffeine, harmine, morphine, hexobarbital, and pentobarbital, compared with those on a normal diet (Seidel and Endell, 1978). The results were attributed to activation of liver metabolism by the flavonoids. The rats on the flavonoid-free diet over 25 wk grew significantly more in body weight and lipid content than did those on the normal diet, but no data were given on the effects of subsequent addition of flavonoid to the animals on the flavonoid-free diet. Silybin, an unusual coniferyl alcohol derivative of dihydroquercetin, if administered very soon after Amanita mushroom poison in rats, mice, and dogs, produces an antitoxic effect apparently by inhibiting uptake of the toxin by the liver and increasing its urinary excretion (Faulstich et al., 1980). Potentiation of catecholamines by interference with their inactivation is one possible mechanism for the reduction of capillary permeability and the spasmolytic activities of flavonoids. This interference appears to occur in more than one manner. As was already noted, phenols in general are often substrates for detoxi-

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cation by methylation. Methylation occurs during the digestion and metabolism of flavonoids and would compete with catecholamines for a limited methyl group pool (Gazave and Parrot, 1973a,b). However, some flavonoids appear to be inhibitors of catechol 0-methyltransferase, and thus prolongers of the catecholamine effects, without serving as substrates (Kuhnau, 1976). A catechol B-ring and aglycone rather than glycoside were more inhibitory, but some activity was shown by all chalcones and flavonoids (Gugler and Dengler, 1973). Feeding of epicatechin to rats on a choline-deficient diet prevented a fall in choline level in blood and liver, normalized the lipid content of the liver, and decreased the cholesterol content (Chaudhari and Hatwalne, 1977). This apparently opposite effect might result from diminished methyl group consumption through 0-methyltransferase inhibition, but serious nervous effects were not reported. Inhibition of oxidation of catecholamine derivatives by flavonoids may also be involved, but interference with the uptake of catecholamines into adrenergic nerve terminals appears not to be affected by flavonoids (Jayasundar et al., 1977). The effects of flavonoids on blood vessels and edema combine to give an anti-inflammatory action (Gabor, 1972, 1974). Reports of the beneficial effects of flavonoids in many experimental conditions have often been followed by clinical application. Conditions include prevention of or aid in recovery from frostbite, myocardial infarction, thrombophlebitis, scalding, “little strokes,” bruising in contact sports, and irradiation damage. Bioflavonoids given orally in the final stages of Rh-factor-threatened pregnancies have been reported to reduce antibody formation by decreasing the contact between fetal and maternal blood. Venous insufficiency and varicosity problems have been treated rather widely in Europe with sizable and prolonged dosage with flavonoids. Only with parenteral administration, and then apparently only when solubility problems are involved, have any serious problems of toxicity appeared. Injected flavonoids have also been reported to produce a transient lowering of arterial blood pressure, a slowing of the heart rate, and yet an increase in the amplitude of the pulses and blood flow. Flavonoids inhibit erythrocyte aggregation (Robbins, 1977) thereby lowering the blood viscosity slightly (Farkas et ( I / . , 1977). Methoxylated flavonoids such as nobiletin and tangeretin (Fig. 3) of citrus fruit appear to be the most active (Robbins e t a / . , 1971), and flavonoids increase survival of rats on thrombogenic diets (Robbins, 1967). Flavonoids and certain other phenols antagonize the spasmogenic activity of prostaglandins or affect the conversion of arachidonic acid to prostaglandins; this is probably also related to their anti-inflammatory activity (Damas et a / . , 1977; Levine and Hong, 1977; Lindgren et d., 1977; Murrari et a / . , 1972). Baumann et a / . ( 1 979) found many phenols, including catechin and the cresols, to be inhibitory, but others, including rutin, dihydrocaffeic acid, and p-hydroxybenzoic acid, to be stimulatory to rat renal medulla prostaglandin synthetase. Dihydrocaffeic acid, which can be a

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TANGERETIN

FIG. 3 . The structure of tangeretin, an example of relatively rare, fully methoxylated flavones of nutritional significance only in the peel of various citrus fruits. (Nobiletin = 3'-methoxytangeretin.)

metabolite of flavonoids in animals, had about the same activity as adrenaline. The dimethoxy derivative was without stimulatory effect. Flavonoid-3-glucosides, but not aglycones, appear to release endogenous histamine when injected (Lecomte, 1971). Quercetin given intraperitoneally at 25- 100 mg/kg protected guinea pigs against bronchospasm from coadministered histamine (Marozzi et al., 1970), but generally flavonoids have not appreciably modified anaphylaxis. Catechin at concentrations of lop3M or less markedly inhibits histidine decarboxylase, the producer of histamine, without inhibiting histamine-inactivating enzymes (Lorenz et u I . , 1973). The incidence of experimental stress ulcers in rats was reduced about 80% by catechin administered intraperitoneally at 1-50 mg/kg, which blocked histamine formation (Reimann et ul., 1977). Unusual isoprenylflavonoids had specific inhibitory effects on experimental gastric ulceration in rats (Sasajima e t a / ., 1978). Histamine appears to be a control mechanism for gastric secretion, and clinical trial to suppress gastric hypersecretion was recommended, since catechin is so low in toxicity. Flavonol aglycones including quercetin strongly inhibit induction of histamine secretion from rat mast cells and appear to act by inhibiting calcium-ion active transport ATPase (Fewtrell and Gomperts, 1977a,b). Calcium entry appears to trigger the histamine release. The effect of flavonols with membrane active transport ATPases may be general; for example, the sodium-potassium ATPase of rat brain microsomes is inhibited (Mirsalikhova and Pakudina, 1977). Wuethrich and Schatzmann ( 1980) found that active transport across human erythrocyte membranes of Ca2+,Mg'+, and Na+-Ki was inhibited by quercetin. ATPases with different characteristics were involved. Spector and co-workers (1980) purified the Nai-Ki ATPase from Ehrlich ascites tumor cells and incorporated it into Iiposomes, which then weakly catalyzed the ATP-dependent influx of N a + . Addition of quercetin at 24 pg/mg of protein plus lipid gave a severalfold increase in the efficiency of pumping Na+ per ATP by the preparation, but had no effect on similar but already efficient ATPase preparations from electric eel or mouse brain. Quercetin may be acting as a regulatory subunit that protects the membrane enzyme against the

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entry of water. The relative inefficiency of the tumor ATPase appears to be related to the high aerobic glycolysis of tumors. Flavones, in general, at relatively high concentrations (800- 1000 nmoYmg of protein) inhibited mitochondrial ATPase (Bohmont and Pardini, 1979). Flavonoids affect carbohydrate and fat metabolism and ATP production in several ways, and generally it is more than a “simple” decoupling of oxidative phosphorylation (Farkas et a/., 1977; Gajdos et a / ., 1969). Tumor cells have an abnormally high aerobic glycolysis, which is regularized by quercetin, which also inhibits tumor cell proliferation at concentrations of 5-20 mg/liter (Suolinna el a / ., 1974, 1975). Bioflavonoids such as quercetin inhibit mitochondria1 hexokinase activity without changing the ATPase activity (Graziani, 1977). Quercetin occurs free and bound in complex with calcium and other metal ions. The enzymic influence of quercetin depends on the metal ion concentration; it appears that the metal complex inhibits whereas the free flavonol stimulates ATP hydrolysis (Mal’yan and Akula, 1977). Flavonoids can have a rather specific action on animal metabolism and membrane transport, frequently involving the carbohydrates. Phlorizin, a botanically unusual dihydrochalcone glucoside, has a specific ‘ ‘diabetes-like” effect on glucose excretion by animals, as is well known (Singleton and Kratzer, 1969, 1973). The rapid excretion by Ehrlich ascites tumor cells of lactic acid via a lactate-proton mechanism is potently inhibited by quercetin and related compounds (Belt et al., 1979). Flavonoids with four or five hydroxyl groups and an intact C-2-C-3 double bond, and without glycosidation, were most active. At about 0.1 pg/mg of protein, quercetin inhibited lactate efflux by 50%. Apparently, a lowered internal cell pH is involved. The inhibition of aldose reductase and the delay of cataracts have already been discussed. Naringenin appears to have direct effects on the membranes as well as general metabolic effects in dog renal cortex slices (Robinson et al., 1979), as measured by oxygen consumption and accumulation of glycine and glucoside, hippuric acid, and nicotinamide derivatives. Cyclic adenosine monophosphate (-AMP) affects many aspects of animal metabolism, notably the mobilization of glucose 1-phosphate from glycogen. Among 17 flavonoids, quercetin and kaempferol were the most active in inhibiting cAMP phosphodiesterase, the enzyme that hydrolyzes cAMP to AMP thus deactivating it (Beretz et a / . , 1978). Their activity approached that of papaverine, and several other flavonoids were active in the micromolar range. Additional studies added cyanidin chloride to the very active list and showed that the effects of the active flavonoids were competitive (Ferrell et a / . , 1979). The related guanylic nucleotide, cGMP, phosphodiesterase was even more inhibited than the cAMP enzyme, with (+)-catechin especially selective between the two. Amentoflavone potency was 100 times as high as that of the other flavonoids, and five to ten times as inhibitory as papaverine in separate tests with

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cAMP (Ruckstuhl et al., 1979). It is believed that the active flavonoids compete with cAMP for a nucleic acid binding site at which stacking occurs (Ferrell et a l . , 1979). Activity among the tested compounds was flavonols > flavones or anthocyanidins > flavanones or flavanonols > catechin (Beretz et a l . , 1978). The dietary significance, if any, of these findings is not yet clear, but cAMP affects many aspects of animal metabolism, and the activity is high. Catecholamines like adrenaline activate adenylate cyclase, resulting in increased CAMP, a I-pmol increase of which, per kilogram of muscle, causes the formation of 25,000 times as much glucose l-phosphate per unit time. A high level of cAMP is also associated with a decreased nerve response to excitatory stimuli. Inhibition of hydrolysis of cAMP would intensify or prolong the reactions. 3 . Mutagenicity

Older reports of treatment of experimental cancer are variable, some reporting inhibition of “take” or growth of transplanted cancers by certain flavonoids, others reporting stimulation or no effect. This situation is common to other phenols, especially coumarins. For example, DiGiovanni et al. (1978) reported that quercetin inhibited by 22%, whereas myricetin, by 54%, and 4’,5,7trihydroxyflavone, by 29%, enhanced skin tumor initiation when applied topically to shaved mice before application of a benzanthracene carcinogen. There has been sufficient indication of antineoplastic and cytotoxic effects, particularly among uncommon flavones (Kuhnau, 1976), to initiate continuing searches among nonfood plants for useful, unusual flavonoids (Dobberstein et u l . , 1977) and for the preparation of synthetic analogs (Slaga et a l . , 1977), but the common flavonoids do not appear to be useful anticancer drugs (Fritz-Niggli and Rao, 1977). The variable results may be due in part to the fact that low levels of flavonoids can stimulate cell proliferation, whereas higher levels, particularly if quinones are formed, inhibit normal cell multiplication, according to in vitro studies (Huot et al . , 1974). Also, only aglycones appear to be potentially active, and glycosides occur more commonly (Kuhnau, 1976). That common flavonoids apparently have little useful or specific effect against neoplasms is some comfort, because many antitumor drugs are carcinogens (Newberne et al.. 1978). The tumor-promoting or cocarcinogenic effect of tea flavonoid derivatives has already been mentioned and has been tentatively attributed to aid in penetration of the active carcinogen to the sensitive site. Somewhat surprisingly, there is evidence that some tris-0-(P-hydroxyethy1)rutoside can penetrate from gel or ointment through human skin (Pratzel, 1972; Wasilewski, 1972). Recently, different groups of workers have reported on the mutagenic activity of flavonoids on Salmonella typhymurium mutants-the Ames test. In one study, naringin, rutin, neohesperidin, hesperetin, dihydroquercetin, and permethyl

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quercetin were inactive with or without liver microsomes. Only quercetin, of those tested, caused a reversion of mutants (that is, mutation) without metabolic activation by liver microsomes (Bjeldanes and Chang, 1977). Microsoma1 activation increased the mutagenicity of quercetin threefold and converted quercetin pentaacetate to a mutagen. Activated quercetin was 0.1-0.001 as active as the recognized potent carcinogens 2-aminofluorene and aflatoxin. Two hydroxyphenylacetic acids, metabolites of quercetin, were inactive. Another group reported kaempferol, quercetin, and galangin to be mutagenic, but removal of a hydroxyl from galangin (3,5,7-trihydroxyflavone)greatly reduced or destroyed the mutagenic activity (Sugimura et a l . , 1977). A third group found frameshift mutation from the flavonols quercetin > myricetin > rhamnetin > kaempferol > fisetin > morin > robinetin > apigenin (Brown et ul., 1977). Taxifolin, dihydroquercetin, fell between fisetin and morin, and diosmetin and galangin were inactive. All the mutagenic flavonoids were significantly activated by microsomes. Glycosides were weakly active, but they increased 10- to 20-fold in mutagenicity when incubated with gut bacterial enzymes (presumably glycosidases included) and microsomes. The bacterial mutagen of a Japanese spice from seeds of sumac has been shown to be quercetin (Seino et a / . , 1978), and that from a Japanese vegetable pickle was kaempferol and isorhametin (Takahashi et ul., 1979). The Japanese pickle mixture was, however, only about one-sixth as mutagenic as a Chinese pickled product it resembled, and the Chinese sample was heavily contaminated with a fungus known to produce nitrosamines. Hardigree and Epler (1978) also found quercetin and myricetin to be active without liver microsomal activation, although more so after, whereas kaempferol, rutin, morin, and fisetin required microsomal activation to show reversion mutation rates above the control rate. Induced reversions with these compounds occurred only with the frameshift mutant bacteria and not with missense mutants. Macgregor and Jurd (1978) tested forty compounds related to quercetin in the Salmonella typhimurium strain TA98 assay and found four (quercetin, myricetin, rhamnetin, and 5,7-di-O-methyI-quercetin) that were unequivocally mutagenic without metabolic activation, and six that were mutagenic with activation (galangin, kaempferol, tamarixetin, morin, 3'0-methylquercetin, and 4' ,7-di-O-rnethylquercetin). They concluded that mutagenicity required a flavonol structure (flavonoid with 4-keto, free 3-OH. 2-3 double bond) and a 5-OH group or other configuration that weakened the hydrogen bonding between the 3-OH and the 4-keto groups, allowing the 3-OH to tautomerize to the 3-keto form. Although free B-ring hydroxyl groups were not essential if a rat liver metabolic activating system was employed, the active form appears to require a B-ring that permits oxidation to a quinoid form. They suggest that quinone methide forms or hydroperoxides may be the proximate mutagen, and note that it is evidently not quercetin itself, owing to considerable

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increased mutagenicity after activation by rat liver microsome preparations. So far, it appears that flavonoids in general are not mutagens in this bacterial test; even flavonols are often weak or inactive mutagens requiring activation with primed microsomes or bacterial enzymes, and activation requires removal of the glycoside groups normally attached to flavonols in the plant. The significance of these results remains to be seen. It is difficult to believe that, as ubiquitous as quercetin is in the normal diet of all plant-eating creatures, it can be a significant carcinogen. Few indications of such an effect have been forthcoming from animal testing, as far as this reviewer knows. There is a report that bladder implants of solid quercetin might give cancer in mice (Boyland et al., 1964). Quercetin and other flavonoids tested did not increase the normal frequency of micronucleated polychromatic erythrocytes in the bone marrow of mice given up to 1 gm/kg by mouth (Macgregor, 1979). The same result was obtained by quercetin injected intraperitoneally. Rat urine after a similar dosage was mutagenic to the Salmonella strains TA98A and TA100, indicating the presence of the mutagen in the animal. Brown (1980) and Sugimura and Nagao (1979) have also reviewed the situation. A study in progress indicates no increases in numbers of tumors in mice on a 2% quercetin diet. More rare types of tumors may be present in the test group (Brown, 1980). It is usually accepted as axiomatic that essentially all mutagenic substances have been or will be shown to be carcinogenic. Certainly, concern and careful investigation are mandated by indications of mutagenicity. Activation by bacterial preparations can involve removal of P-glycoside groups that animals alone cannot hydrolyze. Further extensive metabolic breakdown and/or conjugation, however, would generally follow in the normal dietary situation. The rat liver microsomal preparations used for activating quercetin in these tests were taken from rats injected with polychlorinated biphenyl, P-naphthoflavone, or phenobarbital a few days previously (Sugimura and Nagao, 1979). Would the results be the same if quercetin were used to condition the livers to be utilized as activator for the bacterial mutagens? The bacteria are themselves already mutants produced by introduction of a plasmid (Sugimura and Nagao, 1979) and are nutritionally deficient, so that reversion will lead to growth and recognition. Mutagenesis in this system could be viewed as a repair process. It is conceivable that a substance could aid reversion to wild type-that is, repair-without being a carcinogen in animals. This seems more likely with frameshift mutants than with missense mutants. Frameshift mutations generally involve intercalation of a planar molecule into the DNA base stack, thus generating a mispairing and frameshift when replication occurs (Bjeldanes and Chang, 1977). If equivalent planarity, a few free phenolic hydroxyls, and a modest size are all that are required, one could easily make a long list of natural phenols that should be active+llagic acid and cyanidin, for example. Penetration to the genetic material may be another factor

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of importance. To make quercetin soluble, dimethylsulfoxide was used in many of the experiments with Salmonella. A notable penetrant, it did not show activity with other flavonoids, but it is known to cany solutes into animal tissues. Penetration to the DNA of a prokaryote may not be the same as with a eukaryote, particularly with the barriers posed by the whole animal. Obviously, it is important to elucidate the true risk to humans and other animals of cancers from this source; one hopes that it will be proved as small a risk as now seems probable from the known exposure. At least it appears by the same sensitive Ames tests that most other flavonoids, including some synthetic (“abnormal”) ones, are not mutagenic. 4 . Metabolism of Flavonoids and Further Consideration of Toxicity

The metabolism of flavonoids by animals has been studied in some detail but is not completely understood (DeEds, 1968; Fairbairn, 1959; Farkas et al., 1975, 1977; Griffiths and Barrow, 1972b; Kiihnau, 1976; Mosser et al., 1974; Williams, 1959). Many of the recent studies have used semisynthetic flavonoid derivatives. One of the early problems was that flavonoids were not detected in blood or urine after oral dosage, or were found only in traces after massive doses. It appeared that they were not absorbed. This view has now been corrected by both direct and indirect experiments. By circular dichroism, both free and protein-bound hydroxyethylrutosides could be detected in blood, reaching a maximum several hours after a 4-gm oral dose in man on a flavonoid-free diet and remaining detectable for 24 hr (Farkas et al., 1977). By perfusion of the small intestine under conditions intended to avoid microbial effects, absorption of hydroxyethylrutin into arterial blood was found (Farkas et al.. 1975). In various studies of passive transport across model membranes and biological preparations, it has also been shown that absorption occurs, with the aglycones generally being absorbed most readily and the glycosides less readily. More was absorbed at acid (stomach) pH than at intestinal pH 6, and rutin was absorbed least, quercitrin and isoquercitrin more, and the aglycones (quercetin, kaempferol, myricetin, and rhamnetin) most (Kozjek et al., 1975). Epicatechin-2-sulfonate was absorbed faster than catechin, and trihydroxyethylrutin least, in everted rat intestinal sacs; in all three cases the rate was increased by sodium taurocholate (Crevoisier et al., 1975). Among the reasons that absorption of flavonoids by animals is difficult to show by examining the blood and urine is the fact that the flavonoids appear mainly in the bile, particularly as glucuronides (Griffiths and Barrow, 1972b; Griffiths and Hackett, 1977; Hackett et al., 1979). Another reason is the rapid disappearance of flavonoid from the blood by concentration in the liver and other tissues (Forster, 1977). Disappearance during perfusion of isolated rat liver was more rapid for the less water-soluble-that is, more nearly normal and less

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substituted-members of the mono- through tetrasubstituted hydroxyethylrutin derivatives. Still a third reason absorption of flavonoids is difficult to show is the appearance of only phenolic acid breakdown products in urine, so that it is not possible to say whether the flavonoid was absorbed intact or was broken down first. Oral administration of (+)-catechin, apigenin, myricetin, hesperidin, naringin, rutin, 3',4',7-tri-O-P-hydroxyethylrutoside,and 3',4',5,7-tetra0-j3-hydroxyethylrutoside to germ-free rats was followed by examination of the feces and urine for phenols (Farkas et a l . , 1975; Griffiths and Barrow, 1972a). Phenolic acids, beyond a trace attributed to the diet, were not detected in the urine under circumstances in which they were found in rats with conventional intestinal flora. Conjugates of catechin and naringin were found in the urine from germ-free rats. Large amounts of unchanged rutin and the other glucosides were found in the feces, but no aglycones, corroborating previous reports that glycoside hydrolysis is the result of microbial action in the gut. Catechin was recovered unchanged from feces only in low amounts, indicating nearly complete absorption. Two catechin conjugates were found in both urine and feces in germ-free rats, but in neither in normal rats, indicating microbial breakdown of any conjugates reaching the feces via the bile in normal animals. With the bile duct ligated, rats absorbed 91 % of radioactive hesperetin (Brown et al., 1978). The same authors were interested in preparing analogs of the highly sweet flavonoids that could be used for artificial sweetness in foods without being absorbable or metabolized by gut microflora. Sulfoalkyl derivatives produced by 0-alkylation of the sweet dihydrochalcone with propane- 1,3-sultone were sweet, poorly absorbed, and not degraded by microorganisms. Enzymic extracts of animal organs are reported to break down flavonols to benzoic acids without the intervention of microorganisms (Kuhnau, 1976). Perfusion of animal liver with apigenin gave six metabolic derivatives (Takacs and Gabor, 1975). Two of these appeared to be unidentified conjugates of apigenin, but the others were the 4-hydroxy derivatives of benzoic, cinnamic, phenylacetic, and phenylpropionic acid. Similar perfusion with rutin (Come1 and Laszt, 1972; Takacs and Gabor, 1975) gave eleven metabolites that were claimed, on the basis of paper chromatography and spectra, to include apigenin, luteolin, di- and trihydroxyflavone, isoferulic acid, 3,4-dihydroxyphenyl-y-valerolactone, 3-methoxy-4-hydroxyphenylacetic acid, and 3-hydroxybenzoict 3-hydroxyphenylacetic, and 3-hydroxyphenylpropionic acids. These studies indicate that, without microorganisms, animals can extensively metabolize flavonoids to acidic breakdown products similar to those produced by microorganisms, but in the normal circumstance, gut microflora play a large role. The urinary constituents after oral administration of flavonoids to normal animals have been the most studied, but for the reasons just given the results may be somewhat misleading. Nevertheless, many data clearly indicate that the

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flavonoids, by a combination of microbial and animal metabolism, are generally converted to a series of B-ring derivative acids, depending somewhat on the flavonoid (Farkas et NI., 1975, 1977; Kuhnau, 1976). Different animals give generally similar products, and this may, in part, be due to similar microflora. Flavonoid pigments have been reported to be excreted without change in normal carnivore urine, but not in the urine of herbivores (Bate-Smith, 1954). The A-ring portion of the flavonoid is generally metabolized more or less completely to COP,and as a rule the B-ring acids appear as mixtures of benzoic, phenylacetic, and phenylpropionic acid derivatives, depending on the degree of oxidation and the original flavonoid (Kuhnau, 1976). Catechin. which has been the most completely studied, appears unique in that, in addition to the expected hydroxphenylpropionic and hydroxybenzoic acids, there was a series of hydroxylated and methylated neutral compounds, phenyly-valerolactones. These lactones are apparently produced by oxidation and fission of the catechin A-ring to oxalacetic acid and a &lactone from the heterocyclic ring with the B-ring attached (Fig. 4). This substance is analogous to antibiotically active natural products. The &lactone is unstable and rearranges to the phenyl-y-valerolactone (Fig. 4). In men receiving 4.2-gm oral doses of catechin, no effects were noted or slight diarrhea; 7.5% was excreted unchanged in the urine, and 18.6% in the feces (Das, 1971). Of eleven urinary metabolites, the major ones were 3-hydroxyphenylpropionicacid, S-(3,4-dihydroxyphenyl)-yvalerolactone, and S-(3-hydroxyphenyl)-y-valerolactone.Sulfate and glucuronide conjugates of the catechin and of the three metabolites were also found. A small amount of 6-(3-methoxy-4-hydroxyphenyl)-y-valerolactone glucuronoside was also observed.

OH OH

\ t

COOH I

COOH

OX

FIG. 4. Prohable route of conversion of (+)-catechin to y-valerolactone derivatives by intestinal tlora of animals.

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Catechin’s breakdown in the rat was similar to that in man, but in the guinea pig and rabbit the products were fewer, and the major ones (3-hydroxybenzoic, vanillic, and protocatechuic acids) were not detected in human urine. In the monkey, oral or intraperitoneal administration of radioactive catechin resulted in urinary excretion of 53.5 and 50.2%, respectively, of the dose (Das, 1974). Radioactivity in the feces was 0.8-2.2% of the dose; 4.3% was excreted as 3-hydroxyphenylhydracrylic acid, and 7.6% as 6-(3-hydroxyphenyl)-y-valerolactone, the two major metabolites. The hydracrylic acid arose from P-hydroxylation of 3-hydroxyphenylpropionic acid. Considering that a large part of the flavonoids is excreted via the bile (Griffiths and Barrow, 1972b; Griffiths and Hackett, 1977; Hackett et a l . , 1979), it is perhaps reasonable that oral doses of about 100 mg/kg (and hydroxycinnamates likewise) have been reported to stimulate bile flow, lower the blood cholesterol, and help protect against an atherogenic diet (Kiyasheva, 1974; Lisevitskaya et a / . . 1970). Such an effect from the phenols of red wine (Masquelier, 1961) appears to be variable in rats, depending on their sex and history (Stoewsand and Anderson, 1974). A flavonoid preparation from lespedeza and robinin, a kaempferol glycoside, has been reported to increase the flow of urine and urea output, and to improve experimental renal insufficiency (Berger, 1968; Drumev and Pashov, 1970; Gulyaev, 1972). Protective effects of flavonoids against other xenobiotics by induction of liver microsomal mixed-function oxidases and by other mechanisms have been reported, but mostly by derivatives not known or rare in nature. Flavone and methoxyl (but not hydroxylated) derivatives induce a two- to threefold increase in lung and liver benzpyrene hydroxylase activity (Cutroneo et a / . , 1972; Wattenberg er a / . . 1968). Tangeretin, however, was reported to be inactive. Various flavonoids and semisynthetic modifications have been found able to minimize experimental damage to the liver from such agents as carbon tetrachloride (Heintz et a / . , 1963). This is probably due to free radical scavenging, because the trichloromethyl radical is believed to be the toxic form of carbon tetrachloride activated by cytochrome-450. Vitamin E, for example, also hastens recovery from the injury. Catechin in v i m (and the antioxidant propyl gallate) strongly inhibited the lipid peroxidation system involving carbon tetrachloride, but in vivo no protective action was found (Danni e t a / . , 1977). Catechin as a treatment for hepatic diseases has undergone clinical trials involving 1000 patients at fortyeight centers i n eleven European countries (Alhadeff, 1977). Tolerance at a typical dosage of I .5 gm/day per os was excellent, with slight gastrointestinal side effects and “no known contraindications.” A prophylactic as well as therapeutic effect was claimed in treating partial hepatectomy , effects of alcohol on the liver, liver lesions from prolonged isoniazid, acute viral hepatitis, and toxic hepatitis. A favorable effect in restoring bilirubin levels to normal in alcoholic hepatitis was noted, as well as the effect in experimental animals of

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increasing liver ATP and preventing the fall in ATP due to toxic agents, such as malonate. Anthocyanin preparations from red peanut skins and cashews caused goiter (increased thyroid weight) in rats in 7 weeks when fed at 20 mg/day (Jeney, 1968; Moudgal et a / ., 1958). The effect was countered by the addition of extra iodine to the diet. It appears that phloroglucinol, all flavonoids containing it, and any other aromatic group having electron-donating groups and capable of forming iodine substitutions are weak goitrogens. They apparently compete for the available iodine, and a significant amount of iodine appeared in the phenolic fraction of excreta. This effect might well contribute to the seriousness of the absence of adequate iodine, but the activity is mild compared with that of other types of goitrogens. A toxic effect of flavonoids that probably has benefited man is their inhibitory action on microbes and viruses. The use of tea or wine as a preventive for certain diseases has a long history. It has long been known that these effects are due to the phenolic compounds and not to other constituents, such as the alcohol in wine. They are weak compared with the effects of commercial antibiotics, but they are sufficient to excite new scientific interest periodically, as reported in the public press. Some effects appear to be general in the phenols (Singleton and Kratzer, 1969), and the flavonoids are no exception. In tea, it is the catechins, particularly epigallocatechin and its gallate ester, that are active, green tea infusion being more so than black (Das, 1962). Naringenin and hesperitin are the most active flavonones. Flavonols are active, especially morin (unusual 2’hydroxylation), and dihydroflavonols are more active than the corresponding flavonol (Ramaswamy et a / . , 1972). In wine and related products, attention has centered on anthocyanins, but polymerization to form tannins seems also to be involved, since activity increases for a time as wine ages (Fairbairn, 1959; Masquelier and Jensen, 1953). In general, pyogenic and enteric bacteria of the gram-positive group are most susceptible to the phenols and flavonoids (Das, 1962; Lucia, 1963; Pagenkopf and Woodburn, 1970; Powers et id., 1960; Singleton and Kratzer, 1969). Quercetin, taxifolin, and red wine flavonoids are able to inactivate a wide range of viruses and prevent their infectivity (Bakay et al., 1968; Konowalchuk and Speirs, 1976). Although these activities are puny in a therapeutic sense, historically they were very significant in a preventive sense. Wine was used as an important part of the provisions of the hugely successful Roman armies. Even during our Civil War, the largest loss of life was due to poor sanitation and enteric diseases, such as dysentery, typhoid, and viral hepatitis. Mixing wine and its flavonoids with water and allowing time for the water-borne pathogens to be killed was a treatment well known to the ancients and to the modems in the Mediterranean area (Konowalchuk and Speirs, 1976; Lucia, 1963). It has been shown by experiment that these pathogens do not survive long in such mixtures. Many of our

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forebears probably survived because of flavonoids in wine, and if wine had been available and appreciated in the 1860s, the Civil War might have been less bloody. Anthocyanins have been reported to have a favorable effect on vision in man as well as in experimental animals, particularly with regard to regeneration of visual purple and rapid adaptation to the dark (Alfieri and Sole, 1966; Wegmann et al., 1969). Anthocyanin preparations from myrtle, cassis, and red wine grapes have been used on 8000 patients in Europe, with 73-97% improvement in various vascular conditions of the bioflavonoid-responsive type; these preparations have also been given for 15 years to improve night vision and myopia retinopathy (Pourrat, 1977). The typical dose was 600 mg/day by mouth. Little difference was seen among the various anthocyanins, and tolerance was no problem. If the usual flavonoids are generally low in toxicity, are the less common forms more toxic? The answer appears to be typically, but not invariably, yes, and the more unusual the flavonoid, the more toxic it is. A host of semisynthetic derivatives have been tried for various conditions, and some of them appear to have special activity (Bezanger-Beauquesne, 1977; Bognar and Rakosi, 1977). Many of them, including the tris-3’,4’ ,7-(/3-hydroxyethyl)rutinpreviously mentioned, involve relatively minor additions to the basic natural flavonoid structure. The toxicities have generally remained fairly low for these compounds-some are similar to that of the parent natural compound. Some have additional or intensified physiological effects. Dimefline, 3-methyl-7-methoxy-8-dimethylaminornethylflavone (Fig. 5 ) , for example, is a powerful respiratory stimulant with an effect in humans after an oral dose of the order of 24 mg, and a minimum lethal dose in rats of about 8 mg/kg given intravenously (Scanni et al., 1977; Setnikar and Magestretti, 1964). Unusual flavonoids of natural occurrence are of more interest here. Tangeretin (Fig. 3) and nobiletin are completely methoxylated derivatives of kaempferol and quercetin, respectively, with additional methoxyls in positions 6 and 8. This type of compound is incapable of glycoside formation and is found mainly in the peel oil sacs, with distribution limited primarily to certain citrus fruits and a few essential oils. These and related compounds have strong antimicrobial and antiviral activity, and they are part of the plant’s natural resistance to certain of its diseases (Kuhnau, 1976). Compounds of this series have been found to be cytotoxic in a zebra fish embryo assay, when the usual flavonols were not (Jones et al., 1964). Although not toxic to adult mice when given intraperitoneally or subcutaneously at 450 mg/kg, or to rats or dogs, tangeretin caused 83% of the offspring to be born dead or to die within 3 days when 10 mg/kg per day was given subcutaneously in carboxymethylcellulose to pregnant female rats (Singleton and Kratzer, 1969, 1973; Stout et al., 1964). Diosmin, with one more methoxyl and two fewer hydroxyls than rutin, has bioflavonoid activity and was nontoxic at 60-620 mg/kg per day given orally

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DIMEFLINE C HAMANETIN

-OH

OH

FIG. 5 .

‘0

PORlOLlDE

Examples of unusual and physiologically potent or toxic flavonoids

to dogs (Heusser and Osswald, 1977). Polygalactin, 3-rutosyl-3,7-dimethylquercetin, is also considered to be a bioflavonoid that is low in toxicity, but it shows antimitotic activity in onion cells (Reddy et a l . , 1977). A series of cytotoxic flavones, also not glycosides and 6- or 8-methoxylated, have been isolated from Eupcrrorium species (Kuhnau, 1976) and Lychnophoru aflinis (LeQuesne et al., 1976). They show activity against cell cultures of human nasopharynx carcinoma and in in vitro transplants. It appears from this series that lack of glycosidation (which would affect absorption and microbial breakdown in the gut), decreasing the phenolic character by progressive methoxylation, and further substituting the A-ring by methoxyls, all tend to increase the toxicity. However, it is also clear that the maximum acute toxicity of this series, as represented by nobiletin, is not high. Embryos with not only rapidly multiplying cells, but also weak defenses against toxins metabolized in the liver, seem to have the only significant vulnerability. Rather high acute toxicity is possible in the more unusual flavonoids from nonfood plants, such as chamanetin (Fig. 5 ) and its 6-benzyl analog isochamanetin from Uvaria chamar (Lasswell and Hufford, 1977), and poriolide (Fig. 5) and isoporiolide from Leucothoe keiskei (Ogiso et a l . , 1977). The latter pair had an LD,, in mice of 1 .O and 1.6 mg/kg, respectively, when given intravenously. Rotenone, a botanically rare and complex isoflavonoid, is useful because of its toxicity (Singleton and Kratzer, 1969, 1973). Isoflavonoids, in general, are more

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toxic or disruptive to animals, several acting as estrogens (Harborne, 1979; Singleton and Kratzer, 1969, 1973). However, rather unusual flavonoids-the silymarin constituents, for example (Cavallini et al., 1978; Faulstich et al., 1980)-appear to be antitoxic. F. TANNINS The primary role of tannins in plants appears to be as a defense against attack by bacteria, fungi, or herbivores (Swain, 1979). Tannins have been responsible for outbreaks of fatal poisoning of domestic animals of considerable local commercial importance, particularly with regard to grazing of Quercus hnvardi in the American Southwest and acorn consumption in Europe, when food was so restricted that little else was available to eat. Toxic or deleterious properties associated with excessive feeding of carob, lespedeza, certain sorghums, rapeseed oil meal, grape seed meal, and other high-tannin products are known. Human fatalities have been caused by treatment with tannic acid in enemas or on burned skin. Before reviewing these manifestations of tannin’s toxic potential, let us consider different classes of tannins and some of their collective and differential properties. In regard to their chemical behavior, leather-tanning properties, astringent flavor, and physiological effect, tannins have in common a polyphenolic macromolecular nature, which binds with protein to insolubilize it. It is only fairly recently that specific molecular forms of true tannins have been separated and characterized (Haslam, 1966, 1975). Highly purified and well-characterized pure tannins seldom have been available for animal testing, especially not in the amounts necessary for feeding trials. Many of the data on the toxicity and physiological effects of tannins have been obtained on mixtures that, even when free of nonphenols, contain components representing a considerable range of molecular weight and isomerism. This is not necessarily a serious problem, since the physiological effect of an octamer, say, is not likely to be drastically different from that of a heptamer. The effects at the extreme should be considered, however, and also the considerable difference in the type of phenolic “monomers” making up tannins of different classes-specifically hydrolyzable gallo- and ellagitannins vs condensed flavonoid (and, more rarely, stilbene, etc.) tannins. The minimum molecular weight is about 350 for effective protein precipitation as measured with gelatin, for example. In the condensed series this would begin with dimeric flavonoids. In the hydrolyzable series no fewer than two gallic acid precursor units or one ellagic acid precursor unit would be the theoretical minimum. When the molecular weight is quite large, probably more than 5000, the condensed tannins, particularly, become so poorly soluble in physiological solutions, even in the absence of protein, that they have little leather-forming ability or astringent taste. They also seem relatively inert physiologically, espe-

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cially when modified by heat, etc., into “phlobaphenes,” unless injected, and even then they appear to act mainly as inert, obstructive particles. Phlobaphenes are poorly characterized, additionally polymerized, partly oxidized, and very poorly soluble phenolic macromolecules such as are found in outer tree bark. For our purposes, we shall generally accept Haslam’s description of “tannin” as valid, representing a fairly water-soluble, natural polyphenolic mixture of intermediate molecular weight. Since all tannins are polyphenols and function as protein binders and precipitants, their chemical and physiological properties are somewhat similar. Many authors speak of “tannin” as if it were a single entity. Other poor terminology can confuse readers, particularly that found in the older literature. Digallic or rn-digallic acid is a subunit of gallotannic acids, but it has never been readily available, and tannic acid has been sometimes so misnamed. Tannic acid has frequently been used incorrectly as a general term for tannins. Tannic acid should refer only to specific hydrolyzable gallotannins, but they have been the ones most commonly used in testing biological effects. Tannic acid of commerce has generally been Chinese (from Rhus semiulatu leaf galls) or Turkish (Aleppo tannin, from leaf galls of Quercus infectoriu), both of which are predominantly glucose esterified with 6 to 9 gallic acid units. Tara “tannic acid” (from pods of Cuesulpinu spinosa of South America) has 4 or 5 gallic acid units esterified to quinic acid. Various similar gallotannins are prepared from other plants, notably the tropical fruits called myrobalans, maple, witch hazel, chestnut wood, and oak wood (Haslam, 1966). Ellagitannins also come from myrobalans, valonea (acorn cups of Quercus valonea), divi-divi, algarobilla (other Cuesulpinia species), and walnuts (Haslam, 1966). Except for their different hydrolytic products-gallic and ellagic acids, which have already been discussed-the gallotannins and the ellagitannins are quite similar. They frequently occur together, and many ellagitannins also contain gallate units. The condensed tannins of commerce include quebracho (from heartwood of a South American tree), wattle (wood and bark of certain Acacias), and mangrove bark extracts. The properties of the dimeric flavans, the smallest condensed tannins, bear considerable resemblance to the properties of their monomerscatechin and related bioflavonoids, already discussed. Oligomeric condensed tannins serve as bioflavonoids in increasing the weight gained by guinea pigs on minimal ascorbic acid regimes and extending their survival (e.g., Laparra et ul., 1978). Monomers, however, whether catechins or gallate, may bind to proteins by similar hydrogen-bonding mechanisms, but ordinarily they do not precipitate proteins such as gelatin. This is a fundamental difference between tannins and their component phenols, but it is somewhat a matter of degree. Sometimes hydrolyzable and condensed tannins are produced in the same plant, but generally in separate tissues. Oak and chestnut, for example, produce hydrolyzable tannins in wood and new growth, but condensed tannins in outer bark (Haslam, 1966).

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Hydrolyzable tannins are relatively uncommon in human food, but they do occur in the brown outer skin of walnut meats and probably other nuts, and in leachates from barrels used for beverage storage, and pharmaceutical-grade tannic acid has been added to foods and drugs. Condensed tannins are widespread in fruits and in certain grains, and are high in some less common vegetable foods-grape leaves, for example (Haslam, 1975; Hermann, 1959). Their classification as hydrolyzable or condensed in a poorly studied food often depends on whether or not cyanidin or related anthocyanidins are generated when the astringent tissue or its alcoholic extract is boiled with mineral acid. Astringent and bright red means proanthocyanidin, anthocyanogenic condensed tannin, and astringent without reddening indicates hydrolyzable tannin. Chromatographic identification of cyanidin in the former case and gallic and/or ellagic acid in the latter is considered to be confirmation. The amount of tannin in plant material used commercially as sources is about 5-50% of the dry weight, with about 20% as the usual amount. Nondescript herbage frequently has 1-5% tannin. As was discussed briefly earlier, animals and humans generally avoid consuming appreciable amounts of high-tannin materials (their astringent flavor being a major reason). A reasonably high astringency may be accepted by animals when access to other food is prevented. Under such circumstances, animals may eat materials with 20% tannin, but toxic effects result, as will be discussed shortly. Animals may accept by preference occasional bites of higher-tannin herbage, even when low-tannin food is available. Man consumes a number of foods containing considerable amounts of condensed tannins, especially in beverages such as cider, cocoa, tea, and red wine. Strong versions of these beverages may have as much as a gram of tannin per liter. An adequate level of tannin in these products and some other foods, such as spinach, stuffed grape leaves, persimmons, and bananas, is considered desirable. The intake of dimeric flavans has already been indicated as about 400 mg/day in human diets (Kuhnau, 1976). Total tannin intake would be somewhat higher. Per capita consumption of red wines in some countries would guarantee an average intake of nearly that amount from this source alone. Certain consumers may take in severalfold the average consumption. Since they are uniquely potent binders and precipitators of protein, it is not surprising that tannins are toxic when injected into the blood stream and that they have inhibitory effects on enzymes and microorganisms. It is well known among plant biochemists that, in order to isolate active enzymes from higher plants in good yield, routine competitive binding of the omnipresent tannins is necessary. Periodically reports appear of the tracking of potent enzyme inhibitors in plant material back to tannins. Enzymic activity may still be demonstrable if an enzyme-tannin precipitate is resuspended in substrate solutions, but usually it is considerably reduced. For similar reasons it is the general finding, and clearly a logical one, that tannins can bind to viruses and microorganisms to inhibit their

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activities. Since phenols in general have antimicrobial properties and tannins have the additional property of binding to proteinaceous surfaces, etc., a rather general but nonspecific “antibiotic” effect by tannins is to be anticipated. The role of high tannin content in helping to protect some plants against pathogens is generally accepted. Many articles could be cited describing antibiotic, antiviral, anticellular, antineoplastic, and related effects of tannins, most in vitro, but a few examples will suffice (Alexander, 1965; Chan er a / . , 1978; DeOliveira et a / . . 1972; Herz and Kaplan, 1968; Konowalchuk and Speirs, 1976; Kreber and Einhellig, 1972; Loub et al., 1973). The effect of tannin on microbes can have a dietary influence in animals. In virro or in fistulated animals the microorganisms of the rumen can be inhibited completely by addition of 7.5% or more tannic acid. Phosphate uptake and synthesis of nucleic acids and proteins by the microorganisms were inhibited proportionally by lesser concentrations of Tannin (Sadanandan and Arora, 1976). The breakdown of peanut protein by goat rumen fluid was inhibited 62 and 87% respectively, by addition of 2 or 4 gm of tannic acid per liter (Tripathi, 1978). Field beans with colored flowers contained 4-8% tannins in the testa, versus less than 0.6% in white-flowered varieties. Among 24 bean varieties, the cellulase digestibility of the testa was negatively correlated with tannin content (Griffiths and Jones, 1977). This type of microbial inhibition may have negative or positive effects on the animal, depending on the situation. Microorganism-dependent cellulose digestion is inhibited, and utilization of poor-quality feed by the ruminant is impaired. Sheep fed sorghum silage with 18.7 gm of tannin per kilogram had depressed digestion of crude fiber and less microorganism activity in the rumen when compared with sheep fed maize silage containing 6.6 gm/kg (Ben-Ghedalia and Tagari, 1977). Sometimes, however, the animal can benefit from the minimizing of microbial competition for starch and amino acids (Hatfield, 1970). Treatment of soybean meal with 10% tara tannic acid reduced deamination of the meal by rumen fluid by 90%, but improved the average daily gain and nitrogen balance in lambs (Driedger and Hatfield, 1972). Other tannins had similar effects. Also in ruminants, bloat (a sometimes fatal condition of high rumen pressure) is evidently prevented by sufficient tannin in the feed. The foaming, which causes pressure retention, is prevented by precipitation with tannins of the proteins, which otherwise form stable foam (Jones et d . . 1973). The production of gas by microbial fermentation is also inhibited. The site of nitrogen metabolism is transferred from the rumen to the intestine of sheep by feeding sainfoin with condensed tannins, owing to the formation of protein-tannin complexes that are stable in the rumen (pH 6.5) but not in the abomasum-duodenum (pH 2.5) (Jones and Mangan, 1977). Still another microbial effect with dietary significance may appear in monogastric animals and perhaps in ruminants as well. Tannins at an appropriate

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level apparently can suppress detrimental microflora in the alimentary tract and stimulate growth and good health; the effect is similar to that seen after more potent antibiotics are given (Produits Chemiques et Celluloses Rey, 1964). The acute toxicity of tannins given orally to animals is low, but, as would be predicted for a protein precipitant, it is much greater when they are given parenterally. The LD,,, values for mice, rats, and rabbits after a single large dose of tannic acid given orally are generally between 2.25 and 6 gm/kg body weight (Boyd and Boyd, 1973; Singleton and Kratzer, 1969, 1973). Acute LD,,, values for rectal administration are significantly lower-often about one-half. The LD,,, for intraperitoneal or subcutaneous administration in rats is of the order of 200700 mg/kg, and for intravenous administration, about 20-80 mg/kg (Singleton and Kratzer, 1969). A condensed tannin preparation from hawthorn with small flavonoids removed gave an intraperitoneal LD,,, of 130 mg/kg in mice and a subcutaneous LD,,, of 300 mg/kg (Rewerski and Lewak, 1967). A condensed tannin from grapes was fatal to rats at 150-200 mg/kg given intraperitoneally, and 75 mg/kg given intravenously killed rabbits (Patay et al., 1962). In drinking water 1 gm/liter caused the death of some animals by 19 days. Gastric irritation and hepatic lesions were present at autopsy. When sufficient tannin is injected intravenously, a sudden fall in blood pressure occurs, and death resembles, but can be distinguished from, anaphylactic shock (lack of histamine production, bronchiospasm rather than cardiac arrest) (Fiedler and Hildebrand, 1954; Hildebrand, 1958). A large condensed tannin from lespedeza (14,000-20,000 MW) and a butanol-soluble, oligomeric, condensed tannin from grapes, fed for 120 days and up to 700 days, respectively, to rats at 2% in the diet, produced no detected gross or histopathological effects (Booth and Bell, 1968). Similarly, no toxic effects were found in rats fed tannin at 1000 mg/kg per day for 231 days (Blumenberg and Kessler, 1960). However, in other studies with rats and other animals, 1-5% tannin of various types in the diet was frequently inhibitory to growth or was otherwise toxic. Blakeslee and Williams (1979), with I % tannic acid in the diet of laying hens for 6 wk, found no significant differences in egg production, feed efficiency, or fertility or hatchability of the eggs, although feed consumption was reduced in the second and third weeks. Tannic acid at 2 or 4% decreased feed consumption and egg production. Salseed at 50 gm/kg of diet (about 0.6% dietary tannin) or more gave depressed chick growth and feed conversion (Zombade et ul., 1979). The threshold of toxicity of tannic acid added directly to rumen contents in fistulated animals was 3-5% in cattle but 8-10% in goats, apparently because the goat produced an active tannase in the rumen mucosa (Begovic et (11.. 1978a). Condensed tannins tend to be more inhibitory to enzymes in vitro than does tannic acid (Tamir and Alumot, 1969). Tea tannin (condensed) was toxic at I % , but Chinese tannic acid at only 0.8%, in experiments with worms (Tkabladze,

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1959). Neither type of tannin added as 10%of the diet affected generally feeding grasshoppers, but hydrolyzable and not condensed tannin was deleterious to a graminivorous locust (Bernays, 1978). The hydrolyzable tannin passed through the peritrophic membrane and damaged the epithelia of the gut. Depression of growth in weanling rats was greater after gallotannin was fed than it was after condensed tannins were given (Joslyn and Glick, 1969). Ellagic acid had no effect on the rats or on chick growth (Kratzer rr ul., 1975). On a weight basis, all other phenols tried on the chicks, except methylgallate, were less growthinhibitory than were two samples of tannic acid. On a phenol content basis, two ellagitannins, two gallotannins, and two condensed tannins (eucalyptus and wattle) had essentially similar effects (98-1 17% of tannic acid’s inhibition), but tara tannic acid and the condensed tannins from mangrove and quebracho were less toxic-76, 72, and 39%, respectively (Kratzer et al., 1975). The differences could not be satisfactorily accounted for by relative dialyzability, but there was a tendency for the less dialyzable, larger molecules to be less inhibitory. Tannic acid was more toxic and growth-depressing for chicks than were condensed and other hydrolyzable tannins in other tests (Lapaz et al., 1975; Vohra etal., 1966). It is apparently a matter of relative size, perhaps gut pH, firmness of binding to proteins and ease of partial breakdown as to which tannin is more inhibitory. Condensed tannins were much less toxic than hydrolyzable tannins when used in burn therapy (Wilson and Gisvold, 1956). Subcutaneous injection of condensed tannins produced only local damage, but hydrolyzable tannin produced liver damage as well (Kirby, 1960). The negative results of feeding tannin in large amounts are attributable to the combined effects of several causes in addition to the microbiological ones already mentioned. The astringent flavor makes the diet unpalatable, and an insufficient amount is eaten for maximum growth rate in the young or, less often, for good health in the adult animal. Protein in the diet is bound, and therefore it passes through the animal without being utilized. Digestive enzymes (proteins) are precipitated and prevented from properly digesting the food. In response to inadequate enzymic digestion and the denuding of mucoproteins from the surface of the gut, extra quantities of these materials are synthesized by the animal, thus placing an extra load on its nutritional status and health. All these factors can be involved without a direct toxic effect of the tannin or actual entrance of tannin into the tissues. Direct initant or toxic effects at the surface or within the body are additional reactions to tannin toxicity. Breakdown products of the tannins, flavonoids, or particularly gallic acid, although low in systemic toxicity, can be produced in sizable amounts. The role of astringency in causing waste because the diet is unpalatable is sizable. It can be estimated by paired feeding-keeping the weight of the intake of the animals on the control diet the same as the weight of the food consumed by those on the diet with tannin added. A considerable portion, but generally not all, of the inhibitory effects of tannin is accounted for by such paired feeding of rats

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(Glick and Joslyn, 1970a; Handler and Baker, 1944; Mueller, 1942) or chicks (Vohra et al., 1966). It does not seem fair to consider this effect as “toxicity,” since it is the result of a failure to consume rather than the result of consumption. The effect seems to be larger in young animals (Glick and Joslyn, 1970a), partly because of their proportionally larger dietary needs, but also because they may be less tolerant of unpalatable diets. Much of the remaining deleterious effect of tannin in the diet, once it is actually consumed, is related to tannin’s binding of the dietary protein, thus making the diet inadequate (Vohra et al., 1966). Once formed, most proteintannin complexes are difficult to break down, and they pass out in the feces. The nitrogen content of the feces generally rises in proportion to the amount of tannin fed. Carob pods, when green, contain partly hydrolyzable tannins at about 200 g d k g dry weight and, when ripe, about 70 g d k g dry weight of condensed tannins (Joslyn et al., 1968). Although the ripe pods seemed to be reasonably palatable to rats and food intake was not much lowered, protein was bound, fecal nitrogen went up, and treatment of the feces with urea released phenol from protein binding (Tamir and Alumot, 1970). Finger millet has a tannin content of about 0.05% for the white-grained varieties and up to 2.5% for brown-grained varieties. Protein digestibility in vitro decreased in proportion to an increase in tannin content or with addition of tannic acid to the low-tannin grain (Ramachandra et al., 1977). The tannin content of barley samples, 0.55-1.23%, correlated negatively with protein utilization when the barley was fed to rats (Eggum and Christensen, 1975). The availability of all amino acids declined, but to different degrees. Barley of the same variety grown under carefully matched fertilization schedules had 0.18-0.75% tannin, depending on the northerly (high) or southerly (low) location of the plots (Gohl and Thomke, 1976). Selections of sorghum are bird-resistant or bird-proof because wild birds do not relish their high tannin. They are also more resistant to bad weather and preharvest germination. The red, high-tannin varieties may contain 7-8% condensed tannin, while the whiter, low-tannin varieties have less than 1% (Mabbayad and Tipton, 1975; Martin-Tanguy et al., 1976). The bird-proof types are important, particularly in semiarid areas where competition for food is severe. Sorghum is considered the fourth most important grain for human consumption, and it is primary in dry areas of Africa and Asia. Again, protein and energy digestibility is reduced for rats and chicks fed sorghum with increased tannin content (Featherston and Rogler, 1975). Extraction of the tannin improved the food value, and addition of the extracted tannin to low-tannin grain lowered it. Treatment of high-tannin sorghum grain for several days with water or, better yet, with alkaline solutions of ammonia, potassium carbonate, or sodium bicarbonate decreased the extractable tannin about 50-99%. The treated grain could be nutritionally equivalent, or nearly so, to low-tannin sorghum for rats and chicks (Price et al., 1978, 1979; Reichert el al., 1980). Simple conversion of high-tannin sorghum grain to a batter or cooking the

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grain, even though the extractable tannin was considerably reduced, did not improve weight gain and even lowered it somewhat, compared with results obtained with untreated grain (Price et a / . , 1980). Differences produced in the tannins, perhaps by rapid oxidative polymerization in alkaline solution as well as by hydrolysis, and in the extent and firmness of protein complexing appear to be involved. Cooking low-tannin sorghum did not lower its nutritional quality. Tannic acid treated with oxygen in alkaline solution for 24 hours and fed for 25 days to young rats (Mitjavila et al., 1978) depressed rat growth, but in comparison with untreated tannic acid, it decreased the nitrogen excreted in the feces and increased that in the urine. Field or horse beans (Vicia fuha) with high condensed tannin and the tannin separated from them depress the growth of ducklings, the laying rate of hens, and protein digestibility in chicks (Griffiths and Jones, 1977; Guillaume and Bellec, 1977; Guillaume and Gomez, 1977; Martin-Tanguy el a / . , 1977). Similar effects are also found with common dry beans (Ronnenkamp, 1978). The tannins are concentrated in the seed coat (as is true for most other seeds) and are of the order of 40 g d k g of seed coat with several molecular sizes present (Martin-Tanguy et u / . , 1977). In feeding chicks low-tannin vs high-tannin varieties of fababeans and control diets, the tannin accounted for at least 50% of the growth depression from fababeans. The tannin level in the diet was highly correlated with decreased feed intake, decreased weight gain, increased feed per unit gain, increased fat retention, and decreased retention of protein, dry matter, calcium, and ash (Marquardt and Ward, 1979). Autoclaving the beans gave some improvement in feeding value whether or not tannin was present, suggesting that additional factors are involved. If binding dietary protein were the only direct effect of dietary tannins, then supplementation of the diet with extra protein should eliminate it. Protein supplementation does markedly alleviate the growth-depressing effect of tannic acid (Click and Joslyn, 1970b). If extra casein was added to a 5% tannic acid diet for rats, the fecal nitrogen was no higher than that found in rats given a diet without the casein, indicating complete utilization of the extra casein. In rats, eating tannic acid at 50 g d k g of diet, plus protein at 60 gm/kg, gave the maximum amount of fecal nitrogen (Mitjavila, 1973; Mitjavila et ul., 1974). The indication that about equal weights of tannin and protein are bound agrees with chemical studies. Increased nitrogen and dry weight of feces were produced with increased tannic acid. There was less body weight loss when 6% protein was added in the tannic diet, but it still occurred, whereas without tannic acid it did not. When the tannic acid was oxidized before it was fed to rats, it gave lower fecal nitrogen retention and had less severe effects on the animals (Mitjavila et a / . , 1978). Oxidation converts hydrogen bond donor hydroxyls into acceptor quinone carbonyls. This decreases protein binding by the usual tannin mechanism, but it

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may give substitution reactions with lysine amino or cysteine sulfhydryl groups. Since phenol oxidation is greatly accelerated at alkaline pH, this is probably the reason the addition of calcium hydroxide or sodium carbonate improved the growth of chicks fed tannic acid (Rayudu et al., 1970b). Tannin-binding agents poly(vinylpyrro1idinone) and polysorbate 80 also improved growth. In general, it appears that amino acid supplementation to overcome dietary tannin inhibition is about as effective as the protein for which it substitutes, although relatively little experimentation has been done, except for methionine. Methionine holds a special place for diets containing mainly legume protein, because it is the most deficient amino acid in legume protein for animal nutrition. I t is also considered to have a special role in detoxicative methylation of tannin breakdown products, notably gallic acid and the phenolic acids from condensed tannin flavonoid fragments. Supplementation of tannin-containing diets with the methyl donors methionine and choline has often counteracted a major part of the effects of dietary tannins, particularly with chicks and with both condensed and hydrolyzable tannin (Armstrong et ul., 1974; Featherston and Rogler, 1975; Fuller et ul., 1967; Guillaume and Bellec, 1977; Rayudu et al., 1970a; Vohra et al., 1966). That enzyme inhibition is also involved in dietary tannin’s inhibition of animal growth is indicated by the general finding of inhibition not only of protein digestion, but also of the digestive yield of metabolizable energy (Featherston and Rogler, 1975; Gohl and Thomke, 1976; Griffiths and Jones, 1977). Rapeseed meal has about 3% tannin; extraction of this tannin increased the available metabolizable energy content for 2-week-old chicks from 1 17 1 kcal/kg with tannin to 1844 kcal/kg without (Yapar and Clandinin, 1972). Admission of 0.5% tannin solution to the true stomach of calves decreased the pepsin activity to one-half to one-third, and the rennet activity to one-fifth to one-fifteenth (Lebedev, 1969). A three- to fourfold increase in total production of intestinal proteolytic enzymes was noted in rats fed tannic acid (Glick and Joslyn, 1970b). Griffiths and Moseley (1980) found that the tannin of field bean testa inhibited trypsin and amylase activity in the intestine of rats, but increased lipase activity. The tannin stimulated pancreatic secretion, and the lipase had less affinity with the tannin than did the other enzymes. Treatment of the intestinal contents of tannin-fed rats with polyvinylpyrrolidone-salineextraction resulted in a recovery of trypsin activity similar to that found with the low-tannin diet, showing that trypsin-tannin binding was the major factor. This high level of enzyme production after binding with tannin can contribute to the high nitrogen content of the feces. Tannin consumption causes a loss in body weight, a loss of nitrogen in the feces in proportion to body weight (Mitjavila et al., 1974), and sometimes a greater total excretion of nitrogen in the feces than is taken in in the diet; it is clear that not just dietary protein is involved in tannin binding. The increased excretion of mucoprotein, sialic acid, and

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glucosamine in the feces in the presence of tannic acid (but less with oxidized tannic acid) fed to rats indicates that mucus hypersecretion is the main source of the excess fecal nitrogen (Mitjavila et al., 1977). We have been saying that tannin binds protein and therefore damages the nutritional status of the animal. This statement can be turned around to say that protein binds tannin, and to the extent that it is successful in carrying tannin away in the feces it prevents other serious toxic effects. In living plant material, tannins and proteins are naturally compartmentalized away from each other. During feeding and food preparation, the tannin and protein may contact each other. Heating of field beans, for example, decreases the toxic effect of the tannin, perhaps by enabling tannin-protein complexing to take place (Guillaume and Gomez, 1977). Intimate mixing of protein and tannin prior to their reaching more sensitive portions of the gut should diminish direct membrane effects, absorption, and systemic toxicity. In ruminants the extra mastication, saliva, and rumen fermentation should, therefore, be a safety factor. The apparently greater toxic effect of tannin when administered by stomach tube in acute oral toxicity studies, or in drinking water (Patay et al., 1962) or enemas (Boyd and Boyd, 1973), is believed to occur because “bare” mucosa are in direct contact with the tannin. Tannic acid, so applied, appears to cause a localized destruction of the epithelium of the rat’s gastrointestinal tract (Weinberg et al., 1966). Young rats appear to be more susceptible and less tolerant than older rats. Early effects of tannin’s contact with intestinal mucosa include removal of the mucus covering, irritation, and edema. With continued exposure, ulceration and necrosis may follow. Ulcer-forming action by tannin appears to be countered by tannin’s ability to inhibit gastric secretion and pepsin’s action, and to promote healing of experimental ulcers (Satoru, 1976). Tannin has long been believed to delay passage of food through the intestine and to stop diarrhea (Frey, 1908). Perfusion of the mouse intestine in vivo with tannic acid at I gmhter markedly reduced absorption of glucose and methionine (Mitjavila et al., 1970). The effect is considered a denaturation of the proteins of the membranous surface and presumably corresponds to the early-stage effects just mentioned. This brings us to the question of whether tannin can be absorbed into the system through the intestinal wall. Different answers have been given at different times. Very early workers said that the tannin must be absorbed because it cannot be found in the feces. We now know that this is a false assumption, based on the difficulty of recovering tannin from its protein-bound condition in the feces. Another group said that tannin must be absorbed because phenolic products are excreted in the urine, but they were countered by a group that noted that the breakdown products may have been absorbed, and not the intact tannin. For a long time it was believed that, as precipitating macromolecules, tannins could

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not be absorbed through normal proteinaceous animal membranes. Perhaps the key word is normal, but it does appear that tannin is difficult to absorb. Korpassy and co-workers (195 1) reviewed previous studies and presented experimental evidence that after tannic acid solutions were administered by stomach tube to rabbits and dogs "tannin" could be found in blood by colorimetry of the plasma. Three hr after a dose of a 2.5% tannic acid solution was administered, 20 mg/liter was found in plasma samples, and after 5% solution was given, 40-50 mg/liter was found. However, these workers recognized that their analytical procedure did not distinguish between gallic acid and tannic acid; in fact, administration of 2.5% gallic acid produced the same blood rise as did 5% tannic acid in their tests. Although they concluded that tannic acid can be absorbed intact, they did so on the basis that previous work by their group and others showed that gallic acid given parenterally or orally does not produce liver injury, whereas tannic acid does. This report has been cited (Boyd and Boyd, 1973) as demonstrating the direct absorption of tannic acid, but that is not entirely correct. The most convincing direct evidence encountered to date is based on the absorption of a radioactive dimeric condensed tannin fraction from grapes. Within 10 rnin after oral administration of 2 pCi of such tannin, relatively small but appreciable amounts of radioactivity appeared in mouse blood (Laparra et al., 1977). The blood radioactivity reached a maximum at 1 hr, and considerable excretion occurred via the bile. If the preparation was free of monomeric flavonoids and if no breakdown occurred during preparation or in the acid conditions of the stomach, this proves that absorption of intact dimeric catechin tannins took place. The appearance of radioactivity in the blood was much faster than the appearance of marked CO, in the breath or of phenolic acids in the urine, showing that extensive metabolic breakdown had not occurred (Laparra et al., 1977). The radioactivity, as indicated by organ analysis and radioautography , concentrated in the liver, blood vessels, and bile duct tissues and in the mucous membranes rich in glycosamine glycans. This same pattern, however, was shown by monomeric taxifolin. Rutin was poorly absorbed in comparison tests (Laparra et al., 1977). Histology of animals fed tannic acid at high levels shows effects that are different from those caused by gallic acid alone, and a rather specific type of coagulation necrosis seems common to liver, heart, kidney, and intestine cells (Karim et al., 1978; Panda et al., 1979). Again, failure to distinguish between tannic acid and the different natural polyphenolic mixtures of tea, coffee, and other foods has marred the studies. It is toosoon for an unequivocal answer as to whether large tannin molecules get through normal gastrointestinal tissues to cause systemic effects. Normally there are formidable barriers to this happening, and if any portion gets through it must be very small. It is clear, however, that free tannin in appreciable concen-

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tration can damage the gastrointestinal surface. Exposure of burned skin surfaces or damaged colon tissue has produced fatal liver damage in humans. Incubation of tannic acid with intestinal fluid liberates gallic acid (Kerzanoski, 1966). Although gallic acid at levels equivalent to those of tannic acid does not produce the same effects, since gallic acid is not a protein precipitant, we have yet to prove that the external effects of tannin plus the internal effects of released gallic acid, and perhaps galloylglucose, etc., do not account for the effects of oral tannic acid. Gallic acid at 5% in the diet produced fatty livers in rats, whereas 5% tannic acid did not (Mueller, 1942). In this regard at least, gallic acid was more toxic than tannic acid. Whereas in feeding trials on chicks the growth-inhibiting effect of gallic acid was only 30% that of tannic acid, methyl gallate was twice as active (Kratzer et a/., 1975). Alternative explanations for differences between the toxic effects of high-level tannins and their more obvious breakdown products seem possible. In chronic feeding trials, protein and other nutritional deficiencies caused by tannin, and not by catechin or gallic acid, should contribute to different toxic patterns. In acute studies, the extra gastritis, etc., could so contribute. Breakdown products, particularly of hydrolyzable tannins, may be poor protein precipitants, quite absorbable, and possibly more toxic than gallic acid. The liver is subject to serious damage from tannin injection and, under some circumstances, from oral intake. The gross and ultrastructural changes in liver caused by injected tannic acid are reported to have much in common with changes caused by other hepatotoxins, such as carbon tetrachloride (Arhelger et a / . , 1965; Drill, 1952). The central liver lobule is more involved, chronic doses give diffuse fibrosis, and massive doses cause necrosis. Oral administration of 50-300 gm of tannic acid to horses gave characteristic symptoms, including anemia, jaundice, and severe bilirubinemia (Begovic and Ozegovic, 1959). indicating overloading of the liver detoxication mechanisms. On the other hand, feeding rats 32 gm of tannic acid per kilogram of diet for six months did not affect the liver’s functional status, triglyceride level, or oxidative enzyme level, although growth was retarded (Mitjavila et d., 1971). At cellular and biochemical levels, injection of tannic acid produces liver polyribosome disaggregation, affects microsomal enzymes, and inhibits nucleic acid and protein synthesis (Badawy et a/., 1969; Gaillard et al., 1974; Oler et al., 1976; Reddy e t a / . , 1970). Within 1 hr after intraperitoneal injection, tannic acid was found by solvent extraction and paper chromatography in liver cells, and by 3 hr it was concentrated in the nucleus fraction (Badawy et d., 1969). The time sequence of events was concentration in the nuclei, inhibition of nuclear RNA synthesis, inhibition of protein synthesis, and, finally, production of necrosis. Cellular necrosis was well marked only after 24 hr following injection. Disaggregation of liver polysomal patterns and ultrastructural changes in the rough endoplasmic reticulum of rat liver cells began 3 hr after subcutaneous

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injection of tannic acid at 700 mg/kg and were complete by 4-10 hr; recovery was evident by 72 hours (Reddy e t a / . , 1970). Tannic acid injected intraperitoneally at 25 mg/kg for 4 days produced a reduction in microsomal protein and certain induced detoxication enzymes, but not in others (Gaillard et a / . , 1974). Concentration of cytochrome P-450 was not reduced, and pentobarbitone sleeping time was prolonged. Gallic acid, ethyl gallate, and a series of related acids at 150 mg/kg had no microsomal effect. Oral dosage of 2000 mg/kg of body weight to rats caused hepatocellular changes similar to those observed after 700 mg/kg given parenterally (Oler et al., 1976). There was more inhibition of RNA synthesis in young rats than in mature rats. Tannic acid resulted in degranulation of rough endoplasmic reticulum, inhibition of protein synthesis, and disaggregation of polysomes. However, no evidence of single-strand breakage in DNA was detected. Degranulation of rat liver microsomes has been proposed as a quick test for carcinogenesis (Gupta and Dani, 1979). In this test tannic acid is active, but the connection between this finding and carcinogenesis seems tenuous and unconvincing. Not just the liver is affected by tannic acid, as many studies have shown. Nonlethal injection produced proteinuria in rabbits in 24-96 hr (Boler et a/., 1966). Renal cell and mitochondria1 alterations were confined almost exclusively to the proximal convoluted tubules. Effects were visible by 1 hr, and after 7 days the tissues had returned to normal. The feeding of 2-3% tannic acid to chicks produced “coagulation necrosis” in the liver, kidney, and intestine, as well as various other degenerative changes in these organs, desquamation of the intestinal epithelium, and hydropic degenerations of pancreas islet cells (Karim et a / . , 1978). Metabolism of tannic acid in animals produces gallic acid derivatives, mainly 4-methoxygallate. In chickens, after oral administration of tannic acid, gallic acid occurred in urine but not in feces, and pyrogallol was found in both (Kadirvel et a / . , 1969; Potter and Fuller, 1968). The special ability of methionine and choline to alleviate tannic acid toxicity obviously relates to the availability of methyl groups to methoxylate gallic acid during detoxication . Pyrocatechol also was formed, presumably by decarboxylation and dehydroxylation. The decarboxylation products were produced whether or not the cecum had been removed (Kadirvel et d..1969). Metabolites of condensed tannins have been studied less. Carob tannin produced gallate metabolites in rat urine from the hydrolyzable tannins or epicatechin gallate present, but the catechin tannin portion did not undergo metabolism (Tamir et a / . , 1972). With avocado and cacao preparations of mainly dimeric catechin tannins, intraperitoneal LD,,,’s in mice were, respectively, 340 and 857 mg/kg (DeOliveira et al., 1975). Intraperitoneal injection of either preparation at 80 mg/kg gave an analgesic effect (response of feet to heated surface) similar to that of morphine at 10 mg/kg and prolonged pentothal sleeping time. The animals were back to normal by 90 min. Human patients also reported

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relief of pain after intravenous administration of these substances (DeOliveira et al., 1975). Analgesia produced by narcotics in rats was substantially prolonged by subcutaneous administration of a zinc tannate combination (Brands ef al., 1980). Areca nut (betel) tannin (condensed) potentiated acetylcholine and noradrenaline action on rat tissue preparations in vitro (Sirsi ef al., 1966), and other reports suggest central nervous system reactions to tannins. A condensed tannin of 2000-3000 molecular weight bound to pig myocardial membranes in the order sarcolemma > mitochondria > sarcoplasmic reticulum (DiPietro et af.,1977b). The binding was enhanced by cations, but did not modify energy-linked calcium-ion accumulation in intact mitochondria, nor did it modify respiratory activity. The tannin behaved like a membrane stabilizer and prevented passive efflux of protein and cations. It helped maintain high ATP and creatine phosphate stores by inhibiting ATPase and creatine kinase, but it had no effect on ATP synthesis by oxidative phosphorylation in intact mitochondria (DiPietro et al., 1977a). Inhibition of ATPase depended on access to the enzyme, since inhibition was produced in submitochondrial particles, but not in intact mitochondria. Tannic acid has also been shown to act only with specific segments of the outer surface of human erythrocyte membranes and to be unable to reach enzymes inside the cell (Herz and Kaplan, 1968). Tannins have been reported to have effects on anemia and blood constituents. Roy and Mukherjee ( 1979) found that tannic acid and tannins from Eugena black plum or green banana had somewhat different effects, but when fed at about 0.5 mg/kg of body weight per day they significantly increased retention of iron and its incorporation into heme in rats made anemic by administration of phenylhydrazine. In normal rats iron excretion was enhanced. At 2 mg/kg of tannin per day, absorption of iron was inhibited in both normal and anemic rats. Several mechanisms appear to be involved. The tannin may help keep the iron reduced to the ferrous ion form for ready uptake and metabolism. It may help recover iron in reusable form from metabolic breakdown products of heme. It may protect erythrocytes from lysis. Begovic and co-workers ( 1 978b) found that introduction of 1 liter of 7-10% tannic acid solution into the rumen of fistulated goats caused within 2-3 days erythrocyte lysis, hemoglobinuria, and hematuria. Severe hyperchromic, megalocytic anemia occurred 1 -2 wk after the administration of tannic acid. It could be cured by repeated injections of vitamin B , 2 . A number of other blood elements were also affected by the tannic acid. A diet of dry oak leaves produced some of the same effects, but not others, and the effects were considerably less severe. Instances of the toxicity of tannin to farm animals have been observed for a long time. Periodic episodes of loss of stock from eating acorns have occurred in Europe (Clark and Cotchin, 1956; Wolter, 1974), notably in England in 1868, 1870, 1884, 1900, and 1933. This effect is attributed to the tannin content of the acorn. The acorns of European oak, Quercus pedunculata, contain about 6%

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tannin; they are not willingly eaten by laboratory rats or mice. The tannins of Quercus hurvardi are estimated to have caused annual losses of cattle worth millions of dollars in the American Southwest, owing to the fact that the leaves and twigs are eaten heavily at times when little else is available (Hulbert and Oehme, 1965; Keeler e r a / . , 1978; Singleton and Kratzer, 1969, 1973). In both cases, the oak tannin is hydrolyzable gallotannin. The tannin from shin oak, Q . havardi, reached an oral LDso after 5 days of dosage with 6.9 gm/kg/day in rabbits (Pigeon e t a / . , 1962). With comparable tests, 50% lethality was produced by commercial tannic acid at 3.4 gm/kg/day for 10 days, and symptoms were markedly similar (Dollahite et al., 1962). At 0.5 gm/kg/day for 15 days given orally to rabbits, the oak tannin caused a doubling of liver P-glucuronidase activity and increased the blood concentration of urea, hemoglobin, erythrocytes, and serum transaminase (Camp et a / ., 1967). Tannin was widely used medically as a bum treatment (for coagulating the burned surface, protecting it, and minimizing exudation from it) from 1925 until 1942, at which time it was shown that tannic acid was the cause of liver necrosis in burn therapy. Owing to the lack of communication during World War 11, tannic acid was still used on bums. Its effects in liver damage were later discovered separately in Central European countries. Experiments showed that tannic acid was absorbed into the system from the burned surface. Similar effects on the liver were produced in experimental animals by injection or treatment of burned or otherwise damaged, exuding surfaces. It was found that condensed tannins were not nearly as likely to cause liver necrosis as was tannic acid. In 1946, it was found that incorporation of about 2.5 gm of tannic acid in a barium sulfate enema cleared the mucus from the colon wall, stimulated colon contraction, and promoted adhesion of barium to the lining for improved x-ray examinations. Commercial preparations incorporating tannic acid for this purpose were made available to American radiologists in 1962 and were applied to an estimated 600,000 patients per year. Beginning in 1963, liver necrosis and eventually at least eight fatalities were reported, which were traced to the tannic acid enema (Boyd and Boyd, 1973; Kerzanoski, 1966; Singleton and Kratzer, 1969, 1973). The products and their use were withdrawn in this country in 1964. It was shown that tannic acid is about twice as toxic when administered via the colon as when given by mouth, and if sufficient volume is given, the enema may reach the small bowel (Kerzanoski, 1966). The tannic acid enema can produce extensive local inflammation and mucosal ulceration in the colon and the cecum, acute hemorrhagic hepatic necrosis, and hemorrhagic cystitis (Dukes, 1975). The hepatotoxicity is dose-dependent and is enhanced by repeated tannin enemas, prolonged retention, or increased concentration of the tannic acid. Patients with preexisting ulcerative colitis or other damage to the colon wall (those most likely to be x-rayed) and patients who are juvenile, debilitated, or aged are more susceptible to hepatotoxicity (Dukes,

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1975; Janower et a l . , 1967; Singleton and Kratzer, 1969, 1973). Patients with mild or no effects generally recovered without lingering sequelae, but a few cases of fulminating liver disease appeared to be initiated in children. Serious toxicity from tannic acid in enemas was evidently rare. With proper selection of patients, limiting the tannic acid content to I % , and elimination of repeated colonic application, liver damage from tannic acid in barium enemas is unlikely to be dangerous (Dukes, 1975). A study of 80 patients in terms of several parameters of liver function for 12-72 hr posttreatment led to the conclusion that, properly managed, the procedure was both safe and useful (Kemp Harper et ul., 1973). Tannins have been implicated in carcinogenesis. Experimental production of tumors in rats was reported by Korpassy and co-workers beginning in 1949 (Korpassy, 1961; Korpassy and Mosonyi, 1950). Young albino rats were given tannic acid subcutaneously every 5 days for prolonged periods. Of 28 treated rats, 23 survived for I 0 0 days; 13 of the rats had hepatic tumors, and 15 had cirrhosis. The tumors were generally benign nodules of 2-8 mm. Only by parenteral treatment was cirrhosis, hepatoma, or cholangioma produced. No local tumors were found at the injection site. Skin ulcers formed by repeated subcutaneous injection of 1 5 2 . 5 % tannic acid at the same site healed rapidly and completely when the site was changed. Skin ulcers produced by burning the skin, which was then painted daily for 400 days with 5% tannic acid, showed no local change, nor were there cirrhotic or precirrhotic changes in the liver (Korpassy, 1961). Later reports confirmed the experimental results and suggested caution regarding heavy human consumption of beverages with high tannin content (Korpassy, 1959, 1961). It was concluded that tannic acid was weakly carcinogenic and was influenced by dietetic and endocrine factors. For example, a high-casein diet produced some protection against lethal and hepatotoxic action, even with injection of tannic acid. Males were more susceptible than females. In comparisons of various tannins (Kirby, 1960), subcutaneous injection with 1 ml/wk for 12 wk of a concentration low enough so that little or no ulceration occurred at the injection site caused “sarcomas” at the injection site only for condensed tannins and, in addition, caused liver tumors with hydrolyzable tannins. Gallic acid and catechin were negative at the doses tested. Consumption of the tannins at 0.1-0.5% in drinking water for 3-6 mo gave no tumors. The results suggest that the condensed tannins are bound to body proteins at the injection site, whereas hydrolyzable tannin or fragments thereof are translocated to the liver. After injection of tannic acid at 0.75 mg/kg intramuscularly into mice every second week for 18 mo, no differences were detected between the treated and the control animals (Bichel and Bach, 1968). According to the literature, none of the tannin-induced tumors were transplanted to other animals or were otherwise proved to be malignant. Later work (Oler et al., 1976) verified the typical

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cellular changes in rat liver reported by others (Korpassy, 1961) after single oral doses of 2000 mg/kg or 700 mg/kg administered parenterally. Although RNA and protein synthesis was inhibited and cellular changes included disruption of the endoplasmic reticulum and polysomes, no strand breaks in DNA were detected. The hepatotoxic effects were again demonstrated, but the lack of a DNA effect cast doubt on their carcinogenicity. The daily intake by a 70-kg man of 560 mg of food-grade tannic acid was considered well within safety limits under normal circumstances (Oler cr a/., 1976). The unconditionally acceptable daily intake by humans of food-grade tannins as evaluated by an international expert committee of scientists is up to 0.6 mg/kg body weight for Peruvian tara tannic acid and up to 0.3 mg/kg for Turkish, Chinese, and Sicilian sumac tannic acids (Bigwood, 1973). Epidemiological studies have attempted to evaluate whether tannins have a role in unusual incidences of human cancer. There have been reports correlating unusual consumption of plant material rich in condensed tannin with unusual frequency of cancer of the esophagus and mouth (Morton, 1970, 1972, 1973; Raghava and Baruah, 1958). These correlations appeared to be suggestive, particularly with betel chewing in parts of Asia, consumption of bird-proof sorghums in parts of Africa (particularly the Transkei), and tea (particularly herbal teas) in other specific societies. Unfortunately, such population-diet correlations, even if statistically evaluated as significant, do not of themselves prove any cause-and-effect relationship. The herbal teas are generally poorly known and quite likely to contain other potentially toxic substances, even if they also contain tannin. The appearance of tumors was accelerated, but the overall incidence was the same when benz(a)pyrene in a single dose was followed by application of tea or oak wood extract to mouse skin (Bogovski and Day, 1977). Tannic acid or gallic acid had no effect. The relative per capita consumption of solid fuels, cigarettes, coffee, and tea all have positive or negative correlations with incidences of some form of human cancer (Stocks, 1970). Tea usage, the one with appreciable tannin, gave a negative correlation with incidence of stomach and uterine cancer, but a positive correlation with intestinal (except rectum), lung, and larynx cancer. The incidence of esophageal cancer in Japan correlates positively with the habitual consumption of rice cooked in tea rather than water; however, it is customary to eat this rice at a burning hot temperature, cooked softer and served with more liquid than ordinary rice is (Segi, 1975). In the most exhaustive study of esophageal cancer to date, it was noted that the incidence of this cancer varies geographically more than does that of any other alimentary tract cancer, often over short distances (Warwick and Harington, 1973). There are about 5.8 cases per 100,000 white males per year in the United States and, for the highest incidence known, 110.5 cases per 100,000 in Turmenistan. Environmental influence is indicated and occupations correlate, but heavy smoking, copious consumption of alcoholic

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beverages, poor nutrition, and high use of emetics appear to be better correlated with the incidence of esophageal cancer than does diet. In fact, consumption of sorghum is not high in the Transkei (Warwick and Harington, 1973). The use of alkali in the form of wood ash or lime with the betel or tobacco chew also appears to be predisposing. It is contended that smoking, related tobacco usage, and other agents (including tannin, perhaps) act on the esophageal epithelium. After many sustained insults, it becomes susceptible, resulting in a high malignancy rate. To summarize quickly, tannins (hydrolyzable tannins especially) can be seriously toxic when injected, when consumed in large amounts (more than 1% of the diet) for a considerable time, or when applied in concentrated solution to burned or otherwise damaged tissue. Sufficient application of tannin solutions (again generally more than 1%) to mucosa results in removal of mucus, epithelial edema, irritation, and tissue breakdown, which may in turn facilitate absorption of tannin, thus increasing its toxicity. The carcinogenicity of tannin appears questionable and is certainly weak (Miller and Miller, 1979; Singleton and Kratzer, 1969, 1973). It may involve repeated irritation and cellular damage, rather like continued mechanical irritation, instead of true DNA mutagenic carcinogenesis.

G.

LIGNINS

Lignins are a heterogeneous group of large, insoluble, three-dimensionally linked polymers. The monomeric units are derived mainly from coniferyl alcohol with (depending on the plant) sinapyl or p-hydroxycinnamyl analogs. It adds bulk to the diet, leading to poor digestibility and unpalatable toughness. It is not particularly inhibitory to microorganisms; white-rot fungi preferentially utilize it in wood rotting. It is, however, sufficiently resistant that soil humus (largely lignin residues) can survive in the soil for upward of 2000 yr (Alexander, 1965). Lignin is considered inert in the diet, except that by linkage to plant carbohydrates and perhaps encrustation coverage it lowers the digestibility of plant material. The lignin content is about 2% in immature forage, up to 15% in mature forages, and about 25% in woods such as spruce (Baker et ul., 1975; Pigden and Heaney, 1969). Much of our knowledge about lignin in nutrition relates to the utilization of wood, straw, and related residues for ruminant feed. Frequently a small increase in natural lignin content causes a considerable decrease in utilization of forage feed. Chemical removal of lignin with sodium chlorite increased cellulose digestion by rumen microorganisms from 66% to 91 %, but simple addition of lignin to insoluble cellulose had no effect (Barton and Akin, 1977). High-lignin feeds can be improved in digestibility by very fine grinding, swelling and modification by steam or alkali, or delignification by white-rot fungi or other chemical treatment (Baker et a / . , 1975; Pigden and Heaney, 1969). Lignin itself has been considered indigestible and has been used as a reference

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marker (for that reason) to compare digestive removal of other feed components, even in ruminants (Barton and Akin, 1977; Porter and Singleton, 1971). The actual digestibility of natural lignin in sheep was 1.2-10.2%, but when purified lignin was added to rabbit diets 41-47% was not recoverable in the feces (Lenz and Schurch, 1967). The lignin is modified by passage through the digestive tract of the rabbit or sheep, the modification occurring mainly prior to the intestine. The lignin fraction isolated from rabbit feces had less carbon and methoxyl content and more oxygen and nitrogen than the purified lignin did. In sheep feces the methoxyl content was 19.2% less than in hay and 33.5% less than in straw (Porter and Singleton, 1971). Lignin or lignin fragments have never, to our knowledge, been reported to cause any serious toxic effects. Lignin degradation products as phenolic substances are somewhat inhibitory, and feeds prepared from wood and containing them can be improved by their removal (Britton, 1978). As a phenolic macromolecule, lignin solubilized by alkali has some tannin-like ability to bind and inhibit active enzymes and other proteins (Naess, 1977). This may be the mechanism by which lignin can cause lower nitrogen retention by the body from the diet, as with tannin (Lenz and Schurch, 1967). These effects are relatively mild, however, and lignin appears unimportant in terms of animal consumption except as an indicator of low quality in forage. Lignin as a component of dietary fiber appears to be needed and beneficial in the human diet; its level is probably too low in the modem diet. Much interest has been aroused by the relatively high incidence of colon cancer and coronary disease in developed countries, compared with that in less developed areas. At least part of the difference is attributed to a diet low in residue, high in meat, and high in refined carbohydrate in relatively affluent societies, versus a diet higher in plant material and crude fiber in poorer societies (Huang et al., 1978; Inglett and Falkehag, 1979; Story and Kritchevsky, 1979). The mechanical bulk of the indigestible fibrous plant material contributes to feces bulk and to regularity of defecation. This in turn contributes to a shorter residence time in the bowel and thus may lower the exposure to dietary or microbially generated toxins and carcinogens in the gut. Lignin is a major component of dietary fiber, but its role and the roles of hemicelluloses, pectins, gums, and cellulose components are not yet clear. Carbohydrates and not lignin seem important to the water-holding capacity of the feces. Lignin-r certain lignin fractions-seems to be the most important fiber component in adsorbing bile steroid and dietary cholesterol, in nitrite binding and thus in lowering the formation of nitrosamine carcinogen, and in inhibiting enterohepatic recirculation (Inglett and Falkehag, 1979). Lignin may also be important for its antioxidant behavior and ion exchange or chelation effects. So far, the effects of lignin in the diet appear to be favorable or inert, rather than toxic. Lignin is frequently associated with plant phenols absorbable by animals. Many such phenols have a

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hypercholesteremic action, producing additional excretion of bile. Combining this action with the ability of lignin to adsorb and fix into the feces both dietary cholesterol and bile steroids would enhance the effect. Favorable participation by dietary lignin in the epidemiology of coronary disease as well as colon cancer seems reasonable.

V.

MECHANISMS OF TOXICITY BY PHENOLS

Earlier reviews emphasized unusual phenols having high toxicity (Singleton and Kratzer, 1969, 1973). The actual mechanisms of the toxicities, as far as they were understood, often did not seem to be unique to the unusually toxic phenol. Phenols with diverse structures appeared frequently to have toxic effects and symptoms in common. The low toxicity of common phenols was considered primarily the result of efficient barriers and detoxications in animals. The greater toxicity of an unusually toxic phenol often seemed to be the result of a structure that survived and penetrated the normal barrier mechanisms rather than a qualitatively unique toxic interaction with the animal’s biochemistry. It was concluded that a number of the relatively common chemical and biochemical properties of phenols are involved in producing toxicity, and that common plant phenols have a toxic potential if natural defenses are weakened, overloaded, or evaded. It was also noted that nontoxic common phenols frequently augmented the effect of toxic phenols. The present review has approached the subject from the other direction, studying the reported effects of the plant-derived phenols widespread in animal and human diets. The hypothesis that common plant phenols may exhibit physiological effects and toxicity under special circumstances by mechanisms similar to those found in notably toxic phenols seems to be validated and strengthened. However, the capacity of the animal body to protect itself against such happenings is impressive, and it generally appears to be quite capable of withstanding natural occurrences. Let us consider in order the mechanisms or routes to toxicity that are likely to be involved with common natural phenols. Of course, any one toxic phenol may use several of these mechanisms simultaneously or at different times. Discussion will be limited to what is deemed necessary to clarify without repeating previous comments. Under certain circumstances a potential for toxicity can confer a protective effect. Some of these will be mentioned. Consideration of the structural details that make phenols capable of avoiding detoxication, and therefore presumably more toxic, easily becomes a circular argument. If the common phenols are nontoxic because they have been evolutionarily common, a mutation giving an uncommon structure has a greater chance of being toxic. On the other hand, we generally have too little information

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concerning the effect of intermediate structural modifications to speculate very helpfully on structure-toxicity relationships. The most noted toxic phenolsgossypol and podophyllotoxin, for example-tend to be complex and quite different from common phenols. Some toxic phenolic derivatives-coumarin and safrole, for example-are fairly simple and more similar to common phenols, such as p-coumaric acid and coniferyl alcohol, that are apparently nontoxic. The balance of water-lipid solubility (penetration, membrane interaction?) and protection from normal detoxicative metabolism and conjugation seem important (methylenedioxy substitution, no hydroxyls for sulfate or glucuronoside conjugation, etc.). Here we are focusing on the usual structures, so details of the unusual toxic phenols are left to other reviews (Singleton and Kratzer, 1969, 1973).To illustrate the universality of the various mechanisms, however, uncommon and toxic phenols will be mentioned. A.

MIMIC-INTERACTION WITH NORMAL PHENOL METABOLISM

The most often cited effect of phenols that falls under this heading is the inhibition of catechol-0-methyltransferse (COMT) and competition for methyl groups by phenols that serve as a substrate for this enzyme. As was already noted, certain flavonoids appear to inhibit the enzyme without serving as a substrate and being methoxylated (Kuhnau, 1976). Many catechol derivatives apparently do compete as substrates, deplete the available methyl group supply, and inhibit normal action of the COMT enzyme (Baraboi, 1967;Doms and Dill, 1977).The adrenergic fibers of the sympathetic division of the autonomic nervous system produce action in the innervated organ by liberation of norephinephrine and epinephrine, catecholamines produced from 3,4-dihydroxyphenylalanine (dopa) (Daly, 1967).The stimulus is terminated by methylation of the epinephrines to inactive metanephrines. If the xenobiotic phenol reaches the nerve junction and inhibits or competes for the COMT action, it will have a sympathomimetic effect, prolonging the actions of the epinephrines. If it acts only by seriously depleting the active methyl group supply via liver COMT, it also can prolong the action of catecholamine; but if it inhibits COMT consumption of methyl groups at the liver, it may free activated methyl groups for inactivation of catecholamines at the nerve (Chaudhari and Hatwalne, 1977). Choline in the form of acetylcholine is the effector-initiator in the cholinergic fibers of the parasympathetic division of the autonomic nervous system. Choline, a source for active methyl groups besides methionine, is subject to dietary inadequacy and depletion if there is a high demand for methyl groups. Furthermore, effects on the central nervous system are frequently noted with administration of phenols (Brewster et a l . , 1977),which might result from competition of these or other types possibly involving dopa and its metabolites. Hydroquinone inhibits melanization reactions involving tyrosinase and dopa, and its use results in

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depigmentation of hair and skin of black animals. Nerve transmission might be affected by Na+-K+ ATPase inhibition, which has been reported for some phenols (Mirsalikhova and Pakudina, 1977). Salicylate analgesia apparently has some weak corollaries with more common phenols (DeOliveira et a / ., 1975), and the anti-inflammatory activity is widely shared. Sympathomimetics commonly induce tremor in animals. Tremor, convulsions, and other nerve-related symptoms are very common in poisoning with natural phenolic intoxicants such as tremetole, hypericin, marijuana, mescaline, nutmeg, kava, and safrole (Singleton and Kratzer, 1969, 1973). The related effects with common phenols occur less frequently and are much milder, yet the common phenols should be as great or greater consumers of methyl groups in terms of the metabolic load. This seems to suggest that the involvement of the common phenols with COMT is effectively handled at the liver, and nerve involvement is minimized. The competitive consumption of iodine by common phenols results in hypertrophy of the thyroid (goiter) in the body’s effort to produce thyroxine (Moudgal et af., 1958; Jeney , 1968). This very weak goitrogenic activity probably requires borderline iodine deficiency and high phenol intake to be significant. Such conditions can, however, occur naturally in humans, even today. This is one reason for the common observation that toxic effects are magnified by unsatisfactory diet or poor health. Other mimic-interference toxic effects of certain natural phenols-for example, the estrogenic action of certain isoflavones, coumarins, and phenolic mold products and the antivitamin K effect of dicoumarol-do not appear to be significant for any of the common dietary plant phenols. Protective effects are observed when detoxication of common natural phenols stimulates the production of the microsomal mixed-function oxidase systems and probably the conjugation systems. The induced enzymes are frequently capable of metabolizing other substances and reducing their toxic effect at a given initial dose. This could fairly be considered a mimic-interaction situation, examples of which were cited earlier (Cutroneo et af., 1972; Wattenburg et af., 1968). The role of the common plant phenols is probably more important, since advance exposure is regular, but some special cases that may involve this effect are actively under study. An example is silymarin protection against amanita toxin, although membrane stabilization appears also to be involved (Antweiler, 1977; Faulstich et a / . , 1980). B.

NUTRITIONAL LOAD

The debilitating drag of the necessity to inactivate or detoxicate a sizable intake is probably the most general naturally significant “toxic” role of the common plant phenols. It is usually somewhat less important for the acutely

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toxic but rarer phenols, since it is likely to be manifest only in chronic conditions of marginal or deficient nutrition. The requirement for extra methionine or another methyl-group source when phenols are consumed has just been mentioned, but it is relevant here as well. Dietary supplementation with methionine or choline has often removed a major portion of the negative effects of a highphenol diet, as was mentioned in the discussion of the effects of gallic acid and tannic acid. A lack of methyl-group donors in the diet produces in poultry a characteristic deficiency disease syndrome called perosis, involving hock joint abnormalities that prevent normal walking. Although the relationship is not always recognized, the symptoms seem essentially the same as those produced by high tannin intake, and they can be at least partly overcome by intake of methionine (Breider, 1968; Elkin et a l . , 1978a,b; Rungruangsak et al., 1977). Supplementation with other amino acids may be helpful in dietary toxicity of phenols, but the effects generally appear to be nonspecific and only alleviate poor nitrogen nutrition. Effects of tannins that make dietary protein unavailable by ‘‘tanning” it, that inhibit and cause extra production of digestive enzymes, that cause losses requiring replacement of the mucus lining of the alimentary tract, or that throw animals consuming large amounts into negative nitrogen balance have been discussed. Similar, albeit much smaller, effects appear to be produced by other phenols in proportion to their ability to bind protein. Small phenols as well as larger ones also cause nutritional drag by consuming energy in detoxication reactions (methylation, sulfation, glucuronide formation, etc.) and by the loss of the substance of the methyls or glucuronic acid from more useful metabolism. Stimulation of the flow of bile and urine to excrete the phenolic detoxication products has a potential health value in the removal of cholesterol and other waste products, but in a starvation situation it may also contribute to depletion. The magnitude of common phenol intake necessary to produce a significant nutritional load effect appears to be in the range of 1-5% of the diet, since that is the range that generally causes the first long-term toxic effect of feeding bioflavonoids, tannin, gallic acid, tyrosine, and other common natural phenols. The range is higher (that is, a higher percentage is required to produce toxicity) for insoluble phenols like ellagic acid and lignin, and lower for more specifically toxic phenols. This shows that the normal protection and detoxication mechanisms have a rather large capacity. Ordinarily, innocuous common phenols compete with more toxic phenols and other toxins for much the same maximal capacity of microsomal mixed-function oxidases and other detoxication mechanisms. This situation explains the observation that the isolated active principle is often not as toxic in unadapted animals as is the whole crude source. For example, nutmeg volatiles contain the toxic phenol derivative myristicin, and the nonvolatile phenols are not toxic (Singleton and Kratzer, 1969). However, feeding the two fractions together increases the toxic-

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ity of the myristicin. Synergy-an increased toxic effect caused by the addition of normally nontoxic phenols with a borderline dose of a toxic phenol-is usual. This effect may be obscured under chronic administration if the induction of detoxication enzymes enables them to cope with the total load. If the maximum inducible detoxication is exceeded in normal animals or is lowered by poor health, the common plant phenols will contribute to the overall toxic effect. The diet may be made inadequate in other ways by the presence of phenols. Caffeic acid (Schaller et al., 1977; Somogyi, 1978; Somogyi and Nageli, 1976; Swanowska and Tautt, 1972), tea flavonoids (Rungruangsak et af., 1977; Kositawattanakul et a l . , 1977), and perhaps other common phenols can have antithiamine effects. Adducts are formed that make the thiamine unavailable. Oxidation is involved, and ascorbic acid prevents this antithiamine effect. Tannic acid precipitates vitamin B,2, and the precipitate passes through the rat, making the B,2 unavailable and contributing to anemia (Carrera et al., 1973). This is thought to be one reason for the common anemia found among strict vegetarians. Another factor can be chelation, which renders the iron of the diet unavailable. Supplementation of the diet with extra iron helps counteract the toxic effects of gossypol, tannin, and some other phenols (Singleton and Kratzer, 1969, 1973). The chelation of metals by properly substituted natural phenols can be also helpful. Mice with 2% tannic acid and 3% lead acetate in their diet survived for 13 wk; those receiving only 3% lead acetate all died before 13 wk (Peaslee and Einhellig , 1977). C.

PENETRATION EFFECTS

Most biological membranes at the cellular level have a lipoprotein or related biphasic nature so that substances readily interacting with the membrane need some water solubility, but also some lipid solubility and limited polarity. Glycosides of phenols are generally less toxic when given orally and often when given parenterally. The glycosides are not readily hydrolyzed by the animal unless by the gut flora, and they are not readily absorbed until hydrolyzed to the aglycones. If injected, the phenolic glycoside, like the conjugate glucuronoside, is prone to rapid excretion. Aglycones of flavonoids are readily absorbed, particularly the ones like the catechins, which usually occur free and not as glycosides in plants. Flavonoids and smaller phenolic derivatives, which lack free phenolic groups, are not subject to sulfate or glucuronoside conjugation. These tend to be rarer in nature and more toxic. Urushiol, the toxic allergen of poison ivy and its relatives, penetrates the skin quickly and binds there (Singleton and Kratzer, 1969, 1973). Phenol itself is a protein solvent and facile skin penetrant with appreciable lipid solubility. Feeding myristicin and several other phenolic toxins with lipid or as fine

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dispersions-for example, kava constituents-increases their toxic effect, presumably by increasing their solubility and absorption via chylomicrons with the fat. The toxicity of safrole, coumarin, psoralen, and rotenone may depend partly on their lack of free hydroxyls and their high lipoidal membrane solubility. The collagen of capillary vessel walls is more retentive in the presence of bioflavonoids. Small phenols, like other penetrants, may carry solutes through tissue barriers. Completely methoxylated flavonoids like tangeretin and nobiletin also have high lipid solubility and cannot be conjugated in the usual way. Additional methoxyls on the A-ring presumably make this structure difficult to break down metabolically, when compared with the normal phloroglucinol ring. Yet the high methoxyl content would confer some hydrophilic character. These and related compounds are rather restricted in their botanical occurrence and appear to have unusual but rather weak antifungal and cytotoxic effects, as was already noted. D.

PROTEIN BINDING

Although tannins are protein binders without peer, the same hydrogen-bonding mechanism is available to smaller phenols and appears to be important in some of their physiological effects and toxicity. Salicylates bind to blood proteins (Singleton and Kratzer, 1969). Injected tannins must also combine with blood proteins. Gossypol accumulates in the body by attachment to lysine of body (especially liver) proteins via a carbonylamine Schiff’s base-not because of its phenolic functions, but its action illustrates the process nevertheless (Singleton and Kratzer, 1969, 1973). The hydrogen bond from phenol to protein is stronger, the more acidic the phenol (Wade et al., 1969). The pK, of simple phenols correlates well with their uncoupling effect on oxidative phosphorylation and binding with serum albumin. Although their role is generally reported favorably as a stabilizing effect, catechin and some other common plant phenols have demonstrated in vitro, and sometimes in vivo, activities that bind them with collagen (Come1 and Laszt, 1972), other proteins, enzymes (Niebes, 1973), and membranes (Farkas et al., 1977; Ring et al., 1976). Tangeretin (Robbins, 1977; Robbins e t a l . , 1971) and ellagic acid (Botti et al., 1965; Girolami et af., 1977) have effects on blood cell aggregation and clotting that presumably involves bonding with proteins or cell membranes. The effects of tannin on dietary protein are similar. The antimicrobial and antiviral activities of tannins and often of other phenols appear to involve adsorption and binding to the microbe or virus surface, thereby inhibiting infectivity and growth. The enzyme-inhibiting and mucus-removal effects of tannins and related substances involve tanning-like reactions with enzyme protein, mucoprotein, or glucosamine glycans.

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E. RESPIRATION EFFECTS, UNCOUPLING OF OXIDATIVE PHOSPHORYLATlON Phenols are noted for their uncoupling of oxidative phosphorylation and related effects. Rotenone is widely used in enzyme biochemistry as a specific inhibitor of NAD to coenzyme Q oxidation early in the electron transport respiration series. Synthetic phenols, notably 2,4-dinitrophenol, are active uncouplers of oxidative formation of ATP. Dicoumarol has uncoupling activity and many other natural phenols do also, to a much smaller degree. The phenols with the most activity as uncouplers of oxidative phosphorylation are generally also those that have the greatest acidity, are more lipophilic, and are relatively more toxic, at least among fairly simple phenols (Fujita and Nakajima, 1969; Tollenaere er d., 1976; Wada er d., 1969). The more oxidizable phenols are likely to be more nephrotoxic (Calder et al., 1975). The tendency of vegetable phenols to stimulate the basal metabolic rate (McLaren et al., 1973; Stelig and Qasim, 1973) appears to be related to the effect of phenols on respiration metabolism (Gadjos et al., 1969; Graziani, 1977; Mal'yan and Akula, 1977; Suolinna et al., 1974, 1975). The effects of phenols on ATPases also may play a role (Fewtrell and Gomperts, 1977; Mirsalikhova and Pakudina, 1977). It seems significant that the essential phenols-vitamins E and K-are involved in oxidation-reduction metabolism. Phenols of common types might affect poising, autoxidation, and oxidation-reduction reactions. Some less common but natural phenols and their quinones (for example, hydroquinone, juglone, anthraquinones, and pyrogallol) certainly appear to owe some of their toxic properties and sometimes (as with antioxidants) their beneficial roles to these effects. The bioflavonoid relationship with vitamin C is at least partly the protection of minimal amounts of ascorbic acid from oxidation. The rather specific effects of common flavonoids on CAMP phosphodiesterase, and on some active transport enzymes discussed earlier, have not been noted to be part of the mechanism of any uncommon toxic phenol. They may be part of the normal control mechanisms in plants.

F.

MUTAGENICITY, CARCINOGENICITY

Aflatoxin is a uniquely carcinogenic phenolic toxin. Its carcinogenicity appears to involve activated furan rings, and its structure is quite unlike that of any common plant phenols. Safrole is evidently a rather weak esophageal carcinogen with an unusual methylenedioxy grouping (Singleton and Kratzer, 1969, 1973). Carcinogenicity has not often been a demonstrated feature of phenolic toxicity, but some of the most potent carcinogens-aflatoxin, for example-are microbial phenols '(Miller and Miller, 1979). The putative carcinogenicity of tannins appears to be very weak, if real. It

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seems to be related more to severe and prolonged imtation than to true carcinogenicity. The necessary experimental conditions are very unlikely to occur naturally. The most thorough epidemiology (Warwick and Harington, 1973) and evidence for lack of DNA reactivity cast doubt on any carcinogenic danger from tannins (Korpassy, 1961). The recent indications of mutagenicity for quercetin and related flavonols in the Ames-type Salmonella mutant tests are somewhat balanced by the associated findings that the structural requirements for mutagenicity exonerate most flavonoids (Macgregor and Jurd, 1978). The effect of quercetin was one to three orders of magnitude less than that of known powerful carcinogens, although apparently it was stronger than that of dimethylnitrosamine or benzidine (Sugimura and Nagao, 1979). In view of a considerable experimental and clinical history with quercetin derivatives without evidence of cancer initiation, the risk must be small. Quercetin is commonly stated to be the most widespread of flavonoids if not of phenols in higher plants. Of course, the risk to animals and humans cannot yet be considered nil; more study is clearly required. Naphthols, nitrophenol, and chlorophenols produce “stickiness” and abnormalities in plant chromosome behavior during meiosis (Amer and Ali, 1968), but common plant phenols apparently do not. Damage to DNA as indicated by strand separation was caused by rotenone and 2,4-dinitrophenol, but upon removal of the agent the DNA was repaired (Hilton and Walker, 1977). Perhaps interference with the supply of ATP and the energy requirement for DNA repair was involved. The furanocoumarin, psoralen, intercalates DNA and upon irradiation covalently links to it (Singleton and Kratzer, 1969). The role of phenols as cocarcinogens or as a cause of tumors after an initiating contact with a known carcinogen is apparently variable. In experimental applications of cigarette smoke condensate, catechol was an active cocarcinogen, but phenol was an inhibitor (Van Duuren et al., 1973). The chlorogenic acid content of coffee is considered a cocarcinogen in the sense of catalyzing the formation of N-nitrosamine carcinogens from nitrate and secondary amines (Challis and Bartlett, 1975; Mori and Hirono, 1977). In other studies, however, gallic acid and tannic acid were found to be effective inhibitors of nitrosation at gastric pH (Mirvish et al., 1975). The embryotoxic effects reported for tangeretin may be at least partly due to the undeveloped detoxication mechanisms of the embryo. Of course, cytotoxicity as demonstrated in cell culture cannot involve the liver metabolism etc. of the intact animal. Synthetic phenylethylphenols applied to mouse skin at 25% in olive oil produced no visible effect on the adults, but decreased the frequency of pregnancy and the size of the litter (Broitman et al., 1966). The treatment had more effect on males and was said to show the same trends for three generations after the treatment! In guinea pigs, teratogenic as well as mutagenic effects were observed.

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It is interesting that antioxidants like butylated hydroxyanisole are potent and wide-spectrum inhibitors of carcinogenesis (Wattenberg, 1980). Quercetin and many other common natural phenols are rather good antioxidants. lnduction of increased microsomal mono-oxygenase activity by plant phenols could enhance carcinogenicity, if the carcinogen requires metabolic activation, but more commonly it inhibits carcinogenesis. Penta-0-methylquercetin inhibited experimental pulmonary adenoma induction by benz(a)pyrene (Wattenberg, 1980).

G.

MISCELLANEOUS EFFECTS

Other properties of phenols have been mentioned in connection with phenol toxicity and physiological activity. Chelation of iron and other metal ions by phenols, common or otherwise, can be involved in development of anemia, removal of lead toxicity, etc. Keeping iron ions reduced to the more useful ferrous form can be a role of readily oxidized phenols. The specific inhibition of histamine-forming enzymes has been reported for catechin, and various phenols have been reported to cause release of histamine from mast cells, although others do not. Inhibition of prostaglandin synthesis has been reported for some natural phenols; it may be related to spasmolytic or possibly to toxic effects. Aspirin and polyphloretin phosphate have been particularly active prostaglandin antagonists (Sanner and Eakins, 1976). Phenols are not very allergenic, with the exception of poison ivy urushiol and its relatives. Sensitivity reactions have been reported with vitamin E aerosols and p-hydroxybenzoate ointments. The coffee allergic reactions attributed to chlorogenic acid seem to be caused by contamination with coffee protein. Histamine-release responses to phenol administration are variable and usually weak. Catechin appears specifically to inhibit the enzyme that produces histamine from histidine in the stomach.

VI.

CONCLUSIONS AND ASSESSMENT OF RISKS

The reported incidences of toxicity or appreciable physiological effects in animals caused by common plant phenols have been reviewed and interpreted. A number of conclusions and tentative conclusions may be drawn. Many plants contain secondary metabolites toxic or deterrent to members of the animal kingdom attempting to feed on them. Phenols are an important group of these secondary plant substances; they may be divided into groups according to their common, uncommon, or rare occurrence in nature. Plant phenols that are highly toxic to animals are rarely found. The phenols that occur widely in plants

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or in large amounts in common food plants have very low acute oral toxicity, and most are also of low toxicity when given parenterally. A case has been made, however, that the mechanisms of toxicity of plant phenols in animals are frequently based upon characteristics shared generally by phenols of both high and low toxicity. The difference is attributed in part to efficient detoxication in the animal of the common phenols. Toxic phenols are unsatisfactorily metabolized because of structural features (or inappropriate, abnormal administration) not readily withstood by the normal animal barriers. The toxicity of various types of plant phenols appears to be an important aspect of their evolutionary advantage to plants; minimizing feeding by herbivores certainly can be one advantage. Animals, particularly mammals, have evolved to tolerate ubiquitous secondary plant products, including most common plant phenols. The major detoxicating mechanisms not only improve with challenge during evolutionary contact, but they are often quickly adaptable and may be induced in the exposed individual to cope with extraordinary types and amounts of would-be toxicants. Herbivores, especially ruminants, through a combination of alimentary tract microbes and evolutionary adaptation of their detoxication capacity, tolerate a considerable intake of common plant phenols without evidence of toxic symptoms. Carnivores, and other creatures not adapted to plant diets, appear to be deficient in detoxication capabilities. Man, as far as the data indicate, seems to be rather tolerant to plant phenol toxicity, compared with most experimental animals. Experiments with primates other than man support this conclusion. The human fetus and newborn seem to have a greater and earlier detoxication capacity than do those of laboratory rodents and other experimental animals. Detoxication capacity in the adult is greater in total, is larger in proportion to body weight, and is more likely to have been already induced by previous exposure to the same or similar compounds. One of the advantages of the normal human on an eclectic diet is that such a diverse diet should give regular exposure, adaptation, and relative detoxication tolerance toward all common plant constituents without excessive consumption of any one food source or constituent. Smaller phenols may be a practical risk factor as cocarcinogens, especially in smoking, but even in smoked foods they are present in such small amounts as to appear safe in terms of direct effects from dietary consumption. The common hydroxycinnamates, hydroxybenzoates, and flavonoids are relatively innocuous, even with elevated and prolonged consumption. Most reports, in fact, suggest favorable effects or even “semiessential roles (Kuhnau, 1976). These physiological effects are generally mild, and the low toxicity has led to considerable experimentation and clinical application, particularly of the so-called bioflavonoids. Some iatrogenic deaths and serious negative effects have been produced by rather heroic parenteral administrations-of poorly soluble quercetin ”

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derivatives, for example, causing fatal liver concretion in infants. The risks from these compounds in normal food consumption appear to be nonexistent, with the possible exception of the worrisome mutagenicity of quercetin and a few close relatives in mutant bacteria reversion tests and the experimental toxicity of completely methoxylated flavonoids, which are rare except in citrus peel oil. In both cases, the data suggesting a toxic risk are far removed from human experience. Further testing is required for a more certain conclusion. On both experimental and epidemiological grounds the risks so far appear to be very small. Tannins are sufficiently concentrated in some plants and of sufficiently astringent and bitter taste that they are a deterrent to feeding by animals. Under normal ad lib consumption, if low-tannin feed is available, high-tannin feed is avoided by animals. Tannins quickly combine with proteins in the diet, and with digestive enzymes, mucus secretions, and microorganism or virus coats. This may lead to griping diarrhea or constipation, reinforcing food avoidance reactions in the presence of tannins. The tannins are seen as plant protectants having this sort of “toxic” effect. The risk to humans or unconfined animals having a choice of diets seems very small. It is not small, however, when high-tannin food is the only choice, as has been proved by fatalities in animals. Human deaths as well as experimental toxicity have occurred from application of hydrolyzable gallotannic acid in concentrations of about 1% to burns, from enemas, or from smaller amounts given by injection. Condensed tannins are considerably safer, particularly with regard to liver damage. The barriers that protect against liver damage, possibly including carcinoma, seem formidable, and the direct risk from dietary consumption seems very small, under all but the most severely restricted and high tannic acid conditions. Evidence of tannin involvement in other examples of “naturally” occurring toxic effects, notably esophageal cancer, has been reviewed and considered to be unconvincing. The most practical risk to humans and other animals from high intake of common phenols appears to be from the extra load placed on the body in conditions of inadequate nutrition. Weakened, debilitated, infantile, senile, or traumatized animals on a diet inadequate or barely adequate, and without appreciable plant phenols, will find it even more inadequate with them (unless the most significant shortage is vitamin C). Depending upon the conditions, there are several reasons for this, which have been discussed. When these findings are taken together with some considerations of the uncommon and highly toxic phenols, a fairly coherent picture appears. Common plant phenols are not invariably innocuous. But, when the known conditions necessary to produce deleterious effects are compared with those normally present, potential toxicity problems are unlikely to occur, are easily foreseen, and may be readily avoided.

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22 1

RESEARCH NEEDS

The crucial need, as this article is written, is to know whether or not the demonstrated bacterial mutagenicity of quercetin and a few related flavonols is of practical significance in human and animal carcinogenesis. Tests bearing on this question are known to be in progress with experimental animals. The intense or long-term animal feeding trials so far completed did not indicate appreciable toxicity, but they were not specifically designed to detect carcinogenesis. In view of the widespread occurrence but low content of quercetin in foods from plants, it is important to consider the problem carefully with input from toxicology, food science, statistics, and other disciplines. Shunning of all foods with traces of flavonols would be impractical and would eliminate most natural plant foods. A diet exclusively of refined carbohydrates and animal products is already known to be epidemiologically associated with more risks than a diet with more vegetable matter. Facts and studious evaluation must supplant piecemeal sensational reporting, if the public is to be best served. The majority of the phenols common in foods have very low practical toxicity and are negative in the Ames test, and many have definite or possible dietary or therapeutic benefits. We need to know more about their fate in man. Testing both on physiological effects and on metabolism after oral intake appears safe and should be expanded. Further efforts to distinguish human metabolism from the metabolism of the gut microbes are needed. The careful investigation of useful dietary and therapeutic applications of the natural phenols and their derivatives has been unduly restricted in this country by the excessively negative evaluation of earlier “bioflavonoid” research. It is also clear that medical use in Europe has been occasionally excessive. More and better research is needed. Expanded roles for anthocyanins to replace synthetic food colorants, for dietary fiber including lignin, and for vegetable versus animal foods indicate current trends. Continued research on the contribution of natural phenols in foods is warranted. New dietary compositions and food formulations suggest further research on plant phenols in relation to food quality and healthfulness. The antioxidant, metal sequestering, preservative, and other effects of natural plant phenols, when better understood, may enable them to replace synthetic additives and help further to maintain the healthful quality of the modem diet.

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Voelter, W., and Jung, G., eds. 1978. “0-(P-Hydroxyethy1)rutosides-Experimentaland Clinical Results,’’ p. 237. Springer-Verlag, Berlin and New York. Vogin, E. E. 1960. A review of bioflavonoids. Am. J. Pharm. 132, 363-382. Vohra, P., Kratzer, F. H., and Joslyn, M. A. 1966. The growth-depressing and toxic effects of tannins to chicks. Poulf. Sci. 45, 135-142. Wada, S . , Tomioka, S . , and Moriguchi, I. 1969. Protein binding, 6. Binding of phenols to bovine serum albumin. Chem. Phorm. Bull. 17, 320-323. Wanvick, G . P., and Harington, J. S. 1973. Some aspects of the epidemilogy and etiology of esophageal cancer with particular emphasis on the Transkei, South Africa. Adv. Concer Res. 17, 81-229. Wasilewski, A. 1972. Percutaneous action of 0-(P-hydroxyethy1)-rutoside. (HR). Wien. M e d . Wochenschr. 122, 106-1 10. Wattenberg, L. W. 1980. Inhibitors of chemical carcinogens. J. Environ. Parhol. T m i c o l . 3,35-52. Wattenberg, L. W.. Page, M. A., and Leong, J. L. 1968. Induction of increased benzpyrene hydroxylase activity by flavones and related compounds. Cancer Res. 28, 934-937. Wegmann, R., Maeda. K., Trouche, P., and Bastide. P. 1969. Effets des anthocyanosides sur les photorecepteurs. Aspects cytoenzymologiques. Ann. Hisrochim. 14, 237-256. Weinberg, M. S . . Goldhamer, R. E.. and Carson, S. 1966. Acute oral toxicity of various drugs in newborn rats after treatment of the dam during gestation. Toxirol. Appl. Phormacul. 9, 234239. Westendorf. J . , and Czok, G . 1978. Untersuchungen der Pharmakokinetik von IT-ZimtsaureDerivaten an Ratten. 2. Ernaehrungswiss. 17, 26-36. Willaman, J. J . 1955. Some biological effects of the flavonoids. J . Am. Pharm. Assoc. 44,404-408. Williams, R . T. 1959. “Detoxication Mechanisms, the Metabolism and Detoxication of Drugs, Toxic Substances and Other Organic Compounds,” 2nd ed., p. 806. Chapman & Hall, London (reprinted, 1975). Williams, R . T. 1967. The biogenesis of conjugation and detoxication products. In “Biogenesis of Natural Compounds” (P. Bernfeld, ed.). 2nd ed., pp. 589-639. Pergamon, Oxford. Wilson, B. J . , and Hayes, A. W. 1973. Microbial toxins. In “Toxicants Occurring Naturally in Foods.” 2nd ed., pp. 372-423. Natl. Acad. Sci., Washington, D.C. Wilson, C. 0.. and Gisvold, 0. 1956. “Textbook of Organic Medicinal and Pharmaceutical Chemistry,’’ 3rd ed., pp. 266-299. Lippincott, Philadelphia, Pennsylvania. Wilson, H. K., Hassall, C . , Price-Jones, C., and Hughes, R. E. 1976. Orange-peel flavonoids and the growth and ascorbic acid status of hypovitaminotic C guinea-pigs. P r o r . Nurr. S o r . 35, I20A- 12 I A. Wislocki. P. G., Chang, R. L., Wood, A. W., Levin, W., Yagi, H., Hernandez, 0.. Mah, H. D., Dansette, P. M.. Jerina, D. M., and Conney, A . H. 1977. High carcinogenicity of 2-hydroxybenzo(a)pyreneon mouse skin. Cancer Res. 37, 2608-261 I . Wiss, 0.. and Gloor, U. 1970. Nature and distribution of terpene quinones. In “Natural Substances Formed Biologically from Mevalonic Acid,” ( R . W. Goodwin, ed.), pp. 79-87. Academic Press, New York. Wogan, G . N . , and Busby, W. F., Jr. 1980. Naturally occurring carcinogens. I n “Toxic Constituents of Plant Foodstuffs” (1. E. Liener, ed.), 2nd ed.. pp. 329-369. Academic Press, New York. Wolter, R. 1974. Toxicity of acorns. Rev. M e d . Vet. (Tuuluuse) 125, 1481-1485. Woo, W. S . 1968. p-Methoxycinnamate and its metabolite in rabbit serum. J. Pharm. S r i . 57, 27-30. Wuethrich, A., and Schatzmann, H. J. 1980. Inhibition of the red cell calcium pump by quercetin. Cell Calcium 1, 2 1 -35.

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Yang, S. K.,Roller, P. P.. and Gelbain, H. V . 1977. Enzymic mechanism of benzo(u)pyrene conversion to phenols and diols and an improved high-pressure liquid chromatographic separation of benzo(o)pyrene derivatives. Biorhemisrry 16, 3680-3687. Yapar. Z., and Clandinin, D. R. 1972. Effect of tannins in rape-seed meal on its nutritional value for chicks. Poulr. Sci. 51, 222-228. Zloch, Z. 1974. Der Eintluss regulierter peroraler Gaben von Dehydroascorbinsaure und Bioflavonoiden auf einige biochemische Funktionen des Vitamins C. Inr. J . Viram. Nurr. 44, 466-476. Zloch, Z., and Ginter, E. 1979. The effect of flavonoids on vitamin C activity of misoascorbic acid. Physiol. Eohemoslov. 28, 519-524. Zombade, S . S . , Lodhi, G . N . , and Ichhporani, J. S. 1979. The nutritional value of salseed (Shoreo robusiu) meal for growing chicks. E r . Poulr. Sci. 20, 433-438.

ADVANCES IN FOOD RESEARCH.

VOL. 27

TECHNOLOGY AND FLAVOR CHEMISTRY OF PASSION FRUIT JUICES AND CONCENTRATES D. J. CASIMIR, J. F. KEFFORD, AND F. B. WHITFIELD Division of Food Research, Commonwealth Scient@c and Industrial Research Organisation, North Ryde, New South Wales, Australia

1. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Passion Fruit Pulp and Juice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . A. Extraction of Passion Fruit Pulp B. Passion Fruit Juice.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C. Removal of Starch .................................. D. Preservation of Passion Fruit Pulp and Juice .............. E. Chemical Composition of Passion Fruit Juice 111. Concentration of Passion Fruit Juice . . .............. A. Selection of Evaporators . . . . , . , . .............. B. Effects of Concent C. Recovery and Restoration of Volatile Flavoring Constituents . . . . . . . . . D. Concentration by Reverse Osmosis ............................. E. Passion Fruit Juice Powders IV. Chemistry of Volatile Flavoring Constituents . , . . A. General Composition of Volatile Flavors . B. Unusual Volatile Flavors in Passion Fruit . C. Flavor Impact Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . D. Effects of Variety . . . . . . . . ................... E. Effects of Maturity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Effects of Processing ...................................

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

I.

243 241 241 25 I 252 251 259 265 265 261 261 268 268 269 210 216 282 285 281 281 288 290

INTRODUCTION

Considerable topical interest has been shown in the greater exploitation of less well-known fruits, especially those from tropical and subtropical species; notable examples are the passion fruits. This name does not imply any special properties in the fruits but derives from the fact that they are the fruits of the passion flower 243 Copyright @ 1981 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 012-016127-2

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vines, Passifora species, so called because parts of the flowers resembled articles associated with the Passion of Jesus Christ-the cross, the crown of thorns, etc. Although passion fruit have been grown commercially in South America, Australia, Hawaii, and Africa since the last century, they still are not as widely utilized as their unique flavor characters deserve. In Advances in Food Research, Volume 12, 1963, J . S . Pruthi contributed a comprehensive review on passion fruit, which covered the physiology and storage of the fruit and the chemistry and technology of the pulp, juice, seeds, and peel. More recently, Luh (1971) has written a chapter on passion fruit juice technology, and Chan (1978) has reviewed the flavor and nutrient qualities of passion fruit. The present review aims to present new knowledge that has accumulated on the composition of passion fruit and on the technology of extraction and concentration of passion fruit juice. The most significant new information relates to the volatile constituents that give passion fruit their characteristic flavors. By the application of modem techniques of separation and identification by gas chromatography and mass spectrometry, the flavor composition of the commercially impcrtant passion fruit varieties has been substantially elucidated, and many new flavor compounds have been discovered. Since much of this work was motivated by a desire to improve the retention of volatile flavors in concentrated passion fruit juice, it is placed in the text of this review after the discussion of the technology of passion fruit juice concentration. There are said to be about 400 known species of Passijlora. Martin and Nakasone (1970) have reported on the botanical characteristics and distribution of some 30 of these that bear edible fruit; see also Fouque (1972) and Howell (1976). All are probably indigenous to the American tropics, and many are known only in native markets in South American countries, Mexico, and the West Indies. Very few have achieved significant commercial development. The best known are the purple and the yellow passion fruits. The purple passion fruit vine, Passiflora edulis Sims, bears ovoid fruit that weigh about 35 gm and have a purple-black leathery skin. In South America, the purple passion fruit is called parchaca (Seelkopf er al., 1962); in African countries, grenadilla or granadilla (Haendler, 1965); and in India, kadambo (Swamy et al., 1977). The fruit of the yellow passion fruit, P . edulis var. fluvicarpa Degener, has a yellow skin softer than that of the purple fruit and is usually larger, weighing about 80 gm. Cytological studies (Beal, 1975) indicate that the yellow passion fruit is a mutant form of P . edulis rather than a hybrid. It originated most likely in South America, where it is called maracuju, or possibly in Australia. In Hawaii, the yellow passion fruit is called lilikoi, and in Indonesia, markisa. In addition to skin color, there are important differences between the purple and yellow passion fruits in horticultural performance and fruit character

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(Akamine et al., 1974). The yellow form is more resistant to soil fungi and better adapted to lowland tropical conditions; it bears crops over longer periods and gives higher yields of fruit and pulp. On the other hand, the purple form is more resistant to cold injury and is superior in fruit character, having a pulp with a more intense and pleasing aroma; it is usually higher in acidity than the pulp of the yellow form, but this characteristic varies greatly with country of origin (see Table IV). The purple and yellow forms of passion fruit rarely hybridize in the field, but they are readily crossed manually (Beal, 1975), and a number of successful hybrids have been produced in Queensland, Australia (Leigh, 1970). They are intermediate in skin color and character between the two parents, and they have been selected for agronomic characteristics, including resistance to viral and fungal diseases, high yield, and extended cropping season, and for flavor quality similar to that of the purple form. The hybrids have given yields up to 25,000 kg/ha, which is ten times the average yield of the purple passion fruit. In commercial passion fruit growing in Australia the purple passion fruit has now been entirely displaced by four hybrids, designated Lacey , Purple-gold, Selection 3-1, and Selection 23-E. In Florida also, the purple and yellow forms have been successfully crossed to produce a hybrid that retains resistance to soil-borne diseases and has a juice character superior to that of the yellow parent (Knight, 1972). Interspecific hybridization was also achieved by Ruberte-Torres and Martin (1974), who reported that the fruit of the hybrid between P . edulis var. fluvicarpa and P . afata was superior in flavor to the fruit of both of its parents. Another species of passion fruit mentioned in the present review is P . mollissima, which is called curuba in South America and the banana passion fruit in English-speaking countries because its fruit somewhat resemble small bananas in shape and color. Although the banana passion fruit has a pleasant acidic flavor, it is inferior in character to the purple and yellow passion fruits. World production of passion fruit is small, and the actual amount is uncertain. Brazil is now a major producer; it expected to export 2700 tons of passion fruit juice in 1977-1978 and 8650 tons in 1978-1979 (Landgraf, 1978). Australian production since 1970 has fluctuated between 2000 and 4000 tons annually. Other major producing areas are Sri Lanka (Berth, 1976), Hawaii (1200 tons in 1976), Fiji, Kenya (Anonymous, 1971), New Zealand, Papua-New Guinea, South Africa, South American countries, and Taiwan (Mott, 1969). Since the review of Pruthi (1963), further investigations on the storage of yellow passion fruit have been reported by Cereda et al. (1976), who found that the fruit kept better at ambient temperatures when stored partially ripe, but ripe fruit were better for cool storage at 6-7°C. By storing the fruit in polyethylene bags or by waxing with paraffin wax, the storage life was extended from 4 to 30 days. The usefulness of polyethylene bags was confirmed by Ganapathy and

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Singh (1976), who found that the outturn of purple passion fruit stored in unperforated polyethylene bags (0.042 mm thick) was superior to that of fruit in perforated polyethylene bags or wooden crates after storage for 18 days at 2730°C and 30-50% relative humidity. When several tropical fruits were irradiated at ambient temperature with gamma radiation from a 6oCosource, a treatment for disinfestation against fruit flies, the order of increasing toleration to irradiation was avocado, sour sop, mango, yellow passion fruit, and papaya (Akamine and Goo, 1971). This was also the decreasing order of the initial increase in respiration due to irradiation. The maximum dose of irradiation that the yellow passion fruit can tolerate is about 100 b a d . The peels of yellow and purple passion fruits and also of P . quadrangularis, the giant grenadilla, were investigated as sources of pectin by de Lima (1972). Extraction with sulfurous acid and precipitation with methanol gave yields ranging from 0.4 to 2.7% of pectins with 5-9% methoxyl groups and 74-85% anhydrous galacturonic acid. The edible portion of the passion fruit is the mass of yellow-orange pulp that fills the interior of the fruit surrounding numerous small black seeds attached to peglike funiculi on the inner lining of the ovary wall. By stereomicroscopic examination, Lipitoa and Robertson (1977) observed that each seed is enclosed in a double juice sac; the juice in the inner sac is more yellow (see Section II,E,4) and pulpy than the juice in the outer sac, which has the larger volume. Australian consumers are used to drinking and eating passion fruit products in which the seeds are still present; in fact, the seeds are regarded as evidence of passion fruit content. This has given rise to the inverted situation of products containing artificial passion fruit flavors being adulterated with passion fruit seeds. A high-seed pulp is available commercially for this and similar purposes! When consumed, the seeds are not digested but pass harmlessly through the alimentary tract. Consumers in America and Europe, however, take exception to what the late Dr.V. L. S. Charley (1969) called the “floating frog spawn of black seeds,” and therefore passion fruit products for those markets must have the seeds removed. The seeds contain about 25% of a semidrying oil; the composition and uses of this oil and of the residual meal are subjects of research at the University of Nairobi, Kenya (Henderson et al., 1978). Because of the nature of the edible portion, passion fruit are utilized mainly in the form of the juice and secondary beverages derived from it. However, in Australia, the whole pulp is greatly favored as a flavoring for yogurt, ice cream, and pavlova, a soft meringue topped with whipped cream, which is often called the Great Australian Dessert. In the present review, the term pulp will be used to refer to the total edible portion of passion fruit including the seeds, the term juice to refer to the edible

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portion with seeds removed, and the term serum to refer to the liquid fraction after a large portion of the suspended solids has been removed.

II. A.

PASSION FRUIT PULP AND JUICE EXTRACTION OF PASSION FRUIT PULP

In countries where labor is relatively cheap, passion fruit pulp is extracted manually from the halved fruit by spooning or reaming, or from the whole fruit by suction needles (Hubbard, 1973). Several kinds of mechanical extractors are available, however. I , Centrifugal Extractor

In Hawaii, a centrifugal extractor developed by Kinch (1959) is the favored method for extraction of the pulp from the yellow passion fruit commonly grown there. The fruit is sliced by a gang of rotating knives, and the slices (15 mm) drop directly into a perforated centrifuge bowl, which has sloping sides and four baffles at right angles to the sides. When it is rotated at a speed representing a centrifugal force of 175 g, the pulp and seeds from the fruit slices are thrown out through the holes in the basket, while the residual skins climb the sides of the basket and are discharged over the edge. The pulp and skins are then collected from separate chutes. A typical extractor has a capacity of 1800 kg of passion fruit per hr, and an efficiency of extraction of 94% is claimed.

2 . Converging Cone Extractor The mechanical extractor commonly used in Australia for passion fruit is based on a different principle. It consists of two rotating flat truncated cones, which may be smooth or have a roughened surface. The cones are mounted so that there is a wide clearance at the top but a “nip” at the bottom (Fig. 1). Typical dimensions are 370 mm for the diameter of the cones and a 150” angle between drive shafts; the cones are parallel at the nip. Whole passion fruit are fed at the top and are carried down until squashed at the nip. In practice the skin bursts suddenly and the pulp and seeds are expelled cleanly. The burst skins (Fig. 2) are carried on by the cones and rejected, while the pulp drops between the cones through a coarse screen (6.3-mm openings), which may be a shaking screen, to remove fragments of skin and detached stems. A study was made by Casimir (1974) of the effects of varying some operating parameters on the performance of the converging cone extractor; the results of a

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FIG. I .

Converging cone extractor for passion fruit, with cover removed

series of trials are summarized in Table I . In an experimental extractor the rate of rotation of each cone could be varied independently, and the clearance at the nip between the cones was also variable. The yield of pulp increased as the clearance

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FIG. 2. Burst skins of purple passion fruit discharged from converging cone extractor.

between the cones at the nip decreased from 8.3 to 3 . 2 mm. When both cones were rotated at the same speed, the yield of pulp tended to decrease with increasing speed from 70 to 280 rpm. The best yields were obtained with differential rates of rotation. However, the larger differences in speed (100 and 210 rpm) between the two cones accentuated the amount of skin damage; this was reflected in a darker color in the extracted pulp, presumably owing to extraction of skin components. The yield of pulp by manual spooning from the fruit under test was 42.3%.All the yields by mechanical extraction were less than this, probably because of loss of juice by adherence to the skins and by absorption in the spongy white mesocarp. On the basis of these trials, recommended conditions for operation of the converging cone extractor are rough cone 170 rpm, smooth cone 100 rpm, and clearance at the nip 3.2 mm. Under these conditions the yield of pulp was 83.7% of the potential yield, and the throughput was 3.3 fruit per sec, representing about 500 kg/hr. It may be possible to increase the yield of passion fruit pulp solids by washing the extracted skins and centrifuging, but it would be necessary to ensure that undesirable skin components were not extracted. The converging cone extractor has also been applied successfully to the banana passion fruit, Passflora mollissima. Fruit with an average weight of 58.5 gm

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TABLE 1 PERFORMANCE TRIALS OF A CONVERGING CONE EXTRACTOR OPERATING ON PURPLE PASSION FRUIT"

Rates of rotation (rpm) Clearance at nip (mm)

Rough cone

Smooth cone

8.3 8.3 8.3 8.3 8.3 6.3 6.3 6.3 6.3 6.3 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2

70 70 70 I70 280 70 70 70 I70 280 70 70 70 170 280 170 I70 100

70 I70 280 170 280 70 170 280 I70 280 70 I70 280 I70 280 70 100 170

Comparative pulp yield" (%)

69.6 56.4 58.7 60.2 55.7 76.4 77.4 69.3 70.6 74.5 81.9 82.6 70.0 76.9 68.4 72.2 83.7 82. I

" From Casimir (1974). "

As percentage of the yield by manual spooning, which was 42.3% by weight.

when passed through the cones rotating at 170 rpm with a gap of 6 mm yielded 49.3%pulp, and only a further 3.2% of pulp could be recovered from the skins by hand spooning (Casimir, 1974). Skins of banana passion fruit discharged from the cone extractor are shown in Fig. 3. 3 . Passypress Extractor

The Italian food machinery manufacturers S. A . Bertuzzi (S. A. Bertuzzi, Brugherio 20047, Milano, Italy) have developed an extractor, particularly for the yellow passion fruit, which they call the Passypress extractor and which resembles some citrus juice extractors. The passion fruit are compressed between two rollers, one rubber-covered and the other having stainless-steel teeth. A diaphragm moving up and down ensures that the fruit are caught between the rollers

PASSION FRUIT JUICES

25 1

FIG. 3. Burst skins of banana passion fruit discharged from converging cone extractor.

and the skins are fractured. The toothed roller then presses the broken fruit against a screen so that the pulp flows through. Extractors are available with feed capacities of 1-4 tons/hr.

B.

PASSION FRUIT JUICE

To remove the seeds from passion fruit pulp in order to make passion fruit juice, the pulp is fed to a brush finisher with a screen having holes 0.8 mm in diameter. This screen will also remove foreign fragments such as small pieces of skin or stem. A brushing action during screening is necessary to separate the black seeds cleanly from the glutinous contents of the juice sacs. For maximum yields of juice, the finisher should be adjusted to discharge seeds free from adhering material. Lipitoa and Robertson (1977) have advocated treatment with pectolytic enzymes to break down the pulp and aid the liberation of the seeds, but this additional procedure should not be necessary. A typical mass balance for the extraction of passion fruit pulp and juice is shown in Table 11.

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TABLE I1 MASS BALANCE DIAGRAM FOR EXTRACTION OF PASSION FRUIT PULP AND JUICE"

Purple passion fruit (100 kg) Converging cone extractor with gap 3.2 m m and cones rotating at 100 and 170 rpm

I

Pulp (35.4 kg) Brush finisher with 0.8-mm screen

Skins (64.6 kg)

I

I

I

Juice (26.7 kg)

'I

Seeds (8.7 kg)

From Casimir (1974)

C. REMOVAL OF STARCH Passion fruit juice is one of the few fruit juices that contain appreciable quantities of starch. Pruthi (1963) gives the starch content of purple passion fruit juice from India as ranging from 1 .O to 3.7%, with a mean value of 2.4%. The starch in passion fruit juice increases the viscosity of the juice and thus decreases the rates of heat transfer during operations such as pasteurization and concentration, particularly when the gelation temperature of the starch is exceeded. Because of the increase in viscosity, the practical limit to concentration of passion fruit juice by evaporation is about 40% soluble solids. To reach higher levels of concentration, it is desirable, therefore, to remove the starch. In 1951, Knock reported that enzymic degradation of the starch in passion fruit juice permitted the preparation of a fourfold concentrate that was relatively free-flowing , but he suggested that centrifugal separation of the starch fraction might be more feasible for commercial operations. I.

Comparison of Centrifugal Systems

In a study of centrifugal procedures for removing starch from passion fruit juice, Casimir ( 1974) examined the performance of three centrifugal separators: the Alfa-Lava1 (Alfa-Lava1 AB, Tumba, Sweden) BRPX and VS-160 centrifuges, and the Sharples (Pennwalt Corporation, Sharples-Stokes Division, 955 Mearns Road, Warminster, Pennsylvania, 18974) P-600 Super-D-Canter centrifuge.

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The Alfa-Lava1 BRPX, 309 SGV-34 centrifuge is a bowl-type centrifuge with provision for automatic ejection of solids. While the machine is running at full speed, the upper and lower sections of the bowl may be separated to allow solids to be discharged through the annular slit. The change in hydraulic pressure, which determines the time of the solids’ discharge, may be programmed by means of a timer or a self-triggering device that “shoots” the bowl when the solids reach a predetermined level in the bowl. The Alfa-Lava1 VS- 160 laboratory decanting centrifuge is a horizontal centrifuge with a conical rotor and a scroll for continuous removal of solids. The scroll conveyor rotates in the same direction as the rotor but at a slightly higher speed. The difference in speed between the rotor and the scroll is made infinitely variable between 10 and 80 rpm by means of an hydraulic drive. At the speed setting used, the gravitational force was 2200 g. The depth of liquid in the bowl is controlled by four interchangeable weir rings. The Sharples P-600 Super-D-Canter is a decanting centrifuge similar in construction to the Alfa-Lava1 VS-160. The differential speed of the rotor and scrolls is varied by changing pulleys on the back-drive motor. Operating parameters that may be varied on decanting centrifuges are the speed of rotation, which determines the centrifugal force; the weir height, which determines hold-up volume; the feed rate, which determines the residence time of the juice in the high gravitational field; and the difference between the rotor speed and the scroll speed, which determines the residence time of the solids on the conical discharge ramp from the horizontal bowl-the lower the differential speed, the longer the residence time and hence the drier the solids will be when discharged. Trials with passion fruit juice (Casimir, 1974) indicated that by adjusting the operating conditions in a decanting centrifuge it is possible to remove the starch from passion fruit juice while retaining most of the other suspended solids in the liquid stream. Mass balance diagrams for the removal of centrifugeable solids from passion fruit juice are shown in Table 111 for the Sharples P-600 centrifuge, and for the Alfa-Lava1 VS-160 and BRPX centrifuges used in succession. The amounts of soluble solids, as represented by citric acid, remaining in the solids fractions indicate that backwashing of these fractions should be considered in commercial operations. The solids fraction A (Table 111) from the VS-160 centrifuge was whitish in color, since it consisted mainly of starch grains, but it also contained some skin and seed fragments not removed by the brush finisher and some larger fragments of juice sac material. The solids fraction B (Table 111) from the BRPX centrifuge was bright orange in color, since it contained a high proportion of pigmented cell fragments and mitochondria. It also contained some of the smaller starch grains not removed by the VS-160 centrifuge.

254

D. J. CASIMIR ET AL. TABLE I11 MASS BALANCE DIAGRAMS FOR CENTRIFUGAL REMOVAL OF STARCH

FROM PASSION FRUIT JUICE'

1 . Sharples centrifuge Purple passion fruit juice (100 kg containing 16. I kg dry solids)

I

Decanting centrifuge (P-600)

I

Solids discharge

Liquids discharge

(5.8 kg containing 2.6 kg dry solids)

(94.2 kg containing 13.5 kg dry solids)

2. Alfa-Lava1 centrifuges Purple passion fruit juice (100 kg containing 16.1 kg dry solids and 3.6 kg citric acid) ,

I

Decanting centiifuge (VS-160)

I (fraction A) Solids discharge (3.8 kg containing I .8 kg dry solids and 0.1 kg citric acid)

I

Liquids bischarge (96.2 kg containing 14.3 kg dry solids and 3.5 kg citric acid)

I

Ejecting centrifuge (BRPX)

I I Solids discharge (fraction B) (4.8 kg containing 1.2 kg dry solids and 0.2 kg citric acid)

I

Liquids discharge (91.4 kg containing 13.1 kg dry solids and 3.3 kg citric acid)

" From Casimir (1974). Taste tests indicated that the solids removed by centrifugation did not make a significant contribution to the flavor of passion fruit juice (Casimir, 1974). Four passion fruit drinks were prepared containing 25% juice and added sugar to give a sugar-to-acid ratio of 18, and with the juice component varied as follows: juice containing all the original suspended solids, juice with solids fraction A (Table 111) omitted but fraction B restored, juice with solids fraction B omitted but fraction A restored, and juice with both fractions A and B omitted. A panel of 30 tasters using the procedures described by Gipps and Casimir (1973) showed no significant preference for any of the drinks.

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In preparing passion fruit juice for heat processing, a single-stage centrifugation is adequate to remove the starch, gelation of which causes high viscosity and reduced rates of heat transfer. The decanting centrifuges are particularly suitable for this operation, but in a passion fruit juice concentration plant in New Guinea a BRPX centrifuge alone has been successfully used. 2 . Properties of Passion Fruit Starch Starch grains from passion fruit juice (Fig. 4) tend to be spherical and highly refractive. They exhibit growth rings and have a restricted size range: 4-14 p m in purple passion fruit from Australia and New Guinea (Casimir, 1974); 5-5 p m in yellow passion fruit from Brazil (da Fonseca, 1976). By microscopic examination Mollenhauer (1962, 1963) estimated the number of starch grains in passion fruit juice to be 206,000-230,000 m ~ n - ~and , he suggested that the starch grain count might provide an index of passion fruit content in mixed products. Seidemann (1963) was very critical of the usefulness of this procedure, however, and it is obvious that centrifugal processing treatments would invalidate it.

FIG. 4.

Starch grains from purple passion fruit juice.

256

D. J . CASIMIR ET AL.

Because of the narrow size range and spherical shape of the grains, slurries of passion fruit starch in wet preparations show marked dilatant characteristics, flowing readily under high rates of shear, yet exhibiting brittle fracture patterns. These properties are further demonstrated in the high loads imposed on the scrolls of decanting centrifuges and the peculiarity that the solids may be discharged with a noise like hail on the collectors, and yet flow as a fluid in the collection tank when the high rate of shear is discontinued. The washed starch may be air-dried to a moisture content of about 10%. when it is a fine white powder with a talclike feel. Kwok et af. (1974) examined the starch component in the juices of purple and yellow passion fruit grown in Hawaii and found, in common with general experience, that the purple passion fruit juice had a higher starch content (0.74%)than the yellow passion fruit juice (0.06%);however, both these contents were below the range ( 1 .O-3.7%) previously reported by Pruthi (1963). On the other hand, the amylose contents of the respective starches-5.8% in the purple passion fruit starch and 8.7% in the yellow passion fruit starch-although confirming that the starches were mainly amylopectins, were much higher than the range ( 1 -2%) found by Cillie and Joubert (1950) in South African granadilla (P. edulis) juice. The size range of the starch grains, 4.0-12.2 p m in diameter, was the same for purple and yellow passion fruit and similar to the range found by Casimir (1974). The starches from the two kinds of passion fruit also showed almost identical ranges of gelation temperature, 58.5-67"C, as determined by observing loss of birefringence. Casimir (1974) followed the course of gelation in a passion fruit starch suspension (7.2% by weight) in a Brabender amylograph with the temperature rise programmed at 1.5 deg/min. Gelation commenced at 55.5"C, and the viscosity reached a maximum of 1470 Brabender units at 73.5"C after 12-min heating. The viscosity then decreased to I120 units as the temperature rose to 90°C over a further 11 min. During holding at 90°C for 10 min the viscosity decreased to 850 units, then increased to 920 units during 20-min cooling to 60°C. These observations agree well with the earlier work of Mollenhauer (1954), who reported that on heating passion fruit juice the viscosity began to increase at 55°C and reached a maximum at 87°C. Observations by da Fonseca (1976) on yellow passion fruit starch showed incipient gelation after 5 min at 65°C and complete gelation after 5 min at 80°C. It is because of the low gelation temperature (55°C) of passion fruit starch and the marked increase in viscosity at higher temperatures that major difficulties are encountered in any process entailing heating of passion fruit juice. As Knock (195 1 ) had done earlier, Kwok et a / . (1974) investigated degradation of the starch in passion fruit juice by amylase treatment. However, fungal a-amylase gave an effective reduction in viscosity only when the starch granules were gelatinized by heating. Further, the fungal a-amylase was not active at the

257

PASSION FRUIT JUICES

natural pH (2.8) of the yellow passion fruit juice; it was active at the pH (4.2) of the purple passion fruit juice, but this was an unusually high pH for this juice, for which Pruthi (1963) reports the range pH 2.6-3.2. In contrast to these observations, da Fonseca (1976) reported successful use of commercial fungal amyloglucosidase and diastase preparaticns to hydrolyze the starch in yellow passion fruit juice.

D. PRESERVATION OF PASSION FRUIT PULP AND JUICE From the earliest experience in the processing of passion fruit juice, the elusive nature of its unique fresh character was recognized, as well as the ease with which this was lost during preservation processes involving heating. It follows that passion fruit pulp and juice are best preserved for marketing and distribution by freezing (Pruthi, 1963). Canning, even with an accelerated heat process such as spin pasteurization (Pruthi, 1963; Wang and Ross, 1965), gives a product that departs significantly from fresh quality. A quantitative study of the effects of heat treatment on the flavor of passion fruit was reported by Casimir (1974). Purple passion fruit juice in 301 X 41 1 lacquered cans (0.45 kg fill) was closed under vacuum and spinpasteurized at 150 rpm in steam at 100°C for periods of 0,0.5, 1 , 2 , 4 , 8 , 16, and 128 min, then immediately cooled. A panel of 30 judges tasted the canned juices in comparison with unheated juice, all in the form of passion fruit drinks containing 25% juice and added sugar to bring the sugar-to-acid ratio to 18. Differences in flavor were assessed according to the following scale: 0-no difference, 2-slight, &small, 6-moderate, 8-large, and 10-very large differences. A highly significant logarithmic relation was established between flavor change on heating and the extent of heat treatment, which could be expressed by the equation Mean flavor change = 2.36 log (minutes at 100°C) r = +0.954 ( P < 0.001)

+ 1.27

Substitution in this equation shows that, for example, a heat treatment of 2 min at 100°C causes a flavor change of 2 on the scale described. Passion fruit juice may also be preserved by the addition of chemical preservatives. The effectiveness of the three commonly approved preservativessodium benzoate (0.1 %), potassium sorbate (0.l%), and sodium metabisulfite (0.02%)-for the preservation of yellow passion fruit juice was studied by Freitas Leitao et a / . (1977). Passion fruit juice samples containing the preservatives were inoculated with 10'- 103 cells/ml of seven yeasts: Endomycopsis vini, Torula candida, Kloeckera apiculata, Hanseniaspora valbyensis, Candida catenulata, C . guillermondii, and Saccharomyces cerevisiae. During incubation at 25°C for 23 days, all three additives were effective preservatives, benzoate and

258

D.J. CASIMIR ET AL.

sorbate being somewhat superior to metabisulfite. In particular, metabisulfite had no inhibitory effect on C . catenulara, which, of the yeasts studied, was the most resistant to the action of the preservatives. Formulations for beverage bases based on passion fruit concentrate or blends of passion fruit with guava or pineapple concentrate are given by Brekke (1973). The bases contain added sugar, chemical preservatives, and a suspending agent, and are intended to be diluted for consumption at the rate of 1 volume of base to 5 volumes of water. A typical formulation is passion fruit concentrate (3.3-fold, 47" Brix) 62 Ib, water 38 lb, sugar 103 lb, sodium benzoate 1.6 oz, potassium sorbate 1.6 oz, gum tragacanth 8 0 2 . This base diluted with five volumes of water will give a beverage with a passion fruit content of about 20% and containing I05 ppm of each preservative. To prevent browning of passion fruit squash in clear glass bottles, Ragab (197 1) recommended addition of either sulfur dioxide (200-300 ppm) or ascorbic acid ( 1000 ppm), together with ethylenediaminetetraacetic acid (250-500 ppm), and to avoid sedimentation he advised heat treatment at 200°F to inactivate pectic enzymes and the addition of gum tragacanth or carboxymethylcellulose (0.1-0.5%) to restore viscosity. Passion fruit juice was one of the ingredients in blended tropical f q i t nectars that were examined for acceptability and shelf life by Salomon et al. (1977a,b). Papaya and passion fruit juice blends in the ratios 75:25, 82.5: 17.5, and 87.5 : 12.5 were mixed with equal quantities of 30" Brix syrup, pasteurized, and canned. Of these nectars, the 75 : 25 blend was least preferred and considered to be too acid. A nectar was also prepared based on a blend of pineapple juice (92%) and passion fruit juice (8%). The nectars slowly darkened in color during storage. Among a number of tropical fruit products investigated by Swamy et al. (1977), passion fruit juice was one of those most preferred in beverages and punches. Milk flavored with passion fruit juice was highly acceptable, according to Luck and Rudd (1972); the flavored milk did not curdle at 95"C, provided a stabilizer (carboxymethylcellulose or propylene glycol alginate, 0.2%) was added. The use of passion fruit to flavor yogurt has already been mentioned, and in Australian experience it provides one of the most successful fruit-yogurt combinations. The usual practice is to add pasteurized passion fruit pulp to yogurt with stirring. Australian standards require a minimum level of addition of 3.5% by weight. The protein enrichment of passion fruit juice with dried cheese whey (12% protein) was investigated by Nazare et al. (1979a,b), who found that addition of up to 20.8% dried whey did not affect the color or flavor of the juice. A dry table wine was prepared from yellow passion fruit juice by Muller et al. (1964), and some of its volatile flavoring constituents were identified. Both

PASSION FRUIT JUICES

259

passion fruit juice and frozen concentrated juice were used by Hashizume et al. (1976) as bases for the preparation of passion fruit liqueurs.

E. CHEMICAL COMPOSITION OF PASSION FRUIT JUICE I.

General Composition

In the review already mentioned, Pruthi (1963) has provided comprehensive information about the composition of the fruit of P . edulis and other Passiflora species from several parts of the world. In Table IV is collected later information on passion fruit juices from Africa and South America. Some data on the acidity and total soluble solids, ash, and nitrogen compounds in purple passion fruit juice from Taiwan are reported by Hou et al. (1978), who suggest that the ratio between total N , ammonia N, and amino N contents (100: 7 . 6 :29.9) may serve as an index of the authenticity of passion fruit juice. The growth and ripening of the yellow passion fruit in Egypt was followed by Hussein (1972) from 14 days after setting to 70 days. The fruit increased in weight, diameter, volume of juice, and soluble solids content up to 60 days. Acidity reached a maximum at 30 days and then decreased, and ascorbic acid content was greatest at 52 days. Total and reducing sugar contents increased to maturity. In a similar study in Brazil (Araujo et al., 1974), yellow passion fruit were examined at eight stages from 12 days after flowering to 67 days, when the fruit had fallen. Again the highest content of soluble solids was found at 60 days, while fallen fruit were lower in soluble solids and also in sugar content, acidity, and ascorbic acid content. It is suggested that the customary practice of gathering fallen fruit should be avoided (see, however, Section IV,D). A further report from Brazil (Landgraf, 1978) indicates that passion fruit juice produced in the wet season (April-June) had lower soluble solids contents (14.5-15.3' Brix) and acidity (3.92-4.55 gm per 100 ml) than juice produced in the dry season (15.516.3"Brix and 3.91 -4.19 gm per 100 ml). This finding is supported by Muller et al. (1979). who state that the season of harvest is more important than fertilizer treatment in determining the quality of passion fruit. In contrast to these observations on the maturation of yellow passion fruits, Singh et al. (1978) concluded that purple passion fruits grown in Coorg, India, should be harvested between the eightieth and eighty-fifth days, when the fruits are turning purple and the sugar, acid, and ascorbic acid contents are optimal. In common with fruit juices in general, the principal solids in passion fruit juice are sugars and acids, and the quantitative composition of these fractions has now been determined. The picture regarding several groups of minor constituents has also become clearer. The volatile flavoring constituents of passion fruit juice are discussed in Section IV.

TABLE IV COMPOSITION OF PASSION FRUIT JUICES Country of origin: Variety:

Angola" Purple

Brazilb

Yellow

N

8

Total solids (%) Soluble solids (96) Insoluble solids (a) Crude fiber ( S ) Total sugars (%) Invert sugars ( 8 ) Sucrose (%) Total acids (a) Citric acid (86) Malic acid (%) Tartaric acid (%)

PH Protein (%) Pectin (96) Starch (%)

10-14'

Yellow (maracuja) 18.6 14.5-17

Egypt''

Spring

5.3-7.50 1.6-2.2O 3.2-3.7

7.8-1 1.3 5.9-6.3

16.7 13.8

-

-

11.4 8.3 3.2 0.6

12.1 7.5 4.6 0.6

Purple

3-40

Yellow' (maracuja)

-h

10-14

-

5.3-7.5 I .6-2.2 4.4-5.5 3.0-3.5

-

-

2.8-2.9

3.43 0.63

1.4-2.0" 3-4 1.04-1.60 0.04-0.06

3.9-5.5

-

0.03 2.8

Banana' (curuba)

0.07-0.09 8.5-8.9 6.7-8.3 0.7-1.8 3.2-3.7

-

Purple' (parchaca)

15.4- 16.4 1.2-1.9

-

-

Venezuela

1.75 6.60 3.90 3.44 0.66

-

0.7-0.8

Yellow Autumn

14.9 13.6

-

Kenya" _-

1.1-1.3 0.2 1-0.24 1.1-1.2

1.24

-

0.52 0.18 I .69 I .46 0.39 -

3.60 0.84 -

1.95

h)

F2

Lipid (%) Tannin (%) Minerals (%)

-

K (mg

Na (mg 9%) Ca (mg %)

P (mg %)

%)

Fe (mg %) Form01 value (10 gm) Chloramine value (1 gm) Ascorbic acid (mg %) Total carotenoids (mg %) P-Carotene (mg %) Thiamine (mg %) Riboflavin (mg %) Nicotinic acid (mg %)

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

30

32

-

-

20-40p -

-

29-32 -

-

-

-

-

-

-

-

-

-

-

-

0.05-0.08 0.07-0.08 0.57-0.58 204-227 0.6-1.0 4.2-5.6 15-22 1.9-2.6 15-20 29-45 1.3-1.7 0.6-0.9 -

-

-

-

-

-

-

-

-

-

-

-

-

-

-

0.35 157 16.1

0.75-0.77

5.1

6.5-9.0 7.0-9.7 1.0-1.4 17-20 0.30-0.35 0.08-0.11 0.09-0.13 1.6-1.8

87 42 0.25 -

-

" From Strauss (1974a.b). From Araujo er ul. (1974). da Fonseca (1976), and Landgraf (1978). From Hussein (1972). From Benk (1967a.b. 1968). t. From Seelkopf er ul. (1962); these authors also provide analyses of the juices of P. liguluris (parcha) and P. quudrunguluris (badea). From Seelkopf and Febres (1966): range of three growing areas. Range of the two varieties. Dash means not reported.

'

-

0.56 161 11.4 5.4 55 -

39 -

0.15 -

-

262

D. J. CASIMIR ET AL.

2.

Sugars

Chan and Kwok ( 1 975) applied the procedures of thin-layer chromatography on cellulose, and of gas chromatography of the trimethylsilyl derivatives, to the identification and determination of the sugars of passion fruit. The three sugars fructose, glucose, and sucrose were found in roughly similar concentrations in both purple (3.24, 3.59, and 2.85%, respectively) and yellow (3.04, 3.94, and 3.35%, respectively) passion fruit juices. When these values were compared with analyses by colorimetric copper reduction, it was found that the latter procedure gave higher values for reducing sugars and lower values for total sugars, so that the values for sucrose by difference were much lower than those measured directly by gas chromatography. Earlier, Ogata et al. ( 1972) had examined passion fruit (presumably yellow) in Hawaii for the presence of seven-carbon sugars, since mannoheptulose is known to cause hyperglycemia in man and animals. Only trace amounts (less than 0.01%) of mannoheptulose (D-manno-heptulose) were found in passion fruit, and in unripe fruit trace amounts also of sedoheptulose (D-ultro-heptulose). 3 . Acids

Nonvolatile acids in purple and yellow passion fruit juice, separated on ionexchange columns, and identified by thin-layer chromatography and gas chromatography of the methyl esters, were citric, malic, lactic, malonic, succinic, ascorbic, and galacturonic acids (Chan et al., 1972). Quantitative determination by gas chromatography gave the values shown in Table V. The sample TABLE V ACIDS IN PASSION FRUIT JUICES"

Passion fruit

Acid Citric acid Malic acid Lactic acid Malonic acid Succinic acid Ascorbic acid Volatile acids Total acids Titratable acidity "

Purple (meq/100 gm)

Yellow (meq/100 gm)

13.10 3.86 7.49 4.95

55.00 10.55 0.58 0. I3

2.42

Trace 0.06 0. I 1

0.05 0.12

-

-

3 1.99

66.43 65.83

32.01

From Chan er al. (1972).

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PASSION FRUIT JUICES

of purple passion fruit juice examined was, however, unusually low in total acidity (1.9%). As shown in Table IV, Seelkopf and Febres (1966) reported the presence of 1.4-2% tartaric acid in the juice of the yellow passion fruit; but this identification must be doubted, especially since they failed to report malic acid. Volatile acids in passion fruit juices are listed in Table VIII. 4 . Minor Constituents a . Alkaloids. It has long been known that extracts of the leaves, stems, and roots of Pass@oru species have sedative properties that are due to the presence of alkaloids related to harman (1) (Haendler, 1965).

By a combination of feeding and trapping experiments using compounds with radioactive labels, Slaytor and MacFarland (1 968) showed that harman is formed in P. edulis from tryptophan. The biogenetic pathway involves decarboxylation of tryptophan to tryptamine, which is acetylated to N-acetyltryptamine. The /3-carboline ring is then formed by cyclic dehydration to give harmalan, which is oxidized to harman (1). Several workers have looked for harman alkaloids in passion fruit juices (e.g., Wucherpfennig, 1966). In the most comprehensive study, Lutomski et al. (1975) determined alkaloids, flavonoids, and carotenoids in the juices of purple and yellow passion fruit, with the results shown in Table VI. Seven alkaloids were detected by thin-layer chromatography, and four were identified as harman (l), TABLE VI ALKALOIDS. FLAVONOIDS. AND CAROTENOIDS IN PASSION FRUIT JUICES"

Passion fruit Constituent

Purple

Yellow

Flavonoids (mg%) Carotenoids (mg%) Alkaloids (mg%)

1.060 0.160 0.012

1 .Ooo 0.058

" From Lutomski

et a / . (1975)

0.700

264

D. J . CASIMIR ET AL

harmol, harmin, and harmalin. Pharmacological tests showed that the juices had slight sedative effects. These authors did not detect any tannins or anthocyanins. b. Amino Acids. Free amino acids found in purple passion fruit juice by Pruthi and Srivas (1964) were arginine, aspartic acid, glycine, leucine, lysine, proline, threonine, tyrosine, and valine; methionine, phenylalanine, and tryptophan were not found.

c . Carotenoid Pigments. The deep-yellow color of passion fruit juice is due to carotenoid pigments, notably p-carotene, (-carotene, and phytofluene. A quantitative examination of the distribution of these three carotenoids in different parts of passion fruit was made by Leuenberger and Thommen (1972) by chromatographic separation and spectrophotometric analysis. Their results are shown in Table VII. The pasteurized and frozen juices were not derived from the same fruit as the fresh juice, and moreover they were mechanically extracted, so that the mucilage surrounding the seeds was incorporated in these juices and increased their pigment contents. However, the relative proportion of <-carotene in the pasteurized juice was lower than in the fresh juice, suggesting some loss of this less stable carotenoid during pasteurization. The same workers in a further qualitative chromatographic study of pasteurized passion fruit juice identified, in addition to the three main carotenoids, the pigments p-apo- 12’-carotenal, p-apo-8’-carotenal, kryptoxanthin, mutatoxanthin, and auroxanthin. The presence of crocetin was suspected but not unequivocally confirmed. Two further carotenoids, neurosporene and triphasiaxanthin, have recently been found in yellow passion fruit from Brazil by Z. Farah and J . De Souza LeHo (unpublished, 1979).

TABLE VII CAROTENOID PIGMENTS IN PASSION FRUIT’

Total carotenoids Material

( m g / l w gm)

Fresh juice Skin Mucilage” Pasteurized juice‘ Frozen juice‘

0.66 0.13 1.14 2.0 I .8

p-Carotene (mg/IOO gm) 0.27 0.014 0.32 0.9 0.12

Phytofluene (mg/Iw gm)

0.35 0.003 0.46 0.53 0.43

5-Carotene (mg/Iw gm) 0.32 -

0.50 0.6 0.73

~~

I‘

From Leuenberger and Thommen (1972); the passion fruit from Kenya were presumably purple.

* Thick pulp surrounding the seeds.

Not from the same fruit as the fresh juice.

PASSION FRUIT JUICES

265

d . Enzymes. Little work has been done on the enzyme systems in passion fruit. In yellow passion fruit juice Aung and Ross (1965) failed to find measurable activity of oxidative enzymes except a slight catalase-like activity in the decomposition of hydrogen peroxide, which was readily destroyed by heating. Workers in Brazil, D. C. de Lima and M. A. G. Sommer (unpublished, 1979), have found in yellow passion fruit low phenolase activity that is very heat stable and may give rise to enzymic browning in concentrated passion fruit juice. Two proteases having pH optima 2.3 and 5.7 when casein was the substrate were found in purple passion fruit juice by Hashinaga et al. (1978). e . Cyanogenic Constituents. Purple and yellow passion fruits grown in Kenya were shown by Gondwe (1976) to be cyanogenic. Analyses are reported only for the purple variety, which contained in very immature fruit 467-509 and in ripe fruit 10 pmol CN- per 100 gm of pulp. The amount of cyanide in ripe fruit has no toxicological significance, since the author calculates that consumption of about 1750 fruit would be required to achieve a minimum lethal dose.

Ill. CONCENTRATION OF PASSION FRUIT JUICE The economies in packaging, transport, and storage associated with partial removal of water have always been powerful incentives toward the development of fruit juice concentrates. When water is removed by evaporation with heating, two kinds of quality deterioration may occur: chemical reactions between constituents of the juice, which are usually detrimental to flavor (heat damage), and loss of volatile flavoring constituents, some of which inevitably evaporate together with the water. Early attempts to concentrate passion fruit juices (Pruthi, 1963) did not produce high-quality products, and it was not until the 1970s that acceptable concentrates were produced commercially. The sensitivity of passion fruit juice to thermal degradation of flavor makes it essential that evaporation be carried out with the least possible total heat treatment. Volatile flavoring constituents may be recovered from the vapors evaporated and added back to the concentrate. A.

SELECTION OF EVAPORATORS

The extent of heat damage to quality during concentration of fruit juices is determined by the residence time and temperature characteristics of the evaporator. Because their residence times are known to be short, centrifugal evaporators recommended themselves as likely to be most suitable for concentration of passion fruit juice.

266

D.J . CASIMIR ET AL.

Centrifugal evaporators are single-effect, single-pass evaporators in which the heat transfer surface consists of one or more rotating stainless-steel cones (UNIDO, 1969). The liquid feed is distributed as a film on the internal surfaces of the cones by centrifugal force. The concentrate is skimmed by a pickup tube from a trough at the base of the cone. Fluid flow in a centrifugal evaporator may be considered as consisting of two parts. First there is a section of plug flow where a particular portion of liquid traverses the rotating conical heat transfer surface at a velocity of 0.3-3 d s e c . In order to measure precise residence times on the heated surface of centrifugal evaporators, Casimir (1974) devised a technique in which the feed and pickup tubes of the evaporator served as electrodes of a conductivity cell after being electrically insulated from each other and from the conical heat transfer surface. When a saturated potassium sulfate solution was fed into the evaporator, the times of contacting and leaving the heat transfer surface were recorded on a double-beam oscillograph as sharp square-wave pulses over a time interval of less than 0.2 sec. Residence times in this section of the Alfa-Lava1 CTlB centrifugal evaporator (Alfa-Lava1 AB, Tumba, Sweden) were very short: 0.2-1 .O sec, depending on the rate of rotation and the viscosity of the liquid. The second section of fluid flow occurs when the liquid leaves the heat transfer surface and enters the trough where it is skimmed by the pickup tube for discharge. This trough accounts for the major portion of the residence time. The volume of liquid held up depends upon the adjustment of the pickup tube, and there is considerable short-circuiting of flow in this section. In the Alfa-Lava1 CTlB and CT6 centrifugal evaporators, which have heat transfer surface areas of 0.1 and 2.4 mz, respectively, the mean total residence times for water when no evaporation is taking place are approximately 3 and 7 sec. Removal of suspended solids by centrifugation increases heat transfer rate during evaporation and thus helps to minimize further the adverse effects of heat treatment. When passion fruit juices were concentrated in a centrifugal evaporator from around 15 to 40% soluble solids (Casimir, 1974), the overall transfer coefficients were approximately 1700J sec mP2 K for noncentrifuged juices, and 5300 J sec-' m-2 K - I for centrifuged juices; and 40% soluble solids was the practical limit for concentration of the noncentrifuged juices. Centrifuged juices could, however, be concentrated to 65-70% soluble solids with an overall heat transfer coefficient of 1700 J sec-' m-2 K - I . However, da Fonseca (1976) claimed to have successfully concentrated the juice of yellow passion fruit (maracuja) grown in Brazil to 44 and 56" Brix in a pilot-scale centrifugal evaporator without removal of the starch (2.76%). Examination of the interior of the evaporator showed no deposits of starch. The viscosities of yellow passion fruit juice concentrates from 15.6 to 33.4" Brix, prepared from juices from which the starch and much of the other suspended solids had been removed, are given by Vitali et al. (1974) for various shear rates and temperatures.

PASSION FRUIT JUICES

B.

267

EFFECTS OF CONCENTRATION ON QUALITY

In view of the very short residence times, it would be expected that concentration of passion fruit juice in centrifugal evaporators would have minimal effects on quality. This was confirmed by Casimir (1974) in trials in which concentrate recombined with distillate fractions was made up into a passion fruit drink that could not be differentiated by a tasting panel from a drink prepared from the feed juice. However, a drink prepared from the concentrate without add-back of volatiles was judged to be inferior in quality. It is clear, therefore, that thermal degradation is not a serious problem in the concentration of passion fruit juice in centrifugal evaporators, and that loss of volatile flavoring constituents then becomes the chief cause of flavor deficiency. C.

RECOVERY AND RESTORATION OF VOLATILE FLAVORING CONSTITUENTS

Centrifugal evaporators are normally provided with a single condenser for condensation of the evaporated water and associated volatiles. In order to recover and examine the volatile flavors removed during the concentration of passion fruit juice, Casimir (1974) modified an Alfa Lava1 centrifugal evaporator (CTIB) to provide three condensers of decreasing temperature in series, cooled, respectively, by tap water (lS"C), ice water (O'C), and liquid nitrogen (-196°C). In addition, to avoid entrainment a centrifugal separator was inserted in the vapor line between the evaporator and the condensers. When passion fruit juice was concentrated in this equipment, the condensate from each of the three condensers obviously contained aroma compounds of interest. The contribution of each fraction was therefore examined in an omission-type tasting test. Passion fruit drinks were prepared containing all the volatile fractions, or with one fraction omitted at a time. Panel scores for flavor showed that the order of importance of the fractions for passion fruit flavor was: tap water condensate, ice-water condensate, and liquid nitrogen condensate. Further observations on the volatile flavoring constituents present in these fractions are reported in Section IV,E. A procedure based on these findings was applied to the production of 3.5-fold concentrate (41" Brix) from purple passion fruit juice in Papua-New Guinea. Centrifuged passion fruit juice was concentrated in a centrifugal evaporator (Alfa-Lava1 CT6), and the vapors were condensed in two condensers in series, cooled by tap water and ice water. In order to reduce the volume of condensate to be added back to the concentrate, the tap water condensate was restripped under vacuum in a small centrifugal evaporator (Alfa-Lava1 CTIB). Casimir (1974) had established that both initial stripping of water vapor and volatiles at atmospheric pressure and restripping of the tap water condensate at atmospheric pres-

D.J . CASlMIR ET AL.

268

sure were detrimental to flavor quality. The restripped condensate and the icewater condensate were then added back to the concentrated juice. A taste panel was unable to distinguish between carbonated passion fruit drinks prepared from untreated juice and those prepared from concentrate with the two condensate fractions added back. D.

CONCENTRATION BY REVERSE OSMOSIS

Another approach to the problem of concentrating heat-sensitive fruit juices is concentration by reverse osmosis, which may be carried out at ambient or lowered temperatures. The application of reverse osmosis using porous cellulose acetate membranes to the concentration of a number of fruit juices has been explored by Matsuura et al. (1973, 1974). and Pompei and Rho (1974) have applied this procedure to passion fruit juice. In a laboratory pilot plant with a membrane surface of 0.36 m2, operating at 6°C and pressures of 40, 50, and 70 atm, passion fruit juice from Sri Lanka was concentrated from 16.8% to 26-28% solids. The authors recognize that for commercial-scale concentration of passion fruit juice it would be necessary to achieve higher levels of concentration, and this would require equipment operating at higher pressures with greater turbulence at the solution-membrane interface. In the pilot-plant trials the retention of sugars and other soluble constituents was substantially complete, while the retention of aroma constituents ranged from 42 to 69%. Matsuura et al. (1974) have pointed out the possibility of second-stage reverse osmosis treatment for the recovery of aroma compounds that permeate through the membrane during the primary concentration of fruit juices.

E.

PASSION FRUIT JUICE POWDERS

In his 1963 review, Pruthi reported that passion fruit concentrate mixed with sugar could be dried under vacuum to a powder that reconstituted to a juice that was acceptable but inferior in flavor to fresh juice; better results were obtained by freeze-drying. Bates (1964) investigated the application to passion fruit juice of the technique of foam-mat drying, which was developed at the Western Regional Research Laboratory of the United States Department of Agriculture for the preparation of dehydrated powders from citrus, tomato, and other fruit and vegetable products. Unexpectedly, passion fruit juice appeared to have a specific foam-depressing effect so that it was difficult to prepare a stable form. However, a nectar base, consisting of 100 parts of passion fruit juice and 55 parts of sucrose by weight, with the addition of 0.5% soybean protein as a foam inducer and 0.25% carboxymethylcellulose as a foam stabilizer, could be adequately foamed and dried to powder.

PASSION FRUIT JUICES

269

The advantages of foam drying were combined with freeze-drying in the technique of vacuum-puff freeze-drying applied by Moy (1971) to the juices of passion fruit and other tropical fruits. He confirmed that addition of sucrose to give a soluble solids content in the range 40 2 5% was desirable to achieve a stable puffed structure as a result of expansion under vacuum of air entrapped in the mixture during blending. In the absence of a puffed structure it was difficult to dry the products to completion. About 5-hr drying of puffed passion fruitsucrose mixtures with tray loadings of 0.5-1 .O lb/ft2 yielded powdered products with a moisture content of 1-2% and a bulk density of 0.23 g d m l , which reconstituted as satisfactory nectars or nectar bases. Addition of tricalcium phosphate (0.15%), calcium oxide (0.05%),and calcium silicate or silica (0.05%)to the wet mixture improved the free-flowing properties of the dried powder. The temperature zones for freezing of passion fruit juice-sucrose mixtures ranged from -2 to -5°C at 17% soluble solids, to - 15 to - 16°C at 55% soluble solids. The apparent bound water content in passion fruit juice with 17% soluble solids, or with sucrose added to give soluble solids contents of 26 and 47%, was 3-4% as measured by the difference in time required to remove the latent heat of fusion of the eutectic juice mixture in comparison with water (Moy and Chan, 1971). In a comparative trial, Huet (1974) prepared passion fruit juice powders by three methods: spray-drying, freeze-drying, and continuous-belt vacuum-drying with microwave heating. The starting product for spray-drying and freeze-drying was passion fruit juice (12.5 kg) mixed with maltodextrins (1.75 kg) to give a Brix of 28.2", and for microwave drying, a mixture of equal weights of passion fruit juice and sucrose having a Brix of 59". When the dried powders were compared for retention of volatile flavoring substances by gas chromatographic examination of the reconstituted juices, the descending order of retention was microwave drying, freeze-drying, and spray-drying. In addition to freeze-drying and spray-drying of passion fruit juice, de Almeida (1974) describes a combined process in which the juice is separated centrifugally into suspended solids and serum fractions. The solids are freezedried, and the serum is concentrated or freeze-dried; then these fractions are recombined to give either a superconcentrated juice (60-70" Brix) or a dry juice powder.

IV. CHEMISTRY OF VOLATILE FLAVORING CONSTITUENTS At the time of the review of Pruthi (1963) only the yellow passion fruit had been examined for volatile flavors, and among these only four esters had been identified: ethyl butanoate, ethyl hexanoate, hexyl butanoate, and hexyl hexanoate. In the succeeding 15 years spectacular developments occurred in

D. J . CASIMIR ET AL.

270

techniques and instruments for the separation and identification of volatile substances, notably by linked systems of gas chromatograph, mass spectrometer, and computer, and passion fruit products are among the numerous foods to which the new methods have been applied. A.

GENERAL COMPOSITION OF VOLATILE FLAVORS

Present knowledge of the volatile flavoring constituents of passion fruit is consolidated in Table VIII. The volatile flavoring constituents of the purple passion fruit were first investigated by Parliment (1972), who used as a starting product the aqueous condensate from the concentration in a centrifugal evaporator of passion fruit juice mechanically extracted in New Guinea. From the condensate the organic volatiles were transferred into an ether extract in which 16 compounds were identified in a neutral fraction and six acids in an acid fraction (Table VIII). A more comprehensive study of the volatile flavoring constituents of completely fresh purple passion fruit juice was undertaken by Murray er al. (1972, 1973). The raw material was the juice of Australian purple passion fruit extracted by manual spooning and screened to remove the seeds. The juice was immediately saturated with sodium chloride and distilled under vacuum at low temperature to separate the volatile flavors as a concentrated essence. Fractionation of this essence by gas chromatography on both polar and nonpolar columns and by chromatography on silica gel revealed at least 250 discrete chromatographic peaks, from which 94 components were resolved; of these, 73 were positively identified by agreement of mass spectra, gas chromatographic retention data, and aromas with authentic compounds (Table VIII). A number of the compounds unidentified at the time were identified subsequently. At every stage of component separation and identification in the investigation of Murray et al. (1972, 1973), a procedure of monitoring by nose of by-passed effluent streams was followed in order to locate compounds of high olfactory interest and to aid identification. A major study of the volatile flavoring compounds of yellow passion fruit was reported by Winter and Kloti (1972). The starting product was 539 kg of passion fruit juice described as “guaranteed pure,” of Hawaiian origin but supplied by food brokers in Chicago. Unfortunately, the methods of extraction and preservation of the juice were not stated, but it was possibly preserved with benzoate, since benzoic acid was found and relatively large amounts of benzaldehyde. An aqueous distillate from the juice extracted with ethyl chloride yielded 0.0012% of a yellow oil. In the neutral fraction from this oil 165 compounds were identified by gas chromatography and mass spectrometry (Table VIII). Winter, who died in 1976, and his co-workers evidently carried out a similar examination of the

PASSION FRUIT JUICES

27 1

TABLE VlII VOLATILE FLAVORING CONSTITUENTS OF PASSION FRUIT. OCCURRENCE AND CONCENTRATIONS IN THE JUICE

Purple passion fruit PPm" Alcohols (nonterpenoid) Methanol Ethanol Propanol Butanol 2-Methylpropan- 1-01 Butan-2.3-diol Pentanol Pentan-2-01 2-Methylbutan- 1-01 3-Methylbutan- 1-01 2-Methylbut-3-en-2-01 3-Methylbut-2-en- 1-01 3-Methylbut-3-en- 1-01 Hexanol (Z)-Hex-3-enolV (E)-Hex-3-enolV (E)-Hex-Cenol Heptanol Heptan-2-01 Octanol (Z)-Oct-3-enol (E)-Oct-3-enol Nonan-2-01 (Z)-Non-2-enol Cyclopentanol Benzyl alcohol 2-Phenylethanol 3-Phenylpropanol Carbonyl compounds Acetaldehyde Benzaldehyde 4-H ydroxy-3-methox ybenzaldeh yde Propan-2-one 3-Hydroxybutan-2-one Pentan-2-one Pentan-3-one Pentan-2.4-dione Heptan-2-one 6-Methylhept-5-en-2-one . .

PPm

Yellow passion fruit, PPm"

W"

t"

t W W

-

t 0.09 t t W

1.9 0.1 t W

-

0.1 W

W

t 0.2 0.2 W

1.5 W

W W W W

t W

(continued)

212

D. J . CASIMIR ET AL. TABLE Vlll (continued) Purple passion fruit PPm"

Nonan-2-one Nonan-3-one Nonan-Cone Undecan-2-one

PPmb

Yellow passion fruit, PPm" W W W

-

(E)-6,1O-Dimethylundeca5.9-dien-2-one (Z)- and (E)-6,10Dimethylundeca-3,5.9trien-2-ones Cyclopentanone

W

W

0.2

2,2.6-Trimethylcyclohexan1 -one 1-Phenylpropan-2-one

p-Ionone Dihydro-P-ionone Dihydroionone 4-(4-Hydroxyphenyl)-3methylbutan-2-one I-Phenylbuta- I .3-dione

7,9,9-Trimethylbicyclo[4.4.0)decaI .6-dien-3-one ' 5.5,9-Trimethylbicyclo(4.4.0~decaI ,9-dien-3-one I-Oxa-8-0xo-2,6,10,10-

W

W

t t

W

W

W

W

tetramethylspiro[4.5]dec-6enes (two isomers) Carboxylic acids Acetic Butanoic 2-Methylpropanoic But-2-enoic Pentanoic 3-Methylbutanoic Hexanoic Hexenoic" (Z)-Hex-3-enoic Heptanoic (E)-Hept-Cenoic Octanoic Octenoic" (Z)-Oct-3-enoic (E)-Oct-3-enoic Nonanoic

W

-

d" d d d d d -

d d d d

d d d

273

PASSION FRUIT JUICES TABLE VIll (confinued) Purple passion fruit PPm" Decanoic Dodecanoic Tetradecanoic Pentadecanoic Hexadecanoic Benzoic 2-Phenylacetic Cinnamic Esters Dimethyl carbonate Diethyl carbonate Benzyl formate Methyl acetate Ethyl acetate Butyl acetate Isoamyl acetate Hexyl acetate (Z)-Hex-3-enyl acetate (E)-Hex-3-enyl acetate 2-Heptyl acetate Octyl acetate Benzyl acetate 2-Phenylethyl acetate Ethyl propanoate Hexyl propanoate Methyl butanoate Ethyl butanoate Butyl butanoate 2-Pentyl butanoate Hexyl butanoate (Z)-Hex-3-enyl butanoate (E)-Hex-3-enyl butanoate (Z)-Hex-.l-enyl butanoate (E)-Hex-Cenyl butanoate (Z)-Hexa-3,5-dienyl butanoate 2-Heptyl butanoate Benzyl butanoate Phenylethyl butanoate Ethyl (E)-but-2-enoate Ethyl 3-hydroxybutanoate Ethyl acetoacetate Ethyl 2-furancarboxylate Ethyl pentanoate

-

-

PPmb

Yellow passion fruit, PPm" d d d d d d d d W

t W

t

-

8.0 0.2

-

-

3. I 1.8 t 0.5 t

0.2 t t t t 35 1. I 0.2

8 4.2 t t t dh 4.2 0.2

t

W

W

t W

-

-

t

-

W

0.5 -

0.1 t -

-

W

W W

W W W

(continued)

214

D. J . CASlMlR ET AL. TABLE VIIl (continued) Purple passion fruit PPm"

Methyl hexanoate Ethyl hexanoate Butyl hexanoate Hexyl hexanoate (Z)-Hex-3-enyl hexanoate (E)-Hex-3-enyl hexanoate (Z)-Hex-4-enyl hexanoate (E)-Hex-4-enyl hexanoate 2-Heptyl hexanoate Octyl hexanoate Benzyl hexanoate Phenylethyl hexanoate Ethyl hex-2-enoate Ethyl (Z)-hex-3-enoate Methyl 3-hydroxyhexanoate Ethyl 3-hydroxyhexanoate Ethyl heptanoate Ethyl (Z)-hept-4-enoate Ethyl (E)-hept-4-enoate Ethyl octanoate Ethyl (Z)-oct-3-enoate Ethyl (E)-oct-3-enoate Ethyl (Z)-oct-4-enoate Ethyl (E)-oct-Cenoate Ethyl (Z)-octa-4,7-dienoate Ethyl decanoate Diethyl malonate Methyl salicylate Methyl 4-hydroxybenzoate Ethyl cinnamates, (Z) and ( E ) Ethyl a-acetylcinnamate Lactones of 4-Hydroxybutanoic acid 4-Hydroxy-2-methylbutanoicacid 4-Hydroxyhexanoic acid 4-Hydroxyheptanoic acid 4-Hydroxyoctanoic acid 5-Hydroxyoctanoic acid 4-Hydroxynonanoic acid 4-Hydroxydecanoic acid 4-Hydroxyundecanoic acid 4-Hydroxydodecanoic acid 2,6,6-Trimethyl-2-hydroxy cyclohexylidene acetic acid

PPm"

Yellow passion fruit, PPm" t

1.3 -

0.07 W

-

W W W

W

t

W

W

t -

W

t

t W

W W W

W

W

W t W

W

W W W

t

W

(continued)

275

PASSION FRUIT JUICES TABLE VlIl (continued) Purple passion fruit PPm" Terpenoid compounds Myrcene p-Ocimene, ( E ) and (Z) 1,4(8)-p-Menthadiene Limonene a-Pinene 3-Carene Citronellol Geraniol Linalool a-Terpineol Terpine-4-01 1.8-Cineole Citronellyl acetate (Z)-Linalool oxide (5-ring) (E)-Linalool oxide (5-ring) Other compounds Heptane Toluene 1-Isopropyl-3-methylbenzene 1 -Methyl-4-isopropenylbenzene Naphthalene I , I ,6-Trimethyl- I ,2-dihydronaphthalene I , I-Diethoxyethane I-Ethoxy- I-propoxyethane I , I-Diethoxypropane Phenol 2-Methylphenol 3-Methylphenol 4-Methylphenol 4-Ethylphenol 4-Allylphenol 2 ,4-Dimethylphenol 3.4.5-Trimethylphenol 2-Methoxyphenol 4-Allyl-2-methoxyphenol Furfural 5-Methylfurfural Furfuryl alcohol 2-Furancarboxylic acid Fury1 methyl ketone 5-Isopropenyl-2-methyl-2vinyloxolan

-

Yellow passion fruit,

PPm

PPm'

-

W

(conrinued)

276

D. J . CASIMIR ET AL TABLE VIll (corttirtued) Purple passion fruit PPm"

I-Oxa-2,6,10,IO-tetramethylspiro[4.5]dec-6-ene ( 2 isomers) 2.3-Dimethylmaleic anhydride (Z)-Rose oxide 2-Methylquinoxaline Ethyl 3-methylthiopropanoate 4-Methyl-5-vinylthiazole 3-Methylthiohexan- 1-01 2-Methyl-4-propyl- I ,3-oxathiane 2-Ethoxyethanol' Diethyleneglycol monoethylether' 1-(3,4,5-TrimethoxyphenyI) prop-2-ene'

PPmb

Yellow passion fruit PPm"

t

d W W W

W

d' d' W W

W

From Murray et a / . (1972. 1973). From Parliment (1972), recalculated. From Winter and Kloti (1972). recalculated. Dash means not reported; t means trace (
' ' '

volatile flavoring constituents of purple passion fruit juice obtained in frozen form from Kenya, but the complete results have not been published (see Naf et al., 1977; Winter et al., 1979a). Yellow passion fruit juices from the Ivory Coast, Guadeloupe, and Brazil were examined by Huet (1973). but only a few major volatile constituents were reported. Passion fruit juice presumably of Japanese origin examined by Kadota and Nakamura (1972) yielded 36 ppm of volatile flavoring constituents, of which about 30% was ethyl butanoate, 13% hexyl hexanoate and nonyl alcohol, and 10% butyl hexanoate and linalool. B.

UNUSUAL VOLATILE FLAVORS IN PASSION FRUIT

It is clear that the flavor of passion fruit juice, like most food flavors, is chemically complex, and that its unique character cannot be attributed to any single component. The volatile flavors present in highest concentrations are the

PASSION FRUIT JUICES

277

C-2 to C-8 esters of the C-2 to C-8 fatty acids that occur in many fruits. However, a number of volatile flavoring compounds have been found in passion fruit for the first time in any natural source, and several of these undoubtedly contribute to the unique flavor of this fruit (see Section IV,E). 1 . Esters Among the volatile flavoring constituents of purple passion fruit isolated by Murray et al. (1972) but not completely characterized were an ethyl octadienoate of major flavor significance (see Table XI), and a hexadienyl butanoate. Two esters with these structures have lately been identified by Winter et al. (1979b). Ethyl (Z)-octa-4,7-dienoate had an aroma with a fresh, juicy top note typical of pineapple and a threshold value of about 125 ppb. The aroma of the other ester, (Z)-hexa-3 S-dienyl butanoate, was faintly reminiscent of ripe pears with a heavy tropical fruit undertone, and its threshold value was about 75 ppb. 2.

Volatiles Probably Derived from Carotenoids

As Murray et al. ( 1972) and subsequent workers have pointed out, it is likely that the biosynthesis of a number of the volatile flavoring constituents of passion fruit is associated with the production or degradation of carotenoid pigments. Linalool, p-ionone, dihydro-&ionone, and the lactone of 2-hydroxy-2,6,6trimethylcyclohexylideneaceticacid (dihydroactinidiolide), an oxidation product of p-ionone, are present in both purple and yellow passion fruit; dihydro-ionone and 1,1,6-trimethyl-l,2-dihydronaphthalene (3,4-dehydroionene), a likely degradation product of p-ionone, have been found only in purple passion fruit (Table VIII). In addition to these known compounds, a number of new and interesting flavor volatiles, probably derived from carotenoids, have been found in passion fruit for the first time in a natural source; they are listed in Table IX. a . The Edulans. During the study of the volatile flavor compounds of purple passion fruit by Murray et al. (1972), four trace components were detected having molecular weight 192, identical mass spectra, and attractive aromas. These components were named the edulans, I, 11, 111 and IV, in order of their relative concentrations in fresh passion fruit juice (Whitfield et a l . , 1973). The edulans were isolated from fresh purple passion fruit juice both by vacuum distillation at low temperatures and by preparative gas chromatography of headspace vapors collected on a porous polymer resin. On evidence from these two methods the concentrations of edulans I and I1 in fresh juice were estimated to be 1 and 0.1 ppm, respectively (Whitfield and Stanley, 1977). It is likely, however, that these concentrations represent an equilibrium mixture of stereoisomers and may not be the relative concentrations in intact fruit.

D. J . CASIMIR ET AL

278

TABLE I X NEW IONONE-RELATED FLAVOR COMPOUNDS IN PASSION FRUIT”

Passion fruit

Compound

Formula No.

Edulan I Edulan I1 Edulan I11 Edulan IV Dihydroedulan I Dihydroedulan I1

“

Yellow (PPm)

I 0.1 t

t W

W

4.4a-Epoxy-4,4a-Dihydroedulan 3-Hydroxyedulan 6-(But-2’-enylidene)-I ,5,5-trimethylcyclohex- I-enes: (6E,2’E) isomer (62,2’E) isomer (62,2’Z) isomer (6E.2’Z) isomer I ,3,4,5,6,7-Hexahydro-a .7,7trimethyl- I -isobenzofuranylethanol I-( I ,3,4,5,6,7-Hexahydro-7.7dimethyl- 1-isobenzofuranyl)propan-2-one Megastigma-5,8-(E)-dien-4-one Megastigma-5.8-( Z)-dien-4-one

Purple (PPm)

d d

I I (Fig. 5) 12 (Fig. 5) 13 (Fig. 5 ) 14 (Fig. 5 ) 16 17 18 19

0.04

0.3 W

0.01

d d d d

For references, see Section IV,B,2. See footnote d in Table VlIl for definition of abbreviations.

Structural studies, based mainly on microspectrometry and microreaction gas chromatography because the compounds were available only in microgram quantities, and confirmed by synthesis, indicated that edulans I and I1 are the epimeric 2,5,5,8a-tetramethyl-3,5,6,8a-tetrahydro-2HI -benzopyrans (2) and (3)(Whitfield and Stanley, 1977). Edulans 111 (4) and IV (5) are the epimeric 2,5,5,8a-tetramethyl-3,5,8,8a-tetrahydro-2HI-benzopyrans (Sugowdz and Whitfield, 19741975). The edulans I and I1 have strongly floral, roselike aromas and make a specific contribution to the flavor of fresh passion fruit juice (see Section IV,E). Heat processing causes a lowering of the concentration of the edulans and a corresponding loss of flavor in processed passion fruit juice. Two further trace components of purple passion fruit juice having camphoraceous aromas and present in fresh juice at concentrations of 6 and 1 ppb are the dihydroedulans 1 (6) and I1 (7)-that is, the epimeric 2,5,5,8a-tetramethyl2,3,4,4a,5,6-hexahydro-8aH-l-benzopyrans (Prestwich et al., 1976). Then

279

PASSION FRUIT JUICES

Winter et al. (1 979a) identified two further edulan derivatives isolated from purple passion fruit but reported that they were practically odorless; these compounds were ( 2 R * , 4S*, 4aS*, 8aS*)-4,4a-epoxy-4,4a-dihydroedulan(8), and ( 2 R * , 3S*,8aS*)-3-hydroxyedulan (9). The edulan group of compounds may be related to carotenoid pigments via trimethylcyclohexanylbutanederivatives such as (10). h. The Megastigmatrienes. Another group of volatile flavors found in passion fruit are substituted cyclohexenes. It is unusual to find hydrocarbons playing

b-.. .

i

..

3

L

b.... ..

b.. .

G

7

.......

boH ...

8

9

10

15

280

D. J . CASIMIR ET AL.

an important flavor role. These substances, called megastigmatrienes, were first detected as trace components of the volatiles of purple passion fruit by Murray et al. (1972), then were subsequently isolated in microgram quantities by collection of headspace volatiles from fresh juice on a porous hydrophobic polymer followed by high-resolution preparative gas chromatography (Whitfield et d . , 1977; Whitfield and Sugowdz, 1979) (see Fig. 5). Structural studies, confirmed by synthesis, indicated that the four megastigmatrienes were the stereoisomers of 6-(but-2’-enylidene)- 1,5,5-trimethylcyclohex- 1-ene (11-14), present in fresh juice in concentrations of 0.04, 0.3, <0.01, and 0.01 ppm, respectively. A biogenetic pathway to the trienes may be postulated from p-ionone by way of the readily dehydrated p-ionol; however, p-ionol itself has not been found in either purple or yellow passion fruit. In dilute aqueous solution the (6E, 2’E) (11) and (62,2’E) (12) isomers had aromas variously described as rose-like or raspbemy-like, and odor thresholds of about 1 part in lo7. Both made significant contributions to the flavor of fresh passion fruit (see Section IV,E). The megastigmatrienes are very susceptible to acid-catalyzed oxidation, which converts them into “ionene”-1 , I ,6-trimethyl- 1,2,3,4-tetrahydronaphthalene

11

FIG. 5 . Portion of gas chromatogram of headspace vapors from purple passion fruit juice showing peaks for edulan I and the megastigmatrienes. Conditions: Stainless-steel column (I50m. 0.75-mm internal diameter) wall-coated with Silicone OV-101,isothermal 128”C, flow rate 3.5 mumin N,

PASSION FRUIT JUICES

28 1

(15). It is likely that this reaction occurs during pasteurization of passion fruit juice and accounts in part for the detrimental effects of pasteurization on flavor. The megastigmatrienes could not be detected in pasteurized juice, but 1,1,6trimethyl- 1,2,3,4-tetrahydronaphthalene (15) was present (Whitfield and Sugowdz, 1979). c . Other Ionone Derivatives. In purple passion fruit juice, Naf et al. (1977) found the first examples of a new class of naturally occurring ionone derivatives distinguished by an ether bridge from the C-5 methyl group to carbon C-7. The structures of these compounds, confirmed by synthesis, were (R*,R*)1,3,4,5,6,7-hexahydro- a,7,7- trimethyl- 1- isobenzofuranylethanol (16) and 1(1,3,4,5,6,7-hexahydro-7,7-dimethylI-isobenzofuranyl)propan-2-one(17). The ketone (17) was found also in yellow passion fruit. From the aroma compounds of yellow passion fruit, Demole et al. (1979) (18) isolated by preparative gas chromatography megastigma-5,8-(E)-dien-4-one and the corresponding (Z) isomer (19) in the proportion of about 3 : I , and confirmed their structure by synthesis. The (E) isomer was also prepared by acid-catalyzed dehydration and rearrangement reactions from the monoepoxides of ionol and damascol. This conversion provides support for a probable biogenesis of these flavor compounds from the oxidative degradation of carotenoid precursors. Megastigma- 5,8-(E)-dien-cone is described as having “a very impressive, fine flowery-fruity odor with a tobacco-like note” (Ohloff, 1978).

3 . Sulfur Compounds

The neutral fraction isolated by Winter and Kloti (1972) from yellow passion fruit contained two sulfur compounds, later identified by Winter et a / . (1976); neither had previously been found in a natural source. One compound described as having a green, fatty, and sulfury aroma typical of certain exotic fruits was 3methylthiohexan- 1-01 (20). A homolog, 3-methylthiopropanol, of this compound has been identified in tomatoes, cabernet wine, and soy sauce. The other comthe (Z) (21) and (E) (22) diapound was 2-methyl-4-propyl-l,3-oxathiane, stereoisomers of which were present in proportions of about 9 : l . Both isomers had strong fruity odors with a green and slightly burnt note that was more pronounced in the (Z) isomer. The authors point out that both of these sulfur compounds contain a six-carbon chain bearing an oxygen atom at position 1 and a sulfur atom at position 3, and that methionine and linoleic acid might be intermediates in their biogenesis. Sulfur compounds were also found by Murray et al. (1972) in purple passion fruit; they were not identified but made a significant contribution to flavor (see Table XI).

D.J . CASlMlR ET AL.

282

15

17

20

21

22

C . FLAVOR IMPACT VALUES

Identification of the organic volatiles in passion fruit is only the first stage in elucidating the chemistry of passion fruit flavor. The subsequent steps involve determining which volatiles contribute significantly to that flavor and the magnitudes of their contributions. From studies on passion fruit juice concentration already described (Section 111,C). Casimir and Whitfield (1978) had available a series of fractions that contained different levels of natural volatile flavoring constituents. These fractions were combined together in various ways, as indicated in Table X, to prepare six bases for tasting purposes. Since the bases were unsuitable for direct tasting, they were used to prepare passion fruit drinks containing 25% juice

PASSION FRUIT JUICES

283

TABLE X TASTE SCORES FOR DRINKS MADE FROM FRACTIONS OBTAINED DURING CONCENTRATION OF PASSION FRUIT JUICEn

Drink bases Single-strength juice Concentrate TWD Concentrate + TWD Concentrate + TWD Concentrate + IWD Concentrate

+

+ IWD + LND'

+ IWD

Taste scores" 100 100 65 56 29

0

From Casimir and Whitfield (1978). Adjusted mean scores: The higher the score, the more desirable is the passion fruit flavor; but the zero score for concentrate does not mean complete absence of passion fruit flavor. The evaporator discharged four fractions with the following yields by weight: 22. I % Concentrate (60.5" Brix) Distillate from tap water condenser (25"C)-TWD 64.6% Distillate from ice-water condenser ( 1°C)-IWD 10.0% Distillate from liquid nitrogen condenser ( - 196"C)-LND 3.3% The bases were prepared from the fractions in these proportions. I'

"

equivalent and adjusted to a sugar-to-acid ratio of 18 : 1. These passion fruit drinks were then evaluated for passion fruit flavor by 32 tasters, with the results shown in Table X . Headspace samples from the six passion fruit drinks were examined by gas chromatography to obtain profiles in which the peak heights indicated the relative concentrations of some 300 volatile flavoring constituents. Parallel sniffing tests indicated, however, that only 22 of the peaks appeared likely to contribute to passion fruit flavor. For each of these 22 compounds a linear regression line was then calculated relating the concentration (peak height) of that compound in each drink to the flavor score of that drink. Significant regressions were established for 15 compounds. The slope of the regression line for each compound was designated the flavor impact vulue of that compound; it represents the increment of flavor response per increment of concentration. Flavor impact values for the significant flavor compounds in passion fruit are shown in Table XI. The contribution of a compound to the total passion fruit profile depends on its concentration as well as on its flavor impact value. Table XI also shows the percentage contribution that each compound makes to the total flavor of passion fruit, calculated from its

284

D. J . CASIMIR ET AL. TABLE XI FLAVOR IMPACT VALUES OF PASSION FRUIT VOLATILES"

Compound

Flavor impact value

Concentration in juice" (PPm)

6-(But-2'-enylidene)-1,5,5trimethylcyclohex-I -enef (2)-Hex-3-enylbutanoate Hexyl butanoate Ethyl (Z)-oct-4-enoate" p-Ionone Edulan I' Ethyl (2)-octa-4.7-dienoate' Linalool Ethyl hexanoate Heptan-2-01 (Z)-Hex-3-enol S compounds (unidentified)" HexanoVnonan-2-one Rose oxide Methyl butanoate

79 41 6.8 62 410 23 239 30 I .3 I .7 26 76 I .8 45 0.7

1.1 0.8 4.1 0.4 0.05 0.8 0.06 0.5 7.6 5.3 0.3 0. I 4.0 0.2 8.3

"

"

Contribution to flavor profile (%)

30 II 9 8 7 6 5 5 3 3

3 3 3 2 2

From Casimir and Whitfield (1978). Calculated from relative peak areas. Not included in recombined essence. Previously incorrectly identified as the 3-enoate (cf. Table VIII).

flavor impact value together with its concentration in the juice. Several esters that are present in the juice at relatively high concentrations (Table VIII) had negligible flavor impact values. The validity of the concept of flavor impact values as applied to the investigation of passion fruit flavor was confirmed by Casimir and Whitfield (1978) when they prepared a nature-identical essence by recombining in weight proportions all the compounds in Table XI that were available as synthetic chemicals. The essence was used to make a passion fruit drink that was presented to taste panels for evaluation together with samples of orange juice and apricot nectar, under orange lights to mask differences in appearance. Three panels, including one panel of eleven flavor chemists, identified the drink as "passion fruit" with the same precision as their identifications of the other two juices. Thus, the selected combination of compounds had successfully reproduced a passion fruit flavor profile. Subsequently 6-(but- 2'-eny1idene)- 1,5,5- trimethylcyclohex- 1-ene and edulan I were synthesized, and when added to the essence they were judged to enhance the passion fruit character. Casimir and Whitfield (1978) also calculated the mass distributions of the

285

PASSION FRUIT JUICES

significant flavor volatiles in the concentrate and condensate fractions when passion fruit juice (13.5% solids) was concentrated to 60.5% solids in a centrifugal evaporator. The concentrate retained volatiles responsible for 75% of the total passion fruit profile, while 20,2, and 3% were removed by condensation at 25, 1, and - 196"C, respectively.

D. EFFECTS OF VARIETY It is generally agreed that the aroma and flavor of the purple passion fruit are more pleasing than those of the yellow passion fruit, and the difference is re2

Y

x

V T

V

P ONM

N

H G

K

J

G

A

E

A

FIG. 6. Gas chromatograms of headspace vapors from purple (upper) and yellow (lower) passion fruit juices (F. B. Whitfield and K . E. Murray, unpublished data). Conditions: Juice sample 25 gm, temperature 28°C. collection time 1 hour. Stainless-steel column ( I50 m, 0.75-mm internal diameter) wall-coated with Carbowax 20M, programmed 70°C for 32 minutes, 70" to 160°C at 0.75"C/min, and 160°C for 2 hours. Peaks (the name compounds are the major components in each peak): A, ethyl butanoate; B, butyl acetate; C, ethyl (E)-but-2-enoate; D, myrcene; E,heptan-2-one; F, limonene; G, ethyl hexanoate; H, (2)- and (E)-P-ocimene; I, 2-heptyl acetate; J, hexyl acetate; K. ethyl hex-3enoate; L, (Z)-hex-3-enyl acetate and for purple passion fruit heptan-2-01: M, 2-heptyl butanoate; N, hexyl butanoate and butyl hexanoate; 0, ethyl octanoate; P (Z)-hex-3-enyl butanoate; Q, ethyl (E)-oct-4-enoate; R, ethyl (Z)-oct-3-enoate; S, theaspirane; T, 2-heptyl hexanoate; U , edulan I; V, hexyl hexanoate; W, (62.2'E)-6-(but-2'-enylidene)-1,5,5-trimethylcyclohex-I-ene and octyl butanoate; X, (Z)-hex-3-enyl hexanoate; Y,octyl hexanoate and hexyl octanoate; Z, p-ionone. Scale: 8x-peaks to the left are magnified eight times. 32x-peaks to the left are magnified thirty-two times.

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flected in the composition of the volatile flavors of the two varieties, as shown in Fig. 6 and Table VIII. The purple passion fruit has higher concentrations than the yellow of the major esters-ethyl, butyl, and hexyl butanoates, and butyl and hexyl hexanoates-and of the terpene ketones p-ionone and dihydro-P-ionone. Significant flavor constituents of the purple passion fruit that are absent from the yellow variety are 2-heptyl acetate, butanoate, and hexanoate, the edulans, dihydroionone, I ,1,6- trimethyldihydronaphthalene, and the megastigrnatrienes (Murray and Whitfield, 1974-1975). There are also differences among the terpene hydrocarbons: the yellow variety has relatively high concentrations of ( E ) p-ocirnene, myrcene, limonene, and 1,4(8)-pmenthadiene, while only ( E ) - P ocimene is present in a significant concentration in the purple variety. As was mentioned in Section 1, the purple passion fruit is no longer grown commercially in Australia but has been replaced by four hybrids: Lacey, Purplegold, Selection 3- 1, and Selection 23-E. These hybrids have significant horticulTABLE XI1 MAJOR FLAVOR COMPOUNDS IN THE JUICES OF PARENT AND HYBRID PASSION FRUIT"

Relative concentrations" (pg/lO gm juice)

Compound p-lonone Ethyl (Z)-octa-4.7-dienoate 6-(But-2'-enylidene)-1.5.5trimethylcyclohex- I -ene Ethyl (Z)-oct-4-enoatef' Rose oxide (Z)-Hex-3-enyl butanoate Linalool (Z)-Hex-3-enol Edulan I Hexyl butanoate Hexanol Hept a n - 2- oI Ethyl hexanoate Methyl butanoate Total flavor profile"

Flavor impact value" 410 239 79 62 45 41 30 26 23 6.8 1.8 1.7

Purple

Yellow

Selection 3-1

Selection 23-E

0.7 0.3 0.2

0.02 0.04 0

0. I 0.04 0.07

0. I 0.08

0.08 0.02 0.07 0.9 0.4 0 0.6 3.8 0. I > 10 0.7 90

0.2 0.02 1.8 0.6 1.4 0.3 6.5 4.8 2.9 > 10 0.6 280

0.5 0.06 110

0.9 0.6 4.5

> 10 0.4 0.9

1.3

> 10

0.7

0.5 I060

0.5

0.1 0.07 5.3 1 .o

0.7 0.6 >I0 2.1 2.6 110 1.3 610

Lacey

Purplegold

0.2 0. I 0.07

0.3 0. I 0.03

0. I 0.01

0.08 0.04 0.7 0.5 0.8 0.2 2.3 3.0 I. I > 10 2.3 260

0.5 0.3 0.6 0.3 2.3 2.4 0.7

> 10 2.8 210

Adapted from Whitfield ef a / . ( 1980). See Table XI. ' Relative concentrations, since they are estimated by headspace sampling and are about half the concentrations determined by extraction as in Table VIII. Previously incorrectly identified as the 3-enoate. 2; (Flavor impact values X concentrations). "

'' (I

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tural advantages over the purple parent, but processors complain that they are less satisfactory for processing because they are deficient in characteristic flavor. The volatile flavoring constituents of the four hybrids were compared with those of the purple and yellow passion fruits by Whitfield et a / . (1980). Table XI1 shows the concentrations of the 14 most important constituents, on the basis of flavor impact value, and the calculated values of the total flavor profile for each variety and hybrid. Almost all of the flavor volatiles occur in greatest concentration in the purple passion fruit, and its total flavor profile is the highest. The Selection 23-E comes closest to the purple passion fruit in the composition of its flavor volatiles. By close cooperation between horticulturists and flavor chemists, it should be possible to breed hybrids that combine virus resistance with flavor profiles still closer to that of the purple passion fruit. E. EFFECTS OF MATURITY The information reviewed so far on the volatile flavoring constituents of passion fruit may reasonably be regarded as refemng to fruit harvested at maturity, and the commercial juice samples examined most probably came from fallen fruit collected from the ground. A specific study of the effects of maturity on the composition of passion fruit volatiles was undertaken by Casimir et al. ( 1 9771978). Purple passion fruit were harvested at several stages of maturity as determined by the force required to remove the fruit from the vine. Volatile constituents from the juice of individual fruits were then collected in traps packed with Chromosorb 105 and examined by gas chromatography and mass spectrometry. In immature green fruit the volatile constituents included monoterpene hydrocarbons, aliphatic and terpenoid alcohols, and carbonyl compounds, and the major components in decreasing order of concentration were linalool, hexanal, 2-methylbut-3-en-2-01, and a-terpineol. With advancing maturity of the fruit and development of the purple color in the skin, these four components decreased in concentration, and aliphatic esters and carotenoid-related compounds became the major contituents. The compounds of greatest flavor significance reached maximum concentrations in fruit just fallen from the vine. This observation suggests that the widespread practice of harvesting passion fruit after it falls from the vine is likely to yield fruit of best flavor, provided gathering from the ground is not delayed. Conversely, mechanical harvesting of passion fruit from the vine before the abscission layer becomes sufficiently fragile is likely to yield fruit in which flavor is not fully developed. F. EFFECTS OF PROCESSING As was noted earlier, the flavor of passion fruit is extremely sensitive to change as a result of heat processing. This is well illustrated in Fig. 7, which

288

D. J . CASIMIR ET AL. P

K

D

FRESH PASSION FRUIT JUICE

E

B

PASTEURIZED PASSION FRUIT JUICE

FIG. 7. Gas chromatograms of headspace vapors from fresh (upper) and pasteurized (lower) purple passion fruit juice (F. B. Whitfield and D. J. Casimir, unpublished data). Conditions: as for Fig. 6 . Peaks (the named compounds are the major components in each peak): A, pentan-2-one; B, ethyl butanoate; C, butyl acetate; D, ethyl hexanoate; E, (Z)- and (E)-P-ocimene; F, 2-heptyl acetate; G, hexyl acetate; H. (Z)-hex-3-enyl acetate; I , hexanol; J , 2-heptyl butanoate; K , hexyl butanoate and butyl hexanoate; L, (Z)-hex-3-enyl butanoate; M, ethyl ( Z ) - and (E)-oct-Cenoate; N , 2-heptyl hexanoate; 0, edulan I; P, hexyl hexanoate; Q, (6Z.2’,5)-6-(but-2’-enylidene)-I .5,5-trimethylcyclohex-I-ene and octyl butanoate; R, (Z)-hex-knyl hexanoate.

shows gas chromatograms of headspace vapors from freshly expressed juice from purple passion fruit and from the same juice after heat treatment by spin pasteurization (see Section II,D) for 30 sec at 95°C. In addition to a general lowering of the concentrations of volatiles as indicated by peak heights, the heat treatment has caused a number of specific changes-for example, hydrolysis of esters and acid-catalyzed oxidation of the megastigmatrienes.

V.

NEEDS AND APPLICATIONS FOR RESEARCH AND DEVELOPMENT

Passion fruit recommends itself as a cash crop for developing countries in tropical and subtropical zones. The vines crop quickly, in about a year from planting; the pulp may be extracted by simple mechanical or hand procedures; and then it may be preserved by freezing or by the use of chemical preservatives

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with the minimum of equipment. A comprehensive account of the setting up of a passion fruit industry is given by Whittaker (1972, 1973), and Hubbard (1973) has described his experiences in establishing such an industry on Niue Island to prepare frozen passion fruit pulp for export. On the other hand, surveys by the Tropical Products Institute (Mott, 1969) did not point to an optimistic future for passion fruit products in world trade, largely because of failure to develop markets in America and Europe. It is likely, however, that lack of interest among consumers in those countries, in contrast to Australia for instance, is due in large measure to the fact that they have never been presented with passion fruit products of high quality-that is, with products that have retained the uniquely pleasing flavor of the fresh fruit. In particular, there is a need for high-quality passion fruit concentrates for international trading, and it is now possible to prepare such concentrates by centrifugal evaporation with recovery and restoration of volatile flavoring constituents. A plea for greater interest in Passiflora among plant breeders was made by Martin and Nakasone (1970). They advocated the establishment of germ plasma collections so that the wealth of native species material that is available might be evaluated for horticultural characters and fruit quality. The literature of food science now contains much accumulated information about the flavor of foods, in the form of long lists of volatile flavoring constituents. Few attempts, however, have been made to determine which compounds contribute significantly to the total flavor profile, and the quantitative contributions that these compounds make. The studies by Casimir and Whitfield (1978) on passion fruit flavor leading to the concept of flavor impact values provide a model investigation that might well be extended to other foods. The steps in such an investigation are these: ( 1 ) Identification of volatile flavoring compounds in a food.

(2) Selection of potentially significant compounds by sniffing. (3) Estimation of the concentrations of these compounds in the food. (4) Assessment of the contribution to flavor of the selected individual compounds by tasting systems containing different concentrations and combinations of the compounds within the appropriate flavor regime. Passion fruit juice and concentrate provided Casimir and Whitfield (1978) with a relatively simple and convenient flavor system to be expressed in quantitative terms. Because the flavor impact value of a compound is determined in the presence of other flavor volatiles, it does take account of synergistic and antagonistic effects that are ignored in assessments of flavor significance that are based on odor thresholds. It is likely, therefore, that application of the flavor impact value concept would have been profitable in such studies of fruit flavors

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as those of Guadagni et ul. (1966) on apples, Pyysalo et al. (1977) on arctic berries, Parliment and Scarpellino (1977) on blueberries, and Ahmed et (11. (1978a,b) on orange juice, and also in off-flavor studies such as that of Jeon pt ul. ( 1978) on ultra-high-temperature-processed milk. Flavor impact values may also be useful for establishing relationships between psychophysical response and the chemical structure of flavor compounds. The great interest and novelty of the volatile flavors that have been discovered in passion fruit prompt some observations on the philosophy and motivation of flavor studies by modem chemical techniques. On the one hand, by extending enormously the lists of flavor compounds found naturally, these studies have broadened the palette of the flavorist who seeks to imitate natural flavors (Hassey, 1974). So the imitation product may reproduce more closely the natural product, and the synthetic ingredients acquire respectability by being “nature-identical. Because of such considerations, Australian workers on passion fruit flavors encountered unfavorable reactions from passion fruit growers. On the other hand, modem flavor studies have provided, in a way not possible before, the chemical knowledge necessary to explain flavor changes in foods during processing and storage, and have revealed opportunities to improve the retention and minimise the deterioration of the natural flavors in foods.

”

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Ragab, M. H . H. 1971. Studies on technical problems in the processing of local food products. 111. Control of sedimentation and browning of passion fruit squash during storage. Food Techno/. Res. Dev. Cent. M a l u v s . . Pub/. No. 43, pp. 1-30; F o o d S c i . Techno/.Ahstr. 8 , 1430 (1976). Ruberte-Torres, R., and Martin, F. W. 1974. First generation hybrids of edible passion fruit species. Euphyticu 23(1), 6 1-70: Food Sci. Technol. Ahstr. 6, 53684 (1974). Salomon, E. A. G., de Martin, Z. J . , Kato, K., da Silva, S. D.. Mori, E. E. M., and Bleinroth, E. W. 1977a. Blended tropical fruit nectars. Bol. Inst. Tecnol. Alirnen. (C‘urnpinus, Braz.j No. 50, 103-121; F o o d S c i . Techno/. Abstr. 10, 3H374 (1978). Salornon, E. A. G.. Kato, K.. de Martin, Z. J., da Silva, S. D., and Mori, E. E. M. 1977b. Blending of papaydpassion fruit nectar. Bol. Inst. Tecnol. Aliment. (Cumpinas, Bra:.) No. 51, 165-179; Food Sci. Technol. Ahstr. 10, 3H373 (1978). Seelkopf, C., and Febres, Y. M. 1966. Maracuja, a new raw material for the fruit juice industry. 2. Lebensm.-Unters. -Forsch. 131, 281-284. Seelkopf, C., Gonsalez, D., and Thomson. H. 1962. Investigations on fruits of some South American passion flowers. F r u c h t s i ~ ~ - I n d . 7 ( 293) , 107. Seidemann, J. 1963. Microscopic examination of fruit stomates. Fruchtsaf-Ind. 8(2). 97-99. Singh, H. P., Ganapathy, K. M., and Bhat, D. N . V. 1978. Fixation of optimum maturity standard for harvest of passion fruit. Indian J . Hortic. 35(4), 314-320; F o o d S c i . Technol. Abstr. 12, 23 181 (1980). Slaytor, M., and McFarlane, I. 3. 1968. The biosynthesis and metabolism of harman in Puss(/loru edulis. I. The biosynthesis of harrnan. Phyrochemistry 7(4), 605-61 I . Straws. D. 1974a. Passion fruir-an exotic fruit. Fluess. Ohst. 41(1), 15-16; Food Sci. Techno/. Abstr. 6, lOH1685 (1974). Straws, D. 1974b. Microscopy of foreign fruit. VII. Passion fruit. Dtsch. Lehensm.-Rundisch. 70(4), 1 4 4 1 4 6 Food Sci. Techno/. Ahstr. 6 , 9J1242 (1974). Sugowdz, G., and Whitfield. F. W. 1974-1975. Synthesis of the edulans. Div. F o o d R e s . Rep. Res. (AUSI. C.S.I.R.O.)1974-75, 35. Swamy, V., Gowda, A. R., and Vijayamma, R. 1977. Utilization of unconventional fruits for the preparation of ready-to-drink beverages. Indian Food Pucker 31(3), 38-52. UNlDO (United Nations1 Industrial Development Organization). 1969. “The Use of Centri-therm, Expanding-flow, and Forced-circulation Plate Evaporators in the Food and Biochemical Industries,” Food Ind. Stud. N o . I . United Nations, New York. Vitali. A. A,, Roig, S. M., and Rao, M. A. 1974. Viscosity behaviour of concentrated passion fruit juice. Confructu 19(5), 201-206. Wang. 3. K., and Ross, E. 1965. Spin processing for tropical fruit juices. Agric. Eng. 46(3). 154- 156. Whitfield, F. B., and Stanley, G . 1977. The structure and stereochemistry of edulan I and II and the stereochemistry of the ~.S,S,8a-tetramethyl-3,4,4a.5,6.7,8,8a-octahydro-2~-I-benzopyrans. Aust. J . Chem. 30, 1073-1091. Whitfield, F. B., and Sugowdz, G. 1979. The 6-(but-2’-enylidene)-I,5,5,-trimethylcyclohex-I-enes: Important volatile constituents of the juice of the purple passionfruit. Aust. J . Chem. 32, 891-903. Whitfield, F. B.. Sugowdz, G . , and Casimir, D. J . 1977. (6E.2I.E) and (6Z,2’E)-6-(but-2’enylidene)-l,5.5-trimethyl-cyclohex-I-ene: Important volatile constituents of the juice of the purple passion fruit (Pussifloru edulis Sims). Chem. I n d . (London) No. 12, pp, 502-503. Whitfield, F. B., Last, J. H . . and Bannister, P. A. 1980. Volatile flavor components of hybrid passionfruit cultivars. IN. Cungr. Essent. Oils, Xth, lY80, in press. Whitfield, F. B., Stanley, G . , and Murray, K . E. 1973. Concerning the structures ofedulan 1 and II. Tetrahedron Lett. No. 2. pp. 95-98.

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Whittaker, D. E. 1972. Passion fruit: Agronomy, processing and marketing. Trop. Sci. 14(1), 59-77. Whittaker, D. E. 1973. Passion fruit: Agronomy, processing and marketing. Indiun Food Packer 27(4), 18-32. Winter, M . , and Kloti, R. 1972. The aroma of the yellow passion fruit. Helv. Chim.Actu 55(6), 1916- 1921. Winter, M., Furrer, A,, Willhalm, B., and Thommen, W. 1976. Identification and synthesis of two new organic sulfur compounds from the yellow passion fruit (Pussifloru edulis F. fluvicarpu). H e l p . Chim. Acru 59(5), 1613-1620. Winter, M . , Schulte-Eke, K . H . , Velluz. A.. Limacher, J . , Pickenhagen, W., and Ohloff, G. 1979a. Aroma constituents of the purple passion fruit. Two new edulan derivatives. Helw. Chim.Acfu 62(1). 131-134. Winter, M., Naf, F., Furrer. A . , Pickenhagen, W., Giersch, W., Meister, A , , Willhalm. B., Thommen, W., and Ohloff, G . 1979b. Ethyl (Z)-4,7-octadienoate and (Z)-3,5-hexadienyl butyrate, two new aroma components of the purple passion fruit. Helv. Chim. Arm 62(1), 135-139. Wucherpfennig, K. 1066. Occurrence of an alkaloid in a tropical fruit. Ber. In!. Fruchrsuft-Utlion, Wiss.-Tech.Komm. 7, 117-124.

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ADVANCES I N FOOD RESEARCH, VOL.

27

PHASE TRANSITIONS OF WATER IN SOME PRODUCTS OF PLANT ORIGIN AT LOW AND SUPERLOW TEMPERATURES YANCOGUEGOV Cunning Research Institute, Plovdiv. Bulgaria

I. Introduction . . . . .. . . . . . , . . . . . . . . . .

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

11. Water-The Basic Component of Plant .......................... A. The Structure and Characteristics of Water . . . . . . . . . . . . . . . . . . . . . . . .

111.

IV.

V.

VI.

B. The Structure and Characteristics of Ice ................... C. The Phase Diagram and Some Physical Characteristics of Water and Ice D. Moisture Distribution in Some Products, and Forms of Binding Initial Crystallization of Water in Fruits and Vegetables . . . . . . . . . . . . . . . . . A. Supercooling as a Phenomenon Preced B. Nucleation and Growth of Ice Crystals .......... C. The Cryoscopic Temperature of Fruits Phase Transitions at Low Temperatures (to A. Phase Transitions during Freezing and Thawing of Model Systems . . . . B. Phase Transitions during Freezing and Rewarming of Foods of Plant Origin Phase Transitions at Superlow Temperatures (-70" to -196°C) . . . A . SoIidification into a Glassy State (Vitrification) . . . . . . . . . . . . . . . . . . . . B. Recrystallization, Polymorphous Transformations, and Vitreous Melting ....................... Conclusion. . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

297 299 299 300 302 303 307 307 308 310 315 315 326 344 344 348 349 352

INTRODUCTtON

In recent years interest in the physical problems of the phase transitions of water in foods has increased, mainly in connection with the optimization of freeze-drying technological parameters. This interest has been stimulated also by the development of numerous other methods in refrigeration technology-for example, storage at temperatures near the cryoscopic temperature, cryoconcentration, superlow freezing, freezing of liquid and semiliquid food products, and frozen storage at low temperatures (-25" to -40°C).The classical pictures of the phase transitions have undergone numerous changes, and entirely new techniques are being utilized, thus allowing extensive research on their mechanisms. 297 Copyright @ 1981 by Acnckrnic Ress. Inc. All rights of reproduction in any form reserved. ISBN 0-12-016427-2

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Freezing is the most important phase transition of water in foods during refrigeration processing. The basic principle in modem refrigeration technology is that the cooled product should be stored at the lowest possible temperature that does not allow any ice formation. The development of systems for precise temperature control and maintenance in cold chambers permits a close approach to these border temperatures whenever the physiology of the product of plant origin makes this possible. Thus, it has become necessary to have reliable information about cryoscopic temperatures for a wider range of fruits and vegetables and to elucidate the factors influencing their temperatures. During the freezing of foods the proportion of ice increases as the temperature decreases, and at a certain temperature level a complete crystallization of the so-called “free” water takes place. At a lower temperature of the product the danger of further changes caused by the hypertonic saline solution found in the vegetative cell disappears. Consequently, it is of the utmost importance to determine this temperature, since it represents the upper limit of the temperature field in which the product possesses thermodynamic, and to a large extent chemical, stability. In order to be subjected to freeze-drying, the food product should be in a completely frozen state. This is especially important for fruit juices and concentrates whose structure during sublimation is more easily subjected to destruction in the presence of liquid water fractions. It should be pointed out that fruits, vegetables, and various other products of plant origin represent some of the main raw materials that can be freeze-dried. To determine the freeze-drying parameters of these products is one of the main problems in the field of food technology. The optimization of the freeze-drying parameters through investigation of the phase transitions, first accomplished by L. Rey, is at present the most rational approach for both biological and food products. The importance of this optimization can be illustrated by the fact that a decrease in the sublimation temperature by only 10°C causes a 50% increase in the energy cost (Lorentzen, 1974). All these considerations have motivated the present survey, whose aim is to summarize the scientific achievements in the investigations of the phase transitions of water in model systems, biological media, and food products at negative temperatures. The problem of the phase transitions is treated from the point of view of food technology, with special emphasis on the freeze-drying of food products of plant origin and the refrigerated storage of fruits and vegetables. The terminology adopted is as follows:

I . Temperature scale, according to Lozina-Lozinski (1972), Fikiin (1973), Rey (1959b), and Smith (1954, 1962): Normal temperature-from 20” to 0°C Low temperature-from 0” to -70°C Superlow (very low) temperature-from -70” to -273°C

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2. Velocity scale for freezing, according to Rey (1959b) and Smith (1954, 1962): Very slow-under O.OI"C/sec Slow-from 0.01 to 0.6"C/sec Fast-from 0.6 to 5O"C/sec Superfast-above 5O"C/sec

II. WATER-THE

BASIC COMPONENT OF PLANT TISSUE

Water represents 60 to 95% of the fruit and vegetable mass. It is one of the most important components of plant tissue, being at the same time both medium and participant in the biochemical process. Every reaction that causes changes in the normal relationships between water and the other components of the food product will change the typical characteristics of the product. Freezing, especially, has such an effect. Almost all frozen products contain unfrozen water. Even though the quantity is small, it greatly influences the stability of the product. Modem conceptions of the structure of water, of its solutions, and of its relationships with biological substances often provide a useful source of reference and become the point of departure in the interpretation of numerous phenomena.

A.

THE STRUCTURE AND CHARACTERISTICS OF WATER

In order to elucidate the characteristics of water and ice, we should have certain information about the water molecule. The structure of the water molecule has been investigated and discussed by a number of authors: Barnes ( I 929), Eisenberg and Kauzmann (1975), Fennema and Powrie (1964), Ginsburg (1969), Lerici and Pallotta (19721, Nemethy (1968), Popovski (1975), Quervain (1974), and Simatos et al. (1975a). The existing electrostatic and quantomechanical models assume the water molecule to be asymmetric (dipolic), with the oxygen atom covalently connected to two hydrogen atoms set at a distance of 0.096 nm and forming an angle of 104.5" (Eisenberg and Kauzmann, 1975; Fennema and Powrie, 1964; Lerici and Pallotta, 1972; Pryde and Jones, 1952). The covalent bonds of the oxygen to the hydrogen and its dipolic structure enable the water molecule to form intermolecular hydrogen bonds; this is the ability of the oxygen atoms to receive electrons from the neighboring hydrogen atoms. The geometric structure of the water molecule and its ability to form hydrogen bonds are the preconditions for the formation of aggregates of water molecules. The extraordinary power of attraction between the water molecules accounts

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for the unusual properties of water in comparision with substances that are very close to water in .molecular weight and electronic configuration-CH,, NH,, HF, PH:,, H,S, and HCI. Thus, water possesses extremely high boiling and melting points, specific heat capacity, latent melting and evaporation heat, surface tension and dielectric constant (Fennema and Powrie, 1964; Lerici and Pallotta, 1972). Although at present there is complete agreement about the physical properties and the structure of the water molecule, there is a difference of opinion on the structure of water (that is, the relative location and movement of its molecules). Today we accept in general the tetrahedron model proposed by Bernal and Fowler (according to Ginsburg, 1969). It is based on the potential possibility of every water molecule to be connected with four other water molecules, thanks to its four hydrogen bonds, situated tetrahedrally. The so-called mixed models or models of permanently changing hydrogen bonds have been worked out on this basis (Eisenberg and Kauzmann, 1975; Lerici and Pallotta, 1972; Nemethy, 1968). These authors consider water to consist either of constantly forming and decomposing molecular aggregates (Eisenberg and Powrie, 1975) or of two modifications with different structural and energetic parameters (Popovski, 1975). In one of the models, accepted as a perfect example of this type-that of Samoylov-water is assumed to consist of incessantly changing icelike structures or skeletons, slightly protected by monomer molecules (Popovski, 1975). Soluble substances cause great changes in the structure of water. In aqueous solutions the ions of most electolytes are to a large extent hydrated. The behavior of these hydrates at low temperatures has not been sufficiently investigated (Luyet, 1957). Water molecules do not show any tendency toward interaction with nonpolar molecules-for example, those of carbohydrates. In similar solutions they are kept together by the hydrogen bonds “water-water,’’ thus forming icelike structures. B. THE STRUCTURE AND CHARACTERISTICS OF ICE At present no unanimously accepted theory is available on the geometric and dynamic structure of the crystal lattice of ice. However, the basic structural characteristics of ordinary hexagonal ice (ice I) have been investigated and established (Eisenberg and Kauzmann, 1975; Lebedev and Perel’man, 1973; Lerici and Pallotta, 1972; Nemethy, 1968; Popovski, 1975; Quervain, 1974; Rey, 1959a). It is well known that every oxygen atom in the ice structure is situated in the center of a tetrahedron formed by four oxygen atoms with 0.276 nm between them (0.3 1 nm in the case of water). When such tetrahedrons combine, a hexagonal structure is often formed (Fennema and Powrie, 1964; Lerici and Pallotta,

PHASE TRANSITIONS OF WATER

30 1

1972; Simatos et ul., 1975a). This molecular arrangement leads to the formation of the so-called open crystal lattice, with considerable intramolecular space. Besides the ordinary hexagonal ice (ice I) formed under normal conditions (temperature and pressure), there are eight additional modifications or polymorphous forms, designated by Roman numerals I1 to VIII. They are formed and remain stable only at high pressure (above 100 MPa)-that is, under conditions impossible or hardly probable in food products. Nevertheless, Rey (1959b) does not exclude the possibility that certain ice forms, resistant to high pressure, develop in the tissue during very rapid cooling. It was established that at atmospheric pressure the so-called “cubic ice,” or ice C, as well as “vitreous” ice is formed at temperatures below -70°C (Eisenberg and Kauzmann, 1975; Lebedev and Perel’man, 1973; Lerici and Pallotta, 1972; Nemethy, 1968; Quervain, 1974; Simatos et ul., 1975a; Warren, 1937). Vitreous ice is formed when water vapor condenses on surfaces with a temperature below - 100°C. It almost certainly represents a vitreous form of water, but in fact nothing is known about its structure. During heating the vitreous ice irreversibly changes into ice I “C”. The transition is accompanied by the release of heat-from 870 to 1260 J/mol (Eisenberg and Kauzmann, 1975; Lebedev and Perel ’man 1973; Luyet, 1959; Simatos et al., I975a). Vitreous ice can be formed also by superfast freezing of a thin water film situated in a capillary structure (Luyet, 1959). A vitreous film is considered to be very difficult to isolate from water, and a combination of crystals and glass is more likely to be obtained. The presence of crystals along with vitreous water renders the water extremely unstable, and its crystallization always takes place when the temperature is increased above a certain point. Cubic ice can be obtained by heating vitreous ice, or by vacuum condensation of water vapors (according to some authors) on surfaces with temperatures between - 140” and - 120”C, or by heating ice modifications I1 to VIII. Under further heating cubic ice turns irreversibly into ice I with a slight entropy change (Eisenberg and Kauzmann, 1975; Lebedev and Perel’man, 1973; Nemethy, 1968; Luyet, 1959). It is impossible to obtain cubic ice by cooling ordinary ice (Nemethy, 1968). No definite temperature has been established for the change of vitreous ice into ice I “C”. Several investigators have observed this transition at temperatures of about - 16O”C, others in the range - 139”to - 129”C, still others below - 12O”C, and some even at temperatures above - 120°C. The transition of ice I “C” can be observed also in the wide temperature range-from - 130” to -70°C (Eisenberg and Kauzmann, 1975). According to Luyet (1959), vitreous ice can change into a liquid at a high heating velocity (vitreous melting). The temperature gradient is several thousand degrees per second, which means that the product must be very small in size but with a very large specific surface.

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During slow and fast cooling of water, hexagonal ice (ice I) is always obtained. Lowering the temperature to -196°C does not change its structure (Barnes, 1929; Rey, 1959b). This has been firmly established through x-ray defraction and through calorimetric measurements [the specific heat capacity in the range 0" to -196°C changes almost linearly from 8.4 to 37.4 J/(mol.K)] (Eisenberg and Kauzmann, 1975). C. THE PHASE DIAGRAM AND SOME PHYSICAL CHARACTERISTICS OF WATER AND ICE A phase can be defined as a homogeneous and physically differing part of a system separated from the other parts of the system by fixed borders (Fennema and Powrie, 1964). The conditions for equilibrium between the solid, liquid, and gaseous states of water, depending on the temperature and pressure, can be represented by the phase diagram. The phase diagram is the key point in analyzing the freezing, freeze-drying, cryocrystallization, and vacuum cooling processes. It has been described from this point of view by many authors: Eisenberg and Kauzmann (1975), Fennema and Powrie (1964), Ginsburg (1969), Lebedev and Perel'man (1973), Popovski (1975), and Owades and Dono (1969). Here we shall consider only those aspects having a direct relationship to the problems being discussed. A state of equilibrium of ice, liquid water, and water vapor exists only at the triple point with a pressure of 61 1 Pa and a temperature of 0.0098"C. Below this triple point the ice is transformed directly into a gaseous state (it sublimes). The vapor pressure above the supercooled water is higher than that above the ice at the same temperature. Consequently, the supercooled water is thermodynamically unstable and can easily change into ice (Eisenberg and Kauzmann, 1975). Of the thermophysical parameters discussed, only the specific heat capacity, the heat conductivity, and the density bear a direct relationship to this work. The specific heat capacity of water hardly changes in the range 0" to 100°C; it is about 4.19 kJ/(kg.K). When water freezes, the specific heat capacity decreases to about one-half-2.08 kJ/(kg. K). With a further decrease in temperature, the specific heat capacity of the ice decreases strictly linearly, reaching 0.42 kJ/(kg.K) at -250°C (Fennema and Powrie, 1964). The heat conductivity coefficient, A , of ice is nearly four times as high as that of water: 2.2 versus 0.6 W/m-kJ. When the temperature is decreased from 0" to - 160"C, the heat conductivity of ice increases almost linearly (Fennema and Powrie, 1964). When the temperature is decreased below O"C, the ice density increases exponentially in the range 0" to -3O"C, and at lower temperatures this increase approaches the linear one (Fennema and Powrie, 1964).

PHASE TRANSITIONS OF WATER

D.

303

MOISTURE DISTRIBUTION IN SOME PRODUCTS, AND FORMS OF BINDING

The presence of large quantities of moisture in fruits and vegetables, the characteristics of its distribution, and its bonds with the other chemical components are especially important in refrigeration technology. Generally speaking, plant tissues can be described as polydisperse systems, water being a disperse medium, and the disperse phase including a large number of organic and inorganic substances, forming real (sugars, salts, acids) and colloidal (proteins, pectin) solutions, as well as emulsions (fats, waxes, etc.). Thermodynamically stable molecular and ionic solutions of organic and inorganic substances predominate, some with a substantial degree of dissociation (Ginsburg, 1969; Kuprianoff, 1964; Popovski, 1975; Tchijov, 1971). The widely adopted classification of plant tissues as colloidal-capillary porous bodies, offered by Luikov (according to Ginsburg, 1969; Lebedev and Perel’man, 1973), is not well grounded, although it is acceptable from the point of view of investigating the heat- and mass-exchange processes in them. Voskresenski (1963), for example, assumes that Luikov’s classification excludes meat, fish, and vegetables. The general regularity of the distribution and the state of water in various materials including foods has been discussed by numerous authors: Dumanski (1949, 1960), Fennema and Powrie (1964), Gaurovits (1965), Ginsburg (1969, 1973), Golovkin et ul. (1955), Kazanski (1960), King (1971), Kuprianoff (1964), Lebedev and Perel’man (1973), Lerici and Pallotta (1972), Rey (1960b), Riutov (1976), Sedikh and Sedikh (1967), Simatos et al. (1975a), Tchijov (1971), Volarovitch et ul. (1972), and Voskresenski (1963). It has been established that water is bound in different ways to the dispersion phase and that the patterns of this binding play an important role during freezing and freeze-drying. The investigation of the pattern of moisture binding inside foods can start from either of two points-binding with the hydrophilic particle, or binding with the hydrophilic material (as a whole). The first approach, adopted by Dumanski (1949, 1960) and by Kroit, Bul, and Deriagin (according to Lebedev and Perel’man, 1973; Voskresenski, 1963) recognizes the presence of three water layers of different depths around the surface of the particles-namely, adsorbed (dense), diffused (border), and free layers. The adsorption water is firmly bound to the molecular salt particles of the border surface or to certain radicals of the zones of high-molecular-weight compounds (proteins, cellulose, starch, pectin, etc .). This water possesses considerably modified properties in comparison with ordinary water-different structure and orientation of the molecules, lower vapor pressure, weaker mobility and dissolving ability, higher density, and lower freezing temperature (Dumanski, 1949, 1960; Fennema and Powrie, 1964; Golovkin

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et u / . , 1955; Lerici and Pallotta, 1972; Popovski, 1967; Simatos et al., 1975a). Adsorption water is equivalent to the concept of bound water. The diffusion water is very slightly bound to the particles, or, more precisely, to the adsorption layer, and hardly differs from the free layer. It is significantly thicker than the adsorption layer-in starch, according to Dumanski (1949), 80 nm versus 0.3 nm. There is no difference between free and ordinary water. Some authors-for example, Deriagin (according to Voskresenski, 1963)recognize the existence of a distinct border between diffusion and free water. Dumanski’s view is similar; he does not deny the existence of this border but assumes the diffusion layer to be nonhomogeneous, with the water characteristics in it gradually approaching those of the free water, while the adsorption layer is moving away. The second approach, adopted by Rebinder and Lipatov (according to Volarovitsch et ul., 1972) and later by Luikov and Griaznow (1956), Luyet (1957), Kazanski (1960), Plank (1960), Rey (1960b), Samoylov and Sedikh (according to Sedikh and Sedikh, 1967; Sedikh and Ichmokhametova, 1970) and King (1975), treats the bonds between the water and the dispersed material thermodynamically as a whole, by the amount of the free energy of moisture binding-that is, by the energy necessary for the isothermal separation of a unit mass of moisture from the moist material. This approach recognizes four forms of binding:

a . Chemically bound water, with the highest binding energy-84 to 420 kJ/mol (Volarovitsch et a / . , 1972). This is the water bound by means of ion bonds as a result of chemical reactions. It participates in the solid matter composition of the product and can be released only during the thermal destruction of the material (Ginsburg, 1969; Popovski, 1967, 1975; Tchijov, 1971). b. Adsorprionally hound wuter. This water is held in the force field of the internal and external surfaces of the colloidal particles by the force of their charge (Gaurovits, 1965). The first sorbed moisture layer, one molecule deep, is called the monomolecular layer, or moisture of the monomolecular adsorption. This layer creates its own force field and sorbs a second molecular layer, which in turn attracts a third layer, etc.-these being the polymolecular adsorption layers. The depth of these layers can become 100 times as large as the diameter of the water molecule, the energy of the water and substance binding gradually decreasing while moving away from the colloidal particle surface (Ginsburg, 1969, 1973; Kazanski, 1960; Popovski, 1975)-according to Volarovistch et a / . (1972). from 63 to 21 kJ/mol. The adsorptionally bound water in foods includes both physically and chemically sorbed water (Bayer, 1962).

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c . Capillary-bound water. This is the water included in the macropores of the product (capillary and pendular moisture), as well as in the micropores (Ginsburg, 1973; Kazanski, 1960). Basically this is free water, with the exception of a very thin layer absorbed on the capillary walls, but with a binding energy lower than that of the adsorptionally bound layer (Ginsburg, 1969; Lebedev and Perel’man, 1973)-more precisely, with lower energy than that of the polymolecular moisture (Kazanski, 1960). d . Osmotically retained moisture. This moisture represents the dispersion media of the various cell solutions migrating through the semipervious membranes. It possesses very weak binding energy-2.1 kJ/mol (Volarovitsch et a l . , 1972). A great number of researchers in the field of refrigeration technology and cryobiology-such as Fennema and Powrie (1964), Lebedev and Perel’man

(1973), Lerici and Pallotta (1972), Owades and Dono (1969), Popovski (1967), Rey (1964a), Riutov (1976), Simatos (1964), Tchijov (1971), and Voskresenski (1963)--classify water into two groups, “free” and “bound,” according to its binding with the material. This is an incomplete and simplified but at the same time a very convenient classification. Their argument is that no sufficiently precise and universal methods have been developed to determine the various ways in which the water is bound to the materials. “Bound” water is the water retained by the soluble substance or by certain parts of its molecules-the polar groups. Such binding is especially strong around the ions and the hydrophilic polar groups of the high-molecular-weight compounds. In this case some particles of the dispersion phase hold a certain quantity of the dispersion medium by means of hydrogen bonds and electrostatic forces, and they move along with this quantity in the whole dispersion medium. “Free” water is water that has the ability to migrate into some materials (Voskresenski, 1963; Golovkin et a l . , 1955; Tchijov, 1970). Opinions differ on the amount of bound water in various food products. This is due, to a great extent, to the fact that the amount of bound water in any material varies, depending on numerous external and internal factors-pH, temperature, moisture of the material, etc. (Ginsburg, 1973; Dumanski, 1960). Kazanski (according to Dumanski, 1949, 1960) and Sedikh and Sedikh (1967), on the other hand, have proved that the transition from one form of moisture binding into another is gradual and that a distinct border between them cannot always be established. I agree with the statements of Meryman (1957) and of Smith (1954, 19621, which have been supported by some recent studies, that the term “bound” water has not been sufficiently well defined. The amount, according to Moran (19341, Riedel (1968), and Toledo et al. (1968), is 0.3 to 0.35 gm per gram of dry matter, or about 0.4 gm per gram of dry protein (Riedel, 1968); 8 to

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12% of the whole amount of water in the product, or 5 to 10% of its mass, is estimated to represent bound water (Fennema and Powrie, 1964; Meryman, 1960; Voskresenski, 1963). The data on the amount of bound water in fruits and vegetables are no less contradictory. It is frequently pointed out that bound water represents 5 to 1 I % of the whole mass (Fennema and Powrie, 1964; Guigo et al., 1966; Moy et al., 1971; Popovski, 1975). Kuprianoff (1964) and Palanche (1973) consider that most fruits and vegetables contain less than 6% bound water (in relation to the entire mass). Lerici and Pallotta (1972) have pointed out that this represents 2 to 5% of the entire amount of water; according to Mestcheriakov (1975) it is 20%, but in our opinion that percentage is too high. It has been established that in fruit and vegetable cell membranes and in their immediate vicinity the water is firmly bound (Popovski, 1975). The cellulose and pectic substances building these membranes are highly hydrophilic and are able to bind large amounts of water (Fennema and Powrie, 1964). Larger quantities of proteins and polysaccharides are included in the cell cytoplasm composition of onion and lettuce, and one would therefore expect the presence of larger amounts of bound water than are found in other vegetables. Natural starch does not bind large amounts of water, but in blanched plant tissues its hydrophilic properties increase considerably during gelatinization (Fennema and Powrie, 1964). There is a controversy among investigators about the possibility that bound water freezes in foods and biological products. The opinion prevails that part of this water does not freeze at all, even at liquid nitrogen temperature (Bonjour et a / . , 1975; Duckworth, 1971; Lozina-Lozinski, 1972; Meryman, 1960; Moran, 1926, 1934; Moy etcil., 1971; Popovski, 1967, 1975; Rey, 1964a; Riutov, 1976; Rowe, 1960; Sedikh and Ichmokhametova, 1970; Simatos et af., 1975a; Smith, 1954, 1962; Tchijov, 1971; Tobback and Maes, 1971). This view has been supported by a satisfactory and theoretically acceptable argument (Lebedev and Perel'man, 1973). Its validity for products of plant origin was experimentally substantiated by Duckworth (1971), who proved that some vegetables (celery, peas, green beans, cabbage) contain water fractions that are absolutely unsusceptible to freezing; that is, part of the water is incapable of being transformed into ice, no matter how low the temperature is. Popovski (1975) reached similar conclusions for five types of fruit purees. Fennema and Powrie (1964) assumed that monomolecular adsorption water is unfreezable also. Some authors suggest that part of the adsorptionally bound water, the so-called crystallizable bound water, could freeze, but they give various temperatures for this phenomenon-below - 18°C (Fennema and Powrie, 1964; Toledo et d., 1968), below -40°C (Palanche, 1973; Riedel, 1968), -50" to -70°C 1975a; Susmann and Chin, 1966), below (Ginsburg, 1973; Simatos et d., - 100°C (Popovski, 1975), to - 120°C (Tchijov. 1971; Simatos, 1964).

PHASE TRANSITIONS OF WATER

307

Some investigations using the most up-to-date physical methods have proved that the adsorptionally bound water in the microcapillaries freezes at -90" to - 100°C (Lebedev and Perel'man, 1973; Rey, 1959b).

111.

INITIAL CRYSTALLIZATION OF WATER IN FRUITS AND VEGETABLES

Crystallization, or the transition of water into ice, is the basic phase transition that occurs during the freezing of food. The initial stage of water crystallization determines the lowest temperature at which the refrigerated storage of foods, including most fruits and vegetables, is possible. This stage also determines to a large extent the transition of water into ice in the entire region of negative temperatures. This section discusses some physical regularities of the initial crystallization of water and the influence of the chemical composition and structural features of fruits and vegetables on this crystallization. A.

SUPERCOOLING AS A PHENOMENON PRECEDING FREEZING

Supercooling of water in foods is a complex phenomenon, dependent on a number of physical and thermodynamic factors. We should mention here the chemical composition and concentration of the solids (especially of sugars, acids, and pectin), the variety and physiological state of the product, the viscosity of the protoplasm, the histological structure of the tissues, the dimensions of the cells, the permeability of their membranes acting sometimes as a serious obstacle to crystallization, the geometric dimensions and shape of the object, the temperature of the cooling media, and the heat transfer coefficient (Eisenberg and Kauzmann, 1975; Fennema and Powrie, 1964; Guegov, 1972; Golovkin and Strakhovitsch, 1964; Golovkin and Tschernichev, 1967; LozinaLozinski, 1972; Rey, 1960a; Simatos e r a l . . 1975a; Smith, 1954, 1962; Tchijov, 1956, 197 1). This diversity of factors makes it impossible to present an analytical description of the process. Investigations have demonstrated that crystallization of any liquid is impossible without supercooling, however insignificant (Fennema and Powrie, 1964; Gutschmidt, 1968; Hallet, 1968; Meryman, 1960; Tchijov, 1971). Supercooling can always be observed in plant tissues cooled at the rate of I"C/min (Fennema and Powrie, 1964). My investigations show that, during the freezing of fresh fruits and vegetables whose tissues are intact, at the rate of 40"C/min, supercooling prior to nucleation is observed in all cases (Guegov, 1972, 1979c,d). Only ground fruits and vegeta-

308

YANCOGUEGOV

bles can be frozen without supercooling. During the freezing of stored grapes no precooling was observed in half of the trials (Guegov, 1972). The admissible limit of supercooling is very important. As a result of numerous experiments, Tchijov (1956) determined that, for pure water in macrovolumes with surface coefficient of heat transfer from 4 to 2 160 W/(cm2.K) this limit is 4.2"C.According to the theory of homogeneous nucleation, water can be supercooled to -41°C (Fennema and Powrie, 1964;Quervain. 1974).The supercooling of water droplets (of 1 to 0.1 mm) to -25" to -33°C (Fennema and Powrie, 1964;Ginsburg, 1973;Golovkin et ui., 1955),and of droplets of superpure water of several microns from -40" to -41"C,was determined experimentally (Fennema and Powrie, 1964;Lebedev and Perel'man, 1973;Mazur, 1963; Smith, 1962).Supercooled water cannot exist in a state of equilibrium with ice crystals (Smith, 1962). The maximum limit of supercooling does not exceed 6.6"Cin model systems (agar and gelatin gels rich in water, and protein solutions), egg albumen, and muscle tissue (Tchijov, 1956).In gelatin gels containing less than 35% water, no ice appears, even at very low temperatures (Moran, 1926;Rey, 1959b),and in glycerol solutions with a concentration above 66% not until -78°C (Rey,

1959b). The supercooling temperature of plant tissues cannot be lower than -5" to 1971; Fennema and Powrie, 1964;Guegov, 1972; Mazur, 1963;Popmarinov and Fikiin, 1956;Popovski, 1975;Saint-Hilaire and Solmes, 1973).Normally this supercooling does not exceed 2" to 3°C.According to Popmarinov (1964),fruits can be supercooled to -5" to -6.5"C,and vegetables to -2" to -3°C. At very low cooling velocities, ice crystals do not form in apples until -5" to -8"C,and in grapes not until -7°C (Golovkin and Tschernichev, 1967). My investigations on grapes (Guegov, 1972)demonstrate that the supercooling temperature correlates with the soluble solids content and other conditions being equal. The greater the solids content is, the lower the supercooling temperature. The lowest supercooling temperature obtained in the freezing of grapes is -11°C. The stability of the supercooling state varies with different fruits and vegetables. According to the author's data (Guegov, 1979c,d),the higher the density and heat conductivity of the products, the shorter will be the period for the products to remain in the supercooled state. - 10°C (Amoignon and Floch,

B. NUCLEATION AND GROWTH OF ICE CRYSTALS Knowledge of the processes of nucleation and growth of ice crystals is indispensable for the correct interpretation of the phenomena that occur during the freezing of foods. In this respect, investigations of pure water and simple solutions provide the necessary initial information.

PHASE TRANSITIONS OF WATER

309

Crystallization starts when suitable conditions are reached for the aggregation of a group of molecules into particles of a certain order, called nuclei (centers) of crystallization. These conditions are determined by the correlation between temperature, cooling velocity, solute concentrations, and magnitude of the forces orienting the molecules in the liquid. Other factors-the movement of the cooled liquid and the characteristics of the border phase-may also play an important role (Fennema and Powrie, 1964; Fikiin, 1973; Hallet, 1968; Lerici and Pallotta, 1972; Lozina-Lozinski, 1972; Meryman, 1957; Quervain, 1974). The cooling velocity is a basic factor in obtaining a certain type of crystal structure. It is well known that at very high cooling velocities (from 150" to 1000"C/sec) even amorphous liquid structures can be obtained (Fennema and Powrie, 1964; Mazur, 1963; Meryman, 1957; Rey, 1959b). Generally speaking, an increase in the cooling velocity leads to a slowdown in the growth of the ice crystals (Luyet, 1951, 1957, 1960). In the temperature range of ordinary food freezing, the cooling velocity is more significant for the growth of the ice crystals than the temperature. The growth of the ice crystals ceases at very low temperatures (below -80°C) (Fennema and Powrie, 1964; Luyet, 1957, 1960; Simatos et d.,1975a). The growth velocity of the ice crystals in foods is checked by various inorganic and organic substances, representing their natural constituents+thanol, saccharose, albumins, and some high-molecular-weight substances (Fennema and Powrie, 1964; Rey, 1959b). The mechanism of this phenomenon is not clear yet, but, as we shall see later, it plays an important role in determining the complete solidification of the food product. Two types of initial crystallization are possible: homogeneous and heterogeneous (catalytic). The homogeneous crystallization centers are formed during a random aggregation of water molecules into ice structures up to a critical size. This is almost impossible with pure water at O"C, but the probability reaches 1 at -40" to -41°C; that is, pure water cooled to -41°C cannot remain liquid during a further temperature decrease. Heterogeneous crystallization takes place when solid particles serve as sites for crystal formation. Ordinarily these solid particles are substances that are insoluble in water, and their size, topography, and molecular characteristics determine the character of ice formation (Hallet, 1968; Meryman, 1957, 1960; Quervain, 1974; Rey, 1959b). Heterogeneous crystallization always occurs at temperatures higher than those found in homogeneous crystallization. Since some particles that help in the primary crystallization of ice are present in all liquid solutions, except for ultrapure water, the crystallization taking place during the freezing of foods is heterogeneous. In biological systems the presence of cell membranes interferes substantially with the normal course of the initial crystallization inside the cells (Kuprianoff, 1964; Smith, 1954). If upon freezing the cell wall remains intact, intracell

310

YANCOGUEGOV

crystallization cannot take place before a certain temperature is reached, when the critical radius of the crystal is smaller than the radius of the pores in the cell walls. Thus, the crystallization can “penetrate” into the cell cytoplasm. Mazur (1963) reports that intracell crystallization takes place at a product temperature of -5” to -10°C. C. THE CRYOSCOPIC TEMPERATURE OF FRUITS AND VEGETABLES The cryoscopic temperatures of ideal mono- and bicomponent solutions have been thoroughly investigated and may be described by the well-known law of Rauhl. They depend on the concentration of the solutes, their molecular weight and degree of dissociation, and the characteristics of the solvent (Fennema and Powrie, 1964; Fikiin, 1962, 1973; Markova and Chkol’nikova, 1962; Smith, 1962). The presence of high-molecular-weight and colloidal substances in solutions has almost no effect on this temperature. In contrast, the dissociated and molecularly dispersed substances substantially decrease the initial freezing temperature. The cryoscopic temperatures of simple mono- and bicomponent solutions of natural constituents of foods of plant origin-salts, carbohydrates, albumins, etc.-were investigated by Heiss and Shahinger (l95l), Kondratiuk (1972), Lusena and Cook (1954), and Nemitz (1961). The theory concerning simple solutions does not allow for the freezing temperatures of concentrated and multicomponent solutions and of foods to be determined mathematically. The complexity of the biochemical composition and texture of fruits and vegetables calls for specific experimental investigation of the cryoscopic temperature of each species (Fikiin, 1973; Gasiuk and Zelenskaia, 1965; Golovkin et al., 1955; Koeppe, 1970; Tchijov, 1956). The cryoscopic temperature of fruits and vegetables depends on the same factors as those described for ideal solutions, the solids content being of basic importance. The higher the solids content is, the lower the incipient freezing point will be. Fruit with a higher sugar or acid content will have a lower cryoscopic temperature (Djafarov, 1974; Guegov, 1972, 1978; Golovkin et af.,1955; International Institute of Refrigeration, 1964; Saburov and Antonov, 1962). The supercooling temperature obtained does not influence the value of the cryoscopic temperature (Lozina-Lozinski, 1972). There is a widespread opinion that in practice the cryoscopic temperature of plant tissues is not influenced by the cooling conditions (Danilov, 1974; Djangaliev and Tsziu, 1969; Djeneev, 1968; Fikiin, 1973; Gutschmidt, 1968; Leblond and Paulin, 1968; Lozina-Lozinski, 1972; Mestcheriakov, 1975; Popmarinov, 1964; Saburov and Antonov, 1962). According to some authors this temperature is not absolutely constant, and it cannot be determined as precisely as can, for example, the melting or freezing point of liquids. These authors base

PHASE TRANSITIONS OF WATER

311

their belief on the possibility that moisture migrates through the cell membranes during the cooling process. In their opinion, the cryoscopic temperature should decrease with an increase in the cooling velocity (Fennema and Powrie, 1964; Golovkin et al., 1955; Maksimov, 1957). This decrease has a rather theoretical aspect, and it does not exceed 0.1" to 0.15"C when the temperature of the cooling medium is changed by 10" to 15°C. The cause for this phenomenon should also be studied in relation to the experimental conditions. During a fast cooling, the heat released during freezing may prove insufficient to raise the thermocouple temperature. For this reason, Lozina-Lozinski (1972) believes that the cryoscopic temperature is in fact not influenced by either the cooling velocity or the coolant temperature (in the range -5" to -20°C). The results from some experimental studies show that the cryoscopic temperature of intact tissues is lower than that of the tissue homogenates (LozinaLozinski, 1972; Fikiin and Kuzmanova, 1970). In the tissue homogenates the freezing point reflects only the influence of the chemical composition of the tissue juices, but in our opinion, from the point of view of refrigeration technology, the cryoscopic temperature of intact tissues offers a more precise and useful characteristic. The cryoscopic temperature of most food products is about - I .O"C (Fikiin, 1962, 1973; Golovkin et al., 1955; Heiss and Shahinger, 1951; Kobulachvili, 1961; Tchijov, 1971). The isotonic solutions of the cell liquids of most plants freeze in the range of -5" to -2°C (Popmarinov, 1964; Rey, 1959b); for some plants rich in sugars, the range is -2.0" to -4.O"C (Popmarinov, 1964). The cryoscopic temperature of vegetables (Plank, 1960) is in the range of -0.5" to -2°C (Djafarov, 1974; Fikiin, 1973; Guegov, 1979d; Popmarinov, 1964). Some vegetables, however, such as garlic and horseradish, freeze at lower temperatures: -2.5"C and -3. I"C, respectively (Guegov, 1979d; Popmarinov, 1964). The cryoscopic temperature of fruits is in the range of -1.0" to -2.5"C (Djafarov, 1974; Fikiin, 1973; Guegov, 1979~;International Institute of Refrigeration, 1964; Kaminarskaia et al., 1963; Plank, 1960; Popovski, 1967; Saburov and Antonov, 1962; Tsinman and Ianiuk, 1969). For some apple and pear varieties, it can reach -3°C (Guegov, 197912;Leblond and Paulin, 1968; International Institute of Refrigeration, 1964; Saburov and Antonov, 1962), for plums -3.2"C (Suegov, 1979c), for sour cherries -3.5"C (Djafarov, 1974; Fikiin, 1973; Golovkin et al., 1955; Popmarinov, 1964; Popovski, 1967; Tchijov, 1971), for bananas -3.6"C (Guegov, 1979c), and for grapes -5" to -6°C (Djafarov, 1974; Fikiin, 1973; Guegov, 1972, 1979c; Popmarinov, 1964; Popovski, 1975; Tchijov, 1971). Wright (according to Fennema and Powrie, 1964) found the cryoscopic temperature of 19 vegetable varieties to be in the range of -0.83"to -2.83"C, and for 22 fruit varieties the range was -0.88" to -2.66"C.

312

YANCOGUEGOV

The data available for the cryoscopic temperatures of fruits and vegetables are either the mean results for a certain plant variety (Djangaliev and Tsziu, 1969; Djeneev, 1968; Fennema and Powrie, 1964; Leblond and Paulin, 1968; Maksimov, 1957; Popmarinov, 1964), or the precise values of the cryoscopic temperature, given a certain solids content (Dickerson, 1968; Fikiin, 1962; Kobulachvili, 1961; Kondratiuk and Ginsburg, 1974; Riutov, 1976; Tchubik and Maslov, 1970). The mathematical interpretation of the correlation between the cryoscopic temperature and the chemical composition of fruits and vegetables is of considerable interest. It was established by Fikiin and Kuzmanova (1970) and Guegov (1972) that a linear correlation exists between the soluble solids content and the cryoscopic temperature in grapes. Some of our recent work (Guegov, 1979c,d) shows a linear correlation between the soluble solids content and the cryscopic temperature of all 35 fruit and vegetable species investigated, some of them represented by several varieties. It was proved that the varietal characteristics of a certain plant species do not in fact influence this dependence (Guegov, 1978, 1979e) (Fig. 1). Equations describing the correlations between the cryoscopic temperature and the soluble solids content for each fruit and vegetable variety have been formulated. Whiteman (according to Gutschmidt, 1968) offers an interesting graphical correlation between the cryoscopic temperature and the soluble solids content for 13 fruit and vegetable varieties. The change in the soluble solids in the range of 4 to 22.5% is accompanied by a linear change in the cryoscopic temperature from -0.5" to -3.5"C.

Apples

-1.5

-

Golden Delicious (

R e d Delicious

)

(X)

(+I -2.0

-2.5

'

t

FIG. 1, Correlation between the clyoscopic temperature and the soluble solids content of apples. From Guegov (1978).

PHASE TRANSITIONS OF WATER

313

The investigations of Kondratiuk (1972), Kondratiuk and Ginsburg (1974), and Popovski (1975) on the freezing of fruit purees concentrated from 10 to 70% soluble solids show that the correlation between the cryoscopic temperature and the soluble solids content is linear for concentrations up to 45%. We have established (Guegov, 1978) a general correlative dependence, expressed by the equation tk =

0.36 - 0.1756

which reflects the influence of the soluble solids (6) on the cryoscopic temperature value (rk) of 40 fruit and vegetable varieties, regardless of their species or variety. This equation is valid within the range of 3.5 to 27% solids content, with a confidence interval of the cryoscopic temperature from +0.05" to +0.2"C. The equation for the soluble solids range of 3.5 to 20% was established by summing up the experimental data for 7 to 17 types of products, and for the solids range of 20 to 27%, the data for three types of products (Fig. 2). The confidence interval is represented in the figure by a dotted line. The calculated correlations dependence describes well the experimental data obtained by other authors also. Leblond and Paulin (1968) and Quervain (1974) state that the change in the initial freezing temperature in certain plant tissues is insignificant, depending on their ripeness. When the fruits have adapted themselves to the cold (as a result of a gradual decrease in their temperature), their ability to resist the intracellular crystallization increases, as Golovkin and Tschernichev (1967) point out. Our investigations (Guegov, 1978) with two apple varieties, three pear varieties, and one grape variety show that the cryoscopic temperatures of fresh and cold-stored (for 3 months) fruits with the same soluble solids content are different (Fig. 3). In apples and pears ripening during cold storage, the cryoscopic temperature of the stored fruits is higher than that of fresh fruits with the same solids content. This phenomenon is probably due to changes in the permeability of the cell membranes, changes related to those in the pectic and hemicellulose complexes that facilitate the initial ice formation. With grapes, which do not ripen during cold storage, the cryoscopic temperature of the stored fruit is lower than that of fresh grapes with the same soluble solids content. The cell membranes of grapes undergo comparatively minor biochemical changes during cold storage, but, as a result of an intensive moisture release, which is seven to nine times as high as that of apples (according to Jadan, 1972), and five to six times as high as that of pears, conditions are created for a certain solidification of the membrane skeleton. We suppose that the initial crystallization is thus prevented, and hence the cryoscopic temperature is reduced. The thermograms of fruit and vegetable cooling have been often discussed in

3 I4

YANCOGUEGOV

FIG. 2. General correlation between the cryoscopic temperature and the soluble solids content of fruit and vegetables: I-apples, 2-pears, 3-raspberries, -uinces, 5-bananas, &ranges, 7--chemes, 8-strawberries, 9-red tart cherries, I L p e a c h e s , I I-apricots, 12--lemons, 13plums, I G g r a p e s , I5-grapefruit. I b c a b b a g e , I7-small radishes, I 8-tomatoes, 19-peppers, 2 k g r e e n onions, 21--celery root, 22-potatoes, 23--watermelon, 2Gmushrooms, 25-small pumpkins. 2 k a r r o t s . 27Leggplants. 28-red beets, 29-radishes, 30-kohlrabi, 3 I+nion bulbs, 32--leeks, 33-sugar melon, 34--cucumbers, 35-elery leaves, 3 6 g r e e n beans, 37spinach, 38-auliflower, 39-pumpkins, 40-green peas. From Guegov (1978).

the literature (Fennema and Powrie, 1964; Fikiin, 1973; Guegov, 1979c,d; Golovkin and Strakhovintsch, 1964; Golovkin and Tschernichev, 1967; Kondratiuk, 1972; Lozina-Lozinski, 1972; Saburov and Antonov, 1962). They indicate important parameters of initial crystallizationdegree and stability of supercooling and the cryoscopic temperature. Some scientists have observed two sharp increases in the temperature after supercooling. According to Golovkin and Strakhovitsch (1964) and Golovkin and Tschernichev (1967), the first increase is seen in the intracellular space during the freezing of water, and the second is observed in the cell vacuoles. The authors prove that similar thermograms could be obtained only with juicy fruits containing large, thin-walled cells-apples, plums, grapes, and red currants. Maksimov (1957) explains the first sharp in-

315

PHASE TRANSITIONS OF WATER

-1

’

1 Grapes.var

B o l g a r -fresh

2.Grapes.var

Bolg a r -stored

3.Pears. var Cur:

-2

-fresh

L.Pears, var C u r 6 - s t o r e d

-3.

-L.

FIG. 3 . Correlation between the cryoscopic temperature and soluble solids content of fresh and cold-stored fruit. From Guegov (1978).

crease as due to the freezing of the juice that is released when the thermocouple is inserted into the tissue. It should be pointed out that these results were obtained with a small-dimensional thermocouple-in the range of 0. I to 0.3 mm. The double sharp temperature increase preceding the isothermal retention of the temperature during the initial formation of ice does not influence the cryoscopic temperature values.

IV.

PHASE TRANSITIONS AT LOW TEMPERATURES (TO -70°C)

A.

PHASE TRANSITIONS DURING FREEZING AND THAWING OF MODEL SYSTEMS

The phase transitions in food products of plant origin can be reliably determined and analyzed only on the basis of the regularities of ice formation in nonstructural systems-salt, sugar, high-molecular-weight solutions, gels, etc. (Maltini, 1974a,b, 1977; Rey, 1959b; Simatos et al., 1975a; Willemer, 1974). According to Maltini and Giangiacomo (1976), the phase transitions in solutions take place in the same manner as in solid foods, which possess the same structures as real or pseudo solutions. The following phase transitions taking place at low temperatures are worth mentioning: crystallization and melting (including the eutectic phenomena), vitrification, glass (vitreous) transformation, devitrification (crystallization) following glass transformation, recrystallization preceding melting, and the socalled “antemelting. This last phase transition was established during this decade by using differential thermal analysis (DTA) and differential scanning calorimetry (DSC) for biological solutions and protective media. Its nature has not yet been investigated in detail. The other phase transitions have been well studied and described in the literature on the physics of solid bodies, cryobiology, and food technology. ”

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YANCO GUEGOV

I.

Eutectic Freezing and Melting

The formation of ice in diluted two-component solutions is accompanied by the separation of water as pure ice crystals, by a progressive increase in the concentrations of the dissolved substance, and by a gradual decrease in the melting point of the concentrated solution. In reaching the temperature called eutectic, the water, not frozen until that moment, and the substance dissolved in it crystallize simultaneously in a fixed relationship, forming a mixed conglomerate (hydrate) (Rey, 1959b. 1960b; Simatos et al., 1975a; Steinbach, 1977). The eutectic temperature depends on the nature of the initial salt solution. In a solution containing several salts, the salts crystallize successively according to their eutectic point. The system retains unfrozen water until the salt solution with the lowest eutectic temperature becomes saturated. Additional cooling does not cause structural changes in the cryohydrate or in the eutectic mixture formed (Greaves, 1964; Rey, 1960b; Simatos et al., 1975a). As early as 1949, Flosdorf (according to Davies, 1966, and Greaves, 1954) was the first to pay serious attention to experimental research on the phase transitions and especially on the eutectic temperature at which or about which freeze-drying should be carried out. This conception played a predominating part in the formation of the theoretical basis for the refrigeration treatment of materials subjected to freeze-drying. Numerous phase diagrams have been published for different salts and carbohydrates in binary and (rarely) in ternary systems, but it is impossible for the eutectic phenomena in multicomponent systems to be interpreted on this basis. Among all salts taking part in the food composition, CaCI, possesses the lowest eutectic temperature (Fennema and Powrie, 1964; Fikiin, 1973; Luca and Lachmann, 1973; Willemer, 1974; Young et al., 1952). Ice formation and the eutectic crystallization of different salts and salt mixtures included in the composition of biological media and food products have been investigated and analyzed (curdova, 1969; Farrant and Woolgar, 1970; Fateeva, 1970; Fikiin, 1969; Guegov, 1980c; Greaves, 1954, 1964; Greaves and Davis, 1965; Ito, 1970, 1971; Kazakov er al., 1973; Larouse, 1970; Luca and Lachmann, 1973; Maltini and Anselmi, 1973; Mazur, 1961; Monzini, 1971; Rey, 1959a,b, 1960a,b; Smith, 1954; Stricker, 1970; Tsurinov, 1955). The solutions of NaCl have been investigated most thoroughly. Its eutectic temperature (-21.3"C) is used as the reference point in determining the sublimation parameters of most of the products. In investigating the eutectic freezing of salt solutions, two things are of utmost importance for freeze-drying: the eutectic crystallization mechanism, and proof that the structure of a freezing solution is determined not only by the temperature but also by the way this temperature has been reached. Microscopic observations during the freezing of salt solutions reveal large

317

PHASE TRANSITIONS OF WATER

spaces of frozen pure water and narrow channels of unfrozen concentrated solution forming a continuous interstitial network. As a whole, the capillary system, filled with liquid having a high salt concentration, is relatively permeable to electricity. when the temperature is decreased, the ice crystals gradually increase in size, resulting in a decrease in the amount of liquid that fills the little channels. At a certain low temperature the interstitial channels darken suddenly and intensively, and numerous tiny crystals appear (compared with the ice structures around them), representing a salt hydrate or a mixture of salts and ice. The solution hardens completely, and its electric resistance becomes in effect infinite-eutectic crystallization has taken place (Luyet, 1957; Meryman, 1960; Podol’ski and Lakovskaia, 1974; Rey, 1959b, 1960a,b; Simatos et al., 1975a; Smith, 1962). The studies show that at negative temperatures the salt solutions, as well as all other liquid media, do not possess a fixed type of structure, corresponding to a certain temperature. Their structure is determined by the way the temperature has been reached (Greaves, 1964; Greaves and Davis, 1965; Maltini and Anselmi, 1973; Rey, 1959b, 1960a,b; Vuillard, 1957; Willemer, 1977). This can best be demonstrated by the investigation of the electric conductivity of a solution during cooling and rewarming (Fig. 4). The change in the electric resistance of a 10% NaCl solution (Fig. 4) shows that the freezing and thawing curves do not coincide. Thus, according to Rey (1959b, 1960b) and Vuillard ( 1957), supercooling and metastable equilibria were obtained during freezing, and complete solidification of the system (complete freezing of the water) was brought about at temperatures below -35”C, much Log R

Interstitial cryrtdlhation

b

I! y

I

I a

I I

I \

I

i, -

Ice formation

--

I

1

1

J ’

-

1c :

FIG. 4. Crystallization of aqueous solution of sodium chloride as determined by electric resistance measurements. From Rey (1964a). Courtesy of the author and “ATLAS” Denmark.

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YANCOGUEGOV

lower than the eutectic one (-21.3"C). This temperature is known among scientists as the maximum temperature of complete solidification, Tcs. In contrast, during rewarming, the melting of the ice in the solution or in the liquid product starts when the so-called minimum temperature of incipient melting, Tim, is reached. In a simple solution, such as the investigated one, the meaning and the value of this temperature, Tim, are identical to those of the eutectic temperature, Tt,. Figure 4 shows that at a temperature of -3O"C, for example, two states of the solution are possible: state 1 , corresponding to a not completely solidified (frozen) product with liquid veins unsuitable for freeze-drying, and state 2, corresponding to a completely solidified system, suitable for freeze-drying. At present, it is considered essential to obtain complete solidification during the freeze-drying of liquid media in order to interrupt the metastable states that arise during freezing (Davies, 1966; Fateeva, 1970; Lakovskaia, 1971 ; Larouse, 1970; Mackenzie, 1974; Marcus, 1974; Owades and Dono, 1969; Podol'ski and Lakovskaia, 1971; Rey, 1960b, 1964a). Besides the changes due to the hypertonic solutions, the presence of liquid veins in the product causes (under vacuum) abundant foaming, which leads to the denaturation of the product, hinders drying, and accounts for the unsatisfactory rehydration of the dried product (Greaves, 1954; Monzini, 1971; Palanche, 1973; Rey, 1960a). From this point of view, the sublimation temperature of the systems discussed must always be equal to or lower than the eutectic temperature (Davies, 1966; Greaves, 1954; Rey, 1960a,b). The described supercooling of liquid media, observed in the entire temperature zone of ice formation (unlike that before the cryoscopic temperature), depends, according to Luca and Lachmann (1973) and Simatos et (I/. (1975a), on the freezing velocity, the nature and the concentration of the solutions, and the degree of dispersion. For the ion solutions the supercooling temperature varies from several degrees (according to Kazakov et al., 1973) to 10" to 20°C (Davies, 1966; Gonda and Koga, 1973; Greaves and Davis, 1965; Luca and Lachmann, 1973; Rey, 1960a,b; Stricker, 1970). Supercooling is very weak or does not occur at all in tissues or systems containing substances that are not typical eutectics (for example, carbohydrates) (Davies, 1966; Fateeva, 1970; Greaves and Davis, 1965; Rey, 1959b, 1960a; Simatos et ul., 1975a; Smith, 1962). So far, no satisfactory theoretical explanation has been offered as to how supercooled water could exist in a solution in the presence of ice crystals. The investigations of Greaves and Davis (1965), Monzini (1971), and Rey (1959a,b, 1960a.b) show that the eutectic temperature of mono- or bicomponent salt solutions can be easily established experimentally and that the measurements offer good reproductibility. In three-component and complex salt solutions a single diffused zone of incipient melting was registered instead of separate eutectic points, and eutectic phenomena could only be assumed in it (Greaves, 1954; Greaves and Davis, 1965; Guegov, 1980c; Lakovskaia, 1971; Monzini, 1971; Owades and Dono, 1969; Podol'ski and Lakovskaia, 1971; Rey, 1959a, 1960a).

PHASE TRANSITIONS OF WATER

319

In such systems a minimum temperature of incipient melting, Ti,, could be determined, instead of the eutectic temperature. The larger the number of the components in a salt, according to Davies (1966) and Rey (1960a), the stronger will be the suppression of the eutectic phenomena in the system. The solidification of the interstitial grid in the complex solutions occurs gradually. During rewarming of the same system, eutectic melting is replaced by a slow, progressive melting of the same interstitial grid. The phenomena of ice formation are greatly complicated by the presence of organic substances in the system: crystallization inhibitors (mainly carbohydrates), pectin, organic acids and certain vitamins, gelatin, and various protective media and stabilizing substances-mannitol, alcohols, and glycols (Maltini and Anselmi, 1973; Simatos, 1976). According to Monzini (1971), these substances form a wide eutectic zone, where the incipient melting is accompanied by a mild endothermic effect. The intensive melting is accompanied by a substantial endothermic effect; it can be accurately registered by DTA, DSC, and microscopic techniques, by the measurement of some rheological properties, etc. The problem of establishing the phase transitions in solutions containing natural food constituents-various sugars, sugar-salt mixtures, pectin, organic acids, etc.-is extremely important in food technology. The phase diagram analysis of aqueous sucrose solutions performed by Mathlouti (1974) shows that eutectic crystallization is hardly possible, whereas complete solidification should take place at - 13" to - 14.5"C (Heiss and Shahinger, 1951; King, 1974). Mixed aqueous solutions of saccharose, glucose, and fructose should solidify completely at -25°C (Flink, 1974a; King, 1974; Maltini and Anselmi, 1973). The investigations of Mathlouti (1974) and White and Cakebread (1966) on freezing of the aforementioned solutions point to initial nucleation with a very limited growth of ice crystals, owing to the high viscosity of the solution. The solutions do not fully solidify even at -40°C (BeIIows and King, 1972; turdova, 1969; Davies, 1966; Farrant and Woolgar, 1970; Fateeva, 1970; Greaves, 1964; Ito, 1971; Jabarit, 1970; King, 1974; Mackenzie, 1974; Maltini, 1974b). Luyet and Rasmussen (1968) and Rasmussen and Luyet (1969) established an incipient melting of glucose solutions at a temperature (Tim) of -28" to -31.5"C, and of sucrose solutions at a temperature of -35" to -41.5"C, which are far below their theoretically calculated eutectic points (-5" and - 13.5"C). According to the authors, this means that, during the cooling of sucrose solutions with medium and high concentrations (above 50%), eutectic freezing does not take place. According to Steinbach (1977), the incipient melting of sucrose solutions with 30 to 70% concentration can be observed in the range of -28" to -34°C. Moreira and Simatos (1977) established an incipient melting of fructose and glucose solutions at -42"C, of sucrose solutions at -32"C, and of citric acid at -50°C.

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Rasmussen and Luyet ( 1969) assume that in highly concentrated solutions (above 75% for glucose and above 77% for sucrose) phase transition of water into ice is impossible; they call such solutions unfreezable. Maltini (1977) experimentally determined that, in the cooling of sucrose solutions with a 67 to 72% concentration, ice crystals do not appear. The investigations of Couach et al. (1977) show that in freezing a 25% sucrose solution the relationship attainable between ice and the total amount of water is 0.88, which represents about 0.34 gm of unfreezable water in 1 gm of solid matter. The addition of saccharose to a NaCl solution entirely eliminates the eutectic crystallization. The supercooling shown in Fig. 4 was not obtained, and no eutectic melting occurred (curdova, 1969; Davies, 1966; Greaves, 1964; Greaves and Davis, 1965; Guegov, 1980~).According to Greaves (1964) and Greaves and Davis (1965), the addition of glucose to the same solution causes smaller changes. During heating, a very weak eutectic melting was noticed. The investigations of Guegov (1980~)and of Maltini and Anselmi (1973) indicated that the addition of pectin to salt solutions showing well-defined eutectic phenomena almost entirely eliminates these transformations. In pure pectin solutions, no well-expressed phase transitions are demonstrated, but only melting in the region around the cryoscopic temperature (Fikiin, 1969; Guegov, 1980~). Agar added to chloride solutions does not suppress the eutectic phenomena (Rey, 1960b) and only slightly decreases the eutectic temperature (2" to 5°C). The behavior of citric acid and raw cellulose is similar to that of pectin (Davies, 1966; Fikiin, 1969). The mechanism of ice formation in cell suspensions or tissues is similar to that in complex solutions. The interstitial grid solidifies at about -30" to -45°C (Kazakov et a l . , 1973; Simatos et a l . , 1975a). In the objects pointed out, no eutectic temperature has been established; however, it is possible to determine representing the upper the minimum temperature of the incipient melting, Tim, limit of the temperature zone within which the frozen object is in a fully solidified state (Davies, 1966; Fateeva, 1970; Lakovskaia, 197l ; Lozina-Lozinski, 1972; Luca and Lachmann, 1973; Rey, 1960b; Simatos et a l . , 1975a; Smith, 1962). 2 . Transitions Connected with the Vitreous Structures During the freezing of solutions containing crystallization inhibitors, ice crystals and a supercooled liquid solution are formed, the latter solidifying into a vitreous mass as the temperature is decreased. Typical examples of this phenomenon are the sugar solutions, certain alcohols, glycerol, and protective media solutions, benzene, chloroform, and cyclohexane (Bohon and Conway, 1972; Davies, 1966; Douset, 1964; Fikiin, 1969; Flink, 1974b; Greaves, 1964; Lozina Lozinski, 1972; Luyet and Rasmussen, 1967; Mackenzie, 1974; Mathlouti,

PHASE TRANSITIONS OF WATER

32 1

1974; Rasmussen and Mackenzie, 1968; Simatos, 1976). Even at lower freezing velocities (to 30”C/min), the presence of similar structures was demonstrated by Gonda and Koga ( 1973) and Mackenzie ( 1970) in biological objects (yeasts), and in blood plasma by Simatos et al. (1975b). It was established that, in solutions that do not crystallize eutectically, the interstitial liquids concentrate gradually, which leads to an increase in their viscosity; after it reaches a certain limit ( lOI3 N.seclm*), they solidify into a vitreous metastable structure. The high viscosity hinders crystallization, and that is why these structures are preserved for a long period (Davies, 1966; Greaves, 1964; Greaves and Davis, 1965; Maltini, 1974b). It is difficult to carry out freeze-drying of products possessing a greater amount of these structures. The rewarming of the vitreous material involves two other phase transitions-glass transformation and crystallization (devitrification). The glass (vitreous) transformation is a typical phase transition observed in systems containing carbohydrates (Greaves, 1964; Greaves and Davis, 1965; Ito, 1970, 1971; Luyet, 1970; Luyet and Rasmussen, 1967; Mackenzie, 1974; Maltini, 1977; Rey, 1960b), glycerol, and other protective media (Bohon and Conway, 1972; Dousset, 1964; Greaves, 1964; Greaves and Davis, 1965; Ito, 1971; Luyet and Rasmussen, 1967; Mackenzie, 1974; Rasmussen and Mackenzie, 1968; Rey, 1964a,b). According to Luyet and Rasmussen (1968), it is characteristic of the glass transformation that at a certain temperature the molecules, which could until then perform only vibrational motions, now become able to perform a rotational and translational movement. The glass transformation involves changes in the specific heat capacity of the substance existing until then in a vitreous state, in the refraction index, and in its viscosity; the latter falls abruptly from 1013 N . sec/m2 to 109N~seclm2(Luyet, 1970; Luyet and Rasmussen, 1967, 1968; Vuillard, 1957). This phenomenon can be clearly observed by using DTA and DSC. Rasmussen and Luyet (1969) assume that at temperatures below the glass (vitreous) transformation temperature, T,, a solution can be represented as a system of ice crystals surrounded by an amorphous “matrix.” At a temperature above T,, this matrix changes to a supercooled “liquid” with a very high viscosity. The vitreous transformation can be distinctly observed in highly concentrated sugar solutions, but it can be seen also in the thinner solutions (Fikiin, 1969; Guegov, 1980; Luyet and Rasmussen, 1968; Maltini, 1977). Maltini finds that this transformation affects the “unfreezable” water. It can be observed both in fast-cooled and in slowly cooled solutions. The devitrification or crystallization of the amorphous structures closely follows the vitreous transformation. According to Luyet and Rasmussen (1968), at higher temperature the molecules are rendered sufficiently mobile to change

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from a random arrangement in the vitreous state to the ordered structure of a crystal. The devitrification is accompanied by an exothermic effect and is distinctly observed by use of DTA and DSC (Dousset, 1964; Lozina-Lozinski, 1972; Luyet and Rasmussen, 1967, 1968; Mackenzie, 1974; Maltini and Anselmi, 1973; Rasmussen and Mackenzie, 1968; Rey, 1959a,b, 1964b). The ordinary devitrification temperature, Tdris 10" to 20°C higher than the vitreous transformation temperature, T , (Luyet, 1970; Luyet and Rasmussen, 1967, 1968; Rey, 1959a,b). The devitrification is observed in solutions with mean concentration of 55 to 65%. By extrapolating data for the viscosity of the glucose solutions at positive temperatures, Luyet and Rasmussen (1967, 1968) point out that devitrification starts at a solidified solution viscosity in the range of lo9 cP. Tobback et al. (1978) have proved experimentally that devitrification is always accompanied by a distinct change in viscosity. They have established that, in glucose solutions with various concentrations of NaCl, devitrification starts at a viscosity of the system in the range of 10" to lo7 CP (Fig. 5 ) . Using DSC, Maltini (1977) has determined that sucrose solutions with 60 to 66% concentration do not crystallize during cooling if no crystal nuclei are present, but, upon heating, the existing water crystallizes with a considerable exothermic effect, equal to that of the subsequent melting. This phenomenon is

Time

FIG. 5. Variation in the viscosity of a solution having a cas composition (27.6% glucose and 18.7% NaCI) as a function of time and corresponding temperature. The viscosity is expressed in relative units. From Tobback el ul. (1979). Courtesy of the authors and IPC Science and Technology Press, Ltd.

323

PHASE TRANSITIONS OF WATER

M

D

f i

231

FIG. 6. (A) Thennogram of a glucose solution (40%):A, without rewarming; B , after rewarming to 224°K. (B) Thennogram of a glucose solution (63%).A, without rewarming; B, after rewarming to 233°K. G-vitreous transformation; D-devitrification; AM-antemelting; IM-incipient melting; M-melting. From Couach et al. (1977). Courtesy of the authors and IIF.

irreversible and takes place, depending on the solution concentration, in the range of -80" to -30°C. It is identified by the author as devitrification, although it is not preceded by a glass transformation. A similar irreversible phenomenon was observed by Simatos et al. (1975b) in blood plasma. A picture of the phase transitions discussed is shown in Fig. 6. Figure 6A is typical of solutions in which larger quantities of water have crystallized during freezing; Fig. 6B is an illustration of the thermal behavior and phase transitions upon warming of solutions containing considerable amounts of vitreous structures. How do these phase transitions take place in the basic constituents of foods of plant origin-the carbohydrates?

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The cooling of the concentrated sugar solutions (above 60%) is accompanied by the formation of vitreous structures. Among carbohydrates, sucrose is regarded as the most susceptible to vitrification (Maltini, 1974b). In the sugar solutions discussed, three phase transitions have been experimentally established: vitreous transformation at -60" to -5O"C, devitrification at -40" to -38"C, and a progressively increasing melting of the ice crystals. The character of these transitions depends on the concentration of the systems (Fateeva, 1970; Fikiin, 1969; Flink, 1974a; Guegov, 1979c; Mackenzie, 1974; Maltini, 1974b, 1977). The heating of the completely solidified, highly viscous amorphous structures, especially those of sugar solutions, is associated with a sharp decrease in their viscosity at a critical temperature and the formation of viscous " f l o ~ s " (Bellows et a l . , 1972; Flink, 1974a; Ito, 1970; Mackenzie, 1965, 1974). The material softens and partially melts, this being the reason for the destruction of its structure during freeze-drying-the so-called "structural collapse. " In the presence of vitreous structures in a frozen liquid, freeze-drying at a temperature above that of complete solidification, T,,, and especially at a temperature above the collapse temperature, T,, is associated, according to Ito (1970), with a considerable risk. For eutectic solutions T,. = T,, and for noneutectic ones T , is 2" to 10°C higher than T,, (Ito, 1970). For 5 to 50% sucrose solutions, T , is -25°C (Ito, 1971) or -30°C (Mackenzie, 1975); for glucose it is -40°C (Marcus, 1974). The collapse itself appears in the range of 1" to 2°C (Mackenzie, 1965, 1974). 3 . Antemelting and Recrystallization During studies of sugar solutions, citric acid, and protective media, using DTA and DSC techniques, a phase transition was established, accompanied by an endothermic effect at a temperature, T,,, 5" to 10°C lower than the temperature of initial melting, Ti, (Fig. 5). This transition, like eutectic melting, does not depend on the concentration of the substance in the solution. This phenomenon is called "antemelting" (Couach et a l . , 1977; Mackenzie, 1974; Moreira and Simatos, 1977; Luyet and Rasmussen, 1968; Rasmussen and Luyet, 1969; Rasmussen and Mackenzie, 1968). Antemelting has been observed also during investigations of phase transitions in biological objects (yeasts) (Mackenzie, 1970). Antemelting is observed during melting of quick-frozen as well as slowly frozen solutions with mean concentrations of 20 to 50%. Luyet and Rasmussen (1968) and Rasmussen and Luyet (1969) point out that after a clearly expressed devitrification the antemelting phenomenon cannot be observed, and conversely that antemelting is not preceded (when the material is rewarmed) by devitrification. The antemelting phase transition occurs neither in pure ice nor in highly

PHASE TRANSITIONS OF WATER

325

concentrated solutions, during whose cooling no ice is formed. That is why Rasmussen and Luyet ( 1969) suggest that the transition requires the presence of both phases and most probably takes place at their border area. These authors and later Mackenzie ( 1 974) suggest that this phenomenon is essentially the formation of liquid-like molecular layers on the surface of the ice crystals. Moreira and Simatos (1977) assume that antemelting is the melting of tiny ice crystals, which may have appeared during devitrification inside the interstitial substance in zones with a concentration lower than that at the equilibrium state. Cuoach et al. (1977), using the method of differential enthalpic analysis of sugar solutions, established that antemelting is an endothermic phenomenon, which is partially identical with devitrification. Their investigations, carried out by using electronic paramagnetic resonance and x-ray diffraction methods, confirm the hypothesis that antemelting is accompanied by initial melting of crystal ice structures, causing dilution of the interstitial veins. No definite answer can be given as yet to the problem of whether antemelting is a eutectic melting or is melting of a certain mixture with a melting enthalpy lower than that of ice. In studying the thermal behavior of sucrose solutions with concentrations below 59%, Maltini (1977) established that antemelting is a reversible phase transition, and that is why it cannot be assumed to be crystallization of vitreous structures. The microscopic observations of the same author show that, during the rewarming of the frozen solutions, near the surface of th crystals in the interstitial veins there starts the formation of small crystals whose size increases with the increase in temperature, reaching its maximum at T,,, followed by remelting in the range of several degrees. If after this moment the solution is recooled, its structure remains unchanged and again causes antemelting during a subsequent rewarming. The author quite logically assumes that two simultaneous phase transitions take place in the material in the antemelting intervals-melting of the ice on the one hand, and, on the other, crystallization of part of the amorphous solution, which starts melting intensively at Ti,. If the sample is cooled again, the crystal phase remains in a crystal state, and the liquid phase formed by the melting of the ice can partially crystallize again, causing a supercooled phase to form in the rest of the solution, solidifying into amorphous structures. Couach er al. ( 1977) assume that “macroscopical structural collapse” cannot take place at temperatures lower than the antemelting temperature, Tarn.King (1974) and Moreira and Simatos (1977) prove that the “structural coallapse” can occur during freeze-drying of a solution or a liquid medium at a temperature between T,, and Ti,. According to Maltini (1977), the water formed at the antemelting temperature, T,,, dilutes the concentrated amorphous solutions, which show a sudden decrease in viscosity around that temperature. This can explain the “structural collapse,” and that is why the author expects the collapse to take place near the

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end of the antemelting-that is, near the minimum temperature of initial melting, Tim. According to Luyet and Rasmussen (1968), at temperatures higher than those of the vitreous transformation and devitrification-that is, at a higher mobility of the molecules-the smaller crystals lose stability; their surface-to-volume ratio is too high. These small crystals melt, and their molecules shift toward the large crystals, which thus grow bigger. This represents the recrystallization phenomenon. Recrystallization is observed at medium concentrations of the solutions (Luyet and Rasmussen, 1968). It is not accompanied by a clearly expressed thermal effect and is difficult to establish by DTA or DSC, although in certain investigations phenomena of spontaneous recrystallization connected with a mild exothermic effect were observed (Rasmussen and Mackenzie, 1968). Recrystallization can be associated with changes in the transparency of the frozen object. Recrystallization of a migrational type was traced by Luyet and Rasmussen (1968) and Rasmussen and Luyet (1969) at temperatures, T,, very close to (but lower than) the temperatures of initial melting, Ti,-at about -40°C for glucose solutions and -30°C for sucrose solutions. Recrystallization in its two basic forms, spontaneous and accretive (of growth), takes place during a continuous, slow rewarming. This is why it is of no special importance for the freeze-drying process and is not discussed in detail in this survey. Summing up the results obtained from the investigations carried out by numerous workers with model systems of natural constituents of food products as well as with biological media and solutions, we should point out that the vitreous transformation temperature, T,, and the devitrification temperature, Td, increase with increasing concentration of the solutions. The temperature of recrystallization, T,, of antemelting, Tam, and of incipient melting, Ti,, could be accepted as constant and independent of the concentration. The results from the literary survey analyzed in this section point out that we could expect the “free” water in the solutions of the basic natural chemical constituents in food products of plant origin to freeze at -20” to -40°C. In the range of -45” to -7O”C, part of the “bound” water in the sugar solution freezes, thus forming vitreous structures.

B. PHASE TRANSITIONS DURING FREEZING AND REWARMING OF FOODS OF PLANT ORIGIN I.

Amount of Frozen Water and Eutectic Temperature

Foods of plant origin are complex multicomponent systems, containing various mineral and organic substances. Generally speaking, upon freezing, they behave in a way analogous to that of ideal solutions, forming pure ice crystals

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327

and interstitial liquids (Kobulachvili, 1961; Marcus, 1974; Mestcheriakov, 1975; Popovski, 1975; Rey, 1964a; Tchernichev er al., 1974). Of course, the liquid phase concentration of the foods is a much more complex process than that of two-component solutions. It is determined by the physicochemical characteristics of the product, the freezing velocity, and the temperature. The quality of foods during freezing depends on the quantitative ratio of the frozen to the unfrozen water inside them. This is why all investigations on phase processes that occur during the freezing of foods have so far been concerned mainly with the problem of establishing the amount of frozen water at different temperatures. In analyzing this index, investigators are faced with another problem-at what temperature the ice formation of the so-called “freezable” water ends. This is the “eutectic” temperature of food products. Foods at -10” to -20°C contain, on the average, 10% unfrozen water (Golovkin et al., 1955; Luikov and Griaznov, 1956), but at any given temperature the amount of frozen water differs in foods of different nature. It has been established that in some fruit purees-apricot, apple, red tart cherry, and plum (Lebedev and Perel’man, 1973)-and in peaches (Fennema and Powrie, 1964) 90% of the water freezes at -16.4”, -17.6”, -23.8”, -26.4”, and - 18”C, respectively, and 98% freezes at -32.9”, -35.4”, -45.0”, -47.0”, and -35.0”C, respectively. In the porous capillary skeleton of plant products, according to Lebedev and Perel’man (1973), unfrozen “free” water can be found at -30°C. In vegetables at -2O”C, 96 to 98% of the water is already frozen (Fikiin, 1973). Some authors assume that the manner of freezing and its duration do not influence the amount of frozen water (Mazur, 1963; Mestcheriakov, 1975). The results of experimental and theoretical studies on the change in the amount of frozen water in relation to the temperature have been expressed both graphically and by semiempirical formulas (Fennema and Powrie, 1964; Fikiin, 1973; Heiss, 1933a-c; Parducci and Duckworth, 1972; Popovski, 1967; Riedel, 1957; Tchijov, 1956). It was established that in no fruit does the quantity of frozen water exceed 90% at -25°C. The analytic formulas of Tchijov (1956), Heiss (1933a-c), and Bartlett (according to Fikiin, 1973) do not reflect the influence of the type of product on the amount of frozen water. They present rather different results, which can be used only for an approximate quantitative evaluation of ice formation. The relationship between the amount of frozen water and the temperature of the product could be used as an orientation in the choice of a freeze-drying program. Popovski ( 1975) has analyzed this relationship (given graphically) for some fruit purees and has established two steps in ice formation: first, when the “free” water freezes (about 75%), and second, when part of the “bound” water, called “bound crystallizable” water (from 7 to 26%), freezes. The author accepts as border temperature at which freezing of the “bound crystallizable” water is achieved when freezing a product intended for freeze-drying. For black

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currant and apple purees this temperature is -3O"C, for red tart cherry puree -4O"C, and for plum puree -44°C. For the freeze-drying process, the problem of the complete freezing of the water in foods is of special importance. In their classical works, Moran (1934) and Birdseye and Taylor (according to Love and Elerian, 1965) support the opinion that the water in foods freezes completely at -40" to -60°C. Moran (1934) proves that beef muscle tissue solidifies completely at -37.5"C, whereas fish fillet still contains unfrozen water at this temperature (presumably bound water). Later, Dickerson and Riedel came to the same conclusion. According to them, the entire free water in fruit and vegetables (Dickerson, 1968) and in fish, poultry, eggs, and bread (Riedel, 1950, 1964, 1968) freezes down to -40°C. Riedel (1968) supposes the remaining water to be bound. It is retained in the proteins and carbohydrates and does not freeze even at very low temperatures. Kuprianoff assumes that the free water freezes down to -3O"C, and the bound water freezes at -50" to -60°C (Lebedev and Perel'man, 1973). On the basis of theoretical assumptions, however, without sufficient experimental arguments, Heiss (1933a-c) predicts the eutectic temperature for meat (containing CaCI, and FeCl,) to be about - 55°C. The thermographic investigations canied out indeed established a small exothermic plateau on the cooling curve at -62" to -65°C. which he considers to be the entectic temperature. The author supposes that in meat and fish the free water freezes almost entirely at -2O"C, and in the range of -20" to -65°C the adsorptionally bound water freezes. Through similar investigations Plank ( 1960) established that the eutectic temperature of meat is -56°C. The investigations of Susmann and Chin (1966), using the nuclear magnetic resonance method on frozen cod muscles, show that the liquid water specter exists down to -68°C and disappears at -70°C. The authors assume that at this temperature all the water in the product freezes, including the bound water. The investigations of Heiss and Plank based on the phase law have served as the basis of the present concept for numerous scientists. They maintain that the eutectic temperature in foods is in the range of -55" to -65°C (Danilov, 1974; Fikiin, 1973; Ginsburg, 1973; Golovkin et al., 1955; International Institute of Refrigeration, 1964; Love and Elerian, 1965; Lozina-Lozinski, 1972; Luikov and Griaznov, 1956; Markova and Chkol'nikova, 1962; Mestcheriakov, 1975; Parducci and Duckworth, 1972; Popmarinov, 1964; Popovski, 1975; Tchijov, 1971). Here "eutectic" refers to the temperature at which all the water in the food freezes (Golovkin er a l . , 1955; Markova and Chkol'nikova, 1962; Popmarinov, 1964). It is not yet clear how the bound water is included-completely or partially. Other results differ, however. Riedel (1961) proves that the phenomenon

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329

observed by Heiss (1933a-c) in meat at -62" to -65°C is not eutectic freezing, thus casting a serious doubt on the generally accepted temperature range of the eutectic temperature. Woolrich (1966) asserts that the most complex salt solutions included in foods have freezing temperatures down to -40°C. On the other hand, the author mentions that orange juice does not entirely solidify at -95°C. Voskresenski ( 1963) assumes that the eutectic temperature is that temperature at which no immobilized (free) water remains. For meat it is -20°C and not -55" to -65"C, as is generally accepted. In freezing at lower temperatures all the water present gradually freezes. Love ( 1 968) has noticed a progressive freezing of water in fish at temperatures below -60°C down to very low temperatures ( - 180°C). Analyzing the freezing of five types of fruit puree, Kondratiuk and Ginsburg (1974) established that the water possessing the lowest binding energy freezes at - 15"C, and the firmly bound water freezes at temperatures from - 15" to -80°C. The complete crystallization of the purees is not accomplished even at - 100°C. Investigations carried out by Mank et al. (1975) by means of nuclear magnetic resonance on the phase transitions during freezing of peas show that the free water freezes at about O"C, part of the physicochemical water freezes at temperatures down to - 10°C; a small amount of the bound water remains unfrozen rather at -50°C. According to data presented by the authors, it is possible for water bound with the ions not to freeze even at temperatures down to -80°C. It is evident that the classical assumptions concerning ice formation in foods need serious reevaluation, and that any approach to this problem must take into account the energy as well as the forms of binding of the moisture with the material.

2 . Phase Transitions in Relation to the Temperature Conditions during Freezing It is impossible to establish a fixed eutectic temperature in food products. It is best to use the concept of a eutectic zone, determined by the wide range of eutectic points in the solutes incorporated in the product. Some investigators admit that the determination of this zone for each food product subjected to freezing and freeze-drying is indispensable in the correct choice of the process parameters (Amoignon and Floch, 1971; Corvazier, 1969; Kondratiuk, 1972; Lakovskaia, 1971; Larousse, 1970; Rey, 1964a, 1974; Simatos et a l . , 1975a; Tobback and Maes, 1971; Vitanov and Nikolov, 1971). The lower boundary of the eutectic zone is associated with two specific temperatures-the temperature of complete solidification, T,,, and the minimum

330

YANCOGUEGOV

temperature of incipient melting, TI,. Their meaning and significance have already been discussed. The meaning of the temperature of incipient melting, Tim,is identical with the classical concept of the eutectic temperature, but its value is much higher than the conventional one (-55" to -65°C). It represents essentially the melting temperature of ice in contact with the highest possible temperature of the solute after all the water that could possibly freeze has crystallized (Rasmussen and Luyet, 1969). The temperature of complete solidification, Tcs,reflects the possibility of supercooling during freeze-drying of food products. It is lower than TI,. During rewarming, the food product, having already reached the state of complete solidification, retains this state in the range T,, to TI,, and the initial melting starts at temperatures higher than TI, (Fikiin, 1973; Popovski, 1975; Rey, 1960a, 1964a, 1974; Stephenson, 1960; Stricker, 1970; Voskresenski, 1963). The temperature increase from T(.sto Ti, allows the partial or complete transformation of some metastable vitreous structures into crystal ones. For the freeze-drying of foods, the temperature of complete solidification, T,.,, should always be reached during freezing (Corvazier, 1969; (hrdova, 1969; Kondratiuk, 1972; Lakovskaia, 1971; Larouse, 1970; Lebedev and Perel'man, 1973; Rey, 1974; Vitanov and Nikolov, 1971). This is especially important for fruit juices and concentrates rich in sugars. When a product has reached the temperature T,.,, then all the free water in it has already frozen (Simatos et ul., 1975a; Tobback and Maes, 1971), and a further decrease in the temperature is not feasible. The temperature of complete solidification for various food products is established in the range of -20" to -45"C, and in some cases-for example, fruit juices and concentrates-in the range of -50" to -70°C (Owades and Dano, 1969; Rey, 1964a, 1974; Simatos et al., 1975a; Tobback and Maes, 1971). It is strictly individual for each product. In many vegetables, the temperature T,, in practice coincides with Ti,. For fruits this difference does not exceed several degrees ( h d o v a , 1969; Simatos et al., 1975a). The opinion prevails that the temperature should be lowered by as much as 10°C below Ti, for the complete solidification of the greater part of the plant products to be guaranteed (Amoignon and Floch, 1971; Owades and Dono, 1969; Rey, 1964a, 1974). For fruit concentrates this difference is 15" to 20°C (King, 1971; Rey, 1964a; Rowe, 1960; Saint-Hilaire and Solmes, 1973; Sauvageot, 1967; Simatos er al., 1975a). In freeze-drying plant products it is necessary that they be frozen to the temperature Tc.sand that the sublimation be carried out at a temperature not higher than Ti,,,. The minimum incipient melting temperature, Ti,, is assumed by many scientists to be the optimum theoretical temperature of sublimation at which the initial properties of the product are preserved (Fikiin, 1969; Flink, 1974a; Popovski, 1975; Rey, 1964b, 1974; Sim:ltos et al., 1975a). However,

PHASE TRANSITIONS OF WATER

33 1

this is not always technically accepted or economically feasible. We should like to repeat that, at the low temperatures at which a freeze-dryer operates, a decrease of 10°C in the sublimation temperature causes about a 50% increase in the cost of electricity (Lorentzen, 1974). The experimental investigations show that some food products can be freezedried by allowing a certain amount of unfrozen water to remain in them without causing a visible change in their quality (Fikiin, 1973; Flink, 1974a; Ito, 1971; Mackenzie, 1965, 1974; Maltini and Giangiacomo, 1976; Popovski, 1975; Rey, 1964a; Tobback and Maes, 1971; Voskresenski, 1963). This finding has determined a new temperature limit, called the maximum temperature of incipient melting, Ti,, after which point an intensive melting of the ice starts (Fig. 7). The temperatures Ti, and T i , determine the working zone of sublimation. Drying at Ti, ensures the maximum velocity of the process, but it can spoil the guarantees the quality, but it may appear product. In contrast, drying at Ti,,, economically unprofitable. The two temperature limits can be established only experimentally. Many investigators recognize a partial incipient melting of the ice during the freeze-drying of foods (Amoignon and Floch, 1971; Corvazier, 1969; Fikiin, 1969; Ginsburg and Kondratiuk, 1974; King, 1974; Lebedev and Perel’man, 1973; Mackenzie, 1974; Maltini and Giangiacomo, 1976; Owades and Dono, 1969; Rey, 1964a; Simatos et al., 1975a; Tobback and Maes, 1971; Voskresenski, 1963). King (1974) admits such a possibility even for fruit juices. According to King, the frozen structure of a fruit juice is preserved, while the viscosity of the solidified amorphous mass remains in the range of lo7 to 1Olo N *sec/m2.A “structural collapse” starts at a viscosity below this limit. The

working zone

tT FIG 7

Working zone for industrial freeze-drying. From Rey (1964a). Courtesy of the author.

332

YANCOGUEGOV

author's calculations and experiments show that the "collapse" temperature, T,, is higher than T i , . Another group of investigators does not admit the presence of liquid water fractions, even in very small quantities, in a product undergoing freeze-drying and especially in liquid foods (Davies, 1966; Gane, 1960; Goldblith and Karel, 1969; Greaves, 1963; Kaminarskaia e t a f . , 1963; King, 1971; Kondratiuk, 1972; Larouse, 1970; Maltini, 1974b; Monzini et al., 1974; Ryan, 1965; Saint-Hilaire and Solmes, 1973; Sauvageoyt, 1967; White and Cakebread, 1966). According to them, the interstitial melting could lead to serious chemical injuries, especially in the vitamins, aroma substances, and color, and to the denaturation of the product. The primary structure of the food is injured also. It can shrink and be deformed (Bellows and King, 1972; Blair, 1966; Larouse, 1970; Marcus, 1974). Its constituents start migrating and change their natural locations (Marcus, 1974). Very often this leads to the formation of dense, glazed films on the surface, greatly hindering the mass exchange and rehydration (Bellows and King, 1972; Blair, 1966; Kaminarskaia et d., 1963; King, 1971; Popovski and Ivasink, 1965; Rey, 1974). In liquid plant products the partial melting may cause foaming accompanied by a sharp shrinking of the initial structure-"structural collapse. " The material gets sticky and dark-colored and during storage changes into a compact dense mass (Gane, 1963; Greaves, 1963; King, 1971; Rey, 1964a, 1974). The investigations of the phase transitions in fruit and vegetables aiming at establishing the process temperatures during freeze-drying are a new trend in food technology, and only a limited number of publications have been devoted to this problem. Rey (1964a), the founder of this approach, established the values of the temperatures Ti, and T i , for strawberries (-32" and -2O"C, respectively). Only a few data on the complete solidification temperatures of some fruit and vegetables have been published to date. Thus, for apples a temperature of -26°C is indicated (Forrest, 1963)-quite high in our opinion; for carrots and other vegetables rich in sugar, -33°C (Tucker, 1963); for cherries, -40°C (Gutschmidt, 1968); and for potatoes, celery, green beans, carrots, and cabbage, the eutectic temperature interval is -25" to -40°C. The latter can be established reproduceably by DTA and is not affected by the freezing method (Duckworth, 1971). Other authors indicate the eutectic temperature of vegetables to be about -15"C, and the temperature of complete solidification to be from -15" to -20°C (Tobback and Maes, 1971). Sauvageot ( 1967) investigated the phase transitions of some fruits during freezing and determined the temperatures of incipient intensive melting (actually T i , ) , recommending them for application during industrial freeze-drying. For the various fruit species these temperatures are as follows: apricots, - 18" to -20°C;

PHASE TRANSITIONS OF WATER

333

strawberries and raspberries, - 17" to -20°C; and apples and pears, - 18" to - 24°C. By measuring the electric resistance, p , curdova (1969) determined the eutectic zone (in our opinion, from Ti, to Tf,) of some plant species as follows: apples, -26" to -38°C; strawberries, -20" to -28°C; raspberries, -12" to -27°C; lemons, -23" to -27°C; apricots, -22" to -27°C; peaches, -18" to -22°C; carrots, -26" to -40°C; celery, -21" to -28°C; potatoes, -21" to -28°C; and apple juice, -22" to -36°C. The zone of sharp decrease in p below the value of lo6 R . c m was accepted as eutectic. Using DTA, the following values for the eutectic temperature, Tim,of the same products were established: apples, - 38°C; Strawberries, -30°C; raspbemes, -24.5"C; lemons, -27°C; apricots, -25°C; peaches, -23°C; carrots, -32°C; celery, -29.5"C; and potatotes, -25°C. Generally speaking, the experimental technique is still at an unsatisfactory stage, and the differential thermograms are quite diffused and uncharacteristic. Fikiin ( 1969) studied the phase transitions of raspberry puree and established devitrification phenomena at -94" and -78"C, eutectic melting at -55" to -6O"C, and intensive melting at -44°C. Popovski ( 1 975) investigated the phase transitions in five types of fruit puree. He established three temperature zones of melting: the zone of incipient melting, the transition zone toward incipient intensive melting, and the intensive melting zone. For applesauce the first zone is in the range of -29" to - 13"C, the second is missing, and the third is from - 1 1" to -9°C. For red tart cherry puree the picture is identical: the first zone is from -43" to -22"C, the second is missing, and the third is from -22" to - 14°C. For plum puree, the first zone is from -45" to -25"C, the second is from -28" to -2O"C, and the third is from -20" to - 14°C. Maltini ( 1 972; Maltini and Giangiacomo, 1976) established incipient melting in strawberries at -34°C and intensive melting at - 15°C. According to the same author, these temperatures for bananas are -27" and - IO'C, respectively. The investigations of Fikiin and Guegov (1971a,b) and Guegov (1978, 1979a,b) on phase transitions in fruits and vegetables show that the incipient melting has no connection with the eutectic phenomenon and that it takes place in a rather broad temperature interval of 10" to 15°C. the free water in the different fruit species starts to melt at -45" to -4O"C, and in vegetables at -45" to -27°C. Intensive melting of the ice crystals in the various fruits starts at -28" to -2O"C, and in vegetables at -24" to - 15°C. In some fruits, and very rarely in vegetables, vitreous transformation and devitrification phenomena connected with the formation of vitreous structures during freezing can be observed during rewarming. It may be presumed that the aforementioned phenomena affect part of the bound water, which possesses less binding energy.

334

YANCOGUEGOV

Typical differential thermograms and curves of specific electric resistance, illustrating the,phase transitions in fruits and vegetables, are shown on Fig. 8; the temperature boundaries established for incipient melting are given in Tables I and 11.

For most of the fruits and for all vegetables the incipient melting intensity is relatively low, and in this case the freeze-drying can be carried out at the maximum incipient melting temperature, T',,,,. For red tart chemes, quinces, and oranges, with a medium incipient melting intensity, we suggest freeze-drying in the medium range of the incipient melting temperature zone. For fruits with a relatively high initial rewarming intensity, it is recommended that the freezedrying be camed out at temperatures near (for cherries) or equal to (for lemons) the minimum incipient melting temperature, Ti, (Fikiin and Guegov, 197 la,b; Guegov, 1978). Almost all chemical and structural changes in the frozen products are related to the presence of water fractions in them. At temperatures lower than Ti,,, the amount of uncrystallized water is very small; this is why undesirable changes in the product should be kept to the minimum. Intensive melting starts at temperatures above TI,. The TI,,, should therefore be accepted, according to Guegov (1978), as the point below which the stability of the frozen product during cold storage can be guaranteed.

a

- sour c h e r r y ---- raspberry -- p o t a t o e s peas

.!1 L

5

FIG. 8. Differential thennograms ( a ) and specific electric resistance curves ( b )of some fruits and vegetables. Resistance ( p ) is calculated in R.cm. From Guegov (1978).

335

PHASE TRANSITIONS O F WATER TABLE 1 BOUNDARY TEMPERATURES AND AMOUNT OF THAWED WATER DURING INCIPIENT REWARMING OF FRUITS’

Minimum temperature of incipient melting, TI, (“C)

Product Apples, Golden Delicious Apples, Red Delicious Apples, Jonathan Pears, Bartlett Pears, Clapp’s Favorite Pears, Golden Doyenne Quinces Plums, Renglot Dark blue plums White cherries Red cherries Red tart cherries Peaches, Early Elberta Peaches, Halle Peaches, Mary Gold Apricots Strawberries, ZengaZengana Strawberries, Madame Mouteau Raspberries Grapes, Bolgar Grapes, Muskat of Hamburg Grapes, Dimiat Lemons Oranges Bananas ‘I

From Guegov (1978).

Maximum temperature of incipient melting, T i m (“C)

Determined by DTA

Determined by P

Determined by DTA

Determined by P

-45.5

-43.5

-29.0

-29.0

-39.0 -39.8 -37.5 -43.3

-45.3 -41.5 -42.6 -36.3 -36. I

-38.0 -37.0 -40.0

-41.5 -43.0 -45.0 -42.0 -41.0

-23.4 -27.5 -20.2 -20.7

b b

-28.5 -25.3 -24.1 -21.5 -22.0

6.7

b b

6.0 5.7

-22.0 b -26.0

-36.5

-34.0

-20.4

b

-38.5 -40.9 -39.0

-38.0 -39.0

-21.8 -24.0 -25.0

-28.0

-24.6 -26.8 -24.3

b

b

-28.0

’ The temperature cannot be determined from the experimental curves.

6.6

b b

-24.7 -22.8

-43.5

3.2

5.9

-41.3 -38.0

-44.5

(%)

-28.5 b b

-34.0 -37.0 -40.5 -36.0

-37.6 -45.3 -42.8

Amount of thawed water at temperature Tim

5.5

5.6

4.9 6.8

6.3 6.4

336

YANCO GUEGOV TABLE I1 BOUNDARY TEMPERATURES AND AMOUNT OF THAWED WATER DURING INCIPIENT REWARMING OF VEGETABLES"

Product Early tomatoes Late tomatoes Peppers Eggplants Early potatoes Late potatoes carrots Onions, fresh Onions, bulbs Leeks Small pumpkins Small radishes Green peas Broad beans Green beans Cabbages Celery (leaves) Spinach Parsley Dill Mushrooms I'

"

Minimum temperature of incipient melting, TI,,,("(3

Maximum temperature of incipient melting, Ti, ("C)

Determined by DTA

Determined by DTA

-21.8 -27.3 -39.2 -41.3 -45.8 -42.5 -27.8 -36.5 -31.5 -43.8 -39. I

Determined by P

-26.5 -38.0 -39.0

-27.0 -33.2 -43.7 -44.3

- 16.2 - 14.8 - 19.3

h h

(96'0)

4.2 6.3

-28.0 - 19.5

-34.0 -39.0 -35.0 -31.0 -25.5 -40.0

- 36.3

-39.5

Determined by P

Amount thawed water at temperature TI",

-36.0 -33.0 -34.0 -28.0 -34.0 -44.0

h h

- 18.7

-20.5 - 17.8 -23.8 -21.3 - 16.8 -24.5 -21.5 - 18.8

-17.2 -15.5 -22.2 - 19.5

-24.0 b h

-26.0

- 19.0 -20.0 b - 19.0 -22.0 -24.5

7.1 7.0

4.2 4.6 2.3 7.3 2.3 4.2 6.8

5. I 6.7 5.8 8. I

From Guegov (1978). The temperature cannot be determined from the experimental curves.

The crystallization of fruit juices has been better studied than that of fruit and vegetables. Here, again, the first investigations were made by Rey (1964a). Using DTA, he established the following values for Ti, and T i , for fruit juices: orange juice, -43" and - 18°C; apple juice, -43" and - 18°C; grape juice, -43" and - 27°C; lemon juice, - 22" and - 19°C; peach juice, - 2 1" and - 18°C; and apricot juice, -29" and -22°C. Sauvageot (2967) determined the temperatures for the incipient intensive melting (Ti,) of some juices: peach and apricot juices, -18" to -20°C; strawberry

PHASE TRANSITIONS OF WATER

337

and raspberry juices, - 17" to -20°C; apple and pear juices, - 18" to -24°C; grape juice, -30" to -35°C; and tomato juice, - 16°C. The author recommends these as the optimum processing temperatures for freeze-drying. In some of his other investigations, Sauvageot (1969) shows that in many fruit juices even at -40°C some liquid water still remains. According to the author, orange juice solidifies completely at -75°C. and upon rewarming devitrification was observed at -41.5"C. Devitrification of raspberry juice was observed at -44°C. During the experiments on the phase transitions in orange juice, Rey (1964a) observed devitrification (crystallization) at -42.5"C, followed by incipient melting at -32°C. Complete solidification of the system was obtained at -50°C. Jabarit (1970) investigated the phase transitions in orange juice, pointing out that complete solidification, accompanied by vitrification, was achieved at -60" to -70°C (T(.,).The author has observed devitrification at -5O"C, incipient melting at -32°C (Tim),and incipient intensive melting at -22°C (Ti,). He points out that oranges have a temperature of complete solidification at about -70°C. The investigations of Fikiin and Guegov (1972a) and of Guegov (1978, 1980a) established typical phenomena of vitreous transformation and devitrification in fruit, preceding (during rewarming) the incipient melting. Instead of eutectic phenomena, a progressive melting was registered. Typical differential thermograms and curves of specific electric resistance, illustrating the phase transitions in fruit and vegetable juices, can be seen in Fig. 9, and the temperatures of the various phase transitions are given in Table 111. Flink ( 1974a) and Ito ( 1 97 1) assume that the incipient melting temperature of fruit juices and extracts should be below -25°C in all cases. It can usually be observed in the range of -30" to -45°C. Thus, for citrus juices Ti, is about -4O"C, for grape juice about -40°C (Ginsburg and Kondratiuk, 1974; Popovski, 1973, for coffee extracts -23" to -25°C (Flink, 1974b), and for apple juice -25" to -30°C (Gane, 1960; Ginsburg and Kondratiuk, 1974; Popovski, 1975). The studies on the freezing of fruit juices show that they are kinetically hampered by solidification at the "eutectic" temperature, Ti, (King, 1974). Complete solidification often takes place at very low temperatures, even at -7O"C, whereas during freeze-drying they melt very easily (Jabarit, 1970; Rey, 1964a). Some data are also available for higher temperatures of solidification. Thus, for grape juice complete solidification is established at -40" to -45°C (Heldman, 1972), for most juices and coffee extracts at about -50°C (Tobback and Maes, 1971), and for apple juice at about -30" to -35°C (King, 1971). Saint-Hilaire and Solmes (1973) investigated the influence of the chemical composition on the freeze-drying temperature. The author presumes that two

338

YANCOGUEGOV 1180

-160

-140

r

-120

-100

-80

-60

-LO

-20

a k(('ck30 -70

I

I

-60 -50

-LO

-30

-20

-10

- - apple juice - - - - quince n e c t a r - - tomato j u i c e b

FIG. 9. Differential thermograms ((I) and specific electric resistance curves ( b ) of some fruit and vegetable juices. Resistance @) is calculated in R . c m . From Guegov (1978).

factors exert a basic influence: the ratio of reducing to nonreducing sugars, and acidity. He established the so-called "threshold" temperatures of sublimation, whose meaning is idential to Ti,. For the fruit juices they are as follows: orange -32°C; grapefruit -34.5"C; lemon -39°C; and apple -41°C. Ginsburg and Kondratiuk ( 1 974) determined three temperature zones, corresponding to the phase transitions in apple and grape juices during rewarming from -80" to 0°C. They are the incipient melting zone, the incipient intensive melting zone, and the intensive melting zone. In apple juice ( 1 1.4% solids) the three zones are within the following ranges: -35" to -21"C, -21" to - 1O"C, and - 10" to -2.5"C. For grape juice (16% solids) they are -40" to -3O"C, -30" to - 12"C, and - 12" to -3°C. The authors suggest that the freeze-drying should be carried out at the temperature conditions of the first zone, and the incipient melting at a temperature below the lower boundary of the same zone (-35" and -4O"C, respectively). As has already been pointed out, because of the absence of a solid cell skeleton in fruit and vegetable juices a real danger of structural collapse exists of the freeze-drying temperature is incorrectly chosen. Considering these facts, Fikiin and Guegov (1972a) and Guegov (1978) recommended TI,,, as the processing temperature only for the freeze-drying of cloudy juices and nectars possessing some structural stability in their frozen state. The experiment carried out by Guegov (1978) with freeze-drying of the products givein Table I11 confirm this

TABLE 111 PHASE TRANSITION TEMPERATURES IN FRUIT AND VEGETABLE JUICES"

Incipient temperature of devitrification (crystallization), Td ("C)

Incipient tempera. ture of vitreous transformation, T , ("C)

Product Strawberry juice (clear) Strawberry juice (cloudy) Raspberry juice (clear) Red tart cherry juice (clear) Apple juice (clear) Apple juice (cloudy) Grape juice (clear) Cherry juice (clear) Quince juice (clear) Peach nectar Apricot nectar Quince nectar Tomato juice Red pepper juice Celery juice (leaves) Celery juice (roots)

"

Determined by DTA

Determined by P

-63.5

Determined by DTA

Determined by P

Determined by DTA

Determined by P

b

Maximum tempera ture of incipient

Determined by DTA

Determined by P

-26.0

-40.0

-55.0 b

Minimum temperature of incipient melting. Ti, ("C)

-29.0

-40.0

-67.0 -68.0

-54.0 -70.0

-59.0 -60.0

-46.5 -51.0

-43.0 -46.0

-42.5 -45.0

-26.5 -34.5

-28.0 -34.5

-75.0 -60.0

-55.5

-50.0 -57.0

-51.1

-46.0 -45.5

-44.0

-28.0 -25.0

-30.0

-62.5 b

-64.0 -66.0 -70.0 b

-59.0 -60.0 -62.0 b b

-60.0 -50.0

-51.0 -58.0 -56.0 b

-46.5 -46.0 -47.0 -46.0 -48.0 b b

-45.0 -30.0

-64.0

From Guegov (1978). The temperature cannot be established from the experimental curves.

-53.5

-41.0 -44.5 -45.5 -29.5 -34.0 -26.0

-41.5 -41.5 -42.0 -41.5 -43.0 -44.0

-30.0

-33.0

-27.0 -27.5 -26.0 - 19.0 - 19.0 -20.0

-29.0 -26.5 -28.0 -26.0 -28.0 -27.0 b

-24.5

340

YANCOGUEGOV

recommendation; at the sublimation temperature, Tf,,not a single case of structural collapse was observed. For clear juices the author recommends that freezedrying be carried out at the temperature Tim, or at a temperature no more than 3°C higher. For reasons already stated, Guegov (1978) recommends Tlimas the temperature for the cold storage of frozen fruits and vegetables. Fruit and vegetable concentrates present a very interesting although difficult material for freeze-drying. The investigations of the phase transitions taking place in them are significant from the point of view of both pure science and practical applications. Accurate complex investigations on phase transitions in citrus juices and concentrates, carried out by Maltini (1971, 1974a,b), Maltini and Anselmi (1973), Monzini and Maltini (1971), and Monzini ef al. (1974) by means of DTA, DSC, and dynamometric techniques, established a large number of phase transitions (Fig. 10). In lemon and orange concentrates (10 to 40%), a typical endothermic ISC

Mn

0

a

II

6

4 2

P .-

0

DTC

-60 -50

-20

-

I

FIG. 10. Thermophysical properties of lemon juice: DSC, differential scanning calorimetry; p , electrical resistivity; DTC, dynanometry at controlled temperature; T g . glass transition temperature; Ts, incipient softening temperature; T,, incipient melting temperature of the free crystallization water. From Maltini (1974a). Courtesy of the author and IIR.

PHASE TRANSITIONS OF WATER

34 1

effect was noticed at -65"C, identified as vitreous transformation, whereas at -40°C it is incipient melting. Three zones in which the phase transitions in citrus juices take place were established: the melting zone of free water (above - 18"C), the zone of incipient melting of the concentrated matrix (-40" to - 18"C), and the zone of complete solidification (-65" to -40°C) (Maltini and Anselmi, 1973). Extending these studies, Maltini (1974a,b) and Monzini er al. (1974) determined exactly the devitrification temperature--56" to -57"C, which corresponds to the temperature of complete solidification, Tcs. They point out that vitrification cannot be observed in all experiments. Incipient softening (melting) in the interval -40" to -38°C was established very clearly (in our opinion this corresponds to the temperature Tim)as well as the incipient melting temperature of the free water at -25°C (Tf,). The phase transitions registered are very similar to those in sugar solutions. They appear at the same temperatures regardless of the juice concentration, but their range is directly proportional to the concentration. Maltini (1974b) points out that the presence of large amounts of sucrose in the juices renders the solidification more difficult-for example, in grape, apple, and raspbeny juices. The investigations on lemon juice with 55 to 80% solids show that in concentrations above 68% all the water is bound. In concentrates up to 78% the water freezes in an amorphous structure (glass) in the range of -65" to -75°C. In concentrations wit solids content above 80% the water does not freeze at all (Maltini, 1974b). The dynamometric trials (Fig. 10) show that concentrates do not change their solid state in the range of -70" to -45°C-that is, the phenomena registered by DTA and DSC take place in the solid phase without causing substantial melting. Thus, Maltini and Anselmi (1973) and Maltini (1974b) was the first to prove that the phenomena whose types are identified at present only by analogy with other products indeed represent vitreous transformation and devitrification (crystallization). Fikkin and Guegov (1972b) and Guegov (1978, 1980b) established various phase transitions in fruit and vegetable concentrates, similar to those in sugar solutions. They observed well-defined phenomena of vitreous transformation and devitrification. According to these authors, the temperature interval and the intensity of the phenomena depend on the concentration of the solids. The process of incipient melting was quite intensive, especially in concentrates of 50 to 65%; this is why the temperature Ti, could not be established in them. These investigations support Maltini's stand ( 1977) concerning the existence of certain critical concentrations in which the nature of the phase transitions changes very sharply. The first critical concentration, according to our findings (Guegov, 1978), is about 55%, and the second about 65%. In view of the values

342

YANCOGUEGOV

obtained for Ti,,there is a practical importance in freeze-drying concentrates with solids content below 40%, with lyophilization taking place at Ti, for concentrates without fruit flesh (for example, apple concentrate), or at TI, for concentrates with fruit flesh (for example, tomato concentrate). Typical differential thermograms and specific electric resistance curves illustrating the phase transition in fruit and vegetable concentrates are presented in Fig. 11; the temperatures of the various phase transitions are given in Table IV.

-160

-140 -120

-100

-80

T,PC)

-60

-LO

-20

0-5

=p

a

FIG. I I . Differential thermograms (a) and specific electric resistance curves (b) of apple concentrates. Resistance @) is calculated in Cl.cm. From Guegov (1978).

TABLE IV PHASE TRANSITION TEMPERATURES IN FRUIT AND VEGETABLE CONCENTRATES'

Incipient temperature of vitreous transformation, T , ("C)

Raspberry syrup Raspberry syrup Red currant syrup Apple concentrate Apple concentrate Apple concentrate Apple concentrate Apple concentrate Apple concentrate Apple concentrate Orange concentrate Peach concentrate Peach concentrate Tomato puree Tomato puree Tomato puree Tomato puree "

"

(69 4%) (54 4%) (69 8 1 ) (70 5%) (65 0%) (60.5%) (50 0%) (40 0%) (30 0%) (20 0%) (53 4%) (24 0%) ( I 6 0%) (35 0%) (29 0%) (22 7%) (17 61;)

Incipient temperature of devitrification (crystalliration). Td ("C)

Minimum temperature of incipient melting, T , , ("C)

Maximum temperature of incipient melting, T ; , ("C)

Determined

Determined

Determined

Determined

Determined

Determined

Determined

Determined

by DTA

by P

by DTA

by P

by DTA

by P

by DTA

by P

-77.5

-70.0 -64.0

h

h

h

h

-56.0

-50.0

h h

h

-84.0 h h

h b

h

-91.5

-38.0 -53.0 -45.0 -45.0 -44.5 -43.5 -80.0 -35.5 -31.0 -40.0 -38.5 -37.0 -37.0

-41.5 -50.0 -47.0 -46.5 -43.5 -42.5 -55.0

- 102.0

-84.0 -79.5 -87.5 -96.5 -63.0 -70.5 -69.5 -69.5 - 108.0 -66.5 -68.0 -68.0 -64.0 -64.0 h

-81.0 h h -65.0 -71.5 -71.0 -70.0 b

-52.0 -71.0 -55.0 -56.0 -56.0 -55.5 -88.0 -55.0 -55.0 -54.0 -52.0 -55.0 h

From Guegov (1978). The temperature cannot be established from the experimental curves.

-57.0 -65.0 -51 .O

-69.5 -54.0 h h

h

-40.0 -39.0 -37.5 -37.0

h h h h b

-33.0 -30.5 -28.0 -57.0 -24.5 -21.5 b h

-26.5 -23.5

h h b b h

-31.0 -28.5 -39.0

344

YANCO GUEGOV

V.

PHASE TRANSITIONS AT SUPERLOW TEMPERATURES (-70" TO -196°C)

The phase transitions in certain objects at superlow temperatures are associated with the freezing of water into a vitreous state and the resultant vitreous transformation, devitrification (crystallization), vitromelting, and transition from vitreous into cubic ice during rewarming. One more type-recrystallization-is known. A.

SOLIDIFICATION INTO A GLASSY STATE (VITRIFICATION)

As the theory of homogeneous nucleation shows, at temperatures below a certain point (for water, -150°C) the formation of crystal structures is impossible-the molecules are not mobile enough to form crystal lattices (Luyet, 1951; Simatos et al., 1975a). Consequently, if a product frozen to 150°C still contains water fractions, upon a further decrease of the temperature these water fractions could solidify only to a vitreous state. This could be accomplished theoretically in the range of 0" to - 150°C during ultrafast cooling (Fikiin, 1973; Golovkin et uf., 1955; Luyet, 1951; Simatos et al., 1975a; Smith, 1954). The vitreous state is easier to create in substances with low nucleation velocity, as is the case with a large number of organic substances. In pure water, however, this is extremely difficult, requiring very high cooling velocities (Luyet, 1939a,b; Meryman, 1957; Plank, 1960; Simatos er al., l975a)according to Stephenson (1960), above 5000"C/sec, which means that the object should reach - 100°C in 0.02 sec. This phenomenon was observed in the range - 125" to - 150°C. During rewarming of the vitreous water, intensive crystallization takes place at -129°C (Plank, 1960) or at -132°C (Meryman, 1957)transition into ice I, which is not preceded by vitreous transformation (Bellows and King, 1972; Fikiin, 1973; Meryman, 1957; Plank, 1960; Smith, 1962). This crystallization ends at - 120°C (Plank, 1960). In contrast to pure water, according to Simatos et al. (1975a), many solutions can be partially or completely solidified into a vitreous state. The vitrification of water in glycerol solutions has been investigated most thoroughly (Bohon and Conway, 1972; Lusena, 1960; Luyet, 1957; Rasmussen and Mackenzie, 1968; Rey, 1959a,b; Smith, 1962; Vinograd-Finkel et al., 1973). During rewarming, and depending on the concentration of the solution, these vitreous formations cause various phenomena of vitreous transformation ( - 123" to -86°C) and of devitrification (-98" to -75°C). It should be noted that these phase transitions are at present accepted as references and analogs in interpreting similar phenomena in other objects. The appearance of vitreous structures in the superlow temperature interval was established also in certain protective media (Luyet and Rasmussen, 1967; Ras-

PHASE TRANSITIONS OF WATER

345

mussen and Mackenzie, 1968), concentrated sugar solutions (Fikiin, 1969; Luyet, 1939a,b, 1951, 1957), amino acid solutions, gelatin, albumin, gums (Lusena and Cook, 1954; Luyet, 1957), alcohol and glycol solutions (Luyet, 1939a,b, 1957; Rey, 1964b), and even inorganic salt solutions (Fikiin, 1969; Luyet, 1939a,b; Rey, 1960b; Vuillard, 1957). By means of dielectroscopy,Simatos ( 1965) investigated the behavior of water in starch solutions and casein during freezing. She established typical changes in the dielectric constant at -1OO"C, which according to the author, could be connected only with the phase transition of the bound water. It is commonly accepted that at superlow temperatures the vitreous transformation and devitrification in the solutions take place only once. There are some communications, however, suggesting that two successive transformations of this type could take place. Fikiin (1969) established in citric acid solutions two successive transitions of vitreous transformation-devitrification at - 105°C and at -58°C. The author has also observed devitrification and recrystallization phenomena in pectic solutions of glucose and pectin as well as in raw cellulose solutions in the range of - 106" to -83°C. By means of the accurate DTA technique, Bohon and Conway (1972) established the double transition, vitreous transformation-devitrification, in glycerol solutions. All the above-mentioned substances are part of the composition of food products. The common point in all these examples is that the vitreous transformation and crystallization zone is between - 130" and -80°C. Vitreous transformation and crystallization phenomena in blood plasma have been determined by the DTA and DSC methods (Bonjour et af., 1975; Simatos, 1974; Simatos et al., 1975b). These investigations are very interesting in that, at a certain water content, no crystallization has been observed after the vitreous transformation (this could be explained by the high viscosity of the solution), and a smaller or larger amount of amorphous mass remains in the object until the beginning of the incipient melting, Ti,. The DTA and DSC curves resemble to a large extent those established for beef (Riedel, 1961) or egg albumen (Duckworth, 1971). Bonjour et al. (1975) assume that only part of the bound water freezes into a vitreous state, and that another part is altogether unfreezable. The fact that the solidification of part of the water into a vitreous state can take place during slow freezing or even after a certain period at the devitrification temperature deserves special consideration (Bonjour et al., 1975; Luyet, 1970; Simatos, 1974; Simatos et a l . , 1975a). According to Simatos et al. (1975a), it is not certain that in all these cases we have vitrification in the full sense of the word. It is very hard to draw a distinct line between completely amorphous materials and those containing submicroscopic crystals.

346

YANCOGUEGOV

The volume of the solidified amorphous mass and hence the intensity of the processes of vitreous transformation and devitrification depend on the concentration and nature of the dissolved substances and the cooling velocity, according to Fikiin (1973) and Simatos et af. (1975b). Both DTA and DSC show that, the higher the freezing velocity, the higher the percentage of the amorphous mass is. But it seems, according to Simatos et ul. (1975a), that a minimum amount of amorphous mass always exists in case the nature of the solution permits vitrification even after slow freezing. Part of the vitrified water crystallizes during rewarming at temperatures higher than the pure water devitrification temperatures. Another part, however, remains in the amorphous state and does not devitrify. This vitreous part is formed even during very slow cooling; the amount is constant and does not depend on the initial concentration of the solution. According to Fikiin (1973), the vitrification at superlow temperatures of a certain amount of the water in the same object and under identical conditions takes place at different temperatures. In general, the devitrification temperature increases with the initial concentration of the solution, if the latter has been quickly cooled. If the cooling is slow, the devitrification temperature does not depend on the initial concentration (Bohon and Conway, 1972; Luyet, 1960; Simatos et u l . , 1975a). What are the possibilities for vitrification of intact biological objects? There is a common point of view that this is possible during ultrafast cooling at several hundred degrees per second (Love, 1968; Lozina-Lozinski, 1972; Luyet, 1951; Mazur, 1963; Meryman, 1957, 1960; Shimada et al., 1972; Simatos et al., 1975a; Smith, 1954, 1962; Stephenson, 1960). This means that during freezing in liquid nitrogen the object should be less than 0.1 mm thick (Luyet, 1951). Many articles have been published on the vitrification of numerous biological objects (Greaves, 1963; Lebedev and Perel’man, 1973; Love, 1968; Luyet, 1951; Mathlouti, 1974; Mazur, 1963; Meryman, 1957, 1960; Rey, 1959b; Smith, 1954, 1962). Absolute amorphous structure, however, has been established with certainty in only one object-plant epidermis-according to Greaves (1963) and Luyet (195 1 ) . The unanimous attitude is that “vitrification” of tissues, in the sense of “amorphousness,” cannot be obtained even during ultrafast cooling (Love, 1968; Meryman, 1957, 1960; Smith, 1962). It has been established, by means of x-ray diffraction and electron microscopy, that “vitrified” tissues contain ultrasmall crystals, dispersed in an amorphous medium (Love, 1968; Luyet, 1957; Meryman, 1960; Mohr and Stein, 1969; Shimada et ul., 1972; Stephenson, 1960). According to the old classical concept, solid bodies are either crystal or amorphous. The possibility for the existence of intermediate states has been rejected. Vitrification is considered as the transition from a liquid state into a solid, amorphous one, devitrification from an amorphous into a crystal one, and vitromelting from an amorphous into a liquid state.

PHASE TRANSITIONS OF WATER

347

Recently a certain degree of “crystallity” has been established in glasses, and so-called “crystallites” are often mentioned. Vitrification is the formation of this “crystallite” system, and devitrification is the transformation of a “crystallite” into a clearly shaped crystal (a kind of recrystallization). The latest conceptions indicate that many substances, which we call glasses, possess a semicrystal structure and that their “vitreous” state may be a phase of a complex crystallization process. There are many intermediate stages between the amorphous and crystal states, depending on the degree of arrangement and the amount (percentage) of the material that has reached crystallity (Love, 1968). It is such a state of the vitreous structure-that is, an amorphous mass containing ultrasmall crystals-that can be seen also during ultrafast freezing, whereas a complete amorphous state of a biological object, according to Lozina-Lozinski (1972) and Shimada et al. (1972), has not yet been achieved. This is why, in this discussion, by “vitreous” and “glassy” we do not mean “amorphous” states, since that expression combines a whole group of structures, and not a given state. During slow or fast freezing, according to Smith (1954), complete vitrification of the water in the cell cytoplasm is impossible. At temperatures below - 129”C, however, it is possible for intracellular vitreous water to exist in combination with crystal water (Mazur, 1963; Smith, 1954). Investigations of various food products confirm the possibility that vitreous structures are formed at superlow temperatures. During normal freezing conditions this is brought about by substances that decrease the crystallization velocity-for example, small amounts of alcohols, glycols, sugars, and albumins. During the investigation of the specific heat capacity of muscle tissue, Riedel (1961) observed a vitreous transformation at - I00”C. Duckworth’s (197 1) research by DTA and DSC on the freezing of celery, fish, and egg albumen to - 196°C established phase transitions in the region -80” to - 150°C. Only a certain amount of the bound water takes part in this transition. Probably this represents a transformation followed by crystallization. These phenomena resemble those that Riedel ( 1961) registered by DSC. The devitrification phenomena were observed by Fikiin (1969) in raspberries in the intervals around -94°C and -78°C. He assumed all these effects to be due to the solidification of the bound water. Fikiin and Guegov (1971a.b) and Guegov ( 1978) established that vitreous transformation and devitrification phenomena in some fruits (apricots, pears, lemons, grapes), but very rarely in vegetables (parsley, leeks), occur in the superlow temperature region. These phenomena are not distinctly expressed. The authors assume that they probably reflect the behavior of small fractions of bound water, possessing a large degree of binding energy. The investigations of Fikiin and Guegov (1972a) and of Guegov ( 1978) point out that in fruit and vegetable juices phase transitions are not observed at superlow temperatures. Complete thermodynamic stability exists at temperatures

348

YANCOGUEGOV

below -130°C. The authors explain the absence of phase transitions by the aforementioned products ’ low content of native hydrocolloids and highmolecular-weight compounds, which are able to bind water and which have remained unchanged during the thermal processing (pasteurization) and clarification of the juices. It has been established that the hydrate water can freeze at about -81”C, although theoretically this could take place even at - 120°C (Sedikh and Sedikh, 1967). Investigations of the modulus of elasticity and the destructive impact tension in meat at very low temperatures show that at temperatures from - 196”to - 120°C these two values remain constant, after which they decrease down to -80°C. This phenomenon can be explained by the complete freezing of the chemically bound water at temperatures below - 120°C (Gurvits and Prichedko, 1969). Love and Elerian (1965) have investigated the degree of denaturation of albumin and have proved that the bound water in fish muscles freezes completely at - 180°C. Riedel (1964) supports this finding; he believes that the phenomenon observed by the authors is a “release” of the bound water at very low temperatures. Popovski (1975) points out that a progressive increase in the amount of bound water in some fruit purees (red tart cherry, plum) could be observed at temperatures down to -1OO”C, in a range far beyond the eutectic temperature. The author associates this fact with the freezing of part of the bound water, and he assumes that the rest of it does not freeze at all. Fikiin and Guegov ( 1972b) and Guegov (1978) have observed comparatively distinct phenomena of vitreous transformation and divitrification in apple concentrate (50%), in orange concentrate (53.5%), and in peach concentrate (24%). In all three products, these phenomena take place in the interval -120” to - 110°C.

B . RECRYSTALLIZATION, POLY MORPHOUS TRANSFORMATIONS, AND VITREOUS MELTING From the three forms of recrystallization known-spontaneous (“eruptive”), migratory, and accretive (of growth)--only the second and third have been observed in the superlow temperature region (Fennema and Powrie, 1964; Lozina-Lozinski, 1972; Luyet, 1960). Migratory recrystallization is often considered as the growth of the larger ice crystals at the expense of the smaller ones in the temperature range in which moisture is completely solidified It takes place at constant temperature. In pure ice it can be observed at temperatures between -70” and -96°C (Mathlouti, 1974; Meryman, 1960). It is more typical of objects frozen at high velocities and in the presence of a large number of small crystals. Like every recrystallization,

PHASE TRANSITIONS OF WATER

349

this one also is not associated with any thermal effects. It can be observed by microscope (Lozina-Lozinski, 1972; Luca and Lachmann, 1973; Luyet, 1957; Mathlouti, 1974; Meryman, 1960). At superlow temperatures accretive recrystallization takes place when the temperature changes. It is important during a long storage period, but it does not play a significant role during the process of freezing and rewarming. In its nature it represents the binding of neighboring ice crystals (Lozina-Lozinski, 1972; Luyet, 1960; Mathlouti, 1974; Meryman, 1960). In biological objects, as a result of the presence of membranes, the effect is weaker, especially at temperatures below -70°C (Meryman, 1957). The transition of vitreous into cubic ice can be explained by the theory of ice formation, but the literature contains almost no communications concerning similar transformations in food solutions or biological media and materials. Rey ( 1959b) presumes that a similar phase transition with an insignificant exothermic effect takes place in glycerol solutions. A phase transition, identical in character and temperature interval in 50% glycerol solution, was observed by Luyet and Rasmussen ( 1968). They named this transition "postdevitrification, " without giving any explanation of its nature. The transition into cubic ice, according to Luyet (1960), can be observed in some other aqueous solutions in the range of - 150" to -80°C or from - 120" to - 100°C. Vitromelting means the transition of vitreous ice into a liquid. Being a phase transition, upon warming it follows the vitreous transformation. To bring about this transition, it is necessar that the rewarming be ultrafast-several thousand degrees per second. So far only Luyet (according to Smith, 1954) has offered proof concerning the course of the process in thin membranes of ultrafast-frozen protein, sucrose, and gelatin solutions. This problem is of purely theoretical interest.

VI.

CONCLUSION

This survey of the literature demonstrates the extreme importance of the investigations of the phase transitions at negative temperatures-from the theoretical point of view, as well as for the practical aims of refrigeration technology. These investigations are of special importance for food products of plant origin, containing large amounts of water. The physical transformations connected with the phase phenomena of water are, for this group of products, of utmost significance as far as their quality during freezing is concerned. The picture of ice formation in fruits, vegetables, juices, and concentrates is complicated by the influence of several factors: their complex chemical composition, their special structure, the presence of crystallization inhibitors, the appearance of metastable phases, the formation of complex eutectic mixtures, the solidification of part of the material

350

YANCOGUEGOV

into vitreous structures, and, finally, the presence of a certain amount of unfrozen water (very often constituting a substantial part). The review of the literature on water as a basic structural element in plant tissues shows that the structure and physical characteristics of pure water and ice have been well studied and can be used as the basis for an interpretation of the phase transitions during the freezing of food products of plant origin. During freezing by means of modern technical equipment and units applied in the refrigeration technology, it is theoretically possible for three polymorphous forms of the ice in the product to be obtained: hexagonal, vitreous, and cubic. An analysis of the process of freezing food products should always take into consideration the forms of binding of the moisture with the material. A certain part of the water in ion dispersion systems, containing hydrophilic components, is retained firmly by these components and thus influences the process of ice formation. In food products of plant origin, the water possesses a different binding energy and will be expected to freeze in the temperature range of 0" to -160°C. A certain amount of bound water (probably the monomolecular layer) does not freeze even at very low temperatures. To establish the temperature boundaries within which the different types of bound water either crystallize or solidify is a very important task for the future of food technology, from both the theoretical and the practical point of view. In model solutions of natural chemical components of food products of plant origin-carbohydrates, mineral salts, organic acids, and hydrocolloids-various phase transitions take place in the temperature range -196" to 0°C: vitreous transformation and devitrification (crystallization), recrystallization, antemelting, and eutectic and progressive melting. Analysis of the phase transitions during the freezing of model solutions and biological media and objects allows for the identification and interpretation of the phenomena that occur during the freezing of complex systems such as food products of plant origin. The establishment of an optimum working temperature zone of sublimation during the freeze-drying of food products is of great interest. A survey of the current literature indicates the importance of making a precise choice of the sublimation parameters for each individual product and confirms the feasibility of determining them experimentally. In food products of plant origin, various phase transitions at negative temperatures are possible: vitreous transformation, devitrification, melting, recrystallization, and polymorphous transformations. The nature of these phenomena can best be identified by analogy with the behavior of the model systems investigated. Four "zones" of thermal behavior have been established: a relative thermodynamic stability (below -75"C), a thermodynamic instability (from -75" to

PHASE TRANSITIONS OF WATER

35 1

-46"C), an initial melting (from -46" to -15.5"C) and an intensive melting (below - 15.5"C). The free water freezes completely in the interval from -26.5" to -46"C, and in our opinion these are the temperature boundaries within which the eutectic temperatures should be sought. All phase transitions typical of the model systems and of their chemical components can be observed. Many studies provide evidence of the formation of vitreous structures during the freezing of these foods. In products with a soluble solids content below 50% the formation of such structures takes place at temperatures from -40" to -75"C, and with a content above 50%, at -60" to -90°C. At temperatures below -155°C no phase transformations of the water have been observed. The experimental results obtained by several authors during the investigation of the phase transitions in food products of plant origin help us to draw the following general conclusions concerning freeze-drying: The various foods intended for freeze-drying should be frozen to the following temperatures: fruits, from -35" to -45°C; vegetables, from -25" to -40°C; fruit juices, from -40" to -60°C; vegetable juices, from -30" to -45°C; and fruit and vegetable concentrates (with a soluble solids content above 40%), from -40" to -55°C. The sublimation of the various fruits should be carried out in the temperature range from -20" to -40°C; for vegetables, from - 15" to -35°C; for fruit juices, from -25" to -45°C; for vegetable juices, from -20" to -35°C; and for fruit and vegetable concentrates (with a soluble solids content below 400%), from -25" to -45°C. Specific temperature parameters of freezing and drying in these temperature intervals should be established each product. Food products of plant origin subjected to freeze-drying should have a soluble solids concentration no higher than 40%, with a sublimation temperature above -45°C (considering the technical possibilities of modem industrial freeze-drying equipment). A significant amount of vitreous structures was established in the frozen product when a higher solids content was present, rendering the drying process more difficult and imposing quite lower sublimation temperatures. This literary survey shows that there are problems requiring further elucidation-for example, the degree of eutectic freezing in the living matter, the types of eutectic mixtures participating in this freezing, and the temperature ranges and conditions required for these mixtures to solidify. The widespread concept concerning the cryohydrate point of food products should be revised, and methodical investigations should be initiated, taking into consideration the various forms of binding of the moisture to the material. The phase transitions in the superlow temperature interval are still insufficiently investigated. These transitions are mainly of theoretical interest for the food technologist, but they are an important factor in determining the suitability of some food products of plant origin (juices and concentrates) to be freeze-dried.

352

YANCO GUEGOV

No general theory on supercooling and incipient crystallization of the ice in food products has been submitted as yet. The factors on which these processes depend are numerous, and the relationships between them are extremely complex. The cryoscopic temperature of fruit and vegetables is determined basically by their chemical composition, and first of all by their soluble solids content. Experimental trials on incipient crystallization are necessary for each plant variety so that scientific material can be accumulated and empirical and semiempirical formulas for the influence of some dominating factors can be obtained. The numerous theoretical and experimental studies discussed in this survey support the concept that the analysis of the phase transformations of water at temperatures below the cryoscopic one is the best method for the choice of the final temperature of freezing and of the temperature range of sublimation during freeze-drying. The investigations of the phase transitions during ice formation in food products give us the opportunity to support, from the thermodynamic point of view, the temperature parameters indicated.

REFERENCES Amoignon, J., and Floch, L. 1971. Principes generaux de la lyophilisation des produits biologiques et pharmaceutiques. Vide 26 (156), 217-226. Barnes, W. H. 1929. The original structure of ice between 0°C and - 183°C. Proc. R . Suc. London, Ser. A 125, 670-693. Bayer, V. 1962. “Biophizika” (“Biophysics”). Inostrannaya Literatura, Moscow. Bellows, E. J . , and King, I . C. 1972. Freeze-drying of aqueous solutions: Maximum allowable operating temperatures. Cryobiology 9, 559-561. Blair, J. M. 1966. Temperature control during freeze-drying. ASHRAE J . 7 , 51-54. Bohon, R. L.. and Conway, W. T. 1972. DTA studies on the glycerol water system. Thermochim. Acra 4, 321 -341. Bonjour, E., Couach, M., and Simatos, D. 1975. Etude de la cristallisation de solutions aqueuses de serum lyophilie au moyen de I ‘analyse calorimetrique differentielle. Bull. Insr. Inr. Froid 55(4), 1163. Corvazier, R. 1969. Quelques aspects prltiques de la congelation prealable a la lyophilisation de produits biologiques. Rev. Gen. Froid 60(2), 179-185. Couach, M.,Moreira, T., Pemp. M., Bonjour, E., and Simatos, D. 1977. Etudes sur I’etat physique de solutions de sucres a hasse temp&ature, en relation avec I’affaissement de structure en lyophilisation. Annexe Bull. Insr. Int. Froid 1, 475-486. curdova, M. 1969. Bestimmung der eutektischen Punkten der Gewebeflussigkeit in einigen Lebensmitteln pflanzlichen Ursprung. Sh. Vys. Sk. Chem.-Techno/. P raze, Porraviny E24, 21-31. Danilov, A . M. 1974. “Kholodil’naia tekhnologia. pistchtevikh produktov” (“Refrigeration technology of foods”). Vicha Shkola, Kiev. Davies, J . D. 1966. Thermal analysis in freezing and freeze-drying. In “La lyophilisationRecherches et applications nouvelles” (L. Rey, ed.), pp. 9-20. Hermann, Paris. Dickerson, R. W. 1968. Thermal properties of foods. In “The Freezing Preservation of Foods” (D.

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K. Tressler, W. B. van Arsdel, and M. J. Copley, eds.), Vol. 2, pp. 26-51. Avi, Westport, Connecticut. Djafarov, A. F. 1974. “Tovarovedenie plodov i ovostchtei” (“Fruits and vegetables-general aspects”). Ekonomika, Moscow. Djangaliev, A. D., and Tsziu, A. L. 1969. “Hranenie iablok, iagod i vinograda” (“Storage of applies, berries and grapes”). Kainar, \ha-Ata. Djeneev, S. Iu. 1968. “Hranenie fruktov i ovostchtei v sovkhosakh i kolkhozakh” (“Fruit and vegetable storage in the sovchoses and kolchoses”). Kolos. Moscow. Dousset, M. 1964. Lyophilisation en milieu non-aqueux. These de Docteur, Universitk de Dijon, Dijon. Duckworth, R. B. 1971. Differential thermal analysis of frozen food systems. I. The determination of unfreezable water. J . Food Techno/. 6(3), 3 17-327. Dumanski. A. V. 1949. Hydrophilness of the colloidal systems and its theoretical value. In “Koloidy v pistchtevoi promychlennosti” (A. V . Dumanski, ed.), pp. 8-20. Pistchepromizdat, Moscow. Dumanski, A. V . 1960. “Liofil’nost dispersnykh sistem” (“Lyophilness of disperse systems”). Akad. Nauk USSR, Kiev. Eisenberg, D., and Kauzamann, W. 1975. “Struktura i svoistva vody (“Structure and characteristics of water”). Gidrometeoizdat, Leningrad. Farrant, I . , and Woolgar, A. E. 1970. Possible relationships between the physical properties of solutions and cell damage during freezing. In “The Frozen Cell” (G. E. Wolstenholme and M. O’Connor, eds.), pp. 97-1 19. Churchill, London. Fateeva, M. V. 1970. About the “eutectic temperature” of frozen, rewarmed and freeze-dried yeast suspensions. Mikrobiologia 39(6), 958-964. Fennema, 0.. and Powrie, W. D. 1964. Fundamentals of low-temperature food preservation. Adv. Food Res. 13, 219-347. Fikiin, A. 1962. About the cryoscopic temperature of foods. Nautschni Tr. VIHVP. Plovdiv 9, 177- 182. Fikiin, A. 1969. Thermodynamical phenomena in food media at low and superlow temperatures (0 to -196°C). Naurschni Tr. NIIKP, Plovdiv 6, 109-128. Fikiin, A. 1973. “Hladilni tekhnologitschni protsesi i saorajeniia” (“Refrigeration technology processes and equipment”). Hr. G. Danov, Plovdiv. Fikiin, A,, and Guegov, J . 1971a. Thermophysical phenomena in fruit tissues at low and superlow temperatures (0 to -196°C). Nautschni Tr. VIHVP, Plovdiv 18, Part 111, 213-228. Fikiin. A., and Guegov, J. 1971b. Thermophysical phenomena in vegetable tissues at low and superlow temperatures (0 to -196°C). Naurschni Tr. VIHVP, Plovdiv 18, Part 111, 229-238. Fikiin, A , , and Guegov, J. 1972a. Thermophysical phenomena in fruit and vegetable juices at low and superlow temperatures (0 to - 196°C). Nautschni Tr. NIIKP, Plovdiv 9, 39-56. Fikiin, A,, and Guegov. J. 1972b. Thermophysical phenomena in fruit and vegetable concentrates at low and superlow temperatures (0 to -196°C). Nautschni Tr. NIIKP, Plovdiv 9, 57-74. Fikiin, A,, and Kuzmanova, E. P. 1970. Crioscopic temperature of grapes depending on the cell juice concentration. Naurschni Tr. VIHVP, Plovdiv 17, Part 11, 19-23. Flink, J. M. 1974a. Application of freeze-drying for preparation of dehydrated powders from liquid food extracts. I n “Freeze-Drying and Advanced Food Technology” (S. A. Goldblith, L. Rey, and W. W. Rothmayr, eds.), pp. 309-329. Academic Press, New York. Flink, J. 1974b. The influence of freezing conditions on the properties of freeze-drying coffee. In Freeze-Drying and Advanced Food Technology” (S. A. Goldblith, L. Rey, and W. W. Rothmayr, eds.), pp. 143-160. Academic Press, New York. Forrest, J. C. 1963. Development of the accelerated freeze-drying process. In “Freeze-Drying of Foodstuffs” (S. Corson and D. B. Smith, eds.), pp. 30-49. Columbine Press (Publishers), Ltd., London.

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Simatos, D. 1976. Structure du produit pendant la congelation. la dessication et a I’etat lyophilid. Bull. Inst. Int. Froid 56( I ). 227. Simatos, D., Blond, G . , Dauvois, P., and Sauvageot, F. 1975a. “La lyophilisation. Principes et application. Association Nationale de la Recherche Technique, Paris. Simatos, D.. Faure, M., Bonjour, E.. and Couach, M. 1975b. The physical state of water at low temperatures in plasma with different water contents as studied by differential thermal analysis and differential scanning calorimetry. Cryobiology 12(3), 202-208. Smith, A. U. 1954. Effects of low temperature on living cells and tissues. I n “Biological Application of Freezing and Drying” (R. J. C. Harris, ed.), pp. 7-86. Academic Press, New York. Smith, A. U. 1962. “Biological Effects of Freezing and Supercooling.” Williams & Wilkins, Baltimore, Maryland. Steinbach, G . 1977. Phase equilibria in frozen solutions from refractometric measurements of freezing curves. Anexe Bull. Insr. Int. Froid 1, 53-66. Stephenson, J . L. 1960. Fundamental physical problems in the freezing and drying of biological materials. In “Recent Research in Freezing and Drying” (A. S. Parkes and A. U. Smith, eds.), pp. 121-145. Blackwell, Oxford. Stricker, P. 1970. Das Einfrieren der zu lyophilisierenden Arzneimittel. Kaclre (Hamburg) 23( lo), 511-516. Susmann, M. V., and Chin, L. 1966. Liquid water in frozen tissue: Study by nuclear magnetic resonance. Science 151( I ) . 324-325. Tchernichev, V. M., Babkin. A. F., Golovkina, T. N., Serajutdinov, L. D., and Makaveeva, G. J. 1974. Application of the nuclear proton relaxation method as a characteristic of the freezing processes in foods. Kholod. Tekh. 51(4), 30-35. Tchijov, J. B. 1956. “Voprosy teorii zamorajivaniia pistchevych produktov” (“Theoretical problems of food refrigeration”). Pistchepromizdat, Moscow. Tchijov, J. B. 1970. “Formirovanie kristalov d’la pistchevych produktov pri ikh zamorajivanie” (“Formation of ice crystals in foods during freezing”). TsNIITEl, Moscow. Tchijov, J . B. 1971. “Teplofizitscheskie protsessy v kholodil’noi tekhnologii pistchevych produktov” (“Thermophysical processes in food refrigeration technology”). Pistchevaia promychlennost’, Moscow. Tchubik, I. A., and Maslov, A. M. 1970. “Spravotschnik PO teplofizitscheskim kharakteristikam pistchevykh produktov i polufabrikatov” (“Manual on the thermophysical characteristics of foods and semifinished products”). Pistchevaia promychlennost ’, Moscow, Tobback, P., and Maes, E. 1971. Nieuwetechnicken in de levensmiddelen konservering; de lyofilisatie: Teoretische aspekten. Agricultura Nos. 1-2, pp. 1-24. Tobback, P. P., Swinnen, J.. and Maes, E. 1979. Relation between interstitial fluid stability and volatiles retention in freeze-dried model systems (sugar-salt solutions). Int. J. Refrig. 2(4), 206-210. Toledo, R., Steinberg, M. P., and Nelson, A. J. 1968. Quantitative determination of bound water by NMR. J. FoodSci. 35(3). 315-317. Tsinman, M. M., and Ianiuk. V. Ia. 1969. “Kholodil’niki dlia fruktov” (“Fruit cold stores”). Pistchevaia promyschlennost’, Moscow. Tsurinov, G . G. 1955. Differential thermal and dilatometric analysis at low temperatures and with small amounts of substances. T r . Soveshch. Termogrufi pp. 116-120. Tucker, C. G. 1963. Freeze-drying of vegetables and fruit. In “Freeze Drying of Foodstuffs” ( S . Cotson and D. B. Smith, eds.), pp. 111-1 18. Columbine Press (Publishers), Ltd.. London. Vinograd-Finkel, F. R., Kisilev. A. E., Ginsburg, F. G., Fedorova, L. I . , and Kudriachova, P. N. 1973. Preservation of blood in ultralow temperature. C.R. Congr. Inr. Froid, 13th. 1971 Vol. Ill, pp. 799-805. ”

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B Bioflavonoid, as drugs, 174-181

C Calcium, in mechanically deboned red meat, 84-86 Carotenoid, in passion fruit juice, 277-281 Cholesterol content in mechanically deboned fish, I I 7- I I9 in mechanically deboned poultry, 117-1 19

of processing variables, 120- I22 of storage, 122-123 Food toxicant, see Phenol Freeze-drying, temperature working zone for, 33 I Fruit boundary temperatures for freeze-drying, 335 differential thermograms for, 334 supercooling, 307-3 15 cryoscopic temperature of, 3 10-3 15 Fruit juice, phase transition temperature of, 339

G

D Gallic acid, animal consumption of, 170- 172 Deboning, see Mechanically deboned fish; Mechanically deboned poultry; Mechanically deboned red meat

E Edulan. in passion fruit juice, 277-279 Ellagic acid, animal consumption of, 170-172 Eutectic freezing, 3 16-320

F Fish, see Mechanically deboned fish Flavonoid, 173-191 as drugs, 174-181 metabolism, 184-191 mutagenicity of, I8 1 - I84 nutritional aspects, 173-174 toxicity, 173- 174. 184- 191 Flavor impact value, for passion fruit juice, 282-287 Flavor stability effect of antioxidants, 125-126 of carbon dioxide cooling, I26 of heme pigments, 123-125 of nitrogen cooling, 126

H Hydroxycinnamate, as food toxicant, 167- I70

I Ice phase diagram, 302-307 physical characteristics, 302 StNCtUR, 301 -302 Iron. in mechanically deboned red meat, 86-88

L Lignin chemical characteristics, 208 dietary fiber and, 209-210 Lipid, content, in mechanically deboned red meat. 78-84

M Mechanical deboner, types, 1 I I Mechanical deboning offish, 110-111 ofpoultry, 110-111

363

364

INDEX

Mechanically deboned fish bone particle content, 119-120, 141 bone residue, utilization, 137-138 calcium content, 141 cholesterol content, 141-142 color stability, 127-129 myoglobin and, 128-129 processing variables, influence of, 127- 128 fatty acid content, 117-1 19 flavor stability, 120- I29 effect of antioxidants, 125- 126 of carbon dioxide cooling, 126 of heme pigments, 123- 125 of nitrogen cooling, 126 of processing variables, 120- I22 of storage, 122- I23 fluorine content, 141 functional characteristics, 129- 136 effect of composition. 129- I32 of food additives, 134- I36 of processing variables, 132- 134 heme pigments, 120 lipid content, 141-142 microbial quality, 138- 140 mineral composition. I 15- I I 7 nutritive properties, I I I - 120 proximate composition, 1 13- I 15 structural characteristics. 1 I 1 - 1 13 protein content, 141 protein quality, 1 17 regulations for, 140 research needed, 142-143 Mechanically deboned poultry bone particle content, 119-120. 141 bone residue, utilization, 137- I38 calcium content, 141 cholesterol content. 117-1 19. 141-142 color stability, 127-129 myoglobin and, 128- 129 processing variables, intluence of, 127-128 fatty acid content, I 17- I I9 flavor stability, 120-129 effect of antioxidants. 125-126 of carbon dioxide cooling, 126 of heme pigments, 123-125 of nitrogen cooling. 126 of processing variables, 120- 122 of storage. 122-123 fluorine content, 141 functional characteristics. 129- I36

effect of composition. 129- I32 of food additives, 134- I36 of processing variables, 132- 134 heme pigments, 120 lipid content, 141-142 microbial quality, 138-140 mineral composition, 1 15- 1 I7 nutritive properties. I 1 1-120 proximate composition, I 13- I I5 structural characteristics, 1 I 1 - 1 13 protein content, 141 protein quality, 117 regulations for, 140 research needed, 142-143 Mechanically deboned red meat, 23-95 bone fragments in, 56-62 calcium content, 84-86 composition of, 39-53 fat, 3 9 4 2 mineral content, 42-56 antimony, 50 arsenic, 5 1 ash, 42-43 cadmium, 50-51 calcium, 42-43 cobalt, 49-50 copper, 49-50 fluoride, 51-53 iron, 49 lead, 50 magnesium, 48 mercury, 51 nickel, 49-50 phosphorus, 43. 48 potassium. 48-49 selenium, 50-5 I sodium, 48 tin, 50 zinc, 49 moisture content, 39-42 protein, 39-42 cost impact, 37 definition, 23-24 development of, 26 economic implications, 35-39 employment, 37 social effects, 38-39 functional properties of, 68-74 color, 68-70

INDEX emulsion characteristics, 70-72 pH and water-holding capacity, 72-74 iron content, 86-88 lipid make-up, 78-84 mineral toxicity of, 53-56 fluoride, 54-55 lead, 54 strontium-90, 55-56 palatability. 88-93 and oxidative stability, 88-90 sensory characteristics, 90-93 potential yield, 26-3 I from different meat sources, 28 process of, 24-25 protein quality, 74-78 regulations governing, 3 1-35 in Australia, 32 in Denmark, 32 in the United States, 33-35 U.S. Department of Agriculture and, 33-35 research needs, 93-95 safety, 53-68 and bone particle size, 56-62 microbiological properties, 62-65 mineral toxicity, 53-56 nucleic acids, 65-68 Megastigmatriene, in passion fruit juice, 27928 1 Mineral toxicity, of mechanically deboned red meat, 53-56

N Nucleic acid, content, of mechanically deboned red meat, 65-68 Nutritional value of mechanically deboned fish, 1 11-120 of mechanically deboned poultry, 110-120 of mechanically deboned red meat, 74-88 protein quality, 74-78

P Palatability, of mechanically deboned red meat, 88-93 Passion fruit juice composition, 259-265 acids, 262-263 alkaloids, 263-264 amino acids, 264

365

carotenoid pigments, 264 cyanogenic constituents, 265 enzymes, 265 sugars, 262 concentration of, 265-267 evaporators, selection of, 265-266 powders, 268-269 quality, 267 by reverse osmosis, 268 volatile flavoring, recovery of, 267-268 development, research needs, 288-290 preservation, 257-259 from pulp, 251-252 starch properties of, 255-257 removal of, 252-255 volatile flavors, 269-28 1 carotenoids, 277-28 I edulans, 277-279 ionone derivatives, 28 I megastigmatrienes, 279-28 I effect of fruit maturity, 287 of fruit variety, 285-287 of processing, 287-288 esters, 277 flavor impact values, 282-287 sulfur compounds, 28 1-282 Passion fruit pulp extraction, 247-25 1 by centrifugal extractor, 247 by converging cone extractor, 247-250 by passypress extractor, 250-25 1 preservation, 257-259 Phase transition during freezing and rewarming of foods, 326-343 conditions during freezing, 329-343 eutectic temperature, 326-329 during freezing and thawing of model systems, 315-326 antemelting and recrystallization, 324-326 eutectic freezing, 316-320 vitreous structures, 320-324 at superlow temperatures, 344-349 recrystallization, 348-349 vitrification, 344-348 Phase transition temperature in fruit and vegetable juices, 339 Phenol, see also Ellagic acid; Flavonoid; Gallic acid; Hydroxycinnamate; Lignin; Tannin

366

INDEX

Phenol (conr.) animal adaptation to, 157- 158 animal consumption of, 162-210 ellagic acid. 170-172 flavonoids, 173-191 gallic acid, 170- I72 hydroxycinnamate, 167- I70 lignins, 208-210 tannins, 191-208 animal metabolism of, 158-162 biosynthesis, 151-153 evolution, 153-154 as nutrients, 166- 167 of plant origin, in foods, 140-221 from plants, 154-157 pyrolysis products, 163-166 toxicity, mechanisms of, 210-218 carcinogenicity, 216-21 8 mimic-interaction with metabolism, 21 1212 mutagenicity, 216-218 nutritional load, 212-214 penetration effects, 214-215 protein binding, 215 uncoupling of oxidative phosphorylation, 216 Plant tissue moisture distribution. 303-307 water content. 299 Poultry. see Mechanically deboned poultry Prescott, Samuel Cate, 1-20 books written by, 15, 17-18 Boston Bacteriological Club and, 15 career, early, 5-7 contemporaries, 12-13 contributions to chemistry of coffee, 10- I I to refrigeration and freezing of foods, 8- 10 to thermal processing, 7-8 education, 3-5 honors, awards, and public service, 14-16 Institute of Food Technologists, and, I 1-12 Massachusetts Institute of Technology, and, 16-18

Underwood. William Lyman, work with, 7-8 World War I , during and after, 8 Protein

quality of mechanically deboned fish, 117 of mechanically deboned poultry, 117 of mechanically deboned red meat, 74-78

R Red meat, see ulso Mechanically deboned red meat mechanical deboning of, 23-95

T Tannin, 191-208 animal toxicity of, 195-204 carcinogenesis and, 206-208 chemical behavior, 191-194 medical use. 205-206 metabolism, 203-204 microbial inhibition by, 194

v Vegetable boundary temperatures for freeze-drying, 336 supercooling, 307-315 cryoscopic temperature of, 310-3 15 Vegetable juice, phase transition temperature of, 339 Volatile flavoring, of passion fruit juice, 26928 I W Water absorptionally bound, 304 capillary-bound. 305 chemically bound, 304 content, of plant tissue, 29Y ice. structure. 301 -302 moisture distribution in plants, 303-307 phase diagram, 302-307 physical characteristics, 302 osmotically retained moisture, 305-307 structure, 299-300 supercooling in foods, 307-315 cryoscopic temperatures, 3 10-3 I5 nucleation and growth of crystals, 30H-310

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